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 , Strategies for optical clearing of live cells. b , Transmittance (at 600 nm) of HeLa cell suspension (4 × 10 6 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. The refractive index of the medium was adjusted to 1.365 (except for 5% glycerol). Osmolarity was not adjusted to isotonicity. Data are the median ± interquartile range (IQR). *** P < 0.001; ** P < 0.01; NS, not significant ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). d , The osmolality of candidate chemicals in aqueous solution (refractive index 1.365, in double-distilled water (ddH 2 O; n = 3 each). Sucrose was used as a control. Spherical polymers refer to polymers with highly branched and/or higher-order structure. BSA#1 and BSA#2 represent two examples of different BSA products. 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 × 10 6 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 , h , Growth of HeLa/Fucci2 cells. Cell numbers were measured by fluorescence imaging of cell nuclei ( n = 5 wells). g , Proliferation curve of HeLa/Fucci2 cells in iodixanol, Ficoll70 and BSA#1-containing medium (refractive index, 1.363; 320 mOsm kg −1 ). * P < 0.05; NS ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). 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 < 0.05; NS ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). P values are <0.001 unless otherwise mentioned. i – k , HeLa/Fucci2 cell spheroids cleared with SeeDB-Live. 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. l , Phase contrast image. m , Growth of the intestinal organoids. The sizes of the organoids (areas in the phase contrast images) were determined using Cellpose. 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 Δ F/F 0 images (right) of a cortical organoid labeled with a calcium indicator, Calblyte-650AM ( o ). Spontaneous calcium transients of neurons ( p ). q , Principles of optical clearing of live cells with SeeDB-Live. Maximum transparency was achieved by matching the refractive index of the extracellular medium to that of the cytosol (1.363–1.366). Data with error bars represent the mean ± s.d. Images are representatives of ≥3 trials. See Supplementary Table for detailed statistical data. MW, molecular weight; PG, propylene glycol; PEG, polyethylene glycol; HBCD, hyperbranched cyclic dextrin; PVP, polyvinyl pyrrolidone.

Article Snippet: 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).

Techniques: Suspension, Saline, Imaging, Expressing, Refractive Index, Comparison, Control, Concentration Assay, Fluorescence, Labeling, Molecular Weight