Unveiling the limits of precision in iterative MINFLUX.

In single-molecule microscopy, a big question is how precisely we can estimate the location of a single molecule. Our research shows that by using iterative localisation microscopy and factoring in the prior information, we can boost precision and reduce the number of photons needed. Leveraging the Van Trees inequality aids in determining the optimal precision achievable. Our approach holds promise for wider application in discerning the optimal precision across diverse imaging scenarios, encompassing various illumination strategies, point spread functions and overarching control methodologies.

Modelling 3D supramolecular structure from sparse single-molecule localisation microscopy data.

Single-molecule localisation microscopy (SMLM) has the potential to reveal the underlying organisation of specific molecules within supramolecular complexes and their conformations, which is not possible with conventional microscope resolution. However, the detection efficiency for fluorescent molecules in cells can be limited in SMLM, even to below 1% in thick and dense samples. Segmentation of individual complexes can also be challenging. To overcome these problems, we have developed a software package termed PERPL: Pattern Extraction from Relative Positions of Localisations. This software assesses the relative likelihoods of models for underlying patterns behind incomplete SMLM data, based on the relative positions of pairs of localisations. We review its principles and demonstrate its use on the 3D lattice of Z-disk proteins in mammalian cardiomyocytes. We find known and novel features at ~20 nm with localisations of less than 1% of the target proteins, using mEos fluorescent protein constructs.

Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit.

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the 'super-resolution' limit and what opportunities remain for further improvements in the near future.

Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging.

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.

Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.

Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.

A microfluidic device for the hydrodynamic immobilisation of living fission yeast cells for super-resolution imaging.

We describe a microfluidic device designed specifically for the reversible immobilisation of Schizosaccharomyces pombe (Fission Yeast) cells to facilitate live cell super-resolution microscopy. Photo-Activation Localisation Microscopy (PALM) is used to create detailed super-resolution images within living cells with a modal accuracy of >25 nm in the lateral dimensions. The novel flow design captures and holds cells in a well-defined array with minimal effect on the normal growth kinetics. Cells are held over several hours and can continue to grow and divide within the device during fluorescence imaging.

Optimizing Voronoi-based quantifications for reaching interactive analysis of 3D localizations in the million range.

Over the last decade, single-molecule localization microscopy (SMLM) has revolutionized cell biology, making it possible to monitor molecular organization and dynamics with spatial resolution of a few nanometers. Despite being a relatively recent field, SMLM has witnessed the development of dozens of analysis methods for problems as diverse as segmentation, clustering, tracking or colocalization. Among those, Voronoi-based methods have achieved a prominent position for 2D analysis as robust and efficient implementations were available for generating 2D Voronoi diagrams. Unfortunately, this was not the case for 3D Voronoi diagrams, and existing methods were therefore extremely time-consuming. In this work, we present a new hybrid CPU-GPU algorithm for the rapid generation of 3D Voronoi diagrams. Voro3D allows creating Voronoi diagrams of datasets composed of millions of localizations in minutes, making any Voronoi-based analysis method such as SR-Tesseler accessible to life scientists wanting to quantify 3D datasets. In addition, we also improve ClusterVisu, a Voronoi-based clustering method using Monte-Carlo simulations, by demonstrating that those costly simulations can be correctly approximated by a customized gamma probability distribution function.

Cluster analysis for localisation-based data sets: dos and don'ts when quantifying protein aggregates.

Many proteins display a non-random distribution on the cell surface. From dimers to nanoscale clusters to large, micron-scale aggregations, these distributions regulate protein-protein interactions and signalling. Although these distributions show organisation on length-scales below the resolution limit of conventional optical microscopy, single molecule localisation microscopy (SMLM) can map molecule locations with nanometre precision. The data from SMLM is not a conventional pixelated image and instead takes the form of a point-pattern-a list of the x, y coordinates of the localised molecules. To extract the biological insights that researchers require cluster analysis is often performed on these data sets, quantifying such parameters as the size of clusters, the percentage of monomers and so on. Here, we provide some guidance on how SMLM clustering should best be performed.

Optimising correlative super resolution and atomic force microscopies for investigating the cellular cytoskeleton.

Correlative imaging methods can provide greater information for investigations of cellular ultra-structure, with separate analysis methods complementing each other's strengths and covering for deficiencies. Here we present a method for correlative applications of super resolution and atomic force microscopies, optimising the sample preparation for correlative imaging of the cellular cytoskeleton in COS-7 cells. This optimisation determined the order of permeabilisation and fixation, the concentration of Triton X-100 surfactant used and time required for sufficient removal of the cellular membrane while maintaining the microtubule network. Correlative SMLM/AFM imaging revealed the different information that can be obtained through each microscopy. The widths of microtubules and microtubule clusters were determined from both AFM height measurements and Gaussian fitting of SMLM intensity cross sections, these were then compared to determine the orientation of microtubules within larger microtubule bundles. The ordering of microtubules at intersections was determined from the AFM height profiles as each microtubule crosses the other. The combination of both microtubule diameter measurements enabled greater information on their structure to be found than either measurement could individually.

Analysing errors in single-molecule localisation microscopy.

In single molecule localisation microscopy (SMLM) a super-resolution image of the distribution of fluorophores in the sample is built up from the localised positions of many individual molecules. It has become widely used due to its experimental simplicity and the high resolution that can be achieved. However, the factors which limit resolution in a reconstructed image, and the artefacts which can be present, are completely different to those present in standard fluorescent microscopy techniques. Artefacts may be difficult for users to identify, particularly as they can cause images to appear falsely sharp, an effect called artificial sharpening. Here we discuss the different sources of error and bias in SMLM, and the methods available for avoiding or detecting them.

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

K-Neighbourhood Analysis: A Method for Understanding SMLM Images as Compositions of Local Neighbourhoods.

Single molecule localisation microscopy (SMLM) is a powerful tool that has revealed the spatial arrangement of cell surface signalling proteins, producing data of enormous complexity. The complexity is partly driven by the convolution of technical and biological signal components, and partly by the challenge of pooling information across many distinct cells. To address these two particular challenges, we have devised a novel algorithm called K-neighbourhood analysis (KNA), which emphasises the fact that each image can also be viewed as a composition of local neighbourhoods. KNA is based on a novel transformation, spatial neighbourhood principal component analysis (SNPCA), which is defined by the PCA of the normalised K-nearest neighbour vectors of a spatially random point pattern. Here, we use KNA to define a novel visualisation of individual images, to compare within and between groups of images and to investigate the preferential patterns of phosphorylation. This methodology is also highly flexible and can be used to augment existing clustering methods by providing clustering diagnostics as well as revealing substructure within microclusters. In summary, we have presented a highly flexible analysis tool that presents new conceptual possibilities in the analysis of SMLM images.

Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package "Fluorescence-Lifetime TrackNTrace," which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.

Fractional occupancy of synaptic binding sites and the molecular plasticity of inhibitory synapses.

The postsynaptic density (PSD) at inhibitory synapses is a complex molecular assembly that serves as a platform for the interaction of neurotransmitter receptors, scaffold and adapter proteins, cytoskeletal elements and signalling molecules. The stability of the PSD depends on a multiplicity of interactions linking individual components. At the same time the PSD retains a substantial degree of flexibility. The continuous exchange of synaptic molecules and the preferential addition or removal of certain components induce plastic changes in the synaptic structure. This property necessarily implies that interactors are in dynamic equilibrium and that not all synaptic binding sites are occupied simultaneously. This review discusses the molecular plasticity of inhibitory synapses in terms of the connectivity of their components. Whereas stable protein complexes are marked by stoichiometric relationships between subunits, the majority of synaptic interactions have fractional occupancy, which is here defined as the non-saturation of synaptic binding sites. Fractional occupancy can have several causes: reduced kinetic or thermodynamic stability of the interactions, an imbalance in the concentrations or limited spatio-temporal overlap of interacting proteins, negative cooperativity or mutually exclusive binding. The role of fractional occupancy in the regulation of synaptic structure and function is explored based on recent data about the connectivity of inhibitory receptors and scaffold proteins. I propose that the absolute quantification of interactors and their stoichiometry at identified synapses can provide new mechanistic insights into the dynamic properties of inhibitory PSDs at the molecular level. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Towards single molecule biosensors using super-resolution fluorescence microscopy.

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.