News'n'Clues - SCIENCE Article Summaries

Defining the vascular niche of human adipose tissue across metabolic states

Source: {'href': 'https://www.nature.com', 'title': 'Nature'} Published: 2026-03-12 12:45:00

Heteronuclear dual-atom insertion into oxetanes via frustrated Lewis pair activation

Source: {'href': 'https://www.nature.com', 'title': 'Nature'} Published: 2026-03-12 12:06:27

Uh, Scientists Have Significantly Miscalculated Earth's Sea Levels

Source: {'href': 'https://www.popularmechanics.com', 'title': 'Popular Mechanics'} Published: 2026-03-12 12:00:00

After analyzing 385 studies related to coastal areas and sea level rise, scientists found a significant discrepancy between geoid measurements and actual sea levels, especially in the global south. We may earn commission if you buy from a link. Science is only as good as the foundation of data it's built upon, and a new study from researchers at Wageningen University in the Netherlands suggests that estimates of future sea level rise may have been worryingly underestimated. But the new data snafu stems instead from an inconsistency between how we measure sea levels in science research broadly. Geoids are handy mathematical models that calculate global mean sea level based on gravity and Earth's rotation. Because Earth isn't a perfect sphere, these models help scientists accurately calculate sea level when conducting science along coastal areas—or at least, that's what we thought. A new study published in the journal Nature took a closer look at that assumption by analyzing hundreds of scientific research publications that used geoid models while conducting research along coastlines around the world. One of the big limitations of geoid models is that they assume a calm ocean, which effectively underrepresents important dynamics like winds, tides, and currents. In Northern Europe and the United States—where seas are generally calmer and scientists have more data sources for sea level rise—these discrepancies are minuscule, but in other parts of the world like Southeast Asia and the Indo-Pacific, the gap between assumed sea levels and real sea levels is cause for concern. “Researchers who study land elevation or sea levels try to make their elevation models as accurate as possible,” Wageningen University's Philip Minderhoud, who co-authored the study with his colleague Katharina Seeger, said in a press statement. “Most researchers […] seem to be unaware that it is necessary to use and correctly align measurements of both land and the sea when performing coastal impact assessments.” Minderhoud first became suspicious of geoid model accuracy while conducting research in Vietnam's Mekong Delta in 2015, after discovering that the delta (one of the largest in the world) was surprisingly lower than geoid models suggest. He published these findings in the journal Nature Communications in 2019, writing at the time that “our results imply major uncertainties in sea-level rise impact assessments for the Mekong delta and deltas worldwide, with errors potentially larger than a century of sea-level rise.” That instinct proved correct, as Seeger similarly found inaccuracies while conducting her Ph.D. research along the Ayeyarwady Delta in Myanmar. While such systematic mismeasurement has potentially disastrous implications for these watery delta regions, the study also found that the reverse can be true—sea levels in Antarctica, for example, are lower than scientists assumed. “That is how science works,' Minderhoud said in a press statement. “Now that we have discovered this blind spot, the scientific community can make more accurate assessments for coastal areas and cities around the world.” Earth Will Lose Its Gravity, Says This Wild Theory

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Source: {'href': 'https://www.nature.com', 'title': 'Nature'} Published: 2026-03-12 10:59:39

You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues. Live biological tissues are dynamic by nature. Thanks to various chemical and genetically encoded fluorescent biosensors, we can image and measure dynamic biological phenomena within the live tissues and organs using fluorescence microscopy. However, the imaging depth is often limited by tissue opacity. It has been a long-standing challenge to make live and healthy biological tissues transparent to facilitate live imaging. Two-photon microscopy uses a near-infrared excitation laser instead of visible light to reduce light scattering; however, the imaging depth is limited to a few hundred microns in mammalian tissues in vivo1. Adaptive optics uses a deformable mirror or spatial light modulator to correct aberrations caused by macroscopic refractive index distortions, but it is not effective for highly scattering samples2. For fixed tissues, optical clearing is a powerful approach: light refraction and scattering are minimized by removing high-index components (for example, lipids) and/or by immersing the sample in high-index solutions with refractive indices of 1.43–1.55 (refs. However, most of the clearing agents developed for fixed tissues are toxic to live cells. Less toxic chemicals (for example, glycerol, dimethyl sulfoxide and sugars) have been tested for highly fibrous extracellular structures, such as skin and skull in vivo14,15,16,17,18,19; however, these chemicals interfere with cellular functions. A recent study claimed to have achieved optical clearing of the skin in live animals using a strongly absorbing dye, such as tartrazine18. Therefore, live mammalian cells and tissues have not yet been rendered transparent while maintaining intact cellular functions. Some chemicals have been proposed to be compatible with live cell imaging. Iodinated contrast agents were attractive candidates because of their low osmolarity. However, its toxicity to mammalian cells has not been fully evaluated. Another study attempted to improve transparency of the mouse brain by adding glycerol to drinking water22. However, it is unclear whether the marginal change in transparency was due to an increase in the refractive index in the brain, as glycerol should be easily metabolized once absorbed in the gut. Here we developed SeeDB-Live, a tissue-clearing medium for live mammalian cells and tissues. SeeDB-Live contains bovine serum albumin (BSA), which has exceptionally low osmolarity when dissolved in water and is minimally invasive to live cells. SeeDB-Live improved the imaging depth of spheroids, organoids, acute brain slices and the mouse brain in vivo. Light scattering in tissues is caused by refractive index mismatch between the light scatterer and the medium. Previously, simple immersion-based clearing agents (refractive index, 1.46–1.52) have been developed (for example, fructose, iohexol and tartrazine)8,9,18; however, osmolarity of these clearing agents is extremely high. To make live tissues transparent under isotonic conditions, we would have to use either (i) membrane-permeable or (ii) membrane-impermeable low-osmolarity (that is, high molecular weight) chemicals to reduce the refractive index mismatch (Fig. We have listed membrane-permeable and membrane-impermeable high-molecular-weight chemicals as candidates. Candidate chemicals also need to be highly soluble in water. These chemicals demonstrated a concentration-dependent increase in refractive index when dissolved in water (Extended Data Fig. a, Strategies for optical clearing of live cells. b, Transmittance (at 600 nm) of HeLa cell suspension (4 × 106 cells per ml) in isotonic saline solution with glycerol or iodixanol at different refractive indices. Fixed cells were treated with PFA and saponin. c, Calcium imaging of GCaMP6f-expressing HEK293T cells stimulated with 50 μM ATP. Osmolarity was not adjusted to isotonicity. Data are the median ± interquartile range (IQR). d, The osmolality of candidate chemicals in aqueous solution (refractive index 1.365, in double-distilled water (ddH2O; n = 3 each). Sucrose was used as a control. The osmolality of low-salt BSA (2) was 2.7 mOsm kg−1, consistent with its molar concentration (2.3 mM). e, The optimal refractive index of the extracellular medium was determined in PBS adjusted at different osmolalities. Transmittance of live HeLa cell suspensions (4 × 106 cells per ml) was measured. Left: optimal refractive index (1.369) of iodixanol-containing PBS. Right: optimal refractive index (1.363–1.369) of BSA-containing PBS (n = 3 each). f, Phase contrast images of live HeLa cells in normal and BSA-containing medium (refractive index, 1.363). g, Proliferation curve of HeLa/Fucci2 cells in iodixanol, Ficoll70 and BSA#1-containing medium (refractive index, 1.363; 320 mOsm kg−1). P values are <0.001 unless otherwise mentioned. h, Growth ratio in refractive index-optimized (refractive index, 1.363; 320 mOsm kg−1) medium compared to the control medium. P values are <0.001 unless otherwise mentioned. i, Phase contrast images of HeLa/Fucci2 cell spheroids under normal (left) and SeeDB-Live culture medium (refractive index 1.366, 320 mOsm kg−1; right). j, Growth curve of HeLa/Fucci2 cell spheroids with and without treatment with SeeDB-Live for 4 h per day. NS (P ≥ 0.05; two-sided Wilcoxon rank-sum test combined with Holm–Bonferroni correction). k, Three-dimensional (3D) confocal images of a HeLa/Fucci2 cell spheroid. l,m, Intestinal organoids in Matrigel treated with SeeDB-Live (refractive index, 1.363) for 4 h per day. m, Growth of the intestinal organoids. NS (P ≥ 0.05; two-sided Wilcoxon rank-sum test combined with Holm–Bonferroni correction). n, 3D confocal images of GCaMP6s-expressing EECs in intestinal organoids from ePet-Cre; Ai162 mice before and after SeeDB-Live treatment. o,p, Calcium imaging of cortical organoids (confocal). Basal fluorescence (temporal median; left) and ΔF/F0 images (right) of a cortical organoid labeled with a calcium indicator, Calblyte-650AM (o). Spontaneous calcium transients of neurons (p). Data with error bars represent the mean ± s.d. See Supplementary Table 4 for detailed statistical data. MW, molecular weight; PG, propylene glycol; PEG, polyethylene glycol; HBCD, hyperbranched cyclic dextrin; PVP, polyvinyl pyrrolidone. Next, we sought to determine the optimal refractive index for clearing live mammalian cells. For this purpose, we prepared a suspension of live or paraformaldehyde (PFA)-fixed and membrane-permeabilized HeLa cells (4 × 106 cells per ml). The refractive indices of media used to clear fixed tissues are typically 1.43–1.55 (ref. We tested a membrane-permeable chemical, glycerol, up to a refractive index of 1.43 (~66% wt/vol); however, it was not effective for live mammalian cells (Fig. We also tested a membrane-impermeable chemical, iodixanol; to keep the osmolarity of the buffer isotonic, we mixed isotonic iodixanol solution (60% wt/vol) and phosphate buffered saline (PBS) to prepare isotonic solutions with different refractive indices. PFA-fixed and membrane-permeabilized HeLa cells were most transparent at a refractive index of ~1.42. Paradoxically, however, we found that the live HeLa cells become most transparent at an extracellular refractive index of ~1.37, much lower than the optimal index for fixed cells (Fig. Moreover, the optimal range of the refractive index for live cells was relatively narrow; the transparency of live cells became lower at higher refractive indices (>1.38). We next examined whether intracellular functions remain intact in the presence of candidate chemicals. Using the GCaMP6f calcium indicator, we evaluated the calcium responses of HEK293T cells to 50 μM ATP solution under various clearing media at a refractive index of 1.365 (Fig. Calcium responses were completely abolished in the presence of membrane-permeable chemicals, glycerol (23% wt/vol), dimethyl sulfoxide (DMSO) and propylene glycol, while a lower concentration of glycerol (5%) showed weak responses. These results indicate that membrane-permeable clearing agents impair cellular functions at a refractive index of 1.365. Among the membrane-impermeable, high-molecular-weight chemicals, straight polymers abolished calcium responses (for example, polyethylene glycol and polyvinyl pyrrolidone). These results indicate that some of the membrane-impermeable, high-molecular-weight chemicals could be useful for index matching of the extracellular medium without compromising cellular functions. Membrane-impermeable chemicals will not directly interfere with intracellular functions but may increase osmolarity. Therefore, the ideal chemical should have a low osmolarity when dissolved in water to achieve the optimal refractive index. The increase in osmolarity can be minimized if we use high-molecular-weight chemicals (>1 kDa); however, extremely large particles (>10-nm scale) will cause Rayleigh scattering. Straight-chain polymers had prohibitively high osmolalities, much higher than the theoretical values based on molar concentrations23; the higher osmolality may explain why straight-chain polymers showed cellular toxicity (Fig. In contrast, we found that spherical polymers (polymers with highly branched and/or higher-order structures) have much lower osmolalities (Fig. Among them, low-salt BSA (BSA#2) demonstrated exceptionally low osmolality of only 2.7 mOsm kg−1, consistent with its molar concentration (2.3 mM; Fig. Slightly higher osmolarity for another BSA product (BSA#1) was due to residual salts in the product (Extended Data Fig. 1e), suggesting that BSA itself has very low osmolality. The osmolarity of the saline buffers and culture media for mammalian cells is 230–340 mOsm l−1, but is typically 300–330 mOsm l−1. For both iodixanol (control) and BSA, we further refined the optimal refractive index for this range. The best refractive index was higher at higher extracellular osmolarity, likely because the cytosol is more condensed. When live HeLa cells were incubated with BSA-containing medium (refractive index, 1.363), the plasma membrane was almost invisible under the phase contrast microscopy (Fig. Cell growth was monitored for up to 3 days. Cell growth was comparable to the control DMEM for one of the BSA products (BSA#1; Fig. However, cell growth was lower for iodixanol, highly branched cyclic dextrin, Ficoll70 and some of the BSA products (Fig. BSA is also preferable in terms of lower viscosity (Extended Data Fig. 1j,k) and specific gravity (Extended Data Fig. Other proteins may be similarly useful; however, BSA has exceptional water solubility and is one of the most affordable proteins available. In addition, albumin is the most abundant protein in the serum (4–5% wt/vol) and has been widely used for mammalian cell culture, suggesting that BSA is minimally adverse to the mammalian cells. Albumin buffers divalent cations (for example, Ca2+ and Mg2+)24,25. Earlier biochemical studies indicated that a half of Ca2+ and Mg2+ binds to BSA in this condition, consistent with our results24,26. Primary cultures of mouse cardiomyocytes (Extended Data Fig. 3a–c) and hippocampal neurons (Extended Data Fig. 3d–g) were maintained in the BSA-containing culture medium for at least 3 days without any obvious signs of toxicity. In this way, we established BSA-containing clearing media (15–17% wt/vol) for live mammalian cells, named SeeDB-Live, with optimal refractive index (1.363–1.366), osmolality (230–340 mOsm kg−1) and total Ca2+ and Mg2+ concentrations (4–6 mM and 1.5–2.5 mM, respectively) with saline or culture medium (Supplementary Tables 1 and 2). Recently, strongly absorbing dyes (for example, tartrazine and ampyrone) have been shown to clear the mouse skin18,27, but they work only under prohibitively high osmolality conditions. Under physiological osmolality conditions (~300 mOsm kg−1), they have lower refractive indices and cannot effectively clear live cells (Fig. Another recent study increased refractive index of the extracellular media by only 0.01 using polymer solutions (6% polyethylene glycol (PEG) and 4% dextran)28; however, the refractive index of 1.34–1.35 was far below the optimal range for live mammalian cells (Fig. Thus, SeeDB-Live is currently the only method that achieves optical transparency of live, healthy mammalian cells. We examined whether SeeDB-Live is useful for fluorescence imaging of multicellular structures. We cleared cultured HeLa/Fucci2 cell spheroids29 with SeeDB-Live; the spheroids became quickly transparent without apparent shrinkage or expansion under SeeDB-Live (Fig. Growth of HeLa/Fucci2 spheroids was slightly slower when continuously cultured in SeeDB-Live, possibly due to lower circulation of oxygen (Extended Data Fig. However, daily clearing with SeeDB-Live for 4 h per day did not affect the growth of the spheroid culture (Fig. Next, we tested SeeDB-Live for imaging intestinal organoids cultured in Matrigel. Intestinal organoids were developed from ePet-Cre; Ai162 mice, in which enteroendocrine cells (EECs) express a calcium indicator, GCaMP6s. 1l), and the organoid growth was not affected by daily 4-h clearing with SeeDB-Live (Fig. Nonetheless, the GCaMP6s-positive EECs were visible in deeper areas under SeeDB-Live using confocal microscopy (Fig. Calcium imaging demonstrated robust responses to high potassium stimulation, indicating that their physiological functions are maintained (Extended Data Fig. We also tested SeeDB-Live for confocal calcium imaging of the neuroepithelial and cortical organoids induced from mouse embryonic stem cells (Fig. Thus, SeeDB-Live will be useful for functional assays of organoids. Together, our results indicate that the light scattering in live cells can be greatly reduced by index matching between the cytosol (1.363–1.366) and the extracellular medium. Index matching of the extracellular medium with isotonic medium with BSA (SeeDB-Live) is minimally invasive and powerful for optical clearing of live mammalian tissue (Fig. Volume imaging is in high demand for neuroscience applications. Perfusion with SeeDB-Live/ACSF cleared acute brain slices within 30 min (Fig. We evaluated the performance of SeeDB-Live using acute brain slices from Thy1-YFP-H mice. After the recovery of acute brain slices in oxygenated ACSF, confocal and two-photon images were acquired. The imaging depth was increased ~2-fold for both confocal and two-photon microscopy under SeeDB-Live (Fig. Similar results were obtained for the hippocampus (Extended Data Fig. The optimal refractive index of SeeDB-Live was ~1.363 in acute brain slices (Extended Data Fig. 5d), consistent with our results for cultured cell data (Fig. We did not observe improved transparency with 5% glycerol ex vivo, contrary to a previous report in vivo (Extended Data Fig. a, Preparation of acute brain slices and clearing with SeeDB-Live. Acute brain slices were perfused with SeeDB-Live/ACSF (refractive index, 1.363; 320 mOsm kg−1 in ACSF) at a flow rate of 1.5 ml min−1 in a chamber. 15.6% (vol/vol) 2,2′-thiodiethanol (TDE) in ddH2O (refractive index, 1.363) was used for immersion to minimize spherical aberration. b,c, An acute brain slice (300-μm thick; age, postnatal day 5 (P5); bright-field images) before (b) and after (c) clearing with SeeDB-Live/ACSF. Confocal one-photon (1P) and two-photon (2P) images are shown. Right: normalized fluorescence intensity from cell bodies in x–y fluorescence images of S1 L5ET neurons are shown on the right for each depth. The same sets of neurons were compared before and after clearing. f, Two-photon shadow images in S1 L5 region of acute brain slices (age P18, 300-μm thick). Imaging was performed before and after clearing with SeeDB-Live/ACSF. g, Shadow images (left) and their magnified views (right) in the intermediate (100–115-μm stack) and deeper (185–200-μm stack) regions (inverse look-up table images). Note that the brightness and contrast were adjusted for each image because fluorescence intensities differed across the conditions. h, Line plots showing the raw fluorescence intensity along the orange dashed line in g under control and SeeDB-Live. i, Magnified views of the shadow images showing dense nerve fibers. Data from representative samples of ≥3 trials are shown. See Supplementary Table 4 for detailed statistical data. Previously, shadow imaging of organotypic brain slice cultures with super-resolution, confocal and two-photon microscopy have been proposed for comprehensive structural imaging of the brain, including dense connectomics applications32,33,34; in these techniques, the extracellular space of the brain slices is labeled with a dye solution (for example, calcein). However, the surface of the brain slices (~50 μm) is often mechanically damaged during slice preparation (Extended Data Fig. 5e), making it difficult to image ‘acute' brain slices that represent native in vivo structure. Using SeeDB-Live, the imaging depth possible for the shadow imaging was much improved with two-photon microscopy (Fig. Inverse look-up table images demonstrated morphology of all the cells at higher signal-to-noise ratio under SeeDB-Live (Fig. Thus, SeeDB-Live facilitates comprehensive structural imaging of acute brain slices. For comprehensive recording of neuronal activity with SeeDB-Live, it is important to ensure that neuronal functions remain intact. Using acute mouse brain slices (age, P15–18), we examined membrane properties of layer 5 extratelencephalic-projecting (L5ET) neurons using patch-clamp recording (Fig. Some of the electrophysiological parameters were slightly affected (Fig. However, the firing properties in the frequency–current curve were not affected, possibly because differences in some factors counteracted each other (Fig. We obtained consistent results in older animals (Extended Data Fig. 6f,g) and for fast-spiking interneurons (Extended Data Fig. Samples were analyzed at the same time point after preparation. b, Resting membrane potential, action potential (AP) threshold, AP amplitude and input resistance are shown. n = 17 neurons from four mice and 14 neurons from three mice for control and SeeDB-Live, respectively. c, AP frequency was plotted against injected current amplitude. d,e, Spontaneous currents at the holding potential of −60 mV. Representative traces (d), amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs; e) are shown. f–n, Spontaneous and evoked responses of mitral cells in the olfactory bulb (OB) cleared with SeeDB-Live and imaged with two-photon microscopy. f, GCaMP6f fluorescence images (temporal median) of mitral cells in acute OB slices (age, P11) imaged with two-photon microscopy. Traces for representative neurons (arrows) are shown. Spontaneous activity was imaged before (left), during (middle) and after (right) clearing with SeeDB-Live/ACSF at a depth of 100 μm from the surface of the slice. g,h, Amplitude and frequency of spontaneous activity in the same set of mitral cells in ACSF and SeeDB-Live/ACSF. n = 18 cells from three mice (age, P11–14). NS (multiple comparisons with Bonferroni correction). i–k, 100 μM NMDA and 40 μM glycine (Gly) were applied to the OB slices (Thy1-GCaMP6f, age P15) for 1.5 min. (j) and response amplitudes (k) are shown. The slower decay of the response may be due to slower washout of NMDA/glycine under SeeDB-Live. l–n, Acute OB slices (age, P9–11) were imaged with two-photon microscopy at a depth of 150 μm. l, Basal fluorescence (temporal median) of GCaMP6f and ΔF/F0 images are shown for different time points. Basal fluorescence intensity (m) and ΔF/F0 (n) of mitral cell somata during clearing with SeeDB-Live/ACSF are shown. o, Confocal images of acute OB slices (Thy1-GCaMP6f mouse, age P13) under control and SeeDB-Live conditions. The images of temporal median are shown. Box plots indicate the median ± IQR. See Supplementary Table 4 for detailed statistical data. Patch-clamp recording under SeeDB-Live was extremely difficult because brain slices were almost transparent. We cannot exclude the possibility that unintentional sampling bias has contributed to the difference. It should also be noted that the ionic composition of SeeDB-Live is not identical to that of the control ACSF. The total amount of Ca2+ and Mg2+ is adjusted higher (Extended Data Fig. Cl− concentration is slightly lower because BSA substantially contributed to the net negative charge (Extended Data Fig. BSA may also show the Donnan effect. These factors potentially affect the electrophysiological properties, and further optimization might be needed for more specific experiments. We next evaluated population-level properties of neurons using slice calcium imaging. We used olfactory bulb slices (P11–15), in which mitral/tufted cells show spontaneous activity35. We used Thy1-GCaMP6f mice, in which mitral/tufted cells express GCaMP6f. We imaged mitral cells with two-photon microscopy at a depth of ~100 μm. The frequencies of spontaneous activity were not significantly different between control and SeeDB-Live when Ca2+/Mg2+ concentrations were optimized (Fig. In contrast, spontaneous activity was no longer maintained in the glycerol or iodixanol-containing ACSF (Extended Data Fig. Evoked responses (to 100 μM N-methyl-D-aspartate (NMDA) and 40 μM glycine) of mitral cells were also comparable between control and SeeDB-Live (Fig. Time-lapse imaging of a deeper area (150-μm depth) demonstrated a significant improvement in brightness and ΔF/F0 by SeeDB-Live/ACSF treatment (Fig. Notably, spontaneous activity of mitral cells was clearly visible with one-photon confocal microscopy at a depth of 100 μm under SeeDB-Live, but not with control ACSF (Fig. It should be noted that the superficial ~50 μm of acute brain slices is typically damaged during sample preparation, and intact neuronal activity is only visible in deeper areas, where only two-photon microscopy can access under the normal ACSF. Thus, SeeDB-Live enables calcium imaging of healthy neuronal activity in acute brain slices using conventional confocal microscopy, without using two-photon microscopy systems. SeeDB-Live is based on index matching with a membrane-impermeable molecule, BSA. To clear the mouse brain in vivo in live animals, we performed a craniotomy at the primary somatosensory cortex (S1) and removed the dura mater (durotomy) under anesthesia. We confirmed that fluorescently tagged BSA was infused to a depth of ~500 μm from the surface of the cerebral cortex (Fig. We used a transgenic line, Thy-YFP-H, in which L5ET neurons are labeled with EYFP. Under two-photon microscopy, overall brightness of EYFP signals was increased in the deeper area after incubation with SeeDB-Live for 1 h (Fig. Their basal dendrites, including dendritic spines, were better visualized with SeeDB-Live (Fig. The brightness of L5ET somata, located at a depth of 600–800 μm, was increased ~3-fold by SeeDB-Live treatment (Fig. The cleared part returned opaque once SeeDB-Live is diluted by the CSF circulation and/or by active washout with ACSF (Fig. Thus, SeeDB-Live is a powerful tool for in vivo imaging of live neurons in the brain. a–k, Optical clearing and fluorescence imaging of the cortex in live mice under anesthesia. a, Schematic diagram of surgery and clearing of the mouse cortex with SeeDB-Live/ACSF-HEPES (refractive index, 1.363; 300 mOsm kg−1). Craniotomy and durotomy were made on the right hemisphere. The objective lens was directly immersed in SeeDB-Live/ACSF-HEPES. b,c, The diffusion of fluorescently labeled BSA (1% BSA-CF597 dissolved in SeeDB-Live) into the cortex in anesthetized mice (age, 2–4 months). Frozen sections of non-perfused and unfixed brains were analyzed (b). The relative fluorescence intensity across cortical depth is shown (c). d–k, S1 of a Thy1-EYFP-H mouse was imaged before and after clearing with SeeDB-Live/ACSF-HEPES (1 h after clearing) with two-photon microscopy. d, 3D-rendered images of L5ET neurons (Thy1-YFP-H; age, 6 months). Laser power and photomultiplier tube gain were kept constant across the depths. f, Fluorescence intensity at different depths. g,h, Somata and basal dendrites of L5ET neurons (age, 4 months). Basal dendrites and their dendritic spines could only be clearly visualized after clearing with SeeDB-Live/ACSF-HEPES. k, S1 L5ET neurons of a 4-month-old Thy1-EYFP-H mouse were imaged using two-photon microscopy before, during and after 1 h of clearing with SeeDB-Live/ACSF-HEPES. l, A large cranial window encompassing motor and somatosensory areas was made for the right hemisphere. After craniotomy and durotomy, an optical window was made using a PVDC wrapping film, silicone sealant and a coverslip (center; day −7). SeeDB-Live treatment was performed 7 days after the initial surgery (day 0). The cranial window was replaced with a new one after SeeDB-Live/ACSF-HEPES treatment at day 0 for chronic behavioral assays (n–p). m, Mouse locomotor activity on a treadmill was measured for 10 min during clearing with SeeDB-Live/ACSF-HEPES in head-fixed awake animals. The total distance traveled and the maximum speed of mice treated with control ACSF-HEPES and SeeDB-Live/ACSF-HEPES were compared. Total distances traveled by mice in an open chamber at 1, 4 and 7 days after treatment with control ACSF-HEPES and SeeDB-Live/ACSF-HEPES are shown. NS (P ≥ 0.05; two-sided Wilcoxon rank-sum test). o, Motor function was examined with the wire hanging test55. Fall time of mice in the wire hanging test at 1, 4 and 7 days after treatment with ACSF-HEPES, Rose Bengal and SeeDB-Live/ACSF-HEPES. p, Food consumption of mice treated with control ACSF-HEPES, unilateral ischemia and SeeDB-Live/ACSF-HEPES. Images show representatives of ≥2 trials except for k (single trial). See Supplementary Table 4 for detailed statistical data. Panels a and m created in BioRender. We examined possible toxicity of SeeDB-Live in vivo using a large cranial window on the right cortical surface (Fig. Acute SeeDB-Live treatment of the right cortex, including motor cortices, in awake animals did not affect locomotor activity on a treadmill (Fig. Moreover, SeeDB-Live treatment did not affect locomotor activity, motor function (wire hanging test) and food intake on consecutive days (Fig. We observed no obvious sign of the inflammatory responses in the brain (for example, the number and morphology of neurons and microglia) after clearing with SeeDB-Live (Extended Data Fig. Thus, SeeDB-Live treatment does not induce acute or chronic toxicity in animals. Next, we investigated whether sensory responses are preserved after SeeDB-Live treatment in vivo. We performed a durotomy in the primary visual cortex (V1) and compared the visual responses of layer 4 neurons before and after SeeDB-Live treatment (Fig. 5a), assuming that layer 4 is adequately infused with SeeDB-Live (Fig. Using calcium indicators jGCaMP8m and Cal-520, we recorded the calcium responses of the same sets of neurons to visual grating stimuli of different orientations under anesthesia (Fig. We found that the preferred orientation, response amplitude (ΔF/F0), orientation selective index (OSI) and tuning width of layer 4 neurons were largely preserved (Fig. Thus, SeeDB-Live preserves physiological sensory responses. a–e, Two-photon calcium imaging of L4 neurons expressing jGCaMP8m (AAV-DJ-Syn-jGCaMP8m-WPRE) in V1 before and after clearing with SeeDB-Live/ACSF-HEPES. Drifting gratings of various orientations were presented to anesthetized mice. b, Basal fluorescence of jGCaMP8m without visual stimulation. L4 neurons at a depth of 435 μm. c,d, Responses of a representative L4 neuron (indicated by arrowheads in b) to visual grating stimuli before and after clearing with SeeDB-Live/ACSF-HEPES (c). e, Preferred orientation, maximum responses (ΔF/F0), OSI and tuning width (Sigma) for the same set of L4 neurons (136 neurons from three mice) before (x axis) and after (y axis) clearing with SeeDB-Live/ACSF-HEPES. The comparison was performed as described previously56. f, x–y images of mitral cells in the olfactory bulb of a Thy1-GCaMP6f mouse (4-month-old, anesthetized) was imaged before and after clearing with SeeDB-Live/ACSF-HEPES (1 h after clearing) with two-photon microscopy. g, Odor responses of mitral cells upon 1% valeraldehyde. The odor was delivered to a mouse nose for 5 s at 1 l min−1. Arrows indicate the same sets of neurons. h, Representative excitatory/inhibitory responses of mitral cells indicated in g. i–p, Chronic imaging in awake animals using repeated SeeDB-Live treatment. j, After clearing with SeeDB-Live/ACSF-HEPES for 1 h, the brain surface was covered with the PVDC film. Between the film and the glass coverslip, 1.5% (wt/vol) agarose was applied with and without 19.3% (wt/vol) glycerol (refractive index, 1.363) for the SeeDB-Live and control conditions, respectively. For objective lens immersion, 15.6% (vol/vol) TDE/ddH2O (refractive index, 1.363) and ddH2O were used for SeeDB-Live and the control, respectively. Imaging was performed within 1 h after SeeDB-Live treatment. k, Basal fluorescence (temporal median) of jGCaMP8m-expressing L5 neurons at day 0. m, Representative Ca2+ responses of L5 neurons indicated in k and l during repeated whisker stimulations with air puffs. n–p, Long-term monitoring of neuronal morphology and physiology in awake mice. Data are from a representative animal of four trials. z-stack images (imaging depth: 238–358 μm) of the CyRFP1 fluorescence of L2/3 neurons after SeeDB-Live treatment on days 7, 80, 100 and 120 (n). Mean calcium responses of jGCaMP8m-expressing L2/3 neurons to whisker stimulations (five times) with air puffs (o). Soma size, ΔF/F0 and half-rise time of neurons to whisker stimulation after SeeDB-Live treatment on days 7, 80, 100 and 120 (p). ΔF/F0 and half-rise (τ) time were calculated from the mean responses to five whisker stimulations. NS (P ≥ 0.05; two-sided repeated-measures analysis of variance). Data in c, d and p indicate the mean ± s.d. Images show representative samples of 2–4 trials. See Supplementary Table 4 for detailed statistical data. In the olfactory bulb, we were able to better visualize odor responses in mitral cell somata located at a depth of ~400 μm using GCaMP6f (Fig. Due to the improved brightness, inhibitory responses, represented by a reduction in basal GCaMP6f fluorescence, were better detected using SeeDB-Live (Fig. Voltage imaging is more challenging than calcium imaging in vivo due to the lower signal-to-noise ratio of the signals. However, using SeeDB-Live, we were able to reliably detect action potentials from the somata of layer 5 pyramidal neurons located at a depth of ~560 μm using JEDI-2P indicator (Extended Data Fig. Optical clearing with SeeDB-Live is transient in vivo (~1 h) as BSA is gradually washed out (Fig. To perform chronic calcium imaging with SeeDB-Live in awake animals, we used easily removable plastic films for the cranial window (Fig. A large cranial window (6 × 3 mm2) was made in one hemisphere and SeeDB-Live was applied for 1 h. A polyvinylidene chloride (PVDC) wrapping film was attached onto the window37. Refractive index–matched agarose was placed between the PVDC film and a coverslip for imaging. We obtained stable responses of GCaMP8m in S1 (615-μm depth). We did not find any changes in cytoarchitecture and sensory responses (amplitude and frequency) over 4 months, suggesting that normal neuronal functions are maintained during repeated clearing (Fig. Moreover, we observed minimal inflammatory responses after repeated clearing (Extended Data Fig. This approach could be powerful for chronic imaging of deep cortical regions. Genetically encoded voltage indicators with high signal-to-noise ratios have been developed in recent years. To image fast voltage changes, high-speed epifluorescence imaging is advantageous over point-scanning two-photon microscopy. Here we cleared acute olfactory bulb slices with SeeDB-Live and imaged calcium and voltage signals using GCaMP6f and a fast and sensitive chemigenetic voltage indicator, Voltron2, sparsely introduced to mitral/tufted cells by in utero electroporation (Fig. 6a)38; Voltron2 was visualized with JF549–HaloTag ligand applied to the medium (Voltron2549). After the clearing with SeeDB-Live, the epifluorescence signals of Voltron2549 were clearly visualized at a depth of >150 μm (Fig. Using a high-speed CMOS camera (2 kHz), we recorded voltage changes in different compartments of mitral cell dendrites. We could visualize the backpropagation of action potentials from somata to dendritic tips in single-shot imaging, without averaging (Fig. Thus, the combination of SeeDB-Live and epifluorescence imaging will be a powerful tool for studying subcellular dynamics of voltage signals in acute brain slices. a–f, Voltage imaging of a mitral cells in acute brain slices ex vivo using epifluorescence microscopy. a, Mitral cells in olfactory bulb slices. b, Mitral cells labeled with Voltron2549 at different depths under control and SeeDB-Live conditions (acquired with a high-speed CMOS camera, temporal median). Voltron2 was introduced to mitral cells by in utero electroporation and analyzed at P11. Voltron2 was labeled with Janelia Fluor HaloTag Ligand 549 before the imaging. Ticks indicate the detected action potentials. d, Two-photon image identified a labeled mitral cell (z-stacked, left). Epifluorescence of Voltron2549 (temporal median) is shown on the right. Voltron2 and GCaMP6f were introduced to mitral cells by in utero electroporation. e, Backpropagation of action potentials were imaged at 2 kHz (single-shot images) using a high-speed CMOS camera. f, Spatiotemporal pattern of backpropagating action potentials, averaged from 65 events. The half-rise time of action potentials at soma was defined as 0 ms. Median filtering (4 × 4 pixels) was applied to the images. After durotomy, the olfactory bulb was immersed with ACSF-HEPES containing 50 nM Janelia Fluor HaloTag Ligand 549 for 1 h, followed by a 1-h washout in ACSF-HEPES. SeeDB-Live treatment was then performed for 1 h. We imaged a deeper part of the glomerular layer (90 μm), where mitral/tufted cells form dendritic branches. We used a ×25 objective (NA 1.05) with a short focal depth (1.36 μm) to minimize out-of-focus signals. h,i, Epifluorescence images (temporal median) of dendrites (and some somata) of mitral/tufted cells labeled with Voltron2549 in an anesthetized mouse (4-month-old) before (h) and after (i) clearing with SeeDB-Live (1 h after clearing). F0 images at a depth of 90 μm. Magnified images are shown on the right. j, ROIs were semiautomatically detected from the F0 image using ilastik. Representative traces (−ΔF/F0) from highlighted ROIs (dendrites) are shown on the right. Ticks indicate the detected action potentials. Note that subthreshold activities were also correlated between ROIs within the same glomerulus. k, Cross-correlation matrix for voltage traces. ROIs were clustered using k-means clustering. l, Spatial distribution of ROIs in each cluster. Each color represents a different cluster. m,n, Subclusters based on spike synchronicity between ROIs within a glomerulus (cluster 2; m) and representative traces from indicated ROIs (n). o,p, Comparison of synchronicity between the subclusters in the same or different glomeruli (clusters 2 and 11; o) and representative traces of the indicated subclusters (p). Odor (1% amyl acetate) was delivered to a mouse nose for 5 s (shaded) at 1 l min−1. Data are from representative samples of two trials each. See Supplementary Table 4 for detailed statistical data. Previously, it has been challenging to image genetically encoded voltage indicator signals at deeper parts of the brain using epifluorescence imaging in vivo38,39,40. After the clearing with SeeDB-Live, L2/3 neurons located at a depth of 120–150 μm were better visualized, allowing for reliable detection of spontaneous action potentials in their somata (Extended Data Fig. We also detected backpropagating action potentials from dendrites of Voltron2-expressing L2/3 neurons in awake mice (Extended Data Fig. Next, we performed voltage imaging of mitral/tufted cells in vivo. Epifluorescence voltage imaging has been performed in the olfactory bulb, but not at the single-neuron resolution42,43. We expressed Voltron2 specifically and sparsely in mitral/tufted cells using the Pcdh21-Cre driver and a Cre-dependent adeno-associated virus (AAV) vector, labeled with JF549–HaloTag ligand (Voltron2549). As a result, we were able to detect backpropagating action potentials from ~140 neurites (regions of interest or ROIs) at a depth of ~90 μm using SeeDB-Live and epifluorescence imaging (Fig. In this imaging setup, the theoretical focal depth was ~1.36 μm (Fig. Of course, out-of-focus signals will contribute substantially to the total fluorescence, F0. However, as the fluorescence changes caused by the spikes were all-or-none and up to 2–3% ΔF/F0 (much smaller than calcium imaging), it is unlikely that scattered out-of-focus signals interfere with or contaminate spike detection (that is, −ΔF/F0) in each of the ROIs. Correlation of the voltage traces across ROIs revealed ~11 discrete clusters (Fig. The ROIs within each cluster (11 clusters by k-means) were also spatially clustered (Fig. 6l), demonstrating that neurites within the same glomerulus have similar voltage dynamics (including subthreshold changes; Fig. This makes sense because neurons connecting to the same glomerulus (‘sister' mitral/tufted cells) receive similar synaptic inputs and are electrically coupled within the glomerulus44. When we looked at individual ROIs within the same glomerulus, some pairs, but not all, demonstrated highly correlated backpropagating action potentials, suggesting that these dendritic branches originated from the same neuron (Fig. We, therefore, grouped ROIs with highly synchronized backpropagating action potentials into a subcluster. In this way, we obtained 21 subclusters, each of which most likely represents a single neuron (Extended Data Fig. Subclusters that belong to the same glomerulus (‘sister' mitral/tufted cells) tend to show more synchronized events than those in different glomeruli (Fig. We also observed odor-evoked phase shifts in action potentials relative to the sniff-coupled theta oscillations (Extended Data Fig. Thus, epifluorescence imaging of dendrites combined with SeeDB-Live provides a powerful approach for studying population voltage dynamics in vivo. To date, several studies have achieved optical clearing of live tissues, but only under unhealthy conditions for live cells. In this study, we identified the optimal refractive index and achieved optical clearing of live tissues without affecting osmolarity using BSA. Furthermore, the extracellular ionic condition was largely preserved, which is critical for studying the normal physiology of neurons. This is an advantage of SeeDB-Live over existing methods (Supplementary Table 5). Notably, the SeeDB-Live treatment demonstrated an undetectable level of toxicity to neuronal physiology and animal behavior, providing a powerful new option for imaging-based neurophysiology. Combined with wider field-of-view two-photon microscopy47,48,49,50, targeted one-photon imaging approaches39,40,51 and red-shifted indicators52, SeeDB-Live expands the imaging scale for biological phenomena at the tissue and organ scale both ex vivo and in vivo. In this study, we demonstrated that SeeDB-Live is particularly useful for epifluorescence voltage imaging. Previously, large-scale imaging of voltage changes has been difficult due to the slow scanning speed of two-photon microscopy and light scattering with one-photon microscopy. Epifluorescence imaging of dendritic voltage changes with SeeDB-Live could be a powerful strategy for studying subcellular and/or population-scale voltage dynamics both ex vivo and in vivo. However, some of the induction experiments (for example, optic cup formation31) were unsuccessful for unknown reasons. For the improved culture of organoids, microfluidic culture systems may be useful to improve circulation and/or to exchange the medium30. Alternatively, infusion into the CSF circulation system may be useful for efficient permeation of SeeDB-Live for more extensive clearing of the entire brain in the future studies53. Other organs may be more difficult to clear, and future in vivo applications would require strategies to overcome the accessibility issue. With SeeDB-Live, we can now use confocal microscopy for deep imaging, enabling high-resolution multicolor imaging. The combination of fluorescence imaging and photostimulation will be easier with one-photon and SeeDB-Live than with a multi-photon setup. As optical aberration is minimized, SeeDB-Live should also be very useful for super-resolution imaging of large volume in live tissues9,54. While we have demonstrated shadow imaging with two-photon microscopy, STED microscopy in combination with SeeDB-Live may enable saturated connectomics in acute brain slices, rather than in cultured brain slices33,34. For the best performance, it should be important to use objective lenses optimized for SeeDB-Live (refractive index, ~1.363). Together with ongoing efforts to develop microscopy techniques, our live tissue-clearing approach facilitates our understanding of the tissue-scale and organ-scale dynamics of biological phenomena. ICR and C57BL/6N mice were purchased from Japan SLC. Both males and females were used for our experiments. Mice were kept under a consistent 12-h light–12-h dark cycle (lights on at 8:00 and off at 20:00), with an ambient temperature of 20–26 °C and humidity of 40–70%. To construct pCAG-GCaMP6f, GCaMP6f gene was PCR amplified from pGP-CMV-GCaMP6f (Addgene, 40755) with Q5 High-Fidelity 2X Master Mix (M0492S, NEB). The cDNA was flanked by EcoRI and NotI sites. The GCaMP6f cDNA was subcloned into pCAG vector with a ligation kit (6023, Takara). To make pAAV-CAG-jGCaMP8f-WPRE, jGCaMP8f gene was amplified from pGP-AAV-syn-jGCaMP8f-WPRE (Addgene, 162376) with Q5 High-Fidelity 2X Master Mix. The cDNA contained an extra 20–30-bp overlap regions with pAAV-CAG-tdTomato (Addgene, 59462). The tdTomato was removed from pAAV-CAG-tdTomato by digestion with KpnI and HindIII, and jGCaMP8f cDNA with the extra sequence was subcloned into the vector with NEBuilder HiFi DNA Assembly (E2621S, NEB). pCAG-GCaMP6f plasmid has been deposited to Addgene (no. BSA was dissolved with gentle shaking at 15.6% wt/vol. We found that BSA#1 contained residual salts (~30 mM Na+ and ~1 mM Ca2+ when dissolved at 15% wt/vol; Extended Data Fig. 1e), and this was taken into account. pH was adjusted with sodium hydroxide. BSA is known to chelate Ca2+ and Mg2+. To maintain the free Ca2+ and Mg2+ concentrations the same as ACSF, additional CaCl2 (2 mM) and MgCl2 (1 mM) were supplemented after BSA was fully dissolved in the medium (except for Fig. Saturation of O2 was checked with an O2 sensor (9521, Horiba). Bubbling is not recommended because it produces a lot of foam, and BSA may be denatured. As for the culture medium, BSA#1 was dissolved in ×0.8 culture medium to adjust the osmolarity and CaCl2 (2 mM) and MgCl2 (1 mM) were supplemented. The osmolarity of the BSA solution was measured with a vapor pressure osmometer (VAPRO 5600, Xylem ELITech). The refractive index was measured with an Abbe refractometer (ER-2S, Erma) with a white LED light source. Salts contained in BSA powder were analyzed using Inductively Coupled Plasma Mass Spectrometry (Agilent Technologies, ICP-MS 7700x). We tested the following chemicals during the screening process: glycerol (17018-25, Nacalai), DMSO (043-07216, Fujifilm), propylene glycol (164-04996, Fujifilm), iodixanol (VISIPAQUE 320 INJECTION 50 ml, GE HealthCare), iodixanol (D1556-250ML, Optiprep), iotrolan (Isovist Injection 300, Bayer Pharma Japan), iopamidol (OYPALOMIN, FujiPharma), iopromide (iopromide 370 Injection (FRI), Fujifilm), iohexol (OMNIPAQUE350 INJECTION, GE healthcare Pharma), ioxilan (Imagenil350 Injection, Guerbet Japan), ioversol (Optiray350 Injection, Mallinckrodt), ioxagilic acid (Hexabrix320 Injection, Guerbet Japan), iomeprol (Iomeron400 Bracco-Eisai), Ficoll70 (17031050, Cytiva), Ficoll400 (17030010, Cytiva), HBCD (307-84601, Glico), PVP (P0471, TCI), sucrose (193-00025, Fujifilm), tartrazine (T0388, Sigma-Aldrich), ampyron (017-02272, Fujifilm), polydextrose (polydex300, Nichiga), partially hydrolyzed guar gum (2021092403, Nichiga), methyl-β-cyclodextrin (M1356, TCI), agave inulin (agabe500, Nichiga), PEG8000 (V3011, Promega), PEG10000 (81280, Sigma-Aldrich), RM + stevia (dex-5-500m, Nichiga), resistant maltodextrin from corn (RM1, MK-H108-6T6I, Nichiga), resistant maltodextrin from wheat (RM2, dekisutorin-komugi-400, Nichiga), inulin (inurinn500, Nichiga), isomaltodextrin (Fibryxa, Hayashibara), reduced resistant maltodextrin (kg-nandeki-400, Nichiga), dextran (D1662, Sigma-Aldrich) and stevia (sutebiasw5-150m, Nichiga). A step-by-step protocol and technical tips are available at SeeDB Resources (https://sites.google.com/site/seedbresources/). HeLa S3 cells (JCRB9010, JCRB) were cultured in Dulbecco's Modified Eagle Medium (DMEM high glucose; 043-300085, Fujifilm) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS) at 37 °C, 5% CO2. After centrifugation, the medium was replaced with 50 μl of index-adjusted PBS. Transmittance of the cell suspension in 400–1,100 nm was measured with a ratio beam spectrophotometer (U-5100, Hitachi High-Tech). This measurement was performed quickly because unhealthy cells have a nonoptimal refractive index, which results in reduced transmittance. HEK293T cells (AAVpro 293T, 632273, Takara) were cultured in DMEM (high glucose) supplemented with 1% penicillin–streptomycin and 10% FBS at 37 °C and 5% CO2. Cells seeded in 35-mm glass-bottom dishes (60% confluent) were transfected with pGP-CMV-GCaMP6f (Addgene, 40755) using PEI Max (Polysciences, 24765-1). Two hours after the medium exchange, cells were imaged with an inverted microscope (DMI600B, Leica) equipped with a ×10 NA 0.4 dry objective lens and controlled by LAS AF software (Leica). A final concentration of 50 μM ATP was added to the medium during imaging. For the calcium measurement with a plate reader (TriStar LB941, Berthold), GCaMP6f-expressing HEK293T cells were transferred to a 96-well plate at 4 × 105 cells per ml per well. Mean values during 1–10 s after stimulation were analyzed. To measure the viscosity of the solutions, we measured the time taken for 20 ml of solutions to flow out from a 50-ml syringe (TERMO, SS-50ESZ) with an internal tip diameter of ~2 mm. The plots were fitted by single-exponential fitting. Twenty-four hours after seeding, the medium was replaced with an index-adjusted medium (refractive index, 1.363; 310–320 mOsm kg−1). Fucci2 fluorescence (mVenus and mCherry) was imaged with an inverted fluorescence microscope (DMI600B, Leica) equipped with a ×5 NA 0.1 dry objective lens and controlled by LAS AF software (Leica). Cells were counted based on the nucleus images of Fucci2 fluorescence with ImageJ software (https://imagej.net/ij/). First, the green and red channels were summed. The speckle noise was removed with ‘Despeckle'. The overlapped nuclei were separated with ‘Watershed'. The intensity threshold was determined manually. Finally, the cell number in each well was counted with ‘Analyze particles'. Twenty-four hours after plating, the medium was replaced with an index-adjusted medium (refractive index, 1.363; 320 mOsm kg−1). After trypsinization, the cell number was counted with a hemocytometer. For spheroid formation, HeLa/Fucci2 cells were seeded on an ultralow-attachment 96-well plate (7007, Corning) at 1,000 cells per well. At 24–48 h after plating, the spheroid was incubated in an index-adjusted medium (SeeDB-Live; refractive index, 1.366; 320 mOsm kg−1) for 4 h per day or for all the time. For manual counting, the cells were incubated in a mixture of 50 μl DMEM and 200 μl Trypsin-EDTA for 30 min. The suspension was then centrifuged at 1,000 rpm for 5 min. The HeLa/Fucci2 spheroid was incubated in SeeDB-Live (refractive index, 1.366; 320 mOsm kg−1) for 1 h. Then, the spheroid was mounted on a glass slide and sealed with a 1-mm-thickness silicone rubber spacer (Togawa rubber) and a coverslip (Matsunami). Imaging was performed using an FV1000MPE microscope (Olympus/Evident) with Fluoview FV10-ASW software (Olympus/Evident, RRID: SCR_014215) and a ×25 NA 1.05 objective lens (Olympus/Evident, XLPLN25XWMP). For the two-photon imaging, a femtosecond laser (Insight DeepSee, SpectraPhysics) was tuned to 920 nm for mVenus excitation. A 1,040-nm laser was used for mCherry excitation. For cell detection, flat areas of the images were cropped. The green and red channels were merged to make reference images. A median filter was applied (2 × 2 pixels). ROIs for each cell in a spheroid were created with Cellpose61,62. Cell numbers and the fluorescence intensity were calculated based on the ROIs using MATLAB (MathWorks). 3D-rendered images were made by Imaris Viewer (Oxford Instruments). Intestinal organoids were created following the manufacturer's protocol (VERITAS). Briefly, ePET-Cre; Ai162 mice were euthanized with an overdose of pentobarbital (intraperitoneal (i.p.) injection, 100–150 mg per kg body weight). A small intestine was taken out and cut to expose the lumen side. The lumen was gently washed with cold PBS (−) several times. The small intestine was cut into 2-mm pieces in 10 ml of cold PBS (−). After pipetting three times, the supernatant was replaced with new cold PBS (−). This procedure was repeated >15 times until the supernatant became clear. The supernatant was replaced with 25 ml of Gentle Cell Dissociation Reagent (ST-100-0485, STEMCELL Technologies). The pieces were gently shaken for 15 min at room temperature. The supernatant was replaced with 10 ml of cold 0.1% BSA/PBS. After pipetting three times, the suspension was passed through a 70-µm cell strainer (352350, Corning). This step was repeated to obtain the fraction containing more crypts. After centrifugation at 200g for 3 min at 4 °C, the supernatant was replaced with 10 ml of DMEM/F-12 (11039-021, Thermo Fisher). After centrifugation at 200g, for 5 min at 4 °C, the supernatant was replaced with 150 µl of IntestiCult Organoid Growth Medium (ST-06005, STEMCELL Technologies). Matrigel (150 µl; 356237, Corning) was added to the suspension. After pipetting ten times, 50 µl of the mixture was mounted on the well of a 24-well plate. The plate was incubated for 10 min at 37 °C to gelatinize Matrigel. IntestiCult Organoid Growth Medium (750 µl) was added to the wells carefully. For imaging, the organoids were dissociated from a gel by pipetting with Gentle Cell Dissociation Reagent and transferred to a 15-ml tube. The tube was gently shaken for 10 min at room temperature. After centrifugation at 300g for 5 min at 4 °C, the supernatant was replaced with 150 µl of IntestiCult Organoid Growth Medium (osmolality, 270 mOsm kg−1). Matrigel (150 µl) was added to the suspension, and 50 µl of the mixture was mounted and spread on the glass region of a 35-mm glass-bottom dish. The mixture was gelatinized by incubation for 10 min at 37 °C. IntestiCult Organoid Growth Medium (1 ml) was added to the dish carefully. For clearing, the culture medium was replaced with SeeDB-Live/IntestiCult Organoid Growth Medium (refractive index, 1.363) 2–3 h before imaging. Phase contrast images were taken with an inverted microscope (DMI600B, Leica) equipped with a ×10 NA 0.4 dry objective lens and controlled by LAS AF software (Leica). Fluorescence of EECs in an organoid was imaged with an inverted confocal microscopy (TCS SP8, Leica) equipped with ×20 NA 0.75 multi-immersion lens and LASX software (Leica Microsystems). A 488-nm laser was used to excite GCaMP6s expressed in EECs. To measure the Ca2+ responses of EECs, KCl (+30 mM at final concentrations) was added to the medium during imaging. Mouse embryonic stem cells were maintained as described in the previous study31. The cell line was provided by M. Eiraku at Kyoto University. Cells were maintained as described in the previous study31. For maintenance, cells were cultured in a gelatin-coated 100-mm dish. The dish contained maintenance medium, to which 20 µl of 106 units per ml leukemia inhibitory factor (Sigma-Aldrich) and 20 µl of 10 mg ml−1 blasticidin (14499, Cayman) were added. The maintenance medium consisted of Glasgow's Modified Eagle Medium (G-MEM; 078-05525, Wako) supplemented with 10% Knockout Serum Replacement (KSR; 10828028-028, GIBCO), 1% FBS (GIBCO), 1% Non-essential Amino Acids (NEAA; 139-15651, Wako), 1 mM pyruvate (190-14881, Wako) and 0.1 mM 2-mercaptoethanol (2-ME; M6250, Sigma-Aldrich). The solution was filtered through a 0.2-μm filter bottle, stored at 4 °C, used within 1 month. For organoid induction, the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture method was performed as described in a previous study31. On day 1, Matrigel (354230, Corning) was mixed with the differentiation medium and added to each well to reach a final concentration of 2.0%. This plate was incubated at 37 °C in a 5% CO2 environment. The differentiation medium consisted of G-MEM supplemented with 1.5% KSR, 1% NEAA, 1% pyruvate and 0.1% 0.1 M 2-ME. This solution was filtered through a 0.2-μm filter bottle, stored at 4 °C and used within 1 month. For imaging, Matrigel surrounding the organoids was reduced by pipetting gently in advance. Then the organoids were transferred from the 96-well plate to a 35-mm glass-bottom dish (D11130H, Matsunami) coated with 0.1% (wt/vol) poly-L-lysine solution in H2O (P8920, Sigma-Aldrich) and 2.5 mg ml−1 Cell-Tak (354240, Corning). Images were captured using an LSM 800 (Zeiss) equipped with a ×25 NA 0.8 multi-immersion lens and controlled by Zen software (Zeiss, RRID: SCR_013672). On day 9 in SFEBq culture, 145 images were taken for each organoid at different z-positions with 3-µm intervals within 432 µm. Organoids were incubated for 2 h in SeeDB-Live medium adjusted at 270 mOsm kg−1. Small incisions were made in the organoid by randomly inserting a glass capillary five times to facilitate penetration of SeeDB-Live into the internal vesicle. Cultures for cortical organoid induction were performed as described in the study63. In this method, the cortical organoid differentiation medium consisted of G-MEM supplemented with 10% KSR, 1% NEAA, 1% pyruvate and 0.1% 0.1 M 2-ME. The solution was filtered through a 0.2-μm filter bottle, stored at 4 °C and used within 1 month. On day 0, 3,000 cells were suspended in 100 µl of differentiation medium and placed in each well of a 96-well plate. The plates were incubated at 37 °C in a 5% CO2 environment. On day 7, the aggregates were transferred to a 35-mm bacterial-grade dish containing DMEM/F-12 with Glutamax (10565, Invitrogen) supplemented with N2 (17502-048, Invitrogen) and incubated in a 5% CO2, 40% O2 environment at 37 °C. The medium was changed every 3 days. For Ca2+ imaging, the organoids were incubated with 5 µM Calbryte 630 (20721, AAT Bioquest) for 1 h, transferred to a 35-mm glass-bottom dish and covered with cover glass (Matsunami). Images were captured using an LSM 800 (Zeiss) equipped with a ×25 NA 0.8 multi-immersion lens and controlled by Zen software (Zeiss, RRID: SCR_013672). On day 36 in SFEBq culture, 145 images were taken for each organoid at different z-positions with 3-µm intervals within 432 µm. Organoids were incubated for 1 h in SeeDB-Live medium. AAV-DJ-syn-jGCaMP8m-WPRE vector was generated using pGP-AAV-syn-jGCaMP8m-WPRE (Addgene, 162375), pHelper (AAVpro Helper-free system, Takara), pAAV-DJ (Cell Biolabs) and the AAVpro 293T cell line (632273, Takara) following the manufacturers' instructions. Transfection was performed with PEI Max (24765-1, PSI). AAV vectors were purified using the AAVpro Purification Kit All Serotypes (6666, Takara). AAV.PHP.S-CAG-jGCaMP8f-WPRE vector was generated using pAAV-CAG-jGCaMP8f-WPRE, pHelper, pUCmini-iCAP-PHP.S (Addgene, 103006) and the AAVpro 293T cell line as described previously64. Briefly, the conditioned medium containing AAV vectors was filtered with a syringe filter to remove cell debris at 6 days after transfection. The filtered medium was concentrated and formulated with D-PBS (−) using the Vivaspin 20 column pretreated with 1% BSA in PBS. Viral titers were measured using AAVpro Titration Kit (6233, Takara) or THUNDERBIRD SYBR qPCR Mix (QPS-201, TOYOBO) with StepOnePlus system (Thermo Fisher) or QuantStudio3 real-time PCR system (Applied Biosystems). Primary cultures of cardiomyocytes were prepared from P0 ICR mice as previously described65. The pups were anesthetized on ice and decapitated. The hearts were dissected and washed in PBS (−) containing 20 mM 2,3-butanedione monoxime (B0753, Sigma). The hearts were cut into 0.50–1-mm pieces in Hanks' Balanced Salt solution (HBSS (−); 084-08345, Fujifilm) containing 0.08% Trypsin-EDTA and 20 mM 2,3-butanedione monoxime and shaking at 4 °C for 2 h. L15 medium (128-06075, Fujifilm) containing 1.5 mg ml−1 collagenase/Dispase mix (10269638001, Roche) and 20 mM 2,3-butanedione monoxime was added. Thirty minutes after shaking at 37 °C, the suspension was filtered through a 70-μm cell strainer. The trapped heart tissues were transferred to an L15 medium containing 1.5 mg ml−1 collagenase/Dispase mix and 20 mM 2,3-butanedione monoxime and incubated for 10 min at 37 °C. After centrifugation at 100g for 5 min, the pellet was resuspended with DMEM (high glucose) supplemented with 1% penicillin–streptomycin and 10% FBS. The suspension was mounted on a cell culture dish for 2 h. This helped the removal of highly adhesive cells. After gentle pipetting, the suspension was collected and plated on 35-mm dishes at 1.2 × 105 cells per cm2. The cells were cultured in DMEM (high glucose), without phenol red and glutamine (040-30095, Fujifilm) supplemented with 1% penicillin–streptomycin, 10% FBS and 1% glutamine at 37 °C, 5% CO2. On day 1 in vitro (DIV-1), AAV.PHP.S-CAG-jGCaMP8f-WPRE was added at 2 × 1010 genome copies (GCs) per ml. On DIV-2, the culture medium was exchanged. The spontaneous activity of cardiomyocyte aggregates was measured with a Leica TCS SP8 equipped with a ×20 NA 0.75 multi-immersion lens and LASX software (Leica Microsystems) at DIV-3 to DIV-5. Phase contrast images were taken with an inverted microscope (DMI600B, Leica) equipped with a ×10 NA 0.4 objective lens and controlled by LAS AF software (Leica). Primary cultures of hippocampal neurons were prepared from embryonic day 16 ICR mice. The brain was extracted and put into a cold dissection medium consisting of HBSS (−) supplemented with 20 mM HEPES and 1% penicillin–streptomycin solution. Papain (2 mg ml−1; LS003119, Worthington)/HBSS (−) was activated for 5 min at 37 °C. After filtration, the hippocampi were transferred to papain/HBSS (−) and incubated for 20 min at 37 °C. A total of 1 ml of 150 mg ml−1 DNase I (11284932001, Roche)/HBSS (−) was added to the papain/HBSS (−) containing the hippocampi. The hippocampi were incubated for 5 min at 37 °C. The hippocampi were washed twice with 2 ml of HBSS (−). The cells were dissociated with gentle pipetting using a Pasteur pipette (Iwaki). The cells were then plated on a 35-mm glass-bottom dish coated with poly-D-lysine (P7886, Sigma) at 1.5 × 105 cells on a 12-mm-diameter coverslip and cultured in 5% CO2 at 37 °C. On DIV-2, AAV-DJ-hsyn-jGCaMP8m-WPRE was added at 7 × 1010 GCs per ml after half of the culture medium in the dishes was transferred to a 50-ml tube. On DIV-7, half of the culture medium was transferred to a 50-ml tube. SeeDB-Live (refractive index, 1.363) was made from this culture medium together with the same amount of fresh medium. The spontaneous activity was measured with a Leica TCS SP8 equipped with a ×20 NA 0.75 multi-immersion lens and LASX software (Leica Microsystems) on DIV-8 to DIV-10. Phase contrast images were taken with an inverted microscope (DMI600B, Leica) equipped with a ×20 NA 0.7 objective lens and controlled by LAS AF software (Leica). ICR mice (P5) were anesthetized on ice and euthanized by decapitation. The brain was mounted on a silicone rubber block (Togawa Rubber) and sliced at 300-μm thickness using a microslicer (Dosaka EM). Slices were recovered in O2-saturated ACSF for 1 h at room temperature and then cleared with SeeDB-Live. An upright microscope (Leica, S9E) equipped with a USB camera (Swift, EC5R) was used for image acquisition. Thy1-GCaMP6f mice (P11–15) were used for Ca2+ imaging of the acute olfactory bulb slices. Thy1-YFP-H mice (P17–22) were used for morphological analyses. Mice were euthanized with an overdose of pentobarbital (i.p. injection, 100–150 mg per kg body weight) and decapitated. The brain was mounted on a silicone rubber block (Togawa rubber) and sliced using a microslicer (Dosaka EM) at 300-μm thickness. The slices were placed on a custom-made silicone chamber for imaging using an upright microscope as previously described35,66. For clearing, the brain slices were perfused with SeeDB-Live for 1 h. To remove SeeDB-Live from tissue, ACSF was perfused for >1.5 h. We could record spontaneous activity of the olfactory bulb up to 5 h. The custom-made silicone chamber was set under an FV1000MPE microscope (Olympus/EVIDENT) with Fluoview FV10-ASW software (Olympus/Evident, RRID: SCR_014215) and a ×25 NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP). A perfusion chamber (Warner Instruments, JG-23W/HP, PM-1, SHD-26GH/10) was used for inverted imaging. The correction collar was turned to the appropriate positions (refractive index ~1.34 for control and ~1.363 for SeeDB-Live). For one-photon confocal imaging, a 473-nm laser was used. For two-photon imaging, a femtosecond laser (InSight DeepSee, SpectraPhysics) was tuned to 920 nm. For stimulation, 100 μM NMDA (Nacalai, 22034-1) and 40 μM glycine (Sigma, G7126-100G) in ACSF or SeeDB-Live/ACSF was applied during Ca2+ imaging. Imaging data were analyzed with ImageJ. Briefly, small drifts were corrected by the Image Stabilizer plugin for ImageJ (https://imagej.net/plugins/image-stabilizer) when necessary. After fluorescence intensity was obtained, the data were analyzed with MATLAB software (MathWorks). The F0 was calculated by temporal median filtering (ten-frame window). After the signal was filtered with temporal median filtering (three-frame window), the ‘findpeaks' function was applied for peak detection. Acute brain slices were prepared from wild-type C57BL/6N mice (P18, male and female). The slice was mounted on a custom-made silicone chamber for imaging using an upright microscope as previously described35,66. Calcein (40 μM) was added to the ACSF. The slice was perfused with ACSF containing 40 μM calcein for 1 h. For clearing, the slice was perfused with SeeDB-Live containing 40 μM calcein for 1 h. For imaging, two-photon microscopy (MM201, Thorlabs) equipped with a 25x NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP) and ThorImageLS software (Thorlabs) was used. A 920-nm femtosecond laser (ALCOR 920-4 Xsight, SPARK LASERS) was used. Liquid junction potential (LJP) was determined for ACSF and SeeDB-Live/ACSF as described previously (Extended Data Fig. We filled the recording electrode with internal solution and the reference electrode with 3 M KCl. The two electrodes were sequentially inserted into internal solution, ACSF and SeeDB-Live/ACSF while recording under current-clamp mode to measure potentials in each solution (VIN, Vcontrol-ACSF, VSeeDB-Live/ACSF). C57BL/6J mice (male and female) were purchased from Japan SLC. Mice were deeply anesthetized with isoflurane. For P14–18 mice, brains were quickly removed from mice and put into ice-cold ACSF bubbled with 95% O2 and 5% CO2. Acute coronal slices (300-µm thick) containing S1 were prepared using a vibratome (VT1200S, Leica). The slices were recovered in ACSF for at least 1 h at room temperature (23–24 °C) before recording for the control condition. For the SeeDB-Live condition, following the recovery in ACSF for 1 h, the slices were transferred into SeeDB-Live solution saturated with 95% O2 and 5% CO2 for 1 h at room temperature. For the P15–18 L5ET neurons, all the recordings were performed within 6 h after recovery (4 h after clearing). For the P14–18 fast-spiking interneurons and P28–29 L5ET neurons, all the recordings were performed within 6 h after recovery. Recording was extremely difficult when cleared with SeeDB-Live, even using infrared differential interference contrast. We tried to minimize the sampling bias by limiting the recording period after sample preparation. Consistent sampling (for example, cell type and depth) was confirmed. Neurons were visualized by an infrared differential interference contrast video microscope with a ×60, NA 1.0 water-immersion lens. L5ET neurons (thick-tufted L5 neurons with large cell bodies) and L5 fast-spiking interneurons with high-frequency spiking and little adaptation were analyzed. Neurons whose cell bodies were located deeper than 35 µm from the slice surface were recorded. Neurons were almost invisible under SeeDB-Live. Recordings were performed using MultiClamp700B amplifiers (Molecular Devices), filtered at 10 kHz using a Bessel filter and digitized at 20 kHz with Digidata 1440 A digitizer (Molecular Devices), and stored using pClamp10 (Molecular Devices). A series resistance compensation was not used for recordings. To characterize firing properties, hyperpolarizing and depolarizing square current pulses were injected under current-clamp mode (+50-pA increment, 1 s). In characterizing membrane properties, the membrane potential was clamped at −60 mV, and square pulses (−5 mV, 50 ms) were applied in voltage-clamp mode. sEPSCs were recorded at the holding potential of −60 mV. Transient negative current responses with a peak amplitude of <−10 pA were detected as sEPSCs. The morphologies of the recorded neurons were visualized by staining biocytin with streptavidin-Cy3 (1:1,000 dilution, S6402; Sigma-Aldrich) after recording. Fluorescence images were obtained using confocal microscopy (LSM900, Zeiss) equipped with a ×10 objective lens and controlled by Zen software (Zeiss, RRID: SCR_013672). In utero electroporation was performed as described previously35. To label mitral cells at embryonic day 12 (E12), 1 μg each of pCAG-GCaMP6f and pGP-pcDNA3.1 Puro-CAG-Voltron2 (Addgene, 172909) plasmids were injected into the lateral ventricle. Electric pulses (a single 10-ms poration pulse at 72 V, followed by five 50-ms driving pulses at 40 V with 950-ms intervals) were delivered along the anterior–posterior axis of the brain with forceps-type electrodes (3-mm diameter, LF650P3, BEX) and a CUY21EX electroporator (BEX). Electric pulses (a single 10-ms poration pulse at 72 V, followed by five 50-ms driving pulses at 42 V with 950-ms intervals) were delivered toward the mediolateral axis of the brain with forceps-type electrodes (5-mm diameter, LF650P5, BEX) and an electroporator (CUY21EX, BEX). Voltage imaging with Voltron2 was performed in acute brain slices of P4–11 ICR mice (male and female) subjected to in utero electroporation at E12. Mice were anesthetized in ice and decapitated. The brain was mounted on a silicone rubber block (Togawa rubber) and sliced using a microslicer (Dosaka EM) at 300-μm thickness. The slices were incubated in O2-saturated ACSF containing 50 nM Janelia Fluor HaloTag Ligand 549 (GA1110, Promega) at room temperature for 1 h. After placing on a custom-made silicone chamber, the slices were then washed under the perfusion of O2-saturated ACSF for 1 h (2 h for control experiment) at room temperature. The chamber was set under an FV1000MPE microscope (Olympus/Evident) with Fluoview FV10-ASW software (Olympus/Evident, RRID: SCR_014215) and a ×25 NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP). For epifluorescence imaging, a mercury arc lamp was used. Epifluorescence was imaged with a high-speed CCD camera (MiCAM02-HR or MC03-N256, BrainVision) at 0.3–7 ms per frame. For two-photon imaging of GCaMP6f, a femtosecond laser (InSight DeepSee, SpectraPhysics) was tuned to 920 nm. Image data were analyzed with ImageJ. Small drifts were corrected by the Image Stabilizer plugin. After fluorescence intensity was obtained, the data were analyzed with MATLAB software (MathWorks). The F0 was calculated as temporal median filtering (100-frame window). For peak detection, the ‘findpeaks' function was applied. Voltage changes within a mitral cell (single-trial data) were visualized with BV Workbench (BrainVision). A two-dimensional mean filter (3 × 3 pixels), drift removal (polyfit, degree 3) and a Savitzky–Golay filter (32 points, window size of five frames) were applied. C57BL/6N mice (2- to 4-month-old, male and female) were used. Surgery and imaging were performed using ketamine (Daiichi-Sankyo) and xylazine (Bayer; 80 mg per kg body weight and 16 mg per kg body weight, respectively) anesthesia. During surgery, the depth of anesthesia was assessed by the toe-pinch reflexes, and supplemental doses were added when necessary. Body temperature was maintained with a heating pad (Akizuki, M-08908). The dura matter was carefully removed by a micro hook (durotomy; 10065-15, Muromachi Kikai). BSA-CF594 (1 mg; 20290, biotium) was dissolved in 100 μl SeeDB-Live/ACSF-HEPES. SeeDB-Live/ACSF-HEPES (50–100 μl; 1% BSA-CF594) was mounted onto the brain surface. The solution was stirred for 1 h using a gelatin sponge (Spongel, LTL Pharma). Mice were euthanized with an overdose of pentobarbital (100–150 mg per kg body weight, i.p.) The brain was embedded in OTC compound (4583, Sakura Finetek) and frozen with liquid nitrogen. The frozen sections were made with a cryostat (CM3050S, Leica) and immediately imaged with an inverted microscope (DMI600B, Leica) equipped with a ×5 NA 0.1 dry objective lens and controlled by LAS AF software (Leica). Adult Thy1-YFP-H mice (1- to 6-month-old, male and female) were anesthetized with a mixture of ketamine (Daiichi-Sankyo; 80 mg kg−1 body weight) and xylazine (Bayer; 16 mg kg−1 body weight); 15% mannitol solution (3 ml per 100 g body weight; Sigma-Aldrich, M4125) was then administered intraperitoneally. After the durotomy, bleeding was stopped with a gelatin sponge (LTL Pharma, Spongel). The brain surface was perfused with ACSF-HEPES, then switched to SeeDB-Live/ACSF-HEPES and perfused for 1 h at a flow rate of 1.5 ml min−1. The fluorescence of L5ET neurons was imaged using a two-photon microscope (MM201, Thorlabs) equipped with a ×25 NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP) and ThorImageLS software (Thorlabs). The correction collar was turned to the appropriate positions (refractive index ~1.34 for control and ~1.363 for SeeDB-Live). A 920-nm femtosecond laser (ALCOR 920-4 Xsight, SPARK LASERS) was used. Anesthetized adult C57BL6/J mice (~7-week-old, male and female) were used for in vivo calcium imaging of V1. Under isoflurane anesthesia (2%) together with calprofen (10 mg per kg body weight), atropine (0.3 mg per kg body weight) and dexamethasone (2 mg per kg body weight), a craniotomy and durotomy was performed over the V1 (2.2-mm square). Half of the exposed cortex was covered with a piece of square coverslip (2 × 1 mm), and the other half was directly in contact with an extracellular solution for a subsequent pipette insertion and SeeDB-Live/ACSF-HEPES application. Anesthesia was continued at a lower concentration of isoflurane (0.125–0.5%), supplemented with chlorprothixene (1 mg per kg body weight, Sigma)69. Body temperature was maintained at 37 °C using a feedback-controlled heating pad. Then, the mice were placed under a two-photon microscope (Bermago II, Thorlabs). jGCaMP8m expression was achieved by AAV-DJ-Syn-jGCaMP8m-WPRE (4.1 × 1014 GCs per ml, 150 nl at a depth of 350 μm, three sites 200 μm apart), and jGCaMP8m was excited at 980 nm (MaiTai DeepSee eHP, SpectraPhysics). The Cal-520 AM solution also included 50 μM Alexa Fluor 594 (Sigma) to visualize dye-loading pipettes (at 1,070 nm excitation by Fidelity-2, Coherent). Cal-520 AM solution was pressure injected (150 mbar, 2 min), and after an incubation time of 1 h, two-photon imaging experiments were performed. Two-photon excitation of Cal-520 was at 920 nm (MaiTai DeepSee eHP, SpectraPhysics). Visual stimulation was presented at an LCD monitor (iPad 3/4 Retina Display, Adafruit, refresh rate of 60 Hz, gamma corrected), which was placed contralateral to the craniotomy side, covering an angle of 100° horizontal and 80° vertical in the visual hemifield. The monitor was moved and placed at a position that could evoke maximal full-field calcium response. The stimulation was controlled by MATLAB programs originally developed by Cortex Lab at University College London69. Full-screen sinusoidal drifting gratings were presented randomly at one of the eight directions (0–315°, 45° step) together with a blank condition. After obtaining a visual response to the drifting gratings in the control condition of ACSF-HEPES (more than 15 repeats), solution under the objective lens (~1 ml) was replaced with SeeDB-Live/ACSF-HEPES. After an additional 1-h incubation, we confirmed that baseline brightness was increased for jGCaMP8m and visual response to the same set of drifting gratings was obtained in the presence of SeeDB-Live/ACSF-HEPES. Calcium traces for each cell were computed offline using a Python version of Suite2p62. ROIs were drawn over cells, and fluorescence signal for each ROI was extracted. Neuropil contamination was not corrected, considering correction factor could be different in the presence and absence of SeeDB-Live. Visual response for each cell was further analyzed using MATLAB. First, visual responsiveness was evaluated by Kruskal–Wallis test using calcium response across eight directions plus the blank conditions. Orientation selectivity was judged by Kruskal–Wallis test using the response at eight-direction conditions. Orientation response was fitted with the sum of two Gaussian curves. The tuning width was measured as full width at half maximum height of the fitted curve. The maximum response was average calcium signal at an orientation closest to the preferred orientation. AAV1-Syn-FLEX-Volton2-ST-WPRE (Addgene, 172907-AAV1) was injected into 2-month-old Pcdh21-Cre mice (male and female) for in vivo voltage imaging. Surgery was performed under ketamine (Daiichi-Sankyo) and xylazine (Bayer; 80 mg and 16 mg per kg body weight, respectively) anesthesia. A small circle (~1 mm in diameter) was made with a dental drill over the right olfactory bulb. The AAV1 vector was injected into the center of the dorsal olfactory bulb (200-µm depth), over a 12-min period, using a Nanoject III injector (Drummond Scientific Company). Before imaging, mice were subjected to surgery under ketamine and xylazine (80 mg and 16 mg per kg body weight, respectively) anesthesia. After a craniotomy and durotomy over the right olfactory bulb, a silicone sealant rim was created around the window to maintain fluid over the brain surface. Around 50–100 μl of ACSF-HEPES or SeeDB-Live/ACSF-HEPES was applied onto the brain surface for 1 h. After removing a silicone sealant rim, a circular coverslip (2-mm diameter) was mounted on the brain surface and fixed with a superglue and silicone sealant. Anesthetized adult Thy1-GCaMP6f mice (2- to 4-month-old, male and female) were used for imaging. The fluorescence of mitral cells was imaged using an FV1000MPE microscope (Olympus/Evident) with Fluoview FV10-ASW software (Olympus/Evident, RRID: SCR_014215) and a ×25 NA 1.05 objective lens (Olympus/Evident, XLPLN25XWMP). The correction collar was turned to the appropriate positions (refractive index ~1.33 for control and ~1.363 for SeeDB-Live). Small drifts were corrected by the Image Stabilizer plugin. After fluorescence intensity was obtained, the data were analyzed with MATLAB software (MathWorks). The surface of the olfactory bulb was immersed with ACSF-HEPES containing 50 nM Janelia Fluor HaloTag Ligand 549 to label Voltron2 for 1 h, followed by a 1-h washout in normal ACSF-HEPES. SeeDB-Live treatment was then performed for 1 h. Mice were placed on an FV1000MPE microscope (Olympus/Evident) and a ×25 NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP). For excitation, a mercury arc lamp was used. Epifluorescence was imaged with a high-speed CCD camera (MC03-N256, BrainVision) at 1–7 ms per frame. Imaging data were analyzed using ImageJ. Small drifts were corrected using the Image Stabilizer plugin. ROIs were generated using a machine learning-based image segmentation tool (ilastik)72. Further analysis was performed using MATLAB software (MathWorks). ROIs smaller than five pixels and/or outside the glomeruli were excluded. F0 was calculated by temporal median filtering (25 frames window). k-means clustering was applied with k = 11 (the number of glomeruli). To define subclusters, the spike synchronicity index between ROIs was calculated. Spikes were detected using the findpeaks function. Thresholds were set at mean + 1.5 times the s.d. for the clustering and final spike detection, respectively. A synchronous event was defined when the spike in one ROI was detected within ±7 ms (21-ms window) of the spike in another ROI. The synchronicity index was calculated by dividing the number of synchrony events by half of the total number of spikes in two ROIs. Hierarchical clustering was used to define subclusters. To make a reference for the phase analysis, the averaged ΔF/F0 was calculated from all glomeruli. Odor stimulation using an olfactometer was described previously46. The olfactometer consists of an air pump (AS ONE, 1-7482-11), activated charcoal filter (Advantec, TCC-A1-S0C0 and 1TS-B) and flowmeters (Kofloc, RK-1250). Valeraldehyde (TCI, V0001) and amyl acetate (FUJIFILM-Wako, 018-03623) were diluted at 1% (vol/vol) in 1 ml mineral oil in a 50-ml centrifuge tube. Saturated odor vapor in the centrifuge tube was delivered to a mouse nose with a Teflon tube. Diluted odors were delivered for 5 s at 1 l min−1. After durotomy, bleeding was stopped with a gelatin sponge (Spongel, LTL Pharma). The brain surface was perfused with ACSF-HEPES, then switched to SeeDB-Live/ACSF-HEPES for 1 h at 1.5 ml min−1. A commercially available PVDC wrapping film (Asahi Kasei, Asahi Wrap or Saran Wrap, ~11-μm thick) with a ~1-mm margin was applied to the cranial window and firmly attached to the skull with superglue. Agarose (1.5% wt/vol) with or without 19.3% wt/vol glycerol (refractive index, 1.363) was filled between the film and a glass coverslip (Matsunami 18 × 18 no.1). After imaging, the agarose was replaced with a transparent silicone elastomer (GC, Exaclear) and a glass coverslip was applied on top. The coverslip was then sealed with waterproof film. To minimize inflammation after the surgery, dexamethasone sodium phosphate (4 mg ml−1) was administered intramuscularly into the quadriceps muscle at a dose of 2 µg per gram of body weight immediately before surgery. Additionally, carprofen (0.50 mg ml−1) was administered via subcutaneous injection at a dosage of 5 mg per kg body weight once daily for 3 consecutive days following the surgery to manage postoperative pain and inflammation. jGCaMP8m was introduced by the combination of the injection of 300 nl AAV-DJ-CAG-FLEX-jGCaMP8m-P2A-CyRFP (1.8 × 1011 GCs per ml) to S1 and intravenous injection of 100 μl AAV.PHPeB-mscRE4-minBGpromoter-iCre-WPRE-hGHpA (7.4 × 1010 GCs per ml; Addgene, 163476)73 to the retro-orbital venous sinus. The cranial window with a plastic wrap was carefully removed with a scalpel for repeated clearing experiments. Whisker stimulation was performed with air puffs produced by Picospritzer (Parker). Inflammation should be minimized during the procedure as this can potentially trigger the immune response to BSA. Image data were analyzed with MATLAB (MathWorks). Small drifts were corrected by the rigid motion-correction program (https://github.com/flatironinstitute/NoRMCorre/). For voltage imaging of L2/3 neuron somata from P17–30 ICR mice (male and female) subjected to in utero electroporation at E15, surgery and imaging were performed under urethane (Sigma, U2500) anesthesia (1.9 g per kg body weight). For imaging of the dendrites from the 2-month-old ICR mice, surgery was performed under isoflurane anesthesia (2.5% for induction, 1–1.3% for surgery, flow rate of 0.3–0.6 l min−1). A custom-made headpost was attached with a dental cement (GC Dental Products Corporation, UNIFAST II). After a craniotomy and durotomy (3–5 mm in diameter) over S1, a silicone sealant rim was created around the window to maintain fluid over the brain surface. In total, 50–100 μl of ACSF-HEPES containing 50 nM Janelia Fluor HaloTag Ligand 549 (GA1110, Promega) was applied onto the brain surface for 1 h. After removing the mounted solution, 50–100 μl of ACSF-HEPES was mounted for 1 h (2 h for control experiment). For clearing, SeeDB-Live/ACSF-HEPES was applied onto the brain surface for 1 h. After removing a silicone sealant rim, a circular coverslip (3 mm in diameter, thickness of 0.17 mm for P17–30, 0.34 mm for 2-month-old mice) was fixed with superglue. The mice were placed on an FV1000MPE microscope (Olympus/Evident) and a 25x NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP). For excitation, a mercury arc lamp was used. Epifluorescence was conducted with a high-speed CCD camera (MC03-N256, BrainVision) at 1–3 ms per frame. Image data were analyzed with MATLAB (MathWorks). Small drifts were corrected by the rigid motion-correction program (https://github.com/flatironinstitute/NoRMCorre/). The F0 was calculated by temporal median filtering (100-frame window). For video, a 3D median filter (1 pixel radius) was applied. 105558-AAV1) were injected at a 3:1 ratio into 2-month-old C57BL/6N mice (male and female). Surgery was performed under ketamine (Daiichi-Sankyo) and xylazine (Bayer; 80 mg and 16 mg per kg body weight, respectively) anesthesia. A small circle (~1 mm in diameter) was made with a dental drill over the right S1. The AAV1 vectors were injected into the right S1 (3 mm lateral to the midline, 1 mm caudal to bregma at a depth of 300 μm), over a 12-min period, using a Nanoject III injector (Drummond Scientific Company). A custom-made headpost was attached with a dental cement (GC Dental Products Corporation, UNIFAST II). Before imaging, mice were subjected to surgery under isoflurane anesthesia (2.5% for induction, 1–1.3% for surgery, flow rate of 0.3–0.6 l min−1). After a craniotomy and durotomy over the right S1, 50–100 μl of SeeDB-Live/ACSF-HEPES was applied onto the brain surface for 1–2 h. A circular coverslip (3 mm in diameter, thickness of 0.34 for 2-month-old ICR mice) was mounted on the brain surface and fixed with a superglue. The mice were placed under a resonant two-photon microscope (MM201, Thorlabs) equipped with a ×25 NA 1.05 water-immersion objective lens (Olympus/Evident, XLPLN25XWMP) and ThorImageLS software (Thorlabs). Immersion was performed using 15.6% (vol/vol) TDE/ddH2O (refractive index, 1.363). The correction collar was turned to the appropriate positions (refractive index ~1.33 for control and ~1.363 for SeeDB-Live). A 920-nm femtosecond laser (ALCOR 920-4 Xsight, SPARK LASERS) was used. Image data were analyzed with MATLAB (MathWorks). Small drifts were corrected by the rigid motion-correction program (https://github.com/flatironinstitute/NoRMCorre/). The F0 was calculated by temporal median filtering (15-frame window). Behavioral experiments were performed to evaluate the acute and chronic toxicity of SeeDB-Live treatment. C57BL/6N mice (2-month-old, male and female) were used. We made a large cranial window with a plastic film window (3 × 6 mm2) for the optical clearing of the right cortex as described above. At ≥3 days after surgery, mice were anesthetized with isoflurane (2% for induction, 0.7–1.0% during surgery) and injected with 15% mannitol solution (3 ml per 100 g body weight; Sigma-Aldrich, M4135). The plastic film and silicone were carefully removed. Then, SeeDB-Live/ACSF-HEPES or ACSF-HEPES was perfused onto the brain surface at a rate of 1.5 ml min−1 for 1 h. For acute behavioral experiments, head-fixed mice were placed on a custom-built treadmill 1 h after recovery from anesthesia. The treadmill consisted of a freely rotating roller with eight embedded magnets, which were detected by a sensor to monitor locomotion. Locomotion was recorded at 10 kHz for 10 min using PowerLab (AD Instruments). A transparent silicone elastomer (GC, Exaclear) and a glass coverslip (Matsunami 18 × 18 no.1) were then applied on top of the film. For long-term behavioral experiments, the film was reapplied to the cranial window and firmly secured to the skull with superglue after the application of SeeDB-Live/ACSF-HEPES or ACSF-HEPES for 1 h. A transparent silicone elastomer (GC, Exaclear) and a glass coverslip (Matsunami, 18 × 18 mm, no. 1) were then placed on top of the wrap. A cortical ischemia model was used for control experiments. Photosensitive dye (150 μg per gram of body weight; Rose Bengal) was i.p. injected, and the right cortical surface was irradiated through the skull with a white LED (Leica, KL300LED, 50 W) for 10 min. For locomotion measurements, mouse locomotion in an open chamber (30 × 25 cm) was recorded for 10 min using a USB camera (eMeet, SmartCam C960). Locomotion was tracked and analyzed with ezTrack74 under uniform ambient lighting. To assess changes in motor function, a wire hanging test was performed as described55. Mice were placed on a wire mesh (1-mm diameter wire, 10 × 10-mm grid) and allowed to grip the mesh. Wire hanging requires motor cortex but does not require pretraining. To monitor the food intake, mice were individually housed in cages with ad libitum access to food and water. Mice were kept under a 12-h light–dark cycle. C57BL/6N mice (2-month-old to 4-month-old, male and female) were used. In addition, a 10-month-old mouse was used for the long-term SeeDB-Live treatment experiment. In this mouse, jGCaMP8m was introduced by a 300-nl injection of AAV-DJ-CAG-FLEX-jGCaMP8m-P2A-CyRFP (1.8 × 1011 GCs per ml) into S1 and an intravenous retro-orbital injection of 100 μl AAV. We made a large cranial window with a plastic film window (3 × 6 mm2) on the right cortex as described above. Ten days after the surgery, mice were anesthetized with isoflurane (2% for induction, 0.7–1.0% during surgery) and injected with 15% mannitol solution (3 ml per 100 g body weight; Sigma-Aldrich, M4135). The film and silicone were carefully removed. Then, either SeeDB-Live/ACSF-HEPES or ACSF-HEPES was perfused onto the brain surface at a rate of 1.5 ml min−1 for 1 h. Then, the cranial window was sealed with plastic film, silicone and the coverslip, as described above until the next day. In the mouse with repeated SeeDB-Live treatments, the treatment was initiated immediately after the window surgery and was repeatedly performed over a period of 7 months (day 0, 3, 7, 80, 100, 120 and 218). Mice were deeply anesthetized by an overdose of pentobarbital (i.p. Anesthetized mice were perfused with 4% PFA in PBS. Excised brain samples were then fixed with 4% PFA in PBS at 4 °C overnight. The samples were cryoprotected with 30% sucrose at 4 °C overnight and embedded in OCT compound (Sakura). Frozen brains were cut into 16-μm-thick coronal sections using a cryostat (Leica). Sections were blocked with 10% normal donkey serum or 1% BSA in PBS for 1 h and then incubated with primary antibodies in 10% normal donkey serum or 1% BSA in PBS at 4 °C overnight. After washing three times in PBS with 0.05% Tween20, sections were incubated with secondary antibodies for 2 h at room temperature. Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500 dilution; Thermo Fisher, A21202), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1:500 dilution; Thermo Fisher, A31573) and Alexa Fluor 647-conjugated donkey anti-rat IgG (1:500 dilution; Thermo Fisher, A48272) were used as secondary antibodies. Images were acquired using a spinning disk confocal microscope (Andor, Dragonfly 200) mounted on an inverted microscope (Nikon, Eclipse Ti2) and controlled by Fusion software (Andor). Microglia in the S1 region were analyzed. Microglia morphology was manually traced, and 3D analysis (soma volume, soma roundness, branch number, maximum branch length and total branch length) was performed using Neurolucida software (MBF Bioscience). Roundness was defined as (4 × a / π) / b², where a is the soma area and b is the maximum diameter of the soma in each optical section. Roundness values were calculated at all z-levels per cell, and the median was used for analysis. Cells were selected from the S1 L2/3 region in a blinded manner. MATLAB (2023b) was used for statistical analysis. The number of animals is indicated in figure legends. Welch's t-test was used in Fig. Wilcoxon's rank-sum test was used in Figs. 3f,g, 6e–g,k,l,p,q and 7e. Wilcoxon's signed-rank test was used in Figs. Wilcoxon's rank-sum test combined with Holm–Bonferroni correction was used in Fig. A multiple-comparison test with Bonferroni correction was used in Fig. Dunnett's multiple-comparison test was used in Fig. 1c,g,h and Extended Data Figs. A Tukey–Kramer multiple-comparison test was used in Figs. Repeated-measures analysis of variance was used in Fig. Data inclusion/exclusion criteria are described in figure legends. Numerical data are summarized in Supplementary Table 3. Due to space limitations of the figures and figure legends, all the statistical details (sample size, P values and statistical tests used) are summarized in Supplementary Table 4. A new plasmid generated in this study (pCAG-GCaMP6f) has been deposited to Addgene (no. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Raw image data used in this study has been deposited to the SSBD:repository (https://doi.org/10.24631/ssbd.repos.2025.11.484)76. Detailed protocols and technical tips are described in SeeDB Resources (https://sites.google.com/site/seedbresources/). Requests for additional program codes and data generated and/or analyzed during the current study should be directed to and will be fulfilled upon reasonable request by the corresponding author. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Structural and molecular interrogation of intact biological systems. Dodt, H. U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Erturk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Hama, H. et al. ScaleS: an optical clearing palette for biological imaging. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Ke, M. T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Ke, M. T. et al. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Ueda, H. R. et al. Tissue clearing and its applications in neuroscience. Z., Tuchin, V. V. & Zhu, D. Skin optical clearing by glycerol solutions: mechanism. Cicchi, R., Pavone, F. S., Massi, D. & Sampson, D. D. Contrast and depth enhancement in two-photon microscopy of human skin by use of optical clearing agents. Wang, J., Zhang, Y., Xu, T. H., Luo, Q. M. & Zhu, D. An innovative transparent cranial window based on skull optical clearing. Zhu, D., Larin, K. V., Luo, Q. M. & Tuchin, V. V. Recent progress in tissue optical clearing. Achieving optical transparency in live animals with absorbing molecules. Feng, W., Liu, C. J., Wang, L. S. & Zhang, C. An optical clearing imaging window: realization of mouse brain imaging and manipulation through scalp and skull. Boothe, T. et al. A tunable refractive index matching medium for live imaging cells, tissues and model organisms. Sugimoto, S. & Kinjo, Y. Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging. Iijima, K., Oshima, T., Kawakami, R. & Nemoto, T. Optical clearing of living brains with MAGICAL to extend imaging. Arbelaez-Camargo, D. et al. Osmolality predictive models of different polymers as tools in parenteral and ophthalmic formulation development. Frye, R. M., Lees, H. & Rechnitz, G. A. Magnesium-albumin binding measurements using ion-selective membrane electrodes. Keck, C. H. C. et al. Color-neutral and reversible tissue transparency enables longitudinal deep-tissue imaging in live mice. In vivo optical clearing of mammalian brain. Sakaue-Sawano, A., Kobayashi, T., Ohtawa, K. & Miyawaki, A. Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication. Tan, J. et al. Limited oxygen in standard cell culture alters metabolism and function of differentiated cells. Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Shadow imaging for panoptical visualization of brain tissue in vivo. Tonnesen, J., Inavalli, V. V. G. K. & Nägerl, U. V. Super-resolution imaging of the extracellular space in living brain tissue. Dense 4D nanoscale reconstruction of living brain tissue. Fujimoto, S. et al. Activity-dependent local protection and lateral inhibition control synaptic competition in developing mitral cells in mice. Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Manita, S., Shigetomi, E., Bito, H., Koizumi, S. & Kitamura, K. In vivo wide-field and two-photon calcium imaging from a mouse using a large cranial window. Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator. & Economo, M. N. High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations. Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy. Chong, E. et al. Manipulating synthetic optogenetic odors reveals the coding logic of olfactory perception. Spors, H. & Grinvald, A. Spatio-temporal dynamics of odor representations in the mammalian olfactory bulb. Measuring the olfactory bulb input-output transformation reveals a contribution to the perception of odorant concentration invariance. Schoppa, N. E. & Westbrook, G. L. Glomerulus-specific synchronization of mitral cells in the olfactory bulb. & Rinberg, D. Precise olfactory responses tile the sniff cycle. Iwata, R., Kiyonari, H. & Imai, T. Mechanosensory-based phase coding of odor identity in the olfactory bulb. High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy. Ota, K. et al. Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas. Yu, C. H., Stirman, J. N., Yu, Y. Y., Hira, R. & Smith, S. L. Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry. & Svoboda, K. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Lim, D., Chu, K. K. & Mertz, J. Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy. Yokoyama, T. et al. A multicolor suite for deciphering population coding of calcium and cAMP in vivo. Wichmann, T. O., Damkier, H. H. & Pedersen, M. A brief overview of the cerebrospinal fluid system and its implications for brain and spinal cord diseases. & Hell, S. W. 2,2′-Thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Wang, B. S., Sarnaik, R. & Cang, J. H. Critical period plasticity matches binocular orientation preference in the visual cortex. Dana, H. et al. Thy1-GCaMP6 transgenic mice for neuronal population in vivo. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Swindells, J. F., Snyder, C., Hardy, R. C. & Golden, P. Viscosities of Sucrose Solutions at Various Temperatures: Tables of Recalculated Values, Vol. Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. & Hirai, H. Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB. Ehler, E., Moore-Morris, T. & Lange, S. Isolation and culture of neonatal mouse cardiomyocytes. Aihara, S., Fujimoto, S., Sakaguchi, R. & Imai, T. BMPR-2 gates activity-dependent stabilization of primary dendrites during mitral cell remodeling. Neher, E. Correction for liquid junction potentials in patch clamp experiments. Sun, X. C. et al. Functionally distinct neuronal ensembles within the memory engram. Sato, T. K., Haider, B., Häusser, M. & Carandini, M. An excitatory basis for divisive normalization in visual cortex. Tischbirek, C. H. et al. Functional mapping of a cortical column at single-neuron resolution. Niell, C. M. & Stryker, M. P. Highly selective receptive fields in mouse visual cortex. Berg, S. et al. ilastik: interactive machine learning for (bio) image analysis. Graybuck, L. T. et al. Enhancer viruses for combinatorial cell-subclass-specific labeling. Pennington, Z. T. et al. ezTrack: an open-source video analysis pipeline for the investigation of animal behavior. Long-term imaging of experience-dependent synaptic plasticity in adult cortex. Imaging data in the paper “Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo” https://doi.org/10.24631/ssbd.repos.2025.11.484 (2025). Pertoft, H. & Laurent, T. C. Isopycnic separation of cells and cell organelles by centrifugation in modified colloidal silica gradients. in Methods of Cell Separation (ed. & Tuchin, V. A comparative study of skin optical clearing using two-photon microscopy. SPIE - The International Society for Optical Engineering Vol. SPIE - The International Society for Optical Engineering Vol. & Luo, Q. M. Imaging dermal blood flow through the intact rat skin with an optical clearing method. Choice of cranial window type for imaging affects dendritic spine turnover in the cortex. We thank H. Zeng (Allen Institute, Ai162), J. Sanes (Harvard University, Thy1-YFP-H), K. Svoboda (Allen Institute, Thy1-GCaMP6f) and E. Deneris (Case Western Reserve University, ePet-Cre) for mouse strains; A. Miyawaki (RIKEN) for cell lines (HeLa/Fucci2); M. Eiraku (Kyoto University) for embryonic stem cell lines (EB5); D. Kim & GENIE Project (Janelia Research Campus) for pGP-CMV-GCaMP6f (Addgene plasmid no. 40755); F. St-Pierre (Baylor College of Medicine) for pAAV-EF1a-DIO-JEDI-2P-Kv-WPRE (Addgene viral prep no. 179459-AAV1); J. Wilson (University of Pennsylvania) for pENN.AAV.CamKII 0.4.Cre.SV40 (Addgene viral prep no. 105553-AAV1); GENIE Project (Janelia Research Campus) for pGP-AAV-syn-jGCaMP8m-WPRE, pGP-AAV-syn-jGCaMP8f-WPRE, pGP-pcDNA3.1 Puro-CAG-Voltron2 and pGP-pcDNA3.1 Puro-CAG-Voltron2-ST, pGP-AAV-syn-FLEX-Volton2-ST-WPRE (Addgene plasmid nos. 172907-AAV1); V. Gradinaru (California Institute of Technology) for pUCmini-iCAP-PHP.S (Addgene plasmid no. 103006); B Tasic (Allen Institute) for AiP1010-pAAV-mscRE4-minBGpromoter-iCre-WPRE-hGHpA (Addgene plasmid no. 163476); M -T. Ke and M. Morimoto (RIKEN) for evaluating our earlier versions of the clearing medium; S. Uchida and K. Miyamichi (RIKEN) for sharing reagents; M. Nishihara, E. Nozoe, S. Hamatake, K. Yashiro and S. -H. Chou for technical assistance. We also thank The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences, which was in part supported by Mitsuaki Shiraishi Fund for Basic Medical Research. This work was supported by grants from CREST program (JPMJCR2021 to T.I. ), Kagoshima University Megumikai Medical Research Promotion Fund (to Y.T. ), World Premier International Research Center Initiative (WPI-PRIMe; to K.H. ), the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Uehara Memorial Foundation (to T.I. Shigenori Inagaki, Satoshi Fujimoto, Takahiro Noda, Hikari Takeshima, Koki Ishikawa & Takeshi Imai Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan Nathan Zechen Huynh, Yuki Kambe & Tatsuo K. Sato Rei Yagasaki, Misato Mori, Aki Teranishi & Satoru Okuda Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan Department of Optical Neural and Molecular Physiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan Laboratory of Optical Biomedical Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan Division of Reproductive Systems, Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Japan Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar performed all the experiments for screening and optimizing SeeDB-Live. performed chronic awake in vivo imaging. Correspondence to Shigenori Inagaki or Takeshi Imai. have filed a patent application related to SeeDB-Live. The other authors declare no competing interests. Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. (b) Calcium responses of GCaMP6f-expressing HEK293T cells to 50 μM ATP, measured using a plate reader. Osmolarity was not adjusted to isotonicity (hypertonic, 300-700 mOsm/kg see also c) in this experiment. After incubation with the clearing media for 4 hours, GCaMP6f responses were measured. (c) Calcium responses of GCaMP6f-expressing HEK293T cells to 50 μM ATP in the media with variable osmolality (300-700 mOsm /kg). The osmolality was adjusted with 10x PBS. The osmolalities of high MW media are also indicated. There are several products of BSA with different grades of purity are available from different companies. The osmolality was slightly different among these products, suggesting that salts remain in some of the products. (e) Residual salts contained in BSA products #1 and #2. The amounts of Na+, K+, Ca2+ and Mg2+ in BSA powder were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The Cl− amount was obtained from the product's certificate of analysis, which was measured by ion chromatography. In the preparation of SeeDB-Live, residual salts contained in the BSA powder were taken into account. (f) Transmittance of live HeLa cell suspension (4 × 106 cells/mL, transmittance at 600 nm) in isotonic BSA#1/PBS at refractive index 1.365-1.375. n = 3 each. (g) Growth ratio of HeLa/Fucci2 cells in refractive index-optimized (refractive index 1.363, 320 mOsm/kg) medium of different BSA products. Purification methods are not disclosed for most BSA products. (h) Growth curve of HeLa cells cultured in a 35 mm dish. Cell numbers in suspension was measured with a hemocytometer after trypsinization. The medium was isotonic, and their refractive indices were adjusted to 1.363. n = 3 dishes each. ; not significant (p ≥ 0.05) (Wilcoxon rank sum test corrected with Holm-Bonferroni correction). Cell number was counted based on fluorescence images of the nuclei. ***p < 0.001 (two-sided Dunnett's multiple comparison test). (j) Standard curve for the viscosity measurement using sucrose solution. Flow speed was determined using 50 mL syringes. Plot was fitted with a single-exponential curve. (k) Viscosity of candidate solutions (in ddH2O). (l) Specific gravity of the candidate solutions in PBS. The refractive indices of iodixanol and BSA#1 were 1.366. Specific gravity of cells are cited from previous studies77. (m, n) Some of the BSA products have autofluorescence signals of unknown origin, especially for UV to blue range; therefore, we carefully selected low autofluorescence and low toxicity products from multiple suppliers. Absorbance (m) and fluorescence (n) of fetal bovine albumin (FBS), iodixanol, and BSA from different manufacturers are shown. Except for FBS, they are adjusted to 15% (w/v) in ddH2O. Data with error bars indicate mean ± SD. See Supplementary Table 4 for detailed statistical data. (a, b) Spontaneous activity of mitral cells in the olfactory bulb was measured under different concentrations of divalent cations ex vivo. Amplitude (a) and frequency (b) were analyzed. Thy1-GCaMP6f mice (age, P11-14) were used for two-photon imaging of acute olfactory bulb slices. Note that the amounts of Ca2+ and Mg2+ derived from BSA products are also considered. (c, d) Spontaneous activity of mitral cells in the olfactory bulb was measured under different concentrations of Na+ and Cl⁻ ex vivo. Amplitude (c) and frequency (d) were analyzed. Thy1-GCaMP6f mice (age, P9-11) were used for two-photon imaging of acute olfactory bulb slices. A NaCl stock solution was added to adjust the concentration of Na+ and Cl⁻. Note that the amounts of Na+ and Cl⁻ derived from BSA products are also considered. There are no statistical differences in the amplitude or frequency across all conditions. However, abnormal firing (putative cortical spreading depression) was found at higher Na+ conditions (174 mM) in one trial, where the Cl⁻ concentration was matched to ACSF (134 mM). This slice was excluded in the analysis. n.s., non-significant (p ≥ 0.05) (Bonferroni-corrected pairwise comparisons).The formulation of the ACSF and the isotonic SeeDB-Live/ACSF (in mM) is as follows. See Supplementary Table 4 for detailed statistical data. Cells were cultured in SeeDB-Live medium (refractive index 1.366, 320 mOsm/kg) from DIV2 to DIV5. (b) Primary culture of cardiomyocytes before and after incubation in SeeDB-Live medium. Confocal image (top) and spontaneous calcium signals (bottom) at DIV2. (c) jGCaMP8f signals of cultured cardiomyocytes at DIV3 and DIV5. (d) Primary culture of hippocampal neurons. Neurons were cultured in SeeDB-Live medium (refractive index 1.363, 230 mOsm/kg) from DIV7 to 10. (e) jGCaMP8m signals of cultured hippocampal neurons before and after incubation in SeeDB-Live medium. (f, g) Amplitude and frequency of spontaneous calcium signals for the control (black) and SeeDB-Live (red). n.s., non-significant (two-sided Wilcoxon rank sum test). (h) Transmittance of live HeLa cell suspension (4 × 106 cells/mL) at different wavelengths was compared among PBS, SeeDB-Live/PBS, and 0.6 M tartrazine/PBS18. Due to high absorption of tartrazine, we could not compare transmission below 550 nm. (i) Phase contrast and fluorescence images of GFP-expressing HEK293T cells. The cells were immersed in Control (PBS), SeeDB-Live/PBS (refractive index 1.363, 296 mOsm/kg), and then in 0.6 M tartrazine/PBS (refractive index 1.43, 1407 mOsm/kg). (j) Phase contrast image of HeLa cells before and after immersion in 0.1 M tartrazine solution (RI 1.351), adjusted to isotonic condition (310 mOsm/kg). (k) Refractive indices, osmolalities, and transmittances (at 600 nm) were compared among clearing agents reported for live tissue 18,28. Red indicates ideal ranges for non-invasive optical clearing of live cells. Data with error bars indicate mean ± SD. (a)-(g) show data from representative samples out of 3 trials. (i) and (j) show single-trial data. See Supplementary Table 4 for detailed statistical data. (a) Growth of HeLa/Fucci2 spheroids cultured continuously in SeeDB-Live medium (refractive index 1.366, 320 mOsm/kg). Cell number in suspension was calculated with a hemocytometer after trypsinization. Half of the medium was replaced daily. 17.2% (v/v) 2,2'-thiodiethanol (TDE) in ddH2O (refractive index 1.366) was used for immersion to minimize spherical aberration. (c) Three-dimensional fluorescence images of a HeLa/Fucci2 cell spheroid in the control and SeeDB-Live media (4 hour clearing per day as shown in Fig. Fluorescence intensity indicate the mean intensity of all the cell nuclei in each z plane. 17.2% (v/v) TDE/ddH2O (refractive index 1.366) was used as immersion. (f) Responses of enteroendocrine cells to high potassium stimulation (30 mM at final concentrations). GCaMP6s signals are shown for the intestinal organoids derived from ePet-Cre; Ai162 mice (EEC-GCaMP6s). F0 (left) and ΔF/F0 (right) images (z stack: 0-186 µm) are shown. (g-j) ES cell-derived neuroepithelial organoid culture. (g) Schematic diagram of neuroepithelial organoid sample preparations. The epithelial tissue was broken with a glass capillary to facilitate clearing of the organoid with SeeDB-Live medium. 17.2% (v/v) TDE/ddH2O (refractive index 1.366) was used for immersion. (h) ES cell-derived neuroepithelial organoids (day 9). The bright field images before and after SeeDB-Live treatment. (i) 3D rendered fluorescence images of Lifeact-mCherry-expressing neuroepithelial organoid before and after SeeDB-Live treatment. A representative sample out of three with similar results. Normal (left) and SeeDB-Live medium (refractive index 1.363; right). Small incision was made in the organoid before SeeDB-Live treatment. (j) Fluorescence images of the Lifeact-mCherry-expressing neuroepithelial organoid at different depths before and after SeeDB-Live treatment. Data with error bars indicate mean ± SD. Images show representative samples out of 2-3 trials. See Supplementary Table 4 for detailed statistical data. Panels b, e and g created in BioRender. (a-c) Acute hippocampal slices cleared with SeeDB-Live. Confocal (a) and two-photon (b) images (3D rendering) of acute hippocampal slices cleared with SeeDB-Live/ACSF (refractive index 1.363, 310 mOsm/kg in ACSF). (c) Fluorescence images of the acute hippocampal slices taken at different depths using confocal (left) and two-photon microscopy (right). (d) Fluorescence images of acute brain slices from the primary somatosensory (S1) cortex at different refractive indices. (e) Two-photon shadow images at the superficial region of an acute brain slice from S1. The slice (S1 L5) was perfused with ACSF containing 40 μM Calcein. Bright signals are somata labeled with Calcein. (f, g) Fluorescence intensity from cell bodies in x-y fluorescence images of S1 L5ET neurons treated with 5% glycerol/ACSF. Acute brain slices of Thy1-EYFP-H mice (P19-21) were imaged before and after 5% glycerol/ACSF treatment (f). Mean fluorescence in ROIs are shown (g). Images show representative samples out of 2-3 trials. See Supplementary Table 4 for detailed statistical data. (a) Measurement of liquid junction potential (LJP). We inserted recording electrode filled with internal solution and reference electrode filled with 3 M KCl into internal solution, ACSF, and SeeDB-Live/ACSF sequentially while recording the potential under current-clamp mode. LJPs between internal solution and control ACSF or SeeDB-Live/ACSF were determined by the differences in potentials. (b) Infra-red differential interference contrast (IR-DIC) images of representative Layer 5 extratelencephalic-projecting (L5ET) neurons under electrophysiological recording. (c) Recorded neurons visualized by biocytin staining. (d) Current responses to the test pulse ( − 5 mV, 50 ms). (e) Additional electrophysiological properties of L5ET neurons at P15-18 recorded under ACSF and SeeDB-Live/ACSF. For example, we could easily patch sufficient number of neurons under ACSF. However, it took much longer to patch neurons under SeeDB-Live/ACSF, because neurons are invisible and difficult to find. The box plots indicate median ± interquartile range (IQR). n = 17 neurons from 4 mice and 14 neurons from 3 mice for control and SeeDB-Live, respectively. (f) AP frequency was plotted against injected current amplitude for the more mature L5ET neurons (age, P28-29). (g) Electrophysiological properties of the more mature L5ET neurons (age, P28-29). Results were compared between ACSF and SeeDB-Live/ACSF. Recorded neurons were then confirmed by biocytin staining post hoc. *p < 0.05; n.s., non-significant (Wilcoxon rank sum test). n = 13 cells from 2 mice per group (control and SeeDB-Live). (i) Recorded neurons visualized by biocytin staining. (k) AP frequency was plotted against injected current amplitude for the FS interneurons. (l) Electrophysiological properties of neurons for FS interneurons (age, P14-17). Results were compared between ACSF and SeeDB-Live/ACSF. FS interneurons were identified based on the firing properties ( ~ 100 Hz) upon current injection. Recorded neurons were then confirmed by biocytin staining post hoc. **p < 0.01; n.s., non-significant (two-tailed Wilcoxon rank sum test). n = 12 neurons from 4 mice and 9 neurons from 3 mice for the control and SeeDB-Live conditions, respectively. (m, n) Amplitude (m) and frequency (n) of spontaneous activity in the same set of mitral cells in ACSF and Iodixanol/ACSF (RI1.366). Olfactory bulb slices of Thy1-GCaMP6f mice were imaged. (o-q) (o) Spontaneous activity in the acute olfactory bulb slices was evaluated for glycerol-containing ACSF at a refractive index of 1.366. Mean amplitude (p) and frequency (q) of spontaneous activity in individual mitral cells. n = 24, 33 cells from 3 mice for ACSF and 21% Glycerol/ACSF. ***p < 0.001 (two-tailed Wilcoxon rank sum test). Data with error bars indicate mean ± SD. Images show representative samples out of 2-4 independent trials. See Supplementary Table 4 for detailed statistical data. (a) Layer 5 extratelencephalic-projecting (L5ET) neurons in the primary somatosensory cortex (S1) before and after clearing with SeeDB-Live on day 0 and 7. The S1 of a 4-month-old Thy1-EYFP-H mouse was imaged while the mouse was under anesthesia using two-photon microscopy. (b-k) Evaluation of inflammatory responses in S1 region after SeeDB-Live treatment. A large cranial window was made 10 days prior to the SeeDB-Live treatment because open skull surgery alone is known to cause transient activation of microglia82. SeeDB-Live treatment was performed for 1 hour. Mice were sacrificed 1 day after the treatment. The brain sections were 16 µm thick. (b) Frozen sections of the cerebral cortex were stained with DAPI, anti-NeuN (neuron), anti-Iba1 (microglia), anti-CD16/32 (activated microglia, M1), anti-GFAP (activated astrocyte), anti-Sox9 (astrocyte nucleus), anti-cleaved caspase-3 (apoptosis). (c) Treated and untreated areas of ACSF-HEPES or SeeDB-Live/ACSF-HEPES in the brain. A large cranial window was made only over the treated area in the right cortex. (d, e) Density of Iba1-positive microglia in ACSF-HEPES- and SeeDB-Live/ACSF-HEPES-treated mice. n = 3 mice each for treatment with ACSF-HEPES and SeeDB-Live/ACSF-HEPES. n.s., not significant (p ≥ 0.05) (two-sided Wilcoxon signed-rank test in (d), two-sided Wilcoxon rank-sum test in (e)). (f) Morphology of microglia after treatment with ACSF-HEPES or SeeDB-Live/ACSF-HEPES. (g) Evaluation of microglial morphology in ACSF-HEPES- and SeeDB-Live/ACSF-HEPES-treated mice. Microglial morphology was manually traced in 3D, and quantitative analyses (soma volume, soma roundness, branch number, maximum branch length, and total branch length) were performed. The box plots indicate median ± interquartile range (IQR). (h) Fraction of CD16/32-positive area within Iba1-positive regions (microglia activation). (i) Fraction of GFAP-positive area (astrocyte activation). (j) Density of SOX9-positive cells (all astrocytes). (k) Density of cleaved caspase-3-positive cells (apoptosis). (l) Representative images of the cerebral cortex stained with DAPI, anti-NeuN, anti-Iba, anti-CD16/32, anti-GFAP, anti-SOX9, and anti-cleaved caspase-3 after repeated SeeDB-Live/ACSF-HEPES treatment (day 0, 3, 7, 80, 100, 120, and 218). Representative results from 3 sections of a single mouse are shown. Data with error bars indicate mean ± SD. Images show representative samples out of 2-4 independent trials. See Supplementary Table 4 for detailed statistical data. (a-d) Calcium imaging of L2/3 neurons in the primary visual cortex (V1) before and after clearing with SeeDB-Live/ACSF-HEPES. Drifting gratings of various orientations were presented to anesthetized mice. (b) Basal fluorescence of Cal-520 without visual stimulation. L2/3 neurons at a depth of ~420 μm. (c, d) Responses of a representative L2/3 neuron (indicated by arrowheads in b) to visual grating stimuli before and after clearing with SeeDB-Live. (e) Preferred orientation, maximum responses (ΔF/F0), orientation selective index (OSI), and tuning width (Sigma) for the same set of L2/3 neurons (45-58 neurons in total) before (x-axis) and after (y-axis) clearing with SeeDB-Live. The comparison was performed as described previously56. The fluorescence image of cell somata at 200 μm and 564 μm depth was shown on the right, respectively. The spikes were detected at the somata of L2/3 (g) and L5 neurons (i) indicated in (f) and (h), respectively. Data in (c) and (d) indicate mean ± SD. See Supplementary Table 4 for detailed statistical data. Panels a, f and h created in BioRender. (a-c) Epifluorescence voltage imaging of the olfactory bulb slices. (a) Two-photon image identified a labeled mitral cell (z-stack; left) in acute olfactory bulb slices (P11) also shown in Fig. 6d-f. Epifluorescence of Voltron2549 (temporal median) and ROIs are shown on the right. Cell bodies, shafts of a primary dendrite, and tufted structure within a glomerulus were analyzed. Voltron2 and GCaMP6f were introduced to mitral cells by in utero electroporation. Traces from single shot images (b) and averaged images from 65 events (c) are shown. Ticks indicate the peaks of detected action potentials. (d-f) Epifluorescence voltage imaging of L2/3 neuron somata in anesthetized mice. (d) Epifluorescence images (temporal median) of L2/3 neurons in S1 labeled with Voltron2549-ST in an anesthetized mouse (P17) before and after clearing with SeeDB-Live (1 hour after clearing). (e, f) Action potentials were detected in a L2/3 neuron indicated by the arrow in (d). (g-i) Epifluorescence voltage imaging of L2/3 neuron dendrites in awake mice. (g) Dendrites of a L2/3 neuron in S1 labeled with mTurquoise2 and Voltron2549. We imaged an awake mouse (2 months old) after SeeDB-Live treatment. (i) The spikes were detected at the dendrites of an L2/3 neuron in S1 of an awake mouse indicated in (h). (a-c) is from a representative sample out of 2 slices; (d-f) is a representative sample out of 3 animals; (g-i) is from one sample. (a) Clusters (encircled by dotted lines) and subclusters (shown in different colors) of ROIs (mostly dendrites) of the mitral/tufted cells in the mouse olfactory bulb. The clusters and subclusters were defined based on voltage traces and spike synchronicity of ROIs, respectively. (b) Voltage traces of all subclusters (−ΔF/F0). Amyl acetate was diluted at 1% (v/v) in 1 mL mineral oil in a 50 mL centrifuge tube. Saturated odor vapor in the centrifuge tube was delivered to a mouse nose for 5 second at 1 L/min. Ticks indicate the detected action potentials. (c) The spike timing in each sniff/theta cycle. (d) Spike phase of each cluster against sniff-coupled theta wave before and during odor (1% amyl acetate) stimulation. Data are from a representative sample out of two animals with similar results. Time-lapse phase contrast images of HeLa/Fucci2 spheroids cleared with SeeDB-Live. Phase contrast images of HeLa/Fucci2 spheroids cleared with SeeDB-Live. Confocal images of HeLa/Fucci2 spheroids cleared with SeeDB-Live. Time-lapse transmission imaging of mouse cerebral cortex cleared with SeeDB-Live. A cortical slice (300-μm thick, P5) was cleared with SeeDB-Live. Two-photon imaging of S1 L5ET neurons in acute brain slices prepared from a Thy1-YFP-H mouse. Confocal imaging of pyramidal neurons in hippocampal CA1 in acute brain slices prepared from a Thy1-YFP-H mouse. Two-photon imaging of pyramidal cells in hippocampal CA1 in an acute brain slice prepared from a Thy1-YFP-H mouse. Ahippocampal slice (300-μm thick) from a Thy1-YFP-H mouse (P6) was imaged with two-photon microscopy. Two-photon shadow imaging of acute brain slices. A cortical slice (300-μm thick) from a wild-type mouse (P18) was perfused with 40 μM calcein solution in ACSF or SeeDB-Live. Images were taken with two-photon microscopy. An olfactory bulb slice (300-μm thick) from a Thy1-GCaMP6f mouse (P11) was imaged with two-photon microscopy. Time-lapse two-photon imaging of GCaMP6f in acute olfactory bulb slices cleared with SeeDB-Live. An acute olfactory bulb slice(P11) was imaged with two-photon microscopy at a depth of 150 μm during clearing with SeeDB-Live. An olfactory bulb slice (300-μm thick) from a Thy1-GCaMP6f mouse (P13) was imaged with confocal microscopy. Confocal Ca2+ imaging of spontaneous activity of mitral cells in an olfactory bulb slice at different depths. An olfactory bulbslice (300-μm thick) from a Thy1-GCaMP6f mouse (P5) was imaged with confocal microscopy. A Thy1-EYFP-H mouse (4-month-old) was imaged with two-photon microscopy. Laser power and photomultiplier tube gain were constant across the depths. Time-lapse two-photon imaging of mouse cerebral cortex in a live animal cleared with SeeDB-Live. L5ET neurons in S1 in a Thy1-EYFP-H mouse (4-month-old) were imaged with two-photon microscopy during clearing with SeeDB-Live. Focal depth changes when perfusion is switched from ACSF (refractive index, 1.34) to SeeDB-Live/ACSF (refractive index, 1.363). We imaged multiplez-planes, and the imaging depth was calibrated post hoc to show the same depth. Thy1-GCaMP6f mouse (4-month-old) was imaged with two-photon microscopy. An olfactory bulb slice (300-μm thick, age P11) was imaged with epifluorescence microscopy at 2 kHz. Voltron2 was introduced to mitral cells by in utero electroporation at E12, labeled with JF549. Frames with action potential events are indicated by a white circle in the upper-left corner. ROIs were manually clipped based on two-photon and epifluorescence images. Data were normalized(−ΔF/F0) from Voltron2549 in ROIs. Preparation of SeeDB-Live and other clearing media. Details for all the statistical analyses in this study. Comparison with other in vivo clearing methods18,20,27,28,78,79,80,81. We compared SeeDB-Live and other clearing agents tested in vivo based on several key factors required for live cell clearing. Transparency of live cells was evaluated based on the transmittance of HeLa cell suspension (4 × 106 cells per ml) at 600 nm. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. et al. Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

A repeat expansion in GOLGA8A is a major risk factor for atypical frontotemporal lobar degeneration with ubiquitin-positive inclusions

Source: {'href': 'https://www.nature.com', 'title': 'Nature'} Published: 2026-03-12 10:51:59

Anti-Friedel–Crafts alkylation via electron donor–acceptor photoinitiation

Source: {'href': 'https://www.nature.com', 'title': 'Nature'} Published: 2026-03-12 10:27:19

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. The ubiquity of C–H bonds in organic molecules makes direct C–H functionalization an atom- and step-efficient strategy in synthetic chemistry. However, direct C–H alkylation, particularly of electron-poor aromatic substrates, remains a major challenge because current methods suffer from limited selectivity, functional group tolerance and/or require harsh acidic, pyrophoric or toxic reagents. Here we introduce a selective, scalable and transition-metal-free synthetic strategy for C–H alkylation of electron-poor aromatics under mild conditions, which also exhibits high functional group tolerance applicable to the late-stage functionalization of pharmaceutical compounds. The mechanistic design exploits a redox-active phthalimide ester tag to form an electron donor–acceptor complex that fragments upon photoexcitation to yield a nucleophilic alkyl radical, which selectively alkylates the most electrophilic position of electron-deficient aromatics, thereby exhibiting ‘anti-Friedel–Crafts' selectivity. Mechanistic studies, microkinetic modelling simulations and computational analyses indicate that the reaction then propagates via radical anion autocatalysis. The ‘anti-Friedel–Crafts' selectivity is consistent with theoretical predictions from Fukui indices and machine-learning models that provide the framework necessary to forecast selectivity in previously ‘unseen' substrates, thereby enabling selective alkylation of a wide range of complex molecules and late-stage pharmaceuticals. Despite being a fundamental synthetic disconnection, methods for sp2–sp3 C–H alkylation are particularly limited for electron-deficient aromatics. Friedel–Crafts alkylation reacts preferentially with electron-rich organic aromatics1,2, and other methods for direct C–H alkylation require the use of superstoichiometric organolithium or toxic organomercury reagents (Fig. All these methods are not only limited in selectivity, but also in reaction scope due to the harsh conditions, which severely limit functional group tolerance. Classical Minisci coupling does offer a reliable radical-based route to C–H-alkylated pyridines, typically with acidic activation, but crucially has a limited C2/C4 selectivity5,6. More broadly, some Minisci-type reactions can operate without the presence of acid but remain limited to heteroaromatic alkylation7. The absence of a convenient and reliable direct C–H functionalization strategy requires the installation of a functional handle, such as a halide, followed by traditional palladium-based cross-coupling reactions or the more recently developed photoredox nickel cross-coupling (Fig. This handle reduces the atom economy of the synthesis and introduces further issues with functional group tolerance and selectivity, particularly in late-stage functionalization. The method described in the present work overcomes these issues with an autocatalytic radical mechanism triggered by a mild and targeted photoactivation of an electron donor–acceptor (EDA) complex. a, Literature strategies for the alkylation of electron-poor aromatics4,6,8,10. b, General strategy for neutral radical formation via electron donor–acceptor complex excitation and fragmentation using a redox auxiliary14,15,16. c, This work: a general metal-free, photocatalyst-free approach for sp2–sp3 coupling via an EDA complex-triggered radical mechanism propagated by radical anion autocatalysis. A, carbon or nitrogen; R, alkyl; BET, back electron transfer; SET, single-electron transfer; EWG, electron-withdrawing group; [Ox], oxidant. The generation of radical species through the photoactivation of EDA complexes has been explored since the 1950s, but it has only recently been recognized as a useful tool in organic chemistry12,13,14. Upon photoexcitation, the EDA complex undergoes single-electron transfer (SET) to form a radical ion pair, which subsequently fragments to yield the desired neutral substrate radical (S•) (Fig. EDA complexes have since been exploited in organic chemistry to enable radical formation under mild conditions, with targeted visible photoreactivity achievable through substrate design12,13,14. However, initial applications of EDA catalysis were limited to specific donor–acceptor pairs and typically required a pre-installed leaving group18,19,20. The introduction of redox auxiliaries (RAs), or ‘redox tags', as components of EDA complexes has broadened its synthetic utility and, in the presence of a donor species (D), can facilitate a charge-transfer absorption band in the visible spectrum. These auxiliaries can be attached to a specific functional group of the substrate molecule, imparting more generality to EDA methodology across a wide range of substrates15,16,17. In this Article we exploit EDA methodology to establish a general sp2–sp3 C–H coupling method that selectively alkylates the most electron-deficient position on a broad range of electron-poor aromatics (Fig. This ‘anti-Friedel–Crafts' selectivity addresses a fundamental gap in the synthetic toolbox and has been achieved by utilizing an EDA complex to trigger an autocatalytic radical reaction. This, in turn, enables the reaction to proceed under mild reaction conditions to provide tolerance of a wide range of functional groups, including halide handles, to facilitate further downstream functionalization. The high regioselectivity and functional group tolerance are demonstrated with the late-stage modification of pharmaceuticals and agrochemicals. DABCO (1,4-diazabicyclo[2.2.2]octane) and a redox-active phthalimide ester (RAE) tag form the initial EDA complex to trigger the reaction and generate alkyl radicals, which couple with a wide range of electron-poor aromatics that then serve as electron shuttles to propagate the radical reaction and enable autocatalysis. This dual utility of the aromatic coupling partner overcomes a major limitation in established EDA methodology by eliminating the reliance on either pre-functionalized substrates for intramolecular reactions or a narrow set of ‘radical traps' (for example, silyl enol ethers, isocyanides and vinyl sulfones) as coupling partners15,16,21,22,23,24. The observed ‘anti-Friedel–Crafts' regioselectivity can be readily tuned with aromatic substituents, with reactivity predictable based on density functional theory (DFT) calculations and machine-learning (ML) models developed for this study. These computational insights establish predictive design principles, further broadening the scope and applicability of the methodology. The simple reaction conditions for photoinitiated anti-Friedel–Crafts alkylation require (1) an alkyl substrate functionalized with an RAE to provide the substrate and RA, respectively, (2) a nucleophilic amine to form an EDA complex with the redox auxiliary, and (3) an aromatic sp2 coupling partner for C–H alkylation (Fig. The RAEs were readily synthesized from phthalimide and a range of carboxylic acids to serve as the electron-deficient RA component in the EDA complex. The electron-rich tertiary amine DABCO was selected as the electron donor (D) due to its known but under-utilized activation of redox-active esters (RAEs)25,26. These easily produced and inexpensive components assemble into a visible-light-absorbing EDA complex that undergo photoinduced fragmentation to generate a phthalimide anion and a neutral alkyl radical (S•), with the irreversible loss of CO2 to drive the reaction forward entropically. Phthalonitrile was selected as an example electron-deficient aromatic, which, as well as being a useful synthon in various electrochemical and photochemical transformations, can couple with the generated radical for selective anti-Friedel–Crafts alkylation (Table 1)27,28,29. The reaction thus employs inexpensive and commercially available donor species in conjunction with readily synthesized phthalimide RAEs and avoids the complex donors/acceptors employed in previous EDA methodologies15,17,23. Our standard reaction conditions were therefore as follows: DABCO (50 mol%) methylcylohexyl RAE (1, 1 equiv., 0.15 mmol scale), phthalonitrile (3 equiv.) and blue light-emitting diode (LED) irradiation (λmax = 447 nm) in dimethyl sulfoxide (DMSO) for 16 h at 25 °C under N2 (Supplementary Sections 1 and 2). These conditions enabled alkylation of phthalonitrile with methylcyclohexane at the C4 position to form the desired product, 4-(1-methylcyclohexyl)phthalonitrile (2), which was obtained in 88% assay yield and isolated in 84% yield (Table 1, entry 1). Exclusion controls confirmed the necessity of photolysis of the EDA complex, as no reaction occurred in the absence of light (Table 1, entry 2) or without the electron-rich amine donor (Table 1, entry 3). Alternative amines, such as NEt3, proceeded only with diminished yields (67%; Table 1, entry 4). Only catalytic amounts of amine are required, with no increase in yield observed with stoichiometric amounts of DABCO (87%; Table 1, entry 5). Due to the low extinction coefficient of the EDA complex, 50 mol% of the inexpensive DABCO reagent proved optimal, especially for less reactive substrates. The reaction favoured polar aprotic solvents, with DMF providing yields comparable to DMSO (83%; Table 1, entry 6), whereas protic or less polar solvents substantially reduced the yield (Supplementary Section 3). The inclusion of a prototypical iridium polypyridyl complex as a photocatalyst resulted in lower yields (71%; Table 1, entry 7), confirming the efficacy of the photocatalyst-free system based on the EDA complex. Employing the aryl radical acceptor as the limiting reagent, while having the RAE in excess, also led to a reduced yield (62%; Table 1, entry 8). Irradiation with 405-nm LEDs offered a comparable yield (83%; Table 1, entry 9), in agreement with the broad absorption band of the EDA complex (see ‘EDA fragmentation' section). The radical nature of the reaction was confirmed by the addition of a radical scavenger, TEMPO, which halted reactivity and product formation (Table 1, entry 10). Addition of an auxiliary base in the form of Cs2CO3 or potassium phthalimide to aid deprotonation resulted in yields comparable to those using standard conditions after 16 h at 25 °C (84–88%; Table 1, entries 11 and 12). However, this addition of base resulted in a substantial increase in the reaction rate, which suggests that deprotonation is the rate-limiting step of the reaction (further details are described in the ‘Mechanistic studies' section below). The generality of the anti-Friedel–Crafts alkylation was subsequently explored using various activated substrates utilizing the aforementioned standard conditions, as well as catalytic amounts of an auxiliary base, Cs2CO3 (5 mol%), to accelerate the reaction rates. The reaction proceeded effectively over a wide range of ordinarily deactivating electron-withdrawing substituents, including nitriles (2–7, 13–19 and 23, Fig. 2; for characterization see Supplementary Section 4), ketones (10 and 21), esters (11, 12 and 22), amides (46 and 47, Fig. 2), aldehydes (8, 9), a sulfone (24), as well as electron-poor heteroaromatics (13–25). Electron-poor pyridines and pyrimidines were most successful given their greater aptitude to stabilize negative charge, and therefore their respective radical anion, when compared to their aryl relatives. The reaction with electron-rich aromatics proceeded with either lower or no yields of alkylated product (28, Fig. Experimental conditions: RAE (1) = 1 equiv., 0.15 mmol; DABCO = 0.5 equiv., 75 μmol; Cs2CO3 = 0.05 equiv., 7.5 μmol; DMSO = 1 ml; aromatic acceptor (3–5 equiv. ; cflow conditions (Supplementary Section 4.3); dbatch conditions (Supplementary Section 4.4); eno Cs2CO3. A, carbon or nitrogen; NR, no reaction. Experimental conditions: RAE = 1 equiv., 0.15 mmol; 3-fluoropicolinonitrile (3–5 equiv. or for late-stage functionalization aromatic acceptor (3 equiv. Boc, tert-butyloxycarbonyl; b.r.s.m., based on recovered starting material. Crucially, the photoinitiated anti-Friedel–Crafts alkylation tolerated a wide range of halides (15 and 17–19, Fig. 2) and other transition-metal-sensitive groups (for example, methanesulfonyl and nitrile), which demonstrates the robustness of this methodology to enable downstream or late-stage functionalization. Although fluorinated and trifluorinated aromatics, common in pharmaceuticals and agrochemicals, often deactivate cross-coupling chemistry due to their electron-deficient nature30,31,32, the electron deficiency of these substrates aided the homolytic aromatic substitution in our anti-Friedel–Crafts mechanism (5, 7, 9, 15 and 16, Fig. Extremely electron-poor systems, such as nitrobenzene (26, Fig. 2) or activated pyridine N-oxides (27, Fig. 2), expectedly revealed no alkylated products, as the donor amine forms an alternative EDA complex with these substrates and outcompetes the RAE17,33,34. This confirms the suitable substrate window, with the electron-poor systems being required not to form a more favourable EDA complex than the redox auxiliary. Conversely, to prevent alkylation of the phthalimide, substrates must be better radical acceptors than the phthalimide fragment released upon RAE fragmentation. This can be observed in the absence of an alternative aromatic acceptor or when progressively more electron-rich substrates, such as anisole, are used (28) (Supplementary Section 5.1). To mitigate this competing reaction, an excess of the aromatic coupling substrate (3–5 equiv.) was employed, a strategy common in EDA methodologies using silyl enol ethers, isocyanides and other radical traps15,16,21,22,24,35. This requirement for an excess radical-accepting reagent is not prohibitive, as it can be recovered from the reaction mixture, making the method suitable for expensive or late-stage aromatic acceptor substrates, see the ‘Late-stage functionalization' section (Fig. The radical attack giving anti-Friedel–Crafts regioselectivity was highly selective in the case of aryl aromatic acceptors, with loss of yield mainly arising from alkyl radical quenching and alkylation of the phthalimide fragment of the RAE. Meanwhile, in the heteroaryl case, we observed some minor regioisomer formation, notably at the C4 position on the pyridines. The overall anti-Friedel–Crafts selectivity was retained, and total yield loss was minimized, given the electron-deficient nature of these heteroaryl species as improved acceptors for nucleophilic alkyl radicals (Supplementary Section 5.2). Our methodology operates via a ‘homolytic aromatic substitution' mechanism, in a similar fashion to a classical Minisci reaction, with nucleophilic radical attack of an electron-deficient aromatic acceptor. However, a classical Minisci reaction preferentially functionalizes heteroaromatic bases at the C2/C4 positions7, and the presence of a heteroaromatic substrate is essential in the Minisci reaction mechanistic pathway. In the case of pyridine, it is the activated pyridinium that is the aromatic acceptor, resulting in a cationic radical intermediate. In contrast, our alkylation displays ‘anti-Friedel–Crafts' selectivity, because it selectively alkylates the most electron-deficient site of neutral, electron-poor heteroaromatic and standard aromatic substrates, operating via anionic radical intermediates (Supplementary Section 5.3). ‘Anti-Friedel–Crafts' selectivity has been observed before with palladium-catalysed radical alkylation36, but our methodology exhibits substantial advantages over this precious-metal-catalysed reaction, with a substantially higher selectivity observed alongside broader functional group tolerance in milder, transition-metal-free conditions. The nucleophilic alkyl radicals generated from the RAE demonstrated exceptional scope, with tertiary, secondary and primary radicals successfully coupling with 3-fluorocyanopyridine with a wide functional group tolerance (Fig. Only formation of the methyl radical proved to be too endergonic to fragment and was hence unreactive. As expected, tertiary alkyl radicals proved the most successful, resulting in higher yields due to their nucleophilicity and stability. This provided a straightforward route to highly hindered quaternary carbon centres, common in many natural and biologically active products37. The reaction displayed tolerance to various functional groups, including ketones, aldehydes groups, alkenes, alcohols and esters. Pharmaceutically relevant motifs, such as cyclic ethers and protected amines, were also retained, making this protocol particularly suitable for the late-stage functionalization of complex substrates. The reaction is also scalable to the gram scale (Fig. 2), maintaining similar isolated yields in the alkylation of phthalonitrile from 0.15 mmol (84%) to 5 mmol (82%, 1.23 g) scales. This reaction therefore provides potential for industrial applications by using inexpensive and commercially available catalysts and proceeding through a simple one-step protocol that is scalable in both batch and flow. Building on this versatility and potential for applications, we employed this general C–H anti-Friedel–Crafts strategy to regioselectively functionalize a range of pharmaceutical and agrochemical compounds. Late-stage alkylation with N-Boc-4-methylpiperidine was selected, as piperidines are the most common nitrogen heterocycle found in drug molecules38, including the antiretroviral nevirapine (46), the fungicide boscalid (47), and the steroid biosynthesis inhibitor metyrapone (48) (in moderate yields, Fig. Notably, the excess of electron-poor aromatic radical acceptors was largely recovered, such that all pharmaceutical acceptors were purified in high yield based on recovered starting material (77–88%), thus making the use of excess reagent non-prohibitive for late-stage or expensive substrates. Furthermore, we used an RAE of gemfibrozil, a lipid-regulating fibrate, in the C–H alkylation of 3-fluoropicolinonitrile in good yield (49, 66%). This shows the ability of this methodology not only to alkylate aromatic molecules, but also to furnish carboxylic acid drug molecules with aromatic groups. These results highlight the methodology's practical applicability in the late-stage modification of biologically active compounds, thus offering a valuable tool for drug discovery. DFT calculations were performed to elucidate the mechanistic pathway for this anti-Friedel–Crafts C–H functionalization methodology (Figs. The model system (Table 1, entry 1) was selected based on its optimal reactivity, and the calculations were carried out at the ωB97XD/6-31g(d,p) level of theory (details are provided in Supplementary Sections 6.1 and 6.2). First, the EDA complexation was modelled to validate the photoinduced fragmentation that generates the initial radical species (Fig. The formation of the EDA complex from DABCO and the RAE was calculated to be reversible and slightly endergonic by +0.5 kcal mol−1 (Supplementary Section 6.3). The simulated UV–vis spectrum obtained with time-dependent DFT (TD-DFT) revealed an absorbance peak at 368 nm with a broad band that extends into the visible region, consistent with the experimental UV–vis data (see the ‘Mechanistic insights' section). Gibbs energies (in kcal mol−1), computed via DFT calculations at 298.15 K and 1 atm in DMSO, are given in parentheses (Supplementary Section 6.2). Reduction of the DABCO cationic radical is explored further in Fig. R, methylcyclohexyl; SET, single-electron transfer; BET, back electron transfer. Upon photoexcitation, an electron from the nitrogen lone pair of DABCO is promoted to the RAE's valence π* orbital. The resultant radical cation and anion pair generated can then fragment into a DABCO cationic radical, phthalimide anion, CO2 and the methylcyclohexyl radical (R•). This fragmentation is slightly endergonic, with an overall Gibbs formation energy of +3.9 kcal mol−1 (Fig. Although rapid backward electron transfer (BET) immediately after photoexcited SET can serve as a deactivation pathway, the release of CO2 gas renders the fragmentation irreversible and drives the reaction entropically forward. The alkyl radical R•, formed via the EDA fragmentation, then rapidly and selectively attacks the electron-poor aromatic phthalonitrile at the C4 site to form an aryl radical adduct (I1, ∆G = −2.7 kcal mol−1, Fig. The attack is highly selective for C4, with the alternative attack at the C3 position found to be both kinetically and thermodynamically less favoured (+0.5 and +2.5 kcal mol−1, respectively, Fig. Aryl radical adduct I1 can then proceed through either a hydrogen atom transfer (HAT) or deprotonation to yield the experimentally observed product, 4-(1-methylcyclohexyl)phthalonitrile (Fig. In the HAT pathway, hydrogen abstraction by the DABCO radical cation would lead to the final reaction product (P) in an overall exergonic process by −53.6 kcal mol−1, followed by proton exchange between the phthalimide anion and DABCO(H) to regenerate the DABCO catalyst (−61.4 kcal mol−1). However, this HAT pathway is hindered by a high energy barrier (+29.5 kcal mol−1, TSHAT), suggesting a slow reaction at 25 °C. Conversely, the deprotonation pathway whereby either DABCO or the phthalimide anion acts as a Brønsted base exhibits lower activation barriers of +9.6 and +12.3 kcal mol−1. The calculated transition state (TS) energies follow the order TSHAT > TSPT–Phtl > TSPT–DABCO (for detailed analysis and structures see Supplementary Section 6.4). Gibbs energies (in kcal mol−1), computed via DFT calculations at 298.15 K and 1 atm in DMSO, are given in parentheses (Supplementary Section 6.2). The Gibbs activation barriers, referenced to the prior lowest-energy intermediate, are shown in square brackets with a double-dagger symbol. The unpaired electron in the RAE radical anion is delocalized primarily within the five-membered ring, as indicated by the DFT Mulliken spin densities in Supplementary Fig. The low barriers of TSPT–Phtl and TSPT–DABCO mean that both deprotonation pathways are kinetically accessible at room temperature (r.t.), and both lead to formation of a delocalized aryl radical anion (I2), with deprotonation by the phthalimide anion (−16.8 kcal mol−1) more thermodynamically favoured than with DABCO (−9.0 kcal mol−1). In our optimized substrate scope conditions where we utilize 5 mol% Cs2CO3, the carbonate acts as a sacrificial Brønsted base in the initial stages of the reaction in a highly thermodynamically favoured deprotonation (−45.3 kcal mol−1; Supplementary Section 6.5). The favourable deprotonation accelerates the reaction by enabling further fragmentation and base generation through the propagative chain reaction rather than waiting for the slower EDA fragmentation to produce higher concentrations of base (Fig. a,b, Experimental kinetic studies monitored by 1H NMR spectroscopy. Standard kinetic conditions: RAE (1) = 0.15 M, 1 equiv. ; 450 nm LED; DMSO. The reported data correspond to mean conversions from three kinetic runs with standard deviations. KPhtl, potassium phthalimide; KPhtl-Cl, potassium tert-chlorophthalimide; Phtl-H, phthalimide; Std., standard conditions. c,d, Simulated time and concentration profiles via microkinetic modelling using DFT-computed Gibbs energies (Supplementary Fig. 6.7.1 and Supplementary Table 6.7.2) under standard kinetic conditions (c) and under standard kinetic conditions with the addition of KPhtl (d) (0.2 equiv.). e, Characterization of the EDA complex via UV–vis spectra: RAE (1) = 0.15 M, 1 equiv. ; DMSO; path length = 1 mm. The vertical line at 450 nm represents the irradiative wavelength of the blue LED. Inset: Job plot with absorbance at 450 nm (Supplementary Section 7.3). f, Cyclic voltammograms (100 mV s−1) of phthalonitrile (10 mM), 4-(1-methylcyclohexyl)phthalonitrile (2, 10 mM), methylcyclohexyl RAE (1, 10 mM) and [nBu4N]+[PF6]− (100 mM) in DMSO under N2 in contact with a glassy-carbon-disc working electrode (0.071 cm2) with potential normalized to the ferrocene (Fc/Fc+) redox couple. Cyano-aryl radical anions, analogous to I2, are readily generated and have been demonstrated previously via SET using electrochemistry, being used both as a reagent and as an electron shuttle to reduce a reagent in situ, as in our proposed mechanism29,39,40. Chemical generation of this radical anionic intermediate is known via base-assisted homolytic aromatic substitution with deprotonation of an aryl radical via addition of a superstoichiometric base. However, this strategy has been limited to a highly restricted synthetic scope, and it relies on the use of highly pyrophoric or toxic reagents4,41,42,43,44. The unpaired electron of the aryl radical anion I2 then undergoes SET to form the desired product. Direct reduction of the RAE by I2 has a barrier of only +0.4 kcal mol−1 from Marcus theory, indicating that this SET is diffusion-controlled and the radical anion exists in very low concentrations (Supplementary Section 6.6). The rapid SET to RAE then leads to further fragmentation and chain propagation of the cyclohexyl radical R• (pathway b in Fig. There are two radical-quenching mechanisms that prevent the reaction from being a perfect runaway chain reaction and mean that continued irradiation is required for the reaction to reach completion. The first is that SET can occur from I2 to a DABCO cation (pathway a in Fig. 5), which has an estimated barrier of +9.3 kcal mol−1, and quenches the radical reaction by regenerating DABCO for further EDA complexation, resulting in a net slowing of the reaction rather than termination. An alternative deactivation pathway involves the ester radical anion reducing a DABCO cation to re-form the starting EDA complex (Fig. This process is thermodynamically favourable (−41.3 kcal mol−1) but impractical, as it relies on two short-lived radical species present in low concentrations. The phthalimide anion present within the reaction mixture is formed in situ upon RAE fragmentation and enables this radical anion pathway via deprotonation of I1. Thus, a product of this reaction enables a propagative mechanistic pathway, further accelerating the reaction autocatalytically. However, given the intricacy of the various mechanistic pathways, the precise role of the phthalimide anion has yet to be fully resolved and will require further kinetic and microkinetic studies. The complexity of the deprotonation pathways, as well as their similar kinetic barriers, makes it challenging to directly determine their relative contributions to the overall reaction mechanism. Therefore, the DFT-supported mechanistic hypothesis was probed with experimental kinetic studies (Fig. 6a,b) that were compared with microkinetic modelling (MKM) simulations using the DFT-computed Gibbs energies and activation barriers (Fig. 6c,d and Supplementary Section 6.7). In the experimental kinetic studies, with only DABCO as an additional reagent, we observed two distinct phases of reactivity (Fig. The first phase displayed an initial induction period characterized by very slow reactivity, with less than 3% conversion observed in the first hour. This was followed by a second phase of markedly increased reactivity until completion (Fig. The induction period with slow generation of product P is attributed to the slow and photon-inefficient EDA fragmentation. However, if photolytic EDA fragmentation were the rate-determining step throughout the reaction, the reaction rate should exhibit a slow decline throughout the reaction as the concentration of RAE decreases. Instead, an increasing rate was observed in the second phase, supporting a chain-propagative radical mechanism, which is consistent with previous EDA methodologies45,46. The chain propagation in our proposed mechanism relies on a base to deprotonate the aryl radical adduct (I1 in Fig. This step would be rate-limiting in the initial phase of the reaction, as the basic phthalimide anion is only generated from RAE fragmentation and requires time to accumulate. Thus, adding phthalimide anion into the reaction should increase the rate and eliminate the induction phase. Verifying this hypothesis, the inclusion of phthalimide anion eliminated the slow induction period, resulting in a rapid initial reaction, with higher concentrations of potassium phthalimide correlating with faster reactions (Fig. To confirm that the observed rate increase was due to the availability of a base, the effect of a wider range of inorganic and organic bases was examined, which revealed a clear correlation between increasing reaction rate and increasing base strength (pKaH; Fig. Cs2CO3 enabled the fastest reaction, with completion reached in 3 h (Fig. 6b, black trace), rather than 8 h without base (Fig. 6b, purple trace), all without decreasing the yield. Potassium phthalimide proved slightly slower (Fig. 6b, blue trace), and the less basic tetrachloro-analogue of potassium phthalimide resulted in a less pronounced rate increase (Fig. Conversely, the addition of protonated phthalimide retarded the reaction compared to the addition of no auxiliary base (Fig. Thus, there is a clear correlation between the addition of a base and acceleration of the reaction, which supports a deprotonation step being key to reaction propagation. MKM simulations using the DFT-computed energy barriers replicated the experimental observation of an initial induction phase followed by an acceleration of the reaction rate when no base is added (Fig. Inclusion of the phthalimide anion in the model reduces the induction period and leads to an approximately 50% faster reaction time (Fig. 6d), consistent with the experimental kinetic data (Fig. Furthermore, the simulated concentration profiles from MKM confirm that most product formation arises via the chain-propagation pathway, as radical generation from EDA excitation alone is too slow and photon-inefficient compared to the propagation pathway. The inclusion of base immediately triggers the propagation pathway (Fig. 6d), thereby reducing the reliance on the slow EDA fragmentation until sufficient concentration of base (phthalimide anion) accumulates to enable autocatalysis. Quantum yield experiments also provided further evidence supporting a propagative mechanism, with the estimated chain length of this autocatalytic radical chain reaction calculated to be 17.0 via ferrioxalate actinometry (Supplementary Section 7.1), thus supporting the presence of a highly efficient autocatalytic chain process, consistent with the experimental and computational findings. The combination of the RAE and the amine donor, DABCO, results in EDA complex formation, producing a distinct charge-transfer absorption band. This absorption band tails into the visible range, allowing absorption at 450 nm and consequent photolysis of the RAE (Fig. In contrast, the individual reagents show only negligible absorbance in the visible region (Fig. The measured spectra are consistent with the TD-DFT simulated spectra, showing the EDA complex's absorbance maxima at 368 nm and absorbance tailing well into the visible region (Supplementary Section 6.3). The 1:1 ratio of components within the EDA complex was confirmed via a Job plot, in which the ratio of donor and acceptor is plotted against the maximum absorbance (Fig. Another important mechanistic route considered was the role of the phthalimide anion, formed upon fragmentation of the RAE, as an electron-rich donor that could activate a new EDA complex. This could potentially explain the observed increase in reaction rate upon the addition of KPhtl (Fig. However, DFT calculations indicate that the fragmentation of the putative phthalimide-RAE EDA complex is much less favourable compared with DABCO as the donor (+30.1 versus +3.9 kcal mol−1). The EDA complex with phthalimide anion as a donor has also been shown to have a lower absorbance at 450 nm, resulting in a poorer EDA complex (Supplementary Sections 6.3, 6.5 and 7.2). This is in addition to the fact that the phthalimide anion will not exist in high concentrations during the reaction, as it is protonated over time with the protonated phthalimide-RAE mixture and shows negligible absorbance at 450 nm (Supplementary Fig. Therefore, we find that phthalimide–donor EDA complexes do not play a substantial role in radical formation, especially in the presence of DABCO, which forms a more effective EDA complex chromophore. Additionally, the 5 mol% Cs2CO3 added to accelerate the reaction could similarly act as a donor in a new EDA and not just as a general base. However, the addition of 5 mol% Cs2CO3 does not increase the absorption of the chromophore at 450 nm (Fig. This result, in addition to further control experiments, has led us to conclude that any alternative EDA complexes, formed through the addition of base, do not substantially impact the reaction (Supplementary Sections 6.3, 6.5 and 7.2). The aryl radical anion (I2) is expected to exist only at very low concentrations due to its predicted rapid reaction with RAE, as supported by DFT calculations. The viability of this step was probed by cyclic voltammetry, with phthalonitrile and 4-(1-methylcyclohexyl)phthalonitrile (2) exhibiting half-wave potential reduction potentials (E1/2) of −2.26 V and −2.38 V versus Fc/Fc+, respectively (Fig. Both species are sufficiently negative to reduce the RAE (1), as its reduction potential is −1.62 V versus Fc/Fc+ (Fig. As a result, the radical anion electron can feasibly shuttle from the anionic product (I2) to the phthalonitrile starting material, as evidenced by both the cyclic voltammetry and DFT results (Fig. The subsequent RAE reduction is irreversible and consistent with reduction induced fragmentation. Therefore, the radical anion is sufficiently reducing to undergo SET, reduce the RAE, and thereby propagate the reaction. We also investigated alternative RAEs, although none achieved yields matching our standard phthalimide RAE (Supplementary Section 7.4). In addition, we carried out further mechanistic studies such as TEMPO trapping and a radical clock competition experiment, both of which provided further evidence for the radical-based reaction (Supplementary Section 7.5). Overall, the experimental kinetic data, the role of the base in facilitating reaction propagation (Fig. 6b), the previous literature, DFT calculations (Figs. 4 and 5) and the measured quantum yields (Supplementary Section 7.1) all support the presence of an EDA-triggered autocatalytic radical propagation mechanism via an aryl radical anion intermediate. Typical Friedel–Crafts alkylation proceeds by the attack of a highly electrophilic carbocation towards the most nucleophilic site of an aromatic ring. In contrast, our ‘anti-Friedel–Crafts' selectivity relies on a nucleophilic alkyl radical attacking the most electrophilic site of an aromatic ring1,2. Thus, regioselectivity is largely governed by the ability of each carbon site in the aromatic system to accommodate an additional radical electron, provided steric effects allow the radical approach. We first investigated this using Hammett parameters as electronic descriptors of the aromatic acceptors (Supplementary Section 8.1), but opted for the more precise Fukui indices as a means of predicting selectivity via machine-learning models. The experimental 1H NMR spectroscopy-observed substitution products (Fig. 2) confirm the validity of this approach and can be successfully predicted using Fukui indices, a natural bond orbital (NBO)-based metric that describes the localization of excess electron density at each carbon centre in the aromatic ring, thereby indicating the stability of the corresponding aryl radical intermediate51. A higher Fukui index at a given carbon site corresponds to a greater ability to accommodate an additional electron, making the site more susceptible to attack by the alkyl radical. We note, however, that Fukui indices cannot be quantitatively correlated with selectivity outcomes across different molecules. These indices provide a relative measure of a site's electron-accepting ability within a single molecule, but do not allow for direct comparison between different substrates. Additionally, a limitation of this approach is evident in the case of boscalid (47, Fig. 3), where substantial steric hindrance near the position alpha to the carbonyl group prevents radical attack, despite a high Fukui index at that site. This underscores the need to consider steric effects alongside electronic descriptors to accurately predict regioselectivity. To better account for steric effects, we expanded our dataset to include more aromatic substrates with electron-withdrawing groups, bringing the total to 30 molecules and 124 potentially active sites (Fig. This broader dataset allowed us to assess steric effects on a case-by-case basis and to integrate them alongside electronic factors in our regioselectivity predictions. For each molecule, we defined the alkylation site as the position with the highest Fukui index that is not sterically hindered (for example, ortho to carbonyl or trifluoromethyl groups). Experimental data were then used to validate these predictions, which consistently aligned with the observed regioselectivity. Fukui indices were computed for each carbon position using the total natural population values obtained through NBO analysis (Supplementary Section 8.2). The computed Fukui indices are depicted for each carbon site considered. The sites with the highest Fukui index that are not sterically hindered (for example, by carbonyl or trifluoromethyl groups) are indicated as the predicted alkylation site for each molecule, highlighted with a blue circle. The alkylated position predicted by the XGB classifier, using SOAP features and UFF-optimized geometries, is shown with a dotted circle outline. UFF, universal force field. To automatically incorporate steric effects into the regioselectivity predictions—without requiring manual intervention—we employed ML models. Specifically, we used an eXtreme Gradient Boosting (XGB)52 classifier trained on Smooth Overlap of Atomic Positions (SOAP) descriptors53,54,55. SOAP descriptors are computationally efficient and rely solely on three-dimensional (3D) structures as input, such as those generated from SMILES strings using RDKit (open-source cheminformatics; https://www.rdkit.org). These descriptors encode the precise spatial arrangement and chemical identity of all atoms in the molecule, including the local environment around each carbon site. As previously stated, in our training set, alkylation sites were labelled as the highest Fukui index positions that were not sterically hindered. By providing the model with the full 3D structural context via SOAP descriptors, we enable it to learn this pattern implicitly, predicting the most likely alkylation site only if it satisfies both electronic and steric criteria. This ML-based approach eliminates the need for DFT and NBO calculations in future predictions, offering a faster, automated alternative for regioselectivity prediction (Supplementary Sections 6.1 and 8.2). To evaluate the performance of the ML model, we performed leave-one-group-out cross-validation, using each molecule as an independent group. In this procedure, the classifier was trained on the active sites of all but one molecule, and then used to predict the active site in the excluded molecule. This process was repeated until the alkylation site of every molecule had been predicted once (30 iterations for the 30 molecules). The classifier was trained to assign a probability to each atomic site indicating its likelihood of being reactive; within each molecule, the site with the highest predicted probability was defined as the most likely alkylation site. The XGB classifier correctly predicted the most active site in 28 out of 30 molecules, achieving an accuracy of 93% compared to the experimentally observed alkylation sites (Fig. This high performance is particularly notable given the small dataset of 124 atomic sites and 30 molecules. One incorrectly predicted molecule was 2-(methylsulfonyl)pyrimidine, highlighting a limitation of our dataset—this sulfur-containing molecule was unique, leaving the classifier with no other sulfur-containing examples to learn from, confirming that ML methods perform best for substrates within the same chemical space as the ones on which they have been trained. In addition to XGB, other classification models, including random forest, logistic regression, neural networks and Gaussian process, were tested using the default hyperparameters, achieving comparable or lower performance (Supplementary Section 8.2). Additionally, when comparing the accuracy of these models using SOAP features derived from UFF-optimized geometries with RDKit versus those generated from DFT-relaxed structures (Supplementary Section 8.2), we found no significant difference in performance. Building on the robust performance and efficiency of our model using SOAP features, we next sought to demonstrate the predictive power of the XGB classifier in a real-world setting. For this, we applied the classifier to predict the most active site for four completely new molecules to the model (Supplementary Section 8.2). These predictions, made without the need for DFT calculations, were completed in a matter of seconds on a standard computer using the XGB classifier trained on the 30 known molecules, with SOAP features derived from SMILES strings. Experimental validation using 1H NMR spectroscopy confirmed the accuracy of the model, which correctly identified the most active site for all four new substrates (Supplementary Section 8.2). It is important to note, however, that although the model accurately predicts the most active site, it does not provide information on whether other sites in the molecule may also be active. Overall, our approach demonstrates the potential of ML models to aid in organic synthesis, particularly in predicting regioselectivity for reactions with multiple similar active sites. This opens the door to more efficient and scalable selectivity prediction tools in the future. We have developed a simple, scalable and transition-metal-free method for anti-Friedel–Crafts alkylation of electron-poor aromatics by exploiting the photolytic fragmentation of an EDA complex propagated through radical anion autocatalysis. This autocatalytic mechanism is supported by DFT computation and kinetic evidence, which both demonstrate an increasing reaction rate and an accelerated reaction upon addition of an auxiliary base. Our strategy enables regioselective, photocatalyst-free C–H alkylation using only inexpensive, readily available and non-toxic components. Notably, the catalytic fragmentation of the EDA complex eliminates the need for any exogenous oxidants or reductants. The anti-Friedel–Crafts selectivity observed in this reaction was predicted using DFT-computed Fukui indices. We extended this predictive approach by developing a ML model, which accurately forecasted the regioselectivity of previously ‘unseen' substrates. We envisage further investigations to exploit radical anion autocatalysis for other reactions, as well as the development of alternative propagative mechanisms to exploit EDA fragmentation. The advancement of redox auxiliaries also offers exciting possibilities, both for creating more photon-efficient EDA complexes and for incorporating these complexes productively into catalytic processes, rather than using them merely as disposable redox activators. This photoinitiated anti-Friedel–Crafts alkylation demonstrated excellent functional group tolerance, including in the late-stage modification of pharmaceuticals, while maintaining high regioselectivity. We anticipate that this method will prove to be an effective synthetic strategy, particularly in the context of late-stage modification during drug discovery. A round-bottom flask was charged with N-hydroxyphthalimide (1.1 equiv. ), 4-(dimethylamino)pyridine (DMAP; 0.01 equiv.) and carboxylic acid (1 equiv.) This was followed by portionwise addition of N,N′-dicyclohexylcarbodiimide (DCC; 1.1 equiv.) in DCM (1.0 M) to the stirred round-bottom flask. The reaction mixture was then stirred at r.t. for 18 h. The reaction was filtered, concentrated in vacuo, and purified directly by column chromatography to afford the desired compound. For the anti-Friedel–Crafts alkylation methodology, electron-poor arene (3–5 equiv.) was added to a solution of phthalimide RAE (1 equiv. in DMSO (0.15 M) under N2. The reaction mixture was orbitally stirred in a temperature-controlled photoreactor at 25 °C for 16 h over blue LEDs (λmax = 447 nm). For purification, two separate reaction vials were combined, then H2O (5 ml) and EtOAc (10 ml) were added. The organic layer was separated, and the aqueous layer was extracted twice more with EtOAc (2 × 10 ml). The combined organic layers were washed once with brine (4 ml), dried over Na2SO4, filtered and concentrated in vacuo. The reaction mixture was purified by column chromatography to afford the desired compound. Computational and experimental details, as well as any additional results reported in this work, are provided free of charge in the Supplementary Information, and all of the DFT-optimized structures are openly accessible from the ioChem-BD repository available at https://doi.org/10.19061/iochem-bd-6-417. The primary data supporting the findings of this study are available from the University of Cambridge open-access data repository (https://doi.org/10.17863/CAM.122984). Code and related data for full reproducibility of the machine-learning results are available free of charge from our GitHub repository at https://github.com/CCEMGroupTCD/Fukui-Indices. Friedel, C. & Crafts, J. M. A new general synthetical method of producing hydrocarbons. Shen, Y. et al. Friedel-Crafts alkylation of benzenes substituted with meta-directing groups. Maruoka, K., Ito, M. & Yamamoto, H. Unprecedented nucleophilic addition of organolithiums to aromatic aldehydes and ketones by complexation with aluminum tris(2,6-diphenylphenoxide). Russell, G. A., Chen, P., Kim, B. H. & Rajaratnam, R. Homolytic base-promoted aromatic alkylations by alkylmercury halides. Minisci, F. & Fontana, E. V. F. Recent developments of free-radical substitutions of heteroaromatic bases. Minisci, F., Bernardi, R., Bertini, F., Galli, R. & Perchinummo, M. Nucleophilic character of alkyl radicals—VI: a new convenient selective alkylation of heteroaromatic bases. Recent advances in minisci-type reactions. Han, C. & Buchwald, S. L. Negishi coupling of secondary alkylzinc halides with aryl bromides and chlorides. Sato, Y. et al. Generation of alkyl radical through direct excitation of boracene-based alkylborate. Enabling the cross-coupling of tertiary organoboron nucleophiles through radical-mediated alkyl transfer. Kariofillis, S. K. et al. Using data science to guide aryl bromide substrate scope analysis in a Ni/photoredox-catalyzed cross-coupling with acetals as alcohol-derived radical sources. Mulliken, R. S. Molecular compounds and their spectra. The interaction of electron donors and acceptors. Crisenza, G. E. M., Mazzarella, D. & Melchiorre, P. Synthetic methods driven by the photoactivity of electron donor–acceptor complexes. Wortman, A. K. & Stephenson, C. R. J. EDA photochemistry: mechanistic investigations and future opportunities. Dewanji, A. et al. A general arene C–H functionalization strategy via electron donor–acceptor complex photoactivation. Fu, M.-C., Shang, R., Zhao, B., Wang, B. & Fu, Y. Photocatalytic decarboxylative alkylations mediated by triphenylphosphine and sodium iodide. Davies, J., Booth, S. G., Essafi, S., Dryfe, R. A. W. & Leonori, D. Visible-light-mediated generation of nitrogen-centered radicals: metal-free hydroimination and iminohydroxylation cyclization reactions. & Kornblum, N. Substitution reactions which proceed via radical anion intermediates. Effect of light on electron transfer substitution at a saturated carbon atom. & Kochi, J. K. Annihilation of aromatic cation radicals by ion-pair and radical pair collapse. Unusual solvent and salt effects in the competition for aromatic substitution. Arceo, E., Jurberg, I. D., Álvarez-Fernández, A. & Melchiorre, P. Photochemical activity of a key donor–acceptor complex can drive stereoselective catalytic α-alkylation of aldehydes. Zhao, H., Cuomo, V. D., Rossi-Ashton, J. & Procter, D. J. Aryl sulfonium salt electron donor-acceptor complexes for halogen atom transfer: isocyanides as tunable coupling partners. de Pedro Beato, E., Spinnato, D., Zhou, W. & Melchiorre, P. A general organocatalytic system for electron donor–acceptor complex photoactivation and its use in radical processes. Zhou, W., Wu, S. & Melchiorre, P. Tetrachlorophthalimides as organocatalytic acceptors for electron donor–acceptor complex photoactivation. Wu, J., Grant, P. S., Li, X., Noble, A. & Aggarwal, V. K. Catalyst-free deaminative functionalizations of primary amines by photoinduced single-electron transfer. & Bach, T. 3-Acetoxyquinuclidine as catalyst in electron donor-acceptor complex-mediated reactions triggered by visible light. Hota, S. K. et al. Photoinduced electron donor-acceptor complex-mediated radical cascade involving N-(acyloxy)phthalimides: synthesis of tetrahydroquinolines. McNally, A., Prier, C. K. & MacMillan, D. W. C. Discovery of an α-amino C-H arylation reaction using the strategy of accelerated serendipity. Pirnot, M. T., Rankic, D. A., Martin, D. B. C. & MacMillan, D. W. C. Photoredox activation for the direct β-arylation of ketones and aldehydes. Johnston, B., Loh, D. M. & Nocera, D. G. Substrate-mediator duality of 1,4-dicyanobenzene in electrochemical C(sp2)−C(sp3) bond formation with alkyl bromides. & Shibata, N. Contribution of organofluorine compounds to pharmaceuticals. Britton, R. et al. Contemporary synthetic strategies in organofluorine chemistry. Ogawa, Y., Tokunaga, E., Kobayashi, O., Hirai, K. & Shibata, N. Current contributions of organofluorine compounds to the agrochemical industry. Zon, M. A., Fernández, H., Sereno, L. & Silber, J. J. Electrochemical study of the stability of the electron-donor-acceptor (EDA) complex between N,N,N′,N′-tetramethyl-p-phenylendiamine and m-dinitrobenzene in acetonitrile. & Gryko, D. Pyridine N-oxides as HAT reagents for photochemical C-H functionalization of electron-deficient heteroarenes. Generation of alkoxyl radicals by photoredox catalysis enables selective C(sp3)-H functionalization under mild reaction conditions. Jiao, Z., Lim, L. H., Hirao, H. & Zhou, J. S. Palladium-catalyzed para-selective alkylation of electron-deficient arenes. Ling, T. & Rivas, F. All-carbon quaternary centers in natural products and medicinal chemistry: recent advances. Vitaku, E., Smith, D. T. & Njardarson, J. T. Analysis of the structural diversity, substitution patterns, and frequency of nitrogen heterocycles among US FDA approved pharmaceuticals. Médebielle, M., Pinson, J. & Savéant, J.-M. Electrochemically induced SRN1 substitution of fluorinated aryl halides. Application to the synthesis of fluorinated-aryl heterocycles. Mo, Y. et al. Microfluidic electrochemistry for single-electron transfer redox-neutral reactions. Bowman, W. R. & Storey, J. M. D. Synthesis using aromatic homolytic substitution—recent advances. & Curran, D. P. Organocatalysis and C—H activation meet radical- and electron-transfer reactions. & Trahanovsky, W. S. Homolytic base-promoted aromatic alkylations by alkyl halides. Shirakawa, E., Itoh, K.-I., Higashino, T. & Hayashi, T. tert-Butoxide-mediated arylation of benzene with aryl halides in the presence of a catalytic 1,10-phenanthroline derivative. & Melchiorre, P. Mechanism of the stereoselective α-alkylation of aldehydes driven by the photochemical activity of enamines. Cao, Z.-Y., Ghosh, T. & Melchiorre, P. Enantioselective radical conjugate additions driven by a photoactive intramolecular iminium-ion-based EDA complex. Evans, D. Evans and Ripin pKa Values Compilation, https://organicchemistrydata.org/hansreich/resources/pka/pka_data/evans_pKa_table.pdf Wei, J., Meng, J., Zhang, C., Liu, Y. & Jiao, N. Dioxygen compatible electron donor–acceptor catalytic system and its enabled aerobic oxygenation. Xie, S., Li, D., Huang, H., Zhang, F. & Chen, Y. Intermolecular radical addition to ketoacids enabled by boron activation. Nappi, M., Bergonzini, G. & Melchiorre, P. Metal-free photochemical aromatic perfluoroalkylation of α-cyano arylacetates. Sánchez-Márquez, J. Correlations between Fukui indices and reactivity descriptors based on Sanderson's principle. Chen, T. & Guestrin, C. XGBoost: a scalable tree boosting system. 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining (eds Smola, A. et al.) 785–794 (ACM, 2016). Bartók, A. P., Kondor, R. & Csányi, G. On representing chemical environments. Laakso, J. et al. Updates to the DScribe library: new descriptors and derivatives. Himanen, L. et al. DScribe: library of descriptors for machine learning in materials science. We are grateful for the support of AstraZeneca for a PhD studentship (to D.M.V. ), UK Research & Innovation (UKRI, ERC Advanced Grant EP/X030563/1), the UK's Department of Science, Innovation and Technology and the Royal Academy of Engineering Chair in Emerging Technologies programme (CIET-2324-83), Taighde Eireann – Research Ireland (M.M. SFI-20/FFP-P/8740), the European Commission Horizon Europe Programme under grant agreement no. 101126600, as well as the Research IT Unit of Trinity College Dublin and the DJEI/DES/SFI/HEA Irish Centre for High-End Computing (ICHEC) for the generous provision of computational resources used in this work. We thank R. Phipps, M. Gaunt, L. Castañeda-Losada and D. Kim (Yusuf Hamied Department of Chemistry, University of Cambridge) for helpful discussions. Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK David M. Vahey, Shannon A. Bonke & Erwin Reisner School of Chemistry, CRANN and AMBER Research Centres, Trinity College Dublin, Dublin, Ireland Manting Mu, Timo Sommer & Max García-Melchor Chemical Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Macclesfield, UK Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca, Macclesfield, UK Center for Cooperative Research on Alternative Energy (CIC energiGUNE), Basque Research and Technology Alliance (BRTA), Vitoria-Gasteiz, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar Search author on:PubMed Google Scholar performed and analysed the synthetic experiments. performed and analysed the electrochemical experiments. performed the flow experiments. performed the DFT calculations, microkinetic modelling simulations, and selectivity predictions with Fukui and Hammett parameters. implemented the machine-learning selectivity predictions and drafted the computational sections of the manuscript. co-wrote the manuscript with input from all the co-authors. Correspondence to Max García-Melchor or Erwin Reisner. The authors declare no competing interests. Nature Synthesis thanks Travis Dudding, Trevor Hamlin, Lisa Roy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Peter Seavill, in collaboration with the Nature Synthesis team. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Experimental details, sections 1–10, Supplementary Figures and Supplementary Tables 3.1.1, 6.1.1, 6.1.2, 6.4.1, 6.6.1, 6.7.1, 6.7.2, 7.1.1, 7.1.2, 7.3.1, 7.4.1, 8.2.1, 8.2.2. Source data for Table 1. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Vahey, D.M., Mu, M., Bonke, S.A. et al. Anti-Friedel–Crafts alkylation via electron donor–acceptor photoinitiation. 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The sun and thousands of its twins migrated across the Milky Way just in time

Source: {'href': 'https://www.scientificamerican.com', 'title': 'Scientific American'} Published: 2026-03-12 09:00:00

Now a pair of studies published today in Astronomy and Astrophysics argue that the sun did not make this journey alone. The Milky Way's dense inner regions formed stars faster and accumulated heavy metals far quicker than the outer edges—and a star with the sun's age and chemical components would not have been able to form at its present location. But to get there required crossing a dramatic border. This bar creates a distinct gravitational phenomenon known as the corotation barrier that prevents inner galaxy stars from migrating to the outskirts. Computer simulations suggest that only about 1 percent of stars born at the sun's presumed original location could successfully breach this barrier to reach our current neighborhood within a 4.6-billion-year time frame. And yet Taniguchi and his colleagues discovered that thousands of “solar twin” stars with a mass and a metal makeup similar to those of the sun managed to do so. If you're enjoying this article, consider supporting our award-winning journalism by subscribing. By purchasing a subscription you are helping to ensure the future of impactful stories about the discoveries and ideas shaping our world today. To catalog these stellar migrants, the researchers turned to the European Space Agency's Gaia satellite, an observatory tracking the positions, movements and wavelengths of light from more than two billion stars. The researchers dug up 6,594 solar twins within roughly 1,000 light-years of Earth. When the scientists looked at the age distribution within their catalog, they saw two distinct peaks: one narrow spike of stars around two billion years old that likely formed locally and another broad, massive grouping of stars between six billion and four billion years old that included our sun—“a large population of stars that migrated from their birthplace to their current positions,” Taniguchi proposes. Alice C. Quillen, a physicist and astronomer at the University of Rochester, who was not involved in Taniguchi's study, warns that there's a chance that the broad peak of solar twins might be an artifact generated by the way Taniguchi's team picked this sample—a mere statistical illusion. “The sample is distance-limited, and most of it would be stars that make it into the solar neighborhood,” Quillen says. This factor could favor stars with more oblong orbits, which tend to be older, because younger stars with circular orbits wouldn't have made it to our vicinity yet. But Taniguchi says his team addressed this bias, finding no strong effect of age on the distribution of orbital shape in solar twins. “The field of galaxy dynamics is itself dynamic,” she says. Jacek Krywko is a freelance writer who covers space exploration, artificial intelligence, computer science and all sorts of engineering wizardry. If you enjoyed this article, I'd like to ask for your support. Scientific American has served as an advocate for science and industry for 180 years, and right now may be the most critical moment in that two-century history. I hope it does that for you, too. If you subscribe to Scientific American, you help ensure that our coverage is centered on meaningful research and discovery; that we have the resources to report on the decisions that threaten labs across the U.S.; and that we support both budding and working scientists at a time when the value of science itself too often goes unrecognized. In return, you get essential news, captivating podcasts, brilliant infographics, can't-miss newsletters, must-watch videos, challenging games, and the science world's best writing and reporting. There has never been a more important time for us to stand up and show why science matters. I hope you'll support us in that mission. Subscribe to Scientific American to learn and share the most exciting discoveries, innovations and ideas shaping our world today.

A “ghost” great white shark just reignited a Mediterranean mystery

Source: {'href': 'https://www.sciencedaily.com', 'title': 'ScienceDaily'} Published: 2026-03-12 04:39:58

The young shark measured about 210 cm in length and weighed roughly 80-90 kg. Encounters like this are extremely uncommon in the region, prompting scientists to take a closer look at historical records. Researchers reviewed sightings and reports dating back to 1862, ultimately compiling a comprehensive analysis published in the open access journal Acta Ichthyologica et Piscatoria. The unexpected catch -- considered alongside documented records from the past 160 years -- suggests that great white sharks have never fully disappeared from Mediterranean waters. Instead, scientists describe them as a mysterious or "ghost" population that appears only rarely. Despite their elusive nature, evidence indicates they continue to inhabit the region. Finding younger sharks could indicate that breeding is still taking place somewhere in the Mediterranean, an idea that researchers are eager to investigate further. These sightings remain very rare, but they show a consistent pattern over time. Dr. Báez also addressed the powerful fear that great white sharks often inspire. He explains that better scientific understanding can help change public perception. As populations continue to decline, researchers stress the importance of long term monitoring programs to better understand great white sharks in the Mediterranean. Combining direct sightings with modern tracking technologies could help scientists develop evidence based conservation strategies to protect this iconic predator. "The main idea I want to convey to the public is that these large marine animals have a fundamental role in marine ecosystems. As highly migratory pelagic species, they redistribute energy and nutrients across vast distances. Even in death, their descent to the seafloor provides a critical pulse of nourishment for deep-sea communities," concludes Báez. Bed Bugs Are Terrified of This Simple Thing, Study Finds Scientists Discover Second Pregnancy Rewires the Brain in New Ways Stay informed with ScienceDaily's free email newsletter, updated daily and weekly. Keep up to date with the latest news from ScienceDaily via social networks: Tell us what you think of ScienceDaily -- we welcome both positive and negative comments.

Scientists discover seven strange frog-like insects hidden in uganda's rainforest

Source: {'href': 'https://www.sciencedaily.com', 'title': 'ScienceDaily'} Published: 2026-03-12 00:57:08

These leafhoppers are usually green and have large eyes. They move by jumping with long hind legs that sit alongside their bodies, giving them a frog-like appearance. Details of Dr. Helden's findings were published in the journal Zootaxa. Before this study, scientists had identified only 375 species of Batracomorphus worldwide, with just two documented in the United Kingdom. All seven newly discovered species were collected using light traps in rainforest areas more than 1,500m above sea level in Uganda's Kibale National Park. Leafhoppers in this genus appear almost identical externally, making visual identification extremely difficult. To distinguish them, scientists must examine the insects' genital structures. This is the only reliable way to tell species apart. Leafhoppers reproduce using what scientists call a "lock and key" system. Dr. Helden, an entomologist and member of the Ecology, Evolution and Environment Research Centre at Anglia Ruskin University (ARU), said: "Leafhoppers are beautiful, endearing creatures. Although some can be pests, and are associated with crops such as maize and rice, overall leafhoppers are a really undervalued group of herbivores. "They are an important source of food for birds and other insects, and their presence is a sign of a healthy ecosystem. "Finding these new species has taken a lot of painstaking fieldwork in the rainforest, dealing with heat and humidity, but it is incredibly satisfying to find species previously unknown to science -- it makes all the hard work worthwhile. "I've named six of the leafhoppers, in Greek, after their distinctive features or where they were found. She bought me my first microscope, which I still have, and encouraged my love of science from the very beginning, so naming a species after her feels like the most fitting tribute I could give." Beyond mRNA: Scientists Turn DNA Origami Into a Powerful New Vaccine Platform JWST Detects Evidence of “Monster Stars” That May Have Created the Universe's First Giant Black Holes Stay informed with ScienceDaily's free email newsletter, updated daily and weekly. Keep up to date with the latest news from ScienceDaily via social networks: Tell us what you think of ScienceDaily -- we welcome both positive and negative comments.