Supplementary Figure 1: Optical design of the IsoView light-sheet microscope.

IsoView illumination arms: Beams from multiple lasers (488 nm, 515 nm, 561 nm, 594 nm), led through single mode fibers (SMF), are collimated by fiber collimators (FC) and directed through illumination filter wheels (IFW) and shutters (S). Lens pairs (L1 and L2) de-magnify the beams to attain the intended Rayleigh length of the weakly focused beams in sample space. De-magnified beams are then scanned by dual-axis galvanometer scanners (XY Galvo) to rapidly generate scanned light sheets as well as to laterally translate light sheets in sample space. The planar scanned fields (dotted lines) generated by the f-theta scan lenses (fθ) are reimaged onto the sample plane by the illumination tube lenses (ITL) and objectives (Obj1, Obj2, Obj3 and Obj4), via reflection from dichroic beam splitters (DBS) en route to sample space.

IsoView detection arms: The emitted fluorescence signal collected from the focal planes of the objectives orthogonal to the light sheets is transmitted by dichroic beam splitters located in infinity space between detection tube lenses (DTL) and objectives. The objectives and detection tube lenses image the fluorescent signal emitted by the specimen onto the sCMOS camera chips, following transmission through the detection filter wheels (DFW). Depth-sectioned images are acquired by linearly translating the objectives mounted atop linear piezos (not shown) and synchronously translating the light sheets laterally to match the focal planes of the objectives collecting the emitted fluorescence light.

We note that, in order to reduce the footprint of the microscope, X and Y scan mirrors in the IsoView illumination arms are not conjugated. Depending on which scan mirror is positioned in the focal plane of the f-theta lens, this arrangement is thus non-telecentric for the respective other scan direction. The implications of this design choice are briefly summarized below.

Case 1: When beam scanning is non-telecentric in the direction that sweeps the Gaussian beam over the camera field, the angle between beam and optical axis is at maximum 1.8 degrees (measured across the full field-of-view, i.e. +/- 400 µm). For a Drosophila-sized specimen, this amounts to a vertical beam offset of 6.3 µm at the two ends of the specimen. Since the size of the confocal slit typically used in IsoView mode 2 is 29.3 µm, the slit is large enough to collect all ballistic photons and there is in principle no impact on image quality.

Case 2: When beam scanning is non-telecentric in the direction that sweeps the light sheet across the sample volume, the angle between light sheet and focal plane is at maximum 0.8 degrees across a 400-µm-deep volume. At the end points of a 200-µm-wide volume this corresponds to a maximum shift of +/- 1.4 µm between light sheet and detection plane. For comparison, the light sheet waist is typically on the order of 6.3 µm.