Fig. 4
Reflective imaging at high NA boosts resolution and collection efficiency. a Comparative schematics showing from left to right, 0.8 NA/0.8 NA dual-view on glass coverslip; 1.1 NA/0.7 NA dual-view on glass coverslip; 1.1 NA/1.1 NA dual-view on reflective coverslip; 1.1 NA/0.7 NA quadruple-view on reflective coverslip. Excitation light sheets are represented as blue lines, with arrows indicating the direction of propagation. Virtual light sheets are shown as dashed blue lines, virtual objectives are transparent. Primed (perspective from detection objective) and unprimed (from coverslip) coordinate axes are also shown. b Collection cones for each detection lens geometry corresponding to a, including total solid angle and corresponding collection efficiency. c Cross-sections through optical transfer functions (OTFs) in primed and unprimed coordinate systems, with diffraction limit indicated for 0.8 NA detection (red circle) and 1.1 NA detection (blue dashed circle). d Lateral maximum intensity projections of images of Alexa Fluor 488 immunolabeled microtubules in fixed U2OS cells (top row) derived from each imaging system are shown, as are axial slices (bottom row) corresponding to red dotted lines. Higher-magnification single-plane views at indicated axial depths (e); closer (red dashed rectangular regions in d) and farther (yellow rectangular regions in d) from the coverslip are also shown. Examples of Alexa Fluor 488 phalloidin staining actin in fixed U2OS cells are also provided for 1.1 NA/0.7 NA dual-view geometry on glass coverslip (f) and 1.1/1.1 NA dual-view geometry on reflective coverslip (g) are also shown, with axial depth from coverslip indicated with colorbar. Higher-magnification views in primed (h, j) and unprimed (i, k) coordinate systems corresponding to dashed rectangular regions in f and g are also provided. See also Supplementary Fig. 5, and Supplementary Movies 7 and 8. Scale bars: 5 μm (d); 3 μm (e); 10 μm (f, g); 5 μm (h–k)
