(A) Detection scheme enabling the collection of emitted photons through three different paths. DO detection objective, TL tube lens, PS path selector, EF emission filter. The coordinate system is indicated respect to the horizontal optical table (OT). (B–D) The elements included in the PS are switchable and permits obtaining (B) simultaneous double, (C) sequential double, or (D) single detection schemes. All optical paths converge after the path selector to reach the sCMOS camera chip. The FoV determined by the camera chip can be either optically split (B) between the views, or used as a full (C, D). Figure created with Autodesk Inventor Professional 2015 (www.autodesk.com).

(A) The objectives? configuration enabling multiple illumination/detection schemes. (B) Through the first mounting option the sample is embedded within a cylinder perpendicular to the optical table. The black arrow indicates the scanning direction. (C) The second approach uses the flow of the samples to scan them through the light sheet. The red arrow indicates the flow direction. (D) Through the third approach, the sample flow (red arrow) is used to position the sample in the objective?s FoV of the DO. The scanning of the specimen is obtained instead in the vertical direction (black arrow), either mechanically or optically. IO illumination objective, DO detection objective, LS light sheet, FC fluidic circuit. Figure created with Autodesk Inventor Professional 2015 (www.autodesk.com).

(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).

(A) The setup implementation in the Flow LSFM modality with double simultaneous detection scheme. (B) The imaging chamber embedding two sealed water detection objective (DO) and a FEP tube crossing it at 45°. SF sample flow. (C) Exemplification of the sample mounting and scanning system. The sample flows within the FEP tube through a vertical light-sheet. (D) Depth-colored maximum intensity projection of a fixed tissue spheroid expressing a histone H2B fluorescent nuclear reporter protein (mCherry), obtained through single (left) and double (right) side detection scheme. The fusion of two views (on the right) reveals nuclei that would be otherwise missed through single side detection (see white arrows). Scale bars 100 µm. (E) Series of images during the flow of a Fli-GFP zebrafish embryo through the system. The front view (FV) and the back view (BV) are simultaneously registered on the same camera chip. Scale bar 100 µm. (F) 3D reconstruction view of the same zebrafish showed in (E). Colors represent signal intensity (Fire LUT in Fiji). (A) created with FreeCAD 0.16 (www.freecadweb.org) and (C) created with Autodesk Inventor Professional 2015 (www.autodesk.com).

Setup implementation in the Hybrid LSFM modality with upright detection scheme and examples. The scanning in the depth (z) direction can be performed either (A) through a vertical stage moving the entire chamber or (B) through the displacement of the detection objective with a piezo-element synchronized with galvanometric scanning of the light sheet. Schematic (C) and actual implementation (D) of the imaging chamber for the Hybrid LSFM modality. (E) Exemplification of the sample mounting and scanning system. The sample flows within the FEP tube until the objectives? FoV. There, it can be rotated around the FEP tube?s axis to orient the sample correctly (as shown in F, G) and/or it can be moved in a controlled and stepwise manner along the tube, providing multichannel high-resolution whole larvae imaging by stitching (H). (I) The system allowed high-throughput imaging of zebrafish heart in a single imaging session (18 samples. The showed image was processed through a background correction method to decrease yolk?s auto-fluorescence). Full set of images in Supplementary Fig. 7. The use of a piezo focus control (B) allows fast imaging of macrophage migration in the head (J) and the caudal fin (K) as well as zebrafish brain?s neural activity using calcium indicators (L). More information in Supplementary Videos 5, 6 and 7. (M) Long-term multicolor time-lapse movies of zebrafish embryo development are also achieved. Scale bars: 100 µm. UV upper view, SF sample flow, RC rotational connector, SM stepper motor, W water compartment, PP programmable pump (not shown), BF bright field compartment, IO illumination objective, DO detection objective. (A, B) created with FreeCAD 0.16 (www.freecadweb.org) and (C, E) created with Autodesk Inventor Professional 2015 (www.autodesk.com).