Single-molecule Localization Microscopy Data Research Articles

Detecting continuous structural heterogeneity in single-molecule localization microscopy data.

Fusion of multiple chemically identical complexes, so-called particles, in localization microscopy, can improve the signal-to-noise ratio and overcome under-labeling. To this end, structural homogeneity of the data must be assumed. Biological heterogeneity, however, could be present in the data originating from distinct conformational variations or (continuous) variations in particle shapes. We present a prior-knowledge-free method for detecting continuous structural variations with localization microscopy. Detecting this heterogeneity leads to more faithful fusions and reconstructions of the localization microscopy data as their heterogeneity is taken into account. In experimental datasets, we show the continuous variation of the height of DNA origami tetrahedrons imaged with 3D PAINT and of the radius of Nuclear Pore Complexes imaged in 2D with STORM. In simulation, we study the impact on the heterogeneity detection pipeline of Degree Of Labeling and of structural variations in the form of two independent modes.

DBlink: dynamic localization microscopy in super spatiotemporal resolution via deep learning.

Single-molecule localization microscopy (SMLM) has revolutionized biological imaging, improving the spatial resolution of traditional microscopes by an order of magnitude. However, SMLM techniques require long acquisition times, typically a few minutes, to yield a single super-resolved image, because they depend on accumulation of many localizations over thousands of recorded frames. Hence, the capability of SMLM to observe dynamics at high temporal resolution has always been limited. In this work, we present DBlink, a deep-learning-based method for super spatiotemporal resolution reconstruction from SMLM data. The input to DBlink is a recorded video of SMLM data and the output is a super spatiotemporal resolution video reconstruction. We use a convolutional neural network combined with a bidirectional long short-term memory network architecture, designed for capturing long-term dependencies between different input frames. We demonstrate DBlink performance on simulated filaments and mitochondria-like structures, on experimental SMLM data under controlled motion conditions and on live-cell dynamic SMLM. DBlink's spatiotemporal interpolation constitutes an important advance in super-resolution imaging of dynamic processes in live cells.

Extracting nanoscale membrane morphology from single-molecule localizations

Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that are manually cleaned and curated. Here, we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy data. This makes it possible to study the shape of specifically labeled membranous structures inside cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on single-molecule localization microscopy data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment.

Quantitative Single-Molecule Localization Microscopy.

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

FACAM: A Fast and Accurate Clustering Analysis Method for Protein Complex Quantification in Single Molecule Localization Microscopy

Single molecule localization microscopy (SMLM) enables the analysis and quantification of protein complexes at the nanoscale. Using clustering analysis methods, quantitative information about protein complexes (for example, the size, density, number, and the distribution of nearest neighbors) can be extracted from coordinate-based SMLM data. However, since a final super-resolution image in SMLM is usually reconstructed from point clouds that contain millions of localizations, current popular clustering methods are not fast enough to enable daily quantification on such a big dataset. Here, we provide a fast and accurate clustering analysis method called FACAM, which is modified from the Alpha Shape method (a point dataset analysis method used in many fields). By taking advantage of parallel computation, FACAM is able to process millions of localizations in less than an hour, which is at least 10 times faster than the popular DBSCAN method. Furthermore, FACAM adaptively determines the segmentation threshold, and thus overcomes the problem of user-defined parameters. Using simulation and experimental datasets, we verified the advantages of FACAM over other reported clustering methods (including Ripley’s H, DBSCAN, and ClusterViSu).

DecodeSTORM: A user-friendly ImageJ plug-in for quantitative data analysis in single-molecule localization microscopy

Quantitative data analysis in single-molecule localization microscopy (SMLM) is crucial for studying cellular functions at the biomolecular level. In the past decade, several quantitative methods were developed for analyzing SMLM data; however, imaging artifacts in SMLM experiments reduce the accuracy of these methods, and these methods were seldom designed as user-friendly tools. Researchers are now trying to overcome these difficulties by developing easy-to-use SMLM data analysis software for certain image analysis tasks. But, this kind of software did not pay sufficient attention to the impact of imaging artifacts on the analysis accuracy, and usually contained only one type of analysis task. Therefore, users are still facing difficulties when they want to have the combined use of different types of analysis methods according to the characteristics of their data and their own needs. In this paper, we report an ImageJ plug-in called DecodeSTORM, which not only has a simple GUI for human–computer interaction, but also combines artifact correction with several quantitative analysis methods. DecodeSTORM includes format conversion, channel registration, artifact correction (drift correction and localization filtering), quantitative analysis (segmentation and clustering, spatial distribution statistics and colocalization) and visualization. Importantly, these data analysis methods can be combined freely, thus improving the accuracy of quantitative analysis and allowing users to have an optimal combination of methods. We believe DecodeSTORM is a user-friendly and powerful ImageJ plug-in, which provides an easy and accurate data analysis tool for adventurous biologists who are looking for new imaging tools for studying important questions in cell biology.

Towards an unbiased shape classification of super-resolution microscopy localizations.

Single molecule localization microscopy (SMLM) captures biological structures at nanoscale spatial resolution and has become an important tool for discovery in cell biology in recent years. However, tools that enable robust quantification of complex biological structures have lagged behind. In this study, we developed a new method to quantitatively describe and classify a wide range of diverse biological structures in SMLM data and have applied it to describe the degradation process of neuronal Tau protein aggregates, an important precursor in neurodegenerative diseases.

SMITE (single molecule imaging toolbox extraordinaire), a MATLAB package.

We present SMITE, a MATLAB package that features analysis tools for fluorescence single molecule imaging with an emphasis on single molecule localization microscopy (SMLM) and single-particle tracking (SPT). Some C and CUDA code is embedded in the MATLAB infrastructure to provide CPU/GPU speed-ups for particularly expensive computations. SMITE is organized around two data structures: SMF (Single Molecule Fitting), containing parameters that completely define the data analysis, and SMD (Single Molecule Data), which contains the results of the analysis. The code is divided into namespaces and subclasses that group related concepts and functionality together. SMITE is designed so that low-level tools as well as higher-level application specific interfaces are easy to use, making use of the SMF and SMD data structures whenever possible. Interaction with these tools is via GUIs or scripting. Extensive internal documentation of the interfaces and many usage examples are provided to aid the user. Code coverage includes mature SMLM data analysis techniques (applying gain and offset corrections to raw data, finding localizations, thresholding localizations based on various criteria, frame connection and drift correction), SMLM/SPT simulations, sophisticated SPT analyses, post-processing clustering and statistical analyses (e.g., Bayesian grouping of localizations for removing repeat localizations from the same emitter, diffusion analysis, hidden Markov models for characterizing dimers in SPT results), a variety of visualizations, experimental point spread function creation and characterization. SMITE is available via GitHub: https://github.com/LidkeLab/smite

A framework for evaluating the performance of SMLM cluster analysis algorithms.

Single-molecule localization microscopy (SMLM) generates data in the form of coordinates of localized fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite a range of cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance. Here, we use a systematic approach based on two metrics to score the success of clustering algorithms in simulated conditions mimicking experimental data. We demonstrate the framework using seven diverse analysis algorithms: DBSCAN, ToMATo, KDE, FOCAL, CAML, ClusterViSu and SR-Tesseler. Given that the best performer depended on the underlying distribution of localizations, we demonstrate an analysis pipeline based on statistical similarity measures that enables the selection of the most appropriate algorithm, and the optimized analysis parameters for real SMLM data. We propose that these standard simulated conditions, metrics and analysis pipeline become the basis for future analysis algorithm development and evaluation.

Characterization of unexpected blue-shifted single molecule fluorescence of JF646 and JF669 for SMLM.

JF-Halotag dyes have become increasingly popular for single-molecule localization microscopy (SMLM) because of their favorable photo-physics, ease of use, and compatibility with live-cell imaging. However under a variety of conditions, the excitation and emission wavelengths from individual emitters can deviate significantly from the commonly accepted characterization, leading to artifacts in two color SMLM data. Here I show results from multiple in vivo and in vitro experiments demonstrating 640nm mediated photo switching of JF669 from a conventional far-red signal to a 561nm excitable red single molecule signal. This result is exciting because it enables UV-free PALM experiments. UV radiation causes cell death, increases auto fluorescence, and induces cell stress, which may affect the behavior of labeled proteins or cellular processes of interest. Additionally, I present evidence of a subpopulation of JF646 molecules that can be excited by 561nm light, but emit red instead of far-red light. This unexpected fluorescence causes single molecule cross talk in the red channel, despite the commonly accepted characterization. This phenomenon is more apparent at low dye concentrations as well as high temperatures, suggesting that a self-interaction between JF646 molecules may bias the well characterized fluorescence, while in isolation JF646 may have significantly blue-shifted excitation and emission spectra. Together, these results show potentially useful photo-physical behavior of JF dyes, but also highlight the importance of careful controls when using synthetic dyes for single molecule localization microscopy.

Estimating the localization spread function of static single-molecule localization microscopy images.

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.

Machine learning framework to segment sarcomeric structures in SMLM data

Object detection is an image analysis task with a wide range of applications, which is difficult to accomplish with traditional programming. Recent breakthroughs in machine learning have made significant progress in this area. However, these algorithms are generally compatible with traditional pixelated images and cannot be directly applied for pointillist datasets generated by single molecule localization microscopy (SMLM) methods. Here, we have improved the averaging method developed for the analysis of SMLM images of sarcomere structures based on a machine learning object detection algorithm. The ordered structure of sarcomeres allows us to determine the location of the proteins more accurately by superimposing SMLM images of identically assembled proteins. However, the area segmentation process required for averaging can be extremely time-consuming and tedious. In this work, we have automated this process. The developed algorithm not only finds the regions of interest, but also classifies the localizations and identifies the true positive ones. For training, we used simulations to generate large amounts of labelled data. After tuning the neural network’s internal parameters, it could find the localizations associated with the structures we were looking for with high accuracy. We validated our results by comparing them with previous manual evaluations. It has also been proven that the simulations can generate data of sufficient quality for training. Our method is suitable for the identification of other types of structures in SMLM data.

Recent development of computational cluster analysis methods for single-molecule localization microscopy images

With the development of super-resolution imaging techniques, it is crucial to understand protein structure at the nanoscale in terms of clustering and organization in a cell. However, cluster analysis from single-molecule localization microscopy (SMLM) images remains challenging because the classical computational cluster analysis methods developed for conventional microscopy images do not apply to pointillism SMLM data, necessitating the development of distinct methods for cluster analysis from SMLM images. In this review, we discuss the development of computational cluster analysis methods for SMLM images by categorizing them into classical and machine-learning-based methods. Finally, we address possible future directions for machine learning-based cluster analysis methods for SMLM data.

Introducing Diinamic, a flexible and robust method for clustering analysis in single-molecule localization microscopy.

Super-resolution microscopy allowed major improvements in our capacity to describe and explain biological organization at the nanoscale. Single-molecule localization microscopy (SMLM) uses the positions of molecules to create super-resolved images, but it can also provide new insights into the organization of molecules through appropriate pointillistic analyses that fully exploit the sparse nature of SMLM data. However, the main drawback of SMLM is the lack of analytical tools easily applicable to the diverse types of data that can arise from biological samples. Typically, a cloud of detections may be a cluster of molecules or not depending on the local density of detections, but also on the size of molecules themselves, the labeling technique, the photo-physics of the fluorophore, and the imaging conditions. We aimed to set an easy-to-use clustering analysis protocol adaptable to different types of data. Here, we introduce Diinamic, which combines different density-based analyses and optional thresholding to facilitate the detection of clusters. On simulated or real SMLM data, Diinamic correctly identified clusters of different sizes and densities, being performant even in noisy datasets with multiple detections per fluorophore. It also detected subdomains ("nanodomains") in clusters with non-homogeneous distribution of detections.

Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by Single Molecule Localization Microscopy (SMLM)

The cell as a system of many components, governed by the laws of physics and chemistry drives molecular functions having an impact on the spatial organization of these systems and vice versa. Since the relationship between structure and function is an almost universal rule not only in biology, appropriate methods are required to parameterize the relationship between the structure and function of biomolecules and their networks, the mechanisms of the processes in which they are involved, and the mechanisms of regulation of these processes. Single molecule localization microscopy (SMLM), which we focus on here, offers a significant advantage for the quantitative parametrization of molecular organization: it provides matrices of coordinates of fluorescently labeled biomolecules that can be directly subjected to advanced mathematical analytical procedures without the need for laborious and sometimes misleading image processing. Here, we propose mathematical tools for comprehensive quantitative computer data analysis of SMLM point patterns that include Ripley distance frequency analysis, persistent homology analysis, persistent ‘imaging’, principal component analysis and co-localization analysis. The application of these methods is explained using artificial datasets simulating different, potentially possible and interpretatively important situations. Illustrative analyses of real complex biological SMLM data are presented to emphasize the applicability of the proposed algorithms. This manuscript demonstrated the extraction of features and parameters quantifying the influence of chromatin (re)organization on genome function, offering a novel approach to study chromatin architecture at the nanoscale. However, the ability to adapt the proposed algorithms to analyze essentially any molecular organizations, e.g., membrane receptors or protein trafficking in the cytosol, offers broad flexibility of use.

Maximum-likelihood model fitting for quantitative analysis of SMLM data

Quantitative data analysis is important for any single-molecule localization microscopy (SMLM) workflow to extract biological insights from the coordinates of the single fluorophores. However, current approaches are restricted to simple geometries or require identical structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model to localization coordinates. It extracts meaningful parameters from individual structures and can select the most suitable model. In addition to analyzing complex, heterogeneous and dynamic structures for in situ structural biology, we demonstrate how LocMoFit can assemble multi-protein distribution maps of six nuclear pore components, calculate single-particle averages without any assumption about geometry or symmetry, and perform a time-resolved reconstruction of the highly dynamic endocytic process from static snapshots. We provide extensive simulation and visualization routines to validate the robustness of LocMoFit and tutorials to enable any user to increase the information content they can extract from their SMLM data.

MSDenoiser: Muti-step adaptive denoising framework for super-resolution image from single molecule localization microscopy

Recent developments in single-molecule localization microscopy (SMLM) enable researchers to study macromolecular structures at the nanometer scale. However, due to the complexity of imaging process, there are a variety of complex heterogeneous noises in SMLM data. The conventional denoising methods in SMLM can only remove a single type of noise. And, most of these denoising algorithms require manual parameter setting, which is difficult and unfriendly for biological researchers. To solve these problems, we propose a multi-step adaptive denoising framework called MSDenoiser, which incorporates multiple noise reduction algorithms and can gradually remove heterogeneous mixed noises in SMLM. In addition, this framework can adaptively learn algorithm parameters based on the localization data without manually intervention. We demonstrate the effectiveness of the proposed denoising framework on both simulated data and experimental data with different types of structures (microtubules, nuclear pore complexes and mitochondria). Experimental results show that the proposed method has better denoising effect and universality.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

A density-based enrichment measure for assessing colocalization in single-molecule localization microscopy data

Dual-color single-molecule localization microscopy (SMLM) provides unprecedented possibilities for detailed studies of colocalization of different molecular species in a cell. However, the informational richness of the data is not fully exploited by current analysis tools that often reduce colocalization to a single value. Here, we describe a tool specifically designed for determination of co-localization in both 2D and 3D from SMLM data. The approach uses a function that describes the relative enrichment of one molecular species on the density distribution of a reference species. The function reframes the question of colocalization by providing a density-context relevant to multiple biological questions. Moreover, the function visualize enrichment (i.e. colocalization) directly in the images for easy interpretation. We demonstrate the approach’s functionality on both simulated data and cultured neurons, and compare it to current alternative measures. The method is available in a Python function for easy and parameter-free implementation.

Estimating the localization spread function of static single-molecule localization microscopy images

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.