Eye of a 24 hpf Zebrafish embryo with GFP labelled nuclei – a zoom into the retina behind the lens.

(a), (b): XY cross-section of the same volume acquired with low NA and high NA stitched using ImageJ respectively. Boxes in panel (a) highlight areas where shadows appear due to excitation beam being highly scattered by the eye lens. In images (b) and (d) this effect is minimized by using higher NA excitation (see main text). (c), (d): XZ cross-sections of low NA and high NA (ImageJ stitching) volumes respectively. The shadowing artefacts are highlighted with a box in image (c), while image (d), acquired with high NA excitation, shows improvement in the same area. All scale bars 30 μm. EX arrows indicate excitation beam direction.

Eye of a 24hpf Zebrafish embryo

Eye of a 24hpf Zebrafish embryo with GFP labelled nuclei.(a): 3D view of the eye with planes indicating cross-sections for images (b) and (d) . (b) (c)(d): The contrast analysis images (see main text). These are 5 images with translated 0.3 NA beams (at the top) combined into one based on their contrast and color-coded to correspond to the beam positions. As opposed to figure 5 in the main text the combining of the images was done on a 2D panel basis as opposed to 3D boxes. This gives higher variability in colour-pattern along z direction and allows for better visualisation the effects of scattering in the detection path. Indeed, comparing the panels (b) and (c), at different depths in the stack, the colour pattern is much disrupted in the latter (deeper) one. It can be seen more linearly on the XZ cross section (panel (d), with panel (b) and (c) planes indicated). All scale bars 30μm. EX indicates excitation beam direction.