Fig. 1

Fig. 1 Highly selective near-infrared staining and light-sheet microscopy affords superior imaging at depth in tissue.

a, Maximum intensity projected (MIP) z stacks acquired for a fixed Tg(kdrl:GFP) (vascular marker) zebrafish larva (72 hpf) stained against GFP via conventional indirect immunostaining with AlexaFluor800 (AF800). hpf, hours post fertilization. Visible (GFP) left, NIR (AF800) right. Scale bar, 100 µm. a′, A single superficial z plane from a fixed Tg(h2b:GFP) (nuclear marker) zebrafish larva/embryo (96 hpf) stained against GFP via nanobody-conjugate CF800. Visible (GFP) left, NIR (CF800) right. Scale bar, 100 µm. b,b′, Selected superficial patches shown by the dashed boxes in a,a′, respectively (visible (GFP) top, NIR (AF800/CF800) bottom) and pixelwise correlation plots for all 125 extracted patches. Scale bar, 5 µm. a.u., arbitrary units. c, Multiple deeper z planes acquired for the same nuclear marker (h2b) zebrafish embryo/larva shown in b. Scale bar, 100 µm. d, Pearson correlation and SSIM for the full z stacks acquired for the vascular (kdrl) and nuclear (h2b) marker zebrafish from a,a′, respectively. Dashed lines represent 25/50/75th quartiles. e,e', Selected deeper patches for the vascular (kdrl) and nuclear (h2b) marker zebrafish from a,a′, respectively. Scale bar, 5 µm. f, Pearson correlation and SSIM for all patches extracted at different z planes from the full z stacks of the vascular (kdrl) and nuclear (h2b) marker zebrafish. The z depth provided is the maximum z depth into tissue for each image in the stack. The uncertainty envelope is given by the s.d. and the inner thick line represents the mean value. g, The information content gain (Methods), between the visible (GFP) and IR channels (I_IR/I_GFP) for all patches extracted at different z planes from the full z stacks of the vascular (kdrl) and nuclear (h2b) marker zebrafish. The uncertainty envelope is given by the s.d. and the inner thick line represents the mean value.