Dithered single-objective light sheet for improved super-resolution imaging throughout mammalian cells.

Abstract

Single-molecule super-resolution fluorescence microscopy is a powerful method for imaging detailed biological structures at the nanoscale. However, imaging single molecules precisely in thick samples such as mammalian cells is notoriously difficult due to increased fluorescence background. Light sheet fluorescence microscopy in which the sample is illuminated with a thin plane of light mitigates the problem of increased fluorescence background in thick samples and results in increased signal-to-background ratio and improved localization precision. However, light sheet illumination is sensitive to shadowing artifacts from imperfections in the optical path and scattering throughout the sample. Additionally, most light sheet systems employ two objectives, which may suffer from steric hindrance and drift between the sample and illumination objective. To overcome these disadvantages, we have developed a single-objective tilted light sheet illumination system which employs dithering to reduce stripe artifacts and achieve homogeneous illumination of the sample. Our light sheet has been designed with a width, thickness, and confocal parameter specifically suited for cellular imaging. We further improve the imaging capabilities of our system with a microfluidic device enabling Exchange-PAINT for multi-target imaging without chromatic offsets, using engineered point spread functions for 3D imaging, and employing deep learning algorithms for high-density localization of single molecules. Our approach achieves improved localization precision, imaging speeds, and multi-target accuracy, enabling fast and precise multi-target 3D single-molecule super-resolution cellular imaging. Here, we focus on the design and construction of our single-objective dithered light sheet and its range of applications from nuclear protein investigation to whole cell imaging. We think the improved cellular imaging resulting from this system will impact our understanding of the relationship between organization and function of subcellular structures and help us and others answer relevant biomedical questions involved in disease pathogenesis.