Fig. 2

Comparison of See-Star fluorescence microscopy performance with other clearing methods. A Representative images of Patiria miniata juveniles following different clearing techniques visualized in DIC (A′) or in confocal microscopy after DAPI nuclei staining (Aʺ, Aʺ′). Control samples were imaged in PBS buffer and See-Star cleared samples (4% Acry, 15% Acry, 30% Acry) were imaged in CUBIC mount. For DAPI-stained samples, color-coded (surface: red, base: blue) depth projections indicate the relative depth range for each sample. All samples were imaged from the aboral side of the juveniles, with corresponding orthogonal views shown below (Aʺ). To clearly visualize the entire imaging depth, individual contrast adjustments were done for each condition. Close-ups on surface nuclei in representative areas are shown with identical contrast parameters (A’’’). In A′, Aʺ, scale bar = 200 µm. In Aʺ′, scale bar = 20 µm. B Representative orthogonal projections from the center of the samples shown in Aʺ. C Box plot of average imaging depth at which signal was lost (intensity < average background of 5 a.u. intensity). Grey box indicates the size range of juvenile P. miniata samples, 150–200 µm. D Quantification of mean DAPI fluorescence intensity within nuclei of surface epidermis following different clearing procedures. n = 5 per condition