Supplementary Figure 7: soSPIM imaging of a large cell aggregate.
From: 3D high- and super-resolution imaging using single-objective SPIM
(a) Two-color soSPIM optical sections at different depths of an S180 cell aggregate cultured for 48 h in a 40 × 40 µm2 microwell coated with fibronectin (green, E-cadherin–GFP; red, Lamin-B1–Alexa Fluor 568). (b) Image of a whole cell aggregate of MDCK cells expressing the cytoskeleton protein actin-GFP acquired with a 1.9-µm-thick and 31.4-µm-long light sheet. (c) Image of the cell aggregate in b reconstructed from three images acquired at x0(I1), x0(I2) and x0(I3) positions to create an image with optimized sectioning, higher contrast and reduced shadowing effects without moving the sample. This image was obtained by combining the three images acquired with a 1.6-µm-thick and 25.7-µm-long light sheet as represented in d. (d) Schematic representation of a large cell aggregate within a 60 × 300 µm2 microwell and the positions of a thin light sheet shorter than the whole cell aggregate. The shorter thin light sheet allowed us to image the whole aggregate while optimizing the sectioning performance. The light sheet was defocused at three different positions (x0(I1), x0(I2) and x0(I3)), with three defocus strengths (Ii) to image the whole cell aggregate. (e) Stitching of the three images acquired at three different x0 positions of the light sheet were used to create an image of the whole cell aggregate as represented in c. Upper panels: the masks defined by the equation described in the Online Methods were used to select the image parts acquired with the thinnest part of the light sheet. Lower panels: corresponding images of the cell aggregate after multiplication with the masks represented in the upper panels.
