Single-molecule Localization Research Articles

ECLiPSE: a versatile classification technique for structural and morphological analysis of 2D and 3D single-molecule localization microscopy data

Single-molecule localization microscopy (SMLM) has gained widespread use for visualizing the morphology of subcellular organelles and structures with nanoscale spatial resolution. However, analysis tools for automatically quantifying and classifying SMLM images have lagged behind. Here we introduce Enhanced Classification of Localized Point clouds by Shape Extraction (ECLiPSE), an automated machine learning analysis pipeline specifically designed to classify cellular structures captured through two-dimensional or three-dimensional SMLM. ECLiPSE leverages a comprehensive set of shape descriptors, the majority of which are directly extracted from the localizations to minimize bias during the characterization of individual structures. ECLiPSE has been validated using both unsupervised and supervised classification on datasets, including various cellular structures, achieving near-perfect accuracy. We apply two-dimensional ECLiPSE to classify morphologically distinct protein aggregates relevant for neurodegenerative diseases. Additionally, we employ three-dimensional ECLiPSE to identify relevant biological differences between healthy and depolarized mitochondria. ECLiPSE will enhance the way we study cellular structures across various biological contexts.

The ATTO 565 Dye and Its Applications in Microscopy.

ATTO 565, a Rhodamine-type dye, has garnered significant attention due to its remarkable optical properties, such as a high fluorescence quantum yield, and the fact that it is a relatively stable structure and has low biotoxicity. ATTO 565 has found extensive applications in combination with microscopy technology. In this review, the chemical and optical properties of ATTO 565 are introduced, along with the principles behind them. The functionality of ATTO 565 in confocal microscopy, stimulated emission depletion (STED) microscopy, single-molecule tracking (SMT) techniques, two-photon excitation-stimulated emission depletion microscopy (TPE-STED) and fluorescence correlation spectroscopy (FCS) is discussed. These studies demonstrate that ATTO 565 plays a crucial role in areas such as biological imaging and single-molecule localization, thus warranting further in-depth investigations. Finally, we present some prospects and concepts for the future applications of ATTO 565 in the fields of biocompatibility and metal ion detection. This review does not include theoretical calculations for the ATTO 565 molecule.

Enhanced Performance of the Optimized Dye CF583R in Direct Stochastic Optical Reconstruction Microscopy of Active Zones in Drosophila Melanogaster.

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts.

Real-time data processing in colorimetry camera-based single-molecule localization microscopy via CPU-GPU-FPGA heterogeneous computation.

Because conventional low-light cameras used in single-molecule localization microscopy (SMLM) do not have the ability to distinguish colors, it is often necessary to employ a dedicated optical system and/or a complicated image analysis procedure to realize multi-color SMLM. Recently, researchers explored the potential of a new kind of low-light camera called colorimetry camera as an alternative detector in multi-color SMLM, and achieved two-color SMLM under a simple optical system, with a comparable cross-talk to the best reported values. However, extracting images from all color channels is a necessary but lengthy process in colorimetry camera-based SMLM (called CC-STORM), because this process requires the sequential traversal of a massive number of pixels. By taking advantage of the parallelism and pipeline characteristics of FPGA, in this paper, we report an updated multi-color SMLM method called HCC-STORM, which integrated the data processing tasks in CC-STORM into a home-built CPU-GPU-FPGA heterogeneous computing platform. We show that, without scarifying the original performance of CC-STORM, the execution speed of HCC-STORM was increased by approximately three times. Actually, in HCC-STORM, the total data processing time for each raw image with 1024 × 1024 pixels was 26.9 ms. This improvement enabled real-time data processing for a field of view of 1024 × 1024 pixels and an exposure time of 30 ms (a typical exposure time in CC-STORM). Furthermore, to reduce the difficulty of deploying algorithms into the heterogeneous computing platform, we also report the necessary interfaces for four commonly used high-level programming languages, including C/C++, Python, Java, and Matlab. This study not only pushes forward the mature of CC-STORM, but also presents a powerful computing platform for tasks with heavy computation load.

Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.

A milestone in optical imaging of mechanical forces in cells has been the development of the family of flipper fluorescent probes able to report membrane tension noninvasively in living cells through their fluorescence lifetime. The specifically designed Flipper-CF3 probe with an engineered inherent blinking mechanism was recently introduced for super-resolution fluorescence microscopy of lipid ordered membranes but was too dim to be detected in lipid disordered membranes at the single-molecule level (García-Calvo, J. J. Am. Chem. Soc. 2020, 142(28), 12034-12038). We show here that the original and commercially available probe Flipper-TR is compatible with single-molecule based super-resolution imaging and resolves both liquid ordered and liquid disordered membranes of giant unilamellar vesicles below the diffraction limit. Single probe molecules were additionally tracked in lipid bilayers, enabling to distinguish membranes of varying composition from the diffusion coefficient of the probe. Differences in brightness between Flipper-CF3 and Flipper-TR originate in their steady-state absorption and fluorescence properties. The general compatibility of the Flipper-TR scaffold with single-molecule detection is further shown in super-resolution experiments with targetable Flipper-TR derivatives.

Using Localization Microscopy to Quantify Calcium Channels at Presynaptic Boutons.

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes-expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons. Key features • Super-resolution imaging of live anesthetized C. elegans. • Three-color super-resolution reconstruction of synapses. • Nanodomains and the distribution of proteins. • Quantification of the number of proteins at synapses from single-molecule localization data.

Markov Chain Models of Emitter Activations in Single Molecule Localization Microscopy

Markov Chain Models of Emitter Activations in Single Molecule Localization Microscopy

Multiplexed Nanoscopy via Buffer Exchange.

Understanding cellular functions, particularly in their intricate complexity, can greatly benefit from the spatial mapping of diverse molecules through multitarget single-molecule localization microscopy (SMLM). Existing methodologies, primarily restricting the encoding dimensions to color and lifetime or requiring cyclic staining, often involve broad chromatic detection, specialized optical configurations, or sophisticated labeling techniques. Here, we propose a simple approach called buffer-exchange stochastic optical reconstruction microscopy (beSTORM), which introduces an additional dimension to differentiate between single molecules irrespective of their spectral properties. This method leverages the distinguishable photoblinking responses to distinct buffer conditions, offering a straightforward yet effective means of fluorophore discrimination. Through buffer exchanges, beSTORM achieves multitarget SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color without chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.

Measurement precision bounds on aberrated single-molecule emission patterns

Single-molecule localization microscopy (SMLM) has revolutionized the study of biological phenomena by providing exquisite nanoscale spatial resolution. However, optical aberrations induced by sample and system imperfections distort the single-molecule emission patterns (i.e. PSFs), leading to reduced precision and resolution of SMLM, particularly in three-dimensional (3D) applications. While various methods, both analytical and instrumental, have been employed to mitigate these aberrations, a comprehensive analysis of how different types of commonly encountered aberrations affect single-molecule experiments and their image formation remains missing. In this study, we addressed this gap by conducting a quantitative study of the theoretical precision limit for position and wavefront distortion measurements in the presence of aberrations. Leveraging Fisher information and Cramér-Rao lower bound (CRLB), we quantitively analyzed and compared the effects of different aberration types, including index mismatch aberrations, on localization precision in both biplane and astigmatism 3D modalities as well as 2D SMLM imaging. Furthermore, we studied the achievable wavefront estimation precision from aberrated single-molecule emission patterns, a pivot step for successful adaptive optics in SMLM through thick specimens. This analysis lays a quantitative foundation for the development and application of SMLM in whole-cells, tissues and with a large field of view, providing in-depth insights into the behavior of different aberration types in single-molecule imaging and thus generating theoretical guidelines for developing highly efficient aberration correction strategies and enhancing the precision and reliability of 3D SMLM.

Anisotropic DBSCAN for 3D SMLM Data Clustering.

Single-molecule localization microscopy (SMLM) advanced biological discoveries beyond the diffraction limit. Various implementations enable 3D SMLM to reconstruct volumetric cell images. Yet, the inherent anisotropic point spread function of optical microscopes often limits the localization precision in the axial direction compared to the lateral precision. Such localization anisotropy could also expand spherical cellular structures to ellipsoidal cellular structures. Structure identification, however, is often performed using DBSCAN cluster algorithms, considering an isotropic search volume. Here, we show that an anisotropic DBSCAN search volume identifies anisotropic clusters more reliably using simulated ground truth data sets. Given experimental localization precisions, we suggest optimized search parameters based on an expanded computational grid search and show an enhanced performance of anisotropic DBSCAN amidst variations in localization precision. We demonstrate the capability of anisotropic DBSCAN on experimental data and anticipate that the algorithm allows for a more rigorous identification of clusters in cells, considering the anisotropic localization precisions of astigmatism-based 3D SMLM.

Super-Resolved Protein Imaging Using Bifunctional Light-Up Aptamers.

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

Annotation and automated segmentation of single-molecule localisation microscopy data.

Single Molecule Localisation Microscopy (SMLM) is becoming a widely used technique in cell biology. After processing the images, the molecular localisations are typically stored in a table as xy (or xyz) coordinates, with additional information, such as number of photons, etc. This set of coordinates can be used to generate an image to visualise the molecular distribution, for example, a 2D or 3D histogram of localisations. Many different methods have been devised to analyse SMLM data, among which cluster analysis of the localisations is popular. However, it can be useful to first segment the data, to extract the localisations in a specific region of a cell or in individual cells, prior to downstream analysis. Here we describe a pipeline for annotating localisations in an SMLM dataset in which we compared membrane segmentation approaches, including Otsu thresholding and machine learning models, and subsequent cell segmentation. We used an SMLM dataset derived from dSTORM images of sectioned cell pellets, stained for the membrane proteins EGFR (epidermal growth factor receptor) and EREG (epiregulin) as a test dataset. We found that a Cellpose model retrained on our data performed the best in the membrane segmentation task, allowing us to perform downstream cluster analysis of membrane versus cell interior localisations. We anticipate this will be generally useful for SMLM analysis.

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Abstract Tu005: Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43

Background: Connexin 43 (Cx43) is the body's most widely expressed gap junctional connexin. Researchers have shown evidence of non-canonical functions of Cx43 and the therapeutic promise of Cx43 mimetic peptides in cardiac pathologies. However, some critical barriers, including in vivo stability and potential immunotoxicity of Cx43 mimetic peptides, hinder clinical translation of these pro-drugs. To address these challenges, we are pioneering bovine milk-derived small extracellular vesicles (mEVs) as delivery vehicles for peptidic drugs. While exploring mEVs as drug delivery tools for αCT1 – a peptide mimicking Cx43 carboxyl terminus (CT) undergoing Phase III testing and known for promoting wound healing and cardioprotection - mEVs without loaded drug cargo exhibited similar bioactivity. This study aimed to test the hypothesis that Cx43, particularly CT fragments of Cx43, are naturally present in mEVs. Methods: mEVs were isolated from bovine milk using a novel protocol developed by our lab based on Tangential Flow Filtration and characterized by Western Blotting (WB), Nanoparticle Tracking Analysis, and Transmission Electron Microscopy. To determine the presence of Cx43 in mEVs, we used WB, immunoprecipitation and Single-Molecule Localization Microscopy (SMLM). Results: Characterization of mEVs confirmed relatively pure and ultrastructurally definitive mEVs isolated from milk at high density. WB for Cx43 in mEVs lysates based on two antibodies - one against the cytoplasmic loop domain and the other against CT-most 20 amino acids of Cx43, showed that full-length Cx43 was present in mEVs. Interestingly, the Cx43 CT antibody also detected a band at around 16 KDa, and this was confirmed by WB by using two more Cx43 CT antibodies. We also immunoprecipitated this Cx43 CT polypeptide from mEVs lysates using the CT antibodies. SMLM suggested that a subpopulation of mEVs contain Cx43 and demonstrated vesicle-to-vesicle heterogeneity to Cx43 immunolabeling. Conclusions: We show for the first time that an endogenous Cx43 CT polypeptide is present in mEVs. Our results provide new insight into the role of Cx43 in mEV bioactivity and potentially offer a path to safe, effective, and orally available therapeutic options based on mEVs.

Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media.

Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638nm. An excitation power density of 0.60 kW/cm2 at 638nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.

Self-Blinking Thioflavin T for Super-resolution Imaging.

Thioflavin T (ThT) is a typical dye used to visualize the aggregation and formation of fibrillar structures, e.g., amyloid fibrils and peptide nanofibrils. ThT has been considered to produce stable fluorescence when interacting with aggregated proteins. For single-molecule localization microscopy (SMLM)-based optical super-resolution imaging, a photoswitching/blinking fluorescence property is required. Here we demonstrate that, in contrast to previous reports, ThT exhibits intrinsic stochastic blinking properties, without the need for blinking imaging buffer, in stable binding conditions. The blinking properties (photon number, blinking time, and on-off duty cycle) of ThT at the single-molecule level (for ultralow concentrations) were investigated under different conditions. As a proof of concept, we performed SMLM imaging of ThT-labeled α-synuclein fibrils measured in air and PBS buffer.

Impaired Experience-Dependent Theta Oscillation Synchronization and Inter-Areal Synaptic Connectivity in the Visual Cortex of Fmr1 KO Mice.

Fragile X syndrome (FX) is the most prevalent inheritable form of autism spectrum disorder (ASD), characterized by hypersensitivity, difficulty in habituating to new sensory stimuli, and intellectual disability. Individuals with FX often experience visual perception and learning deficits. Visual experience leads to the emergence of the familiarity-evoked theta band oscillations in the primary visual cortex (V1) and the lateromedial area (LM) of mice. These theta oscillations in V1 and LM are synchronized with each other, providing a mechanism of sensory multi-areal binding. However, how this multi-areal binding and the corresponding theta oscillations are altered in FX is not known. Using iDISCO whole brain clearing with light-sheet microscopy, we quantified immediate early gene Fos expression in V1 and LM, identifying deficits in experience-dependent neural activity in FX mice. We performed simultaneous in vivo recordings with silicon probes in V1 and LM of awake mice and channelrhodopsin-2-assisted circuit mapping (CRACM) in acute brain slices to examine the neural activity and strength of long-range synaptic connections between V1 and LM in both wildtype (WT) and Fmr1 knockout (KO) mice, the model of FX, before and after visual experience. Our findings reveal synchronized familiarity-evoked theta oscillations in V1 and LM, the increased strength of V1→LM functional and synaptic connections, which correlated with the corresponding changes of presynaptic short-term plasticity in WT mice. The LM oscillations were attenuated in FX mice and correlated with impaired functional and synaptic connectivity and short-term plasticity in the feedforward (FF) V1→LM and feedback (FB) LM→V1 pathways. Finally, using 4Pi single-molecule localization microscopy (SMLM) in thick brain tissue, we identified experience-dependent changes in the density and shape of dendritic spines in layer 5 pyramidal cells of WT mice, which correlated with the functional synaptic measurements. Interestingly, there was an increased dendritic spine density and length in naïve FX mice that failed to respond to experience. Our study provides the first comprehensive characterization of the role of visual experience in triggering inter-areal neural synchrony and shaping synaptic connectivity in WT and FX mice.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part I: Super-Resolution’s STED, SIM, SMI, and SMLM

Abstract As technology advances, the field of microscopy offers unique opportunities for scientific exploration. However, the complex terminology used to describe these cutting-edge techniques often appears as a jumble of letters, akin to alphabet soup. Among the plethora of acronyms, some of the most prevalent are associated with super-resolution microscopy, including Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Spatially Modulated Illumination (SMI), and Single-Molecule Localization Microscopy (SMLM). These techniques push past the limitations of conventional light microscopy, achieving resolutions down to the tens of nanometers. This article aims to decode these common super-resolution techniques, fostering a deeper understanding of microscopy and igniting interest in their application.

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.

Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Total internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities—an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combine flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This distinct combination enables quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieve a versatile combination of SMLM and SIM with up to three channels. We deploy this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enables long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties can be correlated with nanoscale cytoskeletal organization and dynamics.