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

Journal: bioRxiv

Article Title: DRP1 induces neuroinflammation via transcriptional regulation of NF-ĸB

doi: 10.1101/2025.09.02.673863

Figure Lengend Snippet: a,b , Primary microglia cultured from Dnm1l +/+ pups were grown on chamber slides and treated with LPS (100 ng/ml) or vehicle for 6h. a , Representative confocal images of IBA1 and DRP1 immunofluorescence, followed by Imaris 3D rendering. b , Nuclear DRP1 (red) and cytoplasmic DRP1 (yellow) were quantified using Imaris. N=5 experiments with 14-17 cells/experiment and analyzed using t-tests. c,d , 3-month old Dnm1l +/+ mice were injected i.p. with a single dose of LPS (5 mg/kg) or saline. c , Midbrain sections were immunostained for SNpc microglia and DRP1, followed by 3D rendering of the nigral microglia and DRP1. d , Nuclear and cytosolic DRP1 puncta were quantified using Imaris, N=4 (2F & 2M) with 10-15 cells/animal, t-tests. e,f , Dnm1l +/+ and Dnm1l +/- primary microglia were treated with 100 ng/ml LPS for 6h followed by nuclear and cytoplasmic fractionation. e , Immunoblotting of DRP1 in the nuclear and cytoplasmic fractions (top panels). Total proteins per lane (bottom panels) were used as loading control and quantified for the levels of DRP1 Nuc and DRP1 Cyto in f . N=4 independent experiments, two-way ANOVA followed by Tukey post-hoc test.

Article Snippet: To test this hypothesis, we performed immunofluorescence, followed by Imaris 3D rendering images to quantify nuclear translocation of DRP1 (red) and those that remain in the cytoplasm (yellow) in primary microglia and mice treated with LPS ( ).

Techniques: Cell Culture, Immunofluorescence, Injection, Saline, Fractionation, Western Blot, Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Bruton Tyrosine Kinase in Lesions of Multiple Sclerosis and 3 of Its Models

doi: 10.1212/NXI.0000000000200413

Figure Lengend Snippet: (A) Low-magnification image of an MS brain section showing demyelination evaluated through LFB and H&E staining with loss of LFB in the middle. The box in (A) is magnified in (B) as a merged image and its individual panels show staining for nuclei with DAPI, microglia/macrophages with Iba1, immune cells with CD45, and BTK. The colocalization of BTK in Iba1 + microglia/macrophages is ascertained by Imaris 3D rendering in (C). BTK = Bruton tyrosine kinase; LFB = Luxol fast blue.

Article Snippet: Staining for Iba1+ microglia/macrophages in the same sample showed a hypercellular region within the lesion in which Iba1 + microglia/macrophages were closely associated with BTK staining , and it was corroborated through Imaris 3D image rendering ( ).

Techniques: Staining