Supplementary Figure 6: 3D volume imaging within microwells.

(a) Top, schematic representation of the microwell architecture enabling image capture of the entire height of cells and cell aggregates within a well. By tuning the duration of deep reactive-ion etching of the silicon wafer, we were able to design the well depth so as to be 5 µm lower than the 45° mirror. This enabled imaging of the bottom of the wells in the soSPIM configuration without having the excitation beam clipped by the base of the 45° mirror. Bottom, soSPIM optical sections of the basal region (displaying stress fibers) to the apical poles of spread hepatocyte cell aggregates labeled with phalloidin-Alexa-647 Fluor 488 within a 40 × 40 µm² well. The sectioning capability of the soSPIM microscope allowed clear identification of the actin stress fibers and structures of the basal region of the spread hepatocyte cell. (b) Microwells aligned along the mirror allow simultaneous imaging of multiple cells. Two-color soSPIM optical sections of three S180 cells expressing the membrane protein E-cadherin–GFP (upper panel) and the cortical actin protein F-Tractin–RFP (middle panel), each positioned in a different 24 × 24 μm² microwell. Each color was acquired sequentially. This capability leads to an increase in the imaging throughput. The scanning light sheet illuminates only the zones defined by the red bars, and the readout speed of the camera is equivalent to that of single-well imaging. The lower panel shows a phase contrast image of the three wells. Scale bars, 5 µm.