Supplementary Figure 3: Technical drawings of IsoView custom optical components.

(a) The IsoView microscope uses three types of custom optical components: objectives for both light-sheet illumination and fluorescence detection, f-theta scan lenses and detection tube lenses. These components have been jointly designed and modeled for optimal combined performance in the illumination and detection arms of the IsoView microscope, minimizing spherical and chromatic aberrations and maximizing telecentricity. The most critical custom components are the objectives, which offer (1) diffraction-limited performance over a wide wavelength range (435-950 nm) for multi-color imaging, (2) small telecentric error (< 0.1º) for efficient confocal line scanning, (3) large field-of-view (800 µm) and long working distance (3.09 mm) for imaging large specimens using various specimen preparation techniques, (4) a light-weight design (361 g) for fast volumetric imaging with objective piezo positioners, (5) a cone angle smaller than 90º, which allows simultaneous use of four objectives aimed at a common focal point in a perpendicular arrangement, and (6) a numerical aperture (0.714) close to the theoretical maximum given the geometrical constrains (Supplementary Fig. 5). Higher numerical apertures can be achieved by sacrificing field-of-view or volumetric scan range, but apertures are generally constrained in four-view imaging by the requirement of an objective cone angle below 90º. (b) Technical drawings of the custom optical components shown in a. (c) System PSFs measured with IsoView custom objectives using fluorescent beads located at the surface of an agarose cylinder. Two representative examples are shown. (d) Gaussian fits and respective three-dimensional FWHM measurements of the IsoView system PSF. When imaging eGFP fluorescence using a 525/50-nm band-pass filter, the average emission wavelength is λ = 0.519 µm. Thus, measured lateral resolution is in good agreement with the theoretical Abbe limit of 0.44 µm (0.61 × λ / 0.714).