Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet

Selective Plane Illumination Microscopy (SPIM) is attractive for its ability to acquire 3D images with high 3D spatial resolution, good optical sectioning capability and high imaging speed. However, tradeoffs have to be made when a large field of view (FOV) is required, results in lower axial resolution or worse optical sectioning capability. Here, we present a novel method for 3D imaging by SPIM that is capable to maintain its high 3D spatial resolution and good optical sectioning capability within a large FOV. Instead of trying to generate a large and uniformly thick excitation light sheet, the method tiles a relative small light sheet quickly to multiple positions within the image plane by defocusing the excitation beam used to create the light sheet, and takes one additional image at each position, so that a large FOV can be imaged by repeating this process and stitching all images together. By implementing this method, light sheets with thin thickness and good excitation light confinement can be used for SPIM imaging with slightly compromised imaging speed. The method was investigated through both numerical simulation and experiments, and the imaging performance was demonstrated by imaging fluorescent particles embedded in agarose gel and live C. elegans embryos.