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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 μm−2) and sensitivity (>86.6 ± 0.9%, 0.1 locs μm−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 μm−2) in dense cytosolic tubulin datasets.

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.

While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase.For this analysis, we have used super-resolution microscopy to image theDrosophila melanogasterY loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops.These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.

Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging., Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.

Funder: W. D. Armstrong Fund (School of Technology, University of Cambridge), 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.

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 μm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended 6 µ m depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.

Single molecule localisation microscopy (SMLM) has opened a new window for imaging fluorescently labelled biological specimens. Common 3D SMLM techniques enable data collection across an axial range of 1 - 5μm with high precision. Despite the success of 3D single molecule imaging there is a real need to image larger volumes. Here we demonstrate, through simulation and experiment, the potential of Single Molecule Light Field Microscopy (SMLFM) for extended depth-of-field super-resolution imaging, extracting 3D point source position by measuring the disparity between localizations of a point emitter in multiple perspective views.

Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.

The use of structured illumination in fluorescence microscopy allows the suppression of out of focus light and an increase in effective spatial resolution. In this paper we consider different approaches for reconstructing 2D structured illumination images in order to combine these two attributes, to allow fast, optically sectioned, superresolution imaging. We present a linear reconstruction method that maximizes the axial frequency extent of the combined 2D structured illumination passband along with an empirically optimized approximation to this scheme. These reconstruction methods are compared to other schemes using structured illumination images of fluorescent samples. For sinusoidal excitation at half the incoherent cutoff frequency we find that removing information in the zero order passband except for a small region close to the excitation frequency, where it replaces the complementary information from the displaced first order passband, enables optimal reconstruction of optically sectioned images with enhanced spatial resolution.

Guided by a general framework for wavefront engineering, experiments demonstrate that in a light field, lines of zero intensity can be shaped into knotted and linked loops of arbitrary topology. Natural and artificially created light fields in three-dimensional space contain lines of zero intensity, known as optical vortices1,2,3. 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 links4 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 condensates6,7.

Structured illumination microscopy (SIM) allows imaging of fluorescently labelled biological samples with a spatial resolution improved by a factor of approximately two compared to traditional optical microscopy techniques. The cost of this resolution improvement is the need to capture a number of raw images of the sample to reconstruct a single SIM image, increasing sample light exposure and limiting the ability of the technique to capture dynamic processes. In this paper we describe image acquisition and reconstruction techniques that allow fast super-resolution imaging within optically thick specimens. By exploiting overlaps between SIM information passbands we are able to generate optically sectioned, super-resolution images from an image sequence acquired in a single focal plane. We consider how single plane super-resolution images may be obtained using 2D and 3D SIM illumination patterns, and compare the resulting images to those obtained using conventional 2D SIM reconstruction methods. By combining a single plane reconstruction algorithm with hardware for high-speed switching between illumination patterns and rapid acquisition of fluorescence images, we demonstrate high speed super-resolution imaging inside biological organisms.

We created superpositions of two different TEM modes in focused beams. Such modes show relative dephasing along the beam axis due to Gouy's phase. This leads to interference effects and significant modifications of a beam's transverse intensity distribution near the beam focus. We investigate the features of the resulting field profiles.

Wavefront sensing in the presence of background light sources is complicated by the need to restrict the effective depth of field of the wavefront sensor. This problem is particularly significant in direct wavefront sensing adaptive optic (AO) schemes for correcting imaging aberrations in biological microscopy. In this paper we investigate how a confocal pinhole can be used to reject out of focus light whilst still allowing effective wavefront sensing. Using a scaled set of phase screens with statistical properties derived from measurements of wavefront aberrations induced by C. elegans specimens, we investigate and quantify how the size of the pinhole and the aberration amplitude affect the transmitted wavefront. We suggest a lower bound for the pinhole size for a given aberration strength and quantify the optical sectioning provided by the system. For our measured aberration data we find that a pinhole of size approximately 3 Airy units represents a good compromise, allowing effective transmission of the wavefront and thin optical sections. Finally, we discuss some of the practical implications of confocal wavefront sensing for AO systems in microscopy.

In this Letter, we present an analysis of the effects of polarization state on the pattern contrast in a structured illumination microscope. Using vectorial ray tracing methods, we show that the contrast varies nonmonotonically with both the numerical aperture of the microscope objective lens and the orientation of the electric field with respect to the meridional plane. By careful selection of these two parameters, high pattern contrast can be obtained without polarization rotation, reducing the cost and complexity of structured illumination imaging systems and increasing light throughput and imaging speed. We present experimental results that show good agreement with theoretical predictions and discuss the implications for super-resolution imaging.

Publisher Summary The widespread availability of spatially and temporally coherent laser sources makes the production of optical vortices inevitable in any experiment involving scattered laser light. The realization of quantized vortices is not specific to optics: these objects occur in all spatial scalar fields. Although optical vortices are often referred to as “points of phase singularity within a cross section of the field,” physical optical fields extend over three dimensions, and the phase singularities are actually lines of perfect destructive interference that are embedded in the volume filled by the light. Optical vortices are examples of the singularity lines within all complicated scalar fields. By comparison, electromagnetic vector fields do not generally have nodes in all components simultaneously. However, vector fields possess singularities associated with the parameterization of elliptical and partial polarization rather than phase. Polarization singularities are present in many situations, ranging from sunlight to the light transmitted by birefringent materials. Their descriptors are more complicated than their scalar counterpart in that they have both handedness and additional categorization. The study of optical vortices and orbital angular momentum has led to a recognition that the energy flow—characterized by the Poynting vector—has features not immediately apparent from the intensity alone, nor from global properties of a beam.

We numerically study the topology of optical vortex lines (nodal lines) in volumes of optical speckle, modeled as superpositions of random plane waves. It is known that the vortex lines may be infinitely long, or form closed loops. Loops are occasionally threaded by infinite lines, or linked with other loops. We find the probability of a loop not being threaded decays exponentially with the length of the loop. This behavior has a similarity to scaling laws studied in superfluid turbulence, and polymers modeled as random walks.

Light emerging from a spiral phase plate with a non-integer phase step has a complicated vortex structure and is unstable on propagation. We generate light carrying fractional orbital angular momentum (OAM) not with a phase step but by a synthesis of Laguerre-Gaussian modes. By limiting the number of different Gouy phases in the superposition we produce a light beam which is well characterised in terms of its propagation. We believe that their structural stability makes these beams ideal for quantum information processes utilising fractional OAM states.

The 3D structure of randomly polarized light fields is exemplified by its polarization singularities: lines along which the polarization is purely circular (C lines) and surfaces on which the polarization is linear (L surfaces). We visualize these polarization singularities experimentally in vector laser speckle fields, and in numerical simulations of random wave superpositions. Our results confirm previous analytical predictions [M. R. Dennis, Opt. Commun. 213, 201 (2002)] regarding the statistical distribution of types of C points and relate their 2D properties to their 3D structure.

Propagating three-dimensional speckle “elds are threaded by random networks of nodal lines (optical vor-tices). We review our recent numerical superpositions of simulations of random plane waves modelling speckle(OHolleran et al. Phys. Rev. Lett. in press), in which the nodal lines and loops were found to have the fractalstructure of brownian random walks. We discuss this result, and its comparison with the discrete vortices of theZ 3 lattice model for cosmic strings. We argue that the scaling depends on the geometry of small vortex loopsand avoided crossings. The analytic statistics of these events, along with related singularities are discussed, andthe densities of vorticity-vanishing points and anisotropy C lines are found explicitly.Keywords: Optical vortex, speckle, three-dimensional, random walk, fractal 1. INTRODUCTION Our understanding of optical speckle, as a natural interference phenomenon, has concentrated historically onthe properties of the bright speckle regions, and much is known of the statistical properties of bright speckle.

Random optical speckle patterns in three dimensions contain complex tangles of optical vortices. We investigate the knotting and linking of these lines in computer simulations of optical fields using tools from knot theory.

Natural light fields are threaded by lines of darkness. For monochromatic light, the phenomenon is familiar in laser speckle, i.e., the black points that appear in the scattered light. These black points are optical vortices that extend as lines throughout the volume of the field. We establish by numerical simulations, supported by experiments, that these vortex lines have the fractal properties of a Brownian random walk. Approximately 73% of the lines percolate through the optical beam, the remainder forming closed loops. Our statistical results are similar to those of vortices in random discrete lattice models of cosmic strings, implying that the statistics of singularities in random optical fields exhibit universal behavior.

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.

Light shaping is usually concerned with shaping the bright part of the beam. Here we investigate a technique for shaping a light beam's vortex lines, which are completely dark.

We report a camera system, associated motorized opto-mechanics, and data handling routines for imaging the three-dimensional structure of singularities within scalar and vector optical fields. Using Fourier techniques to analyse a series of cross-sectional images, we measure either the relative phase or state of polarization of monochromatic fields and analyse this for the identification and mapping of their associated singularities.

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.