POLCAM: Instant molecular orientation microscopy for the life sciences

Abstract

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software; a napari plugin for visualization of high-dimensional diffraction-limited polarization camera datasets, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study both alpha-synuclein fibrils and the actin cytoskeleton of mammalian cells. To demonstrate that POLCAM also allows diffraction-limited imaging, we demonstrate POLCAM imaging actin in fibroblast-like cells and the plasma membrane of live human T cells.

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“…“Microscope 2” was a widefield fluorescence microscope (Eclipse Ti-E, Nikon), with the illumination entering the microscope body through the back illumination port, similar to a microscope described in Bruggeman et al 33 Specifically, in Bruggeman et al, it was described as Microscope 3. The beams from five lasers (Cobolt C-FLEX combiner with 405, 488, 515, 561, and two 638 nm lasers, free space) were coupled into a square-core multimode fiber (05806-1 Rev.…”

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