Figure 3

Figure 3

(A, B) The setup implementation in the Classic LSFM modality, for (A) simultaneous and (B) alternate double side detection (FV: front view, BV: back view), using a sCMOS scientific camera (sCMOS). The path selector is a right-edge prism (REP) in (A) and a rotating mirror (RM) in (B). (C) Photo of the 316L steel imaging chamber, with the two air illumination objectives (IO) and the two sealed water detection objective (DO). MTS medium temperature sensor, RS refilling system, CO2 IN CO₂ inlet, HB heater block, HTS heater temperature sensor. (D) Exemplification of the sample mounting and scanning system. The sample position can be controlled in 3D and rotated (XYZA) around its vertical axis. V-LS vertical light-sheet. (E) Maximum intensity projection of a fixed zebrafish embryo at 90% epiboly, labeling actin (green) and tubulin (magenta), showing multicolor capability and achieving homogenous excitation thanks to the double illumination scheme. Scale bar 200 µm. (F) Maximum intensity projections from 3 time points of a time lapse movie recording an unconstrained developing zebrafish embryo (actin-GFP) from blastula to segmentation stages. The REP was used to register the front (top) and back (bottom) views onto the same camera chip, at the same time. A double illumination scheme was used for excitation. Scale bars 200 µm. (G) Depth colored intensity projection of an E-Cadherin-GFP mouse embryo at E4.5 stage (left) and after about 8 h (right) of imaging. Temperature and pH were conditioned within the imaging chamber, enabling cells proliferation and embryo’s development. The entire volume of the mouse embryo has been imaged every 10 min, through the RM, i.e. alternating the detection of front and back views. The showed images are obtained from the fusion of the two views. Scale bars 100 µm. (A, B) created with FreeCAD 0.16 (www.freecadweb.org) and (D) created with Autodesk Inventor Professional 2015 (www.autodesk.com).