Journal: Nature Methods

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

doi: 10.1038/s41592-026-03023-y

Figure Lengend Snippet: ( a-d ) HeLa/Fucci2 spheroids cleared with SeeDB-Live. ( 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. n = 3 spheroids each. Half of the medium was replaced daily. ( b ) Schematic diagram of fluorescence imaging of spheroids. 17.2% (v/v) 2,2’-thiodiethanol (TDE) in ddH 2 O (refractive index 1.366) was used for immersion to minimize spherical aberration. The correction collar of objective lens was turned to the appropriate position. ( 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. ). ( d ) Depth-dependent fluorescence intensity of cell nuclei in the central part of the spheroids. Fluorescence intensity indicate the mean intensity of all the cell nuclei in each z plane. n = 3 spheroids. ( e, f ) Intestinal organoid culture. ( e ) Schematic diagram of fluorescence imaging of intestinal organoids in Matrigel. 17.2% (v/v) TDE/ddH 2 O (refractive index 1.366) was used as immersion. The correction collar was in the optimal position. ( 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). F 0 (left) and ΔF/F 0 (right) images (z stack: 0-186 µm) are shown. Magnified image of the inset is shown on the right. ( 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/ddH 2 O (refractive index 1.366) was used for immersion. The correction collar was in the optimal position. The organoids were fixed on the glass surface coated with poly-L-lysine and Cell-Tak. ( 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 for detailed statistical data. Panels b , e and g created in BioRender. Imai, T. (2026) https://BioRender.com/gyynf4j .

Article Snippet: 3D-rendered images were made by Imaris Viewer (Oxford Instruments).

Techniques: Cell Culture, Refractive Index, Suspension, Fluorescence, Imaging, Control, Derivative Assay, Expressing

Journal: Nature Methods

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

doi: 10.1038/s41592-026-03023-y

Figure Lengend Snippet: 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 brain surface was perfused with SeeDB-Live/ACSF-HEPES and perfused for 1 h under anesthesia. The objective lens was directly immersed in SeeDB-Live/ACSF-HEPES. The correction collar of the objective lens was turned to the best position. 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). The mice were euthanized either immediately (0 h) or 24 h after treatment. Frozen sections of non-perfused and unfixed brains were analyzed ( b ). The relative fluorescence intensity across cortical depth is shown ( c ). n = 3 mice for each time point (0 h and 24 h after treatment). 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. L5ET neurons are labeled. 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. Depths were 0–700 μm. e , x–y images at different depths. f , Fluorescence intensity at different depths. n = 3 mice. 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. Depth was 495 μm. i , j , Time-lapse images of L5ET neurons in S1 during in vivo clearing with SeeDB-Live/ACSF-HEPES ( i ). j , Quantification of fluorescence for the same sets of neurons. Depth was 590 μm. 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 – p , Toxicity assay using animal behavior. 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). In the acute behavioral experiments, SeeDB-Live/ACSF-HEPES was maintained on the brain surface during the behavioral test. 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. n = 5 mice. NS (Wilcoxon signed-rank test). n , Locomotion assay. 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). n = 4 mice per group. o , Motor function was examined with the wire hanging test . We used a unilateral cortical ischemia model as a control. 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. n = 4 mice per group. *** P < 0.0001; NS ( P ≥ 0.05; two-sided Tukey–Kramer multiple-comparison test). p , Food consumption of mice treated with control ACSF-HEPES, unilateral ischemia and SeeDB-Live/ACSF-HEPES. n = 4 mice per group. *** P < 0.001; ** P < 0.01; NS ( P ≥ 0.05; two-sided Tukey–Kramer multiple-comparison test). Graphs show the mean ± s.d. or median ± IQR. Images show representatives of ≥2 trials except for k (single trial). See Supplementary Table for detailed statistical data. Panels a and m created in BioRender. Imai, T. (2026) https://BioRender.com/gyynf4j .

Article Snippet: 3D-rendered images were made by Imaris Viewer (Oxford Instruments).

Techniques: Fluorescence, Imaging, Refractive Index, Diffusion-based Assay, Labeling, Microscopy, In Vivo, Activity Assay, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin ligase Nedd4-2 promotes localization of DNMBP/Tuba to P-bodies under hyperosmotic stress

doi: 10.1016/j.jbc.2025.110738

Figure Lengend Snippet: DNMBP puncta colocalize with Processing bodies (P-bodies) markers DCP1A and DCP1B under hyperosmotic stress . A , Manders colocalization coefficient was used to determine the proportion of cellular trafficking markers that colocalize with DNMBP puncta. Representative images are shown in A . B and C , HeLa cells were transfected with mCherry-DNMBP ( red ) and GFP-DCP1A or GFP-DCP1B ( green ) for 24 h and treated with iso- or hyper-osmotic cellular medium for 15 min. Representative 60X Confocal images for each condition are shown above. Scale bars are 10 μm. 3D rendering image of DNMBP ( red ) and DCP1B ( green ) colocalization was generated using Imaris software, with scale bar = 5 μm. D , quantification using Manders Colocalization Coefficient shows the fraction of DCP1 puncta (in green ) that colocalizes with DNMBP puncta (in red ) per cell. p -values were calculated using unpaired two-tailed Student’s t test. E , WT or DNMBP KO HeLa cells were transfected with GFP-DCP1B overnight and then treated with iso- or hyper-osmotic solutions for 15 min. Representative 60X confocal IF images for each condition are shown. Scale bars are 10 μm. F , quantification of DCP1B puncta (in green ) per cell for each condition. p -values were calculated using two-way ANOVA with Tukey’s multiple comparison test. All data are mean ± sd, N = 3 independent experiments, >60 cells quantified for each condition. p -values: Not significant (n.s.) > 0.05; ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001; ∗∗∗∗ < 0.0001.

Article Snippet: The 3D rendering image was produced using Imaris (version 10.1.1, Oxford Instruments).

Techniques: Transfection, Generated, Software, Two Tailed Test, Comparison