Lossless Three-Dimensional Parallelization in Digitally Scanned Light-Sheet Fluorescence Microscopy

We introduce a concept that enables parallelized three-dimensional imaging throughout large volumes with isotropic 300–350 nm resolution. By staggering high aspect ratio illumination beams laterally and axially within the depth of focus of a digitally scanned light-sheet fluorescence microscope (LSFM), multiple image planes can be simultaneously imaged with minimal cross-talk and light loss. We present a first demonstration of this concept for parallelized imaging by synthesizing two light-sheets with nonlinear Bessel beams and perform volumetric imaging of fluorescent beads and invasive breast cancer cells. This work demonstrates that in principle any digitally scanned LSFM can be parallelized in a lossless manner, enabling drastically faster volumetric image acquisition rates for a given sample brightness and detector technology.