Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to α-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to α-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.
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.
Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 +/- 0.1%, 0.1 locs um-2) and sensitivity (> 86.6 +/- 0.9%, 0.1 locs um-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs um-2) in dense cytosolic tubulin datasets.
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