POLCAM: instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which 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 that can operate over 1,000-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 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 α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells

Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets

Funder: W. D. Armstrong Fund (School of Technology, University of Cambridge)Abstract: Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues

Illustrations of optical vortices in three dimensions

Optical vortices (phase singularities) arise from interference and are threads of darkness embedded within light fields. Although usually visualised in terms of their points of intersection with an observation plane, appreciation of their true form requires a view in three dimensions. Through numerical simulation we re-examine three situations where optical vortex lines evolve as an additional parameter is varied. Our specific examples are: the addition of a fourth plane wave of varying amplitude into a superposition of three plane waves, the increase of height of a non-integer spiral phase step, and finally the perturbation that creates, and then dissolves, a vortex link in a specific combination of Laguerre-Gauss modes

Isolated optical vortex knots

Natural and artificially created light fields in three-dimensional space contain lines of zero intensity, known as optical vortices. Here, we describe a scheme to create optical beams with isolated optical vortex loops in the forms of knots and links using algebraic topology. The required complex fields with fibred knots and links are constructed from abstract functions with braided zeros and the knot function is then embedded in a propagating light beam. We apply a numerical optimization algorithm to increase the contrast in light intensity, enabling us to observe several optical vortex knots. These knotted nodal lines, as singularities of the wave’s phase, determine the topology of the wave field in space, and should have analogues in other three-dimensional wave systems such as superfluids5 and Bose–Einstein condensates

Topology of optical vortex lines formed by the interference of three, four, and five plane waves

When three or more plane waves overlap in space, complete destructive interference occurs on nodal lines, also called phase singularities or optical vortices. For super positions of three plane waves, the vortices are straight, parallel lines. For four plane waves the vortices form an array of closed or open loops. For five or more plane waves the loops are irregular. We illustrate these patterns numerically and experimentally and explain the three-, four- and five-wave topologies with a phasor argument

High-density volumetric super-resolution microscopy

Abstract 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 biological throughput and applicability by requiring lower labeling 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 SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm−2) 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 μm−2) in dense cytosolic tubulin datasets

High-density volumetric super-resolution microscopy

Acknowledgements: This work was supported by The Royal Society grant (SFL, RGF/EA/181021). We would like to thank Jeremy Graham at CAIRN Research for providing the hexagonal microlens array, Alexander Collins for useful discussions, and Gregory Chant and James McColl for optimizing the dSTORM buffer. We also thank Janelia Materials for providing the PA-JF646 Halo-Tag used for 3D-SPT.AbstractVolumetric 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 biological throughput and applicability by requiring lower labeling 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 SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( &gt; 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( &gt; 86.6 ± 0.9%, 0.1 locs μm−2) 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 μm−2) in dense cytosolic tubulin datasets.</jats:p