A Self Aligning Macroscopic Selective Plane Illumination Microscope With Near Uniform Axial Resolution

A key compromise in selective plane illumination microscopy (SPIM) is the tradeoff between optical confinement, axial resolution, and field of view (FOV). Recent advancements have demonstrated that high axial resolution can be maintained over a large FOV by optical engineering of the light sheet or by scanning the thinnest portion of a Gaussian focus across the FOV. These methods require precise synchronization between the translating focus of the exciting Gaussian to the programmable shutter of a scientific CMOS camera. This synchronization depends on the optical path length between the objective and focal plane within the sample, which commonly varies throughout the sample. A standard solution to maintaining this precise synchronization is a time consuming manual calibration of all microscope parameters throughout the imaging volume before the acquisition. To aid the adoption of uniform axial resolution imaging, we here present an open-source, adaptable, self-aligning, and low-cost SPIM design using an electrotunable lens, a high-efficiency industrial CMOS camera, and structured illumination based autofocusing. We provide an open-source microscope design, control software, and post-processing software to computationally fuse multiple images into one image with near-uniform axial resolution. We demonstrate the versatility of this approach by integrating it into three different SPIM designs. We provide a detailed characterization of the aberrations induced due to the use of an ETL for translating the waist of the light sheet. Using 3D Fourier Ring Correlation, we demonstrate that computational fused SPIM generates similar image quality to axially scanned light sheet microscopy for low to moderate numerical aperture light sheets. Finally, we apply our SPIM approach to the imaging of samples spanning a developing Zebrafish embryo to an entire cleared mouse lung.