Versatile, High-Resolution, and Large Field-of-View Single-Molecule Imaging by Oblique Plane Microscopy

Abstract

Building on recent advancements in oblique plane microscopy (OPM), also known as single objective light sheet, we demonstrate a versatile, high resolution, and large field-of-view OPM with single-molecule resolution. We achieve 300-nm lateral resolution and 800-nm axial resolution, without deconvolution, through the use of a bespoke solid immersion objective for remote focusing. We extend the imaging volume by rapidly translating the sample through the oblique light sheet using optimized stage scanning and digital control of the light sheet to eliminate motion blur. Because OPM requires only one objective at the sample, we can perform high-resolution light sheet imaging using standard sample mounting methods, including microfluidics for flow cytometry and fluidic chambers for iterative multiplexed labeling. We quantitatively evaluated and verified the instrument's volume performance using calibration samples and multiple imaging-based resolution metrics. As constructed, we can image 1-cm x 1-cm x 60-micron (x,y,z) per color per hour, with single-molecule resolution, in real samples. To demonstrate the instrument's capability, we quantified the co-expression of RNA and protein markers for the SARS-CoV-2 entry genes ACE2 and TMPRSS2 in alveolar epithelial type II cells across centimeter-scale fixed human lung tissue slices. In contrast to currently used imaging methods for spatial transcriptomics, our OPM approach provides the correct spatial sampling required to perform rigorous 3D single-molecule localization. Importantly, we achieve this high sampling rate and light collection efficiency at volume imaging rates that are orders of magnitude faster than existing approaches. Beyond spatial -omics, we additionally performed 3D single-molecule localization microscopy in fixed cells and live-cell imaging with with no modifications to the instrument. We envision the increase in resolution, sampling, and speed of high numerical aperture OPM versus traditional high numerical aperture microscopes will greatly aid high-throughput single-molecule imaging.