Fig. 1
Reflective imaging in light-sheet microscopy. a Schematic indicating reflective light-sheet microscopy (symmetric diSPIM) geometry. Sample, objectives, and light sheets are indicated above the mirrored coverslip, “virtual” copies shown below the coverslip. In this example, the light sheet is introduced from the left and reflected by the mirror. Four views, indicated A, A′ (mirror image of A), B (produced by reflected light sheet), and B′ (mirror image of B) are simultaneously obtained as the sample is translated through the stationary light sheets. In other words, A′ and B′ appear to originate from virtual sources below the mirrored coverslip (see also Supplementary Fig. 1a). The reference coordinate system (from the perspective of the right hand objective) is also indicated. Maximum intensity projections of raw volumetric views of EGFP-histone-labeled nuclei in a live nematode embryo, as collected from either objective as the sample is translated through the stationary light sheets are shown in b and c. Data from view A are transformed (deskewed) so that it is shown as if the light sheets are scanned through a stationary sample. d Naive deconvolution results in the reconstruction shown in e but better modeling of the imaging process results in the reconstructions shown in f (shown in g from the same perspective as in d). Note that epifluorescence contamination (green arrows) is markedly reduced in f, g (compared to b–e) and resolution isotropy restored (compare d, g, especially nuclei highlighted with orange arrows). Scale bars: 10 μm. See also Supplementary Movies 1 and 2
