Single-molecule Localization Research Articles

Improving STED microscopy with SUPPOSe: enhancing resolution from a single-image

Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is ∼107 nm with 8 corners ∼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to be R = 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560 , PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561nm MINFLUX imaging.

Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions.

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins, such as I-BAR- and F-BAR-domain-containing proteins, have emerged as upstream regulators that link membrane interactions to actin regulators such as formins and proteins of the Ena/VASP family. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. To explore the significance of the F-BAR domain-containing neuronal membrane adaptor TOCA-1 (also known as FNBP1L) in filopodia we performed a quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Increasing the density of TOCA-1 enhances Ena/VASP protein binding in vitro, and an accumulation of TOCA-1, as well as its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single-molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion and this depends on a functional TOCA-1 SH3 domain and activation of Cdc42, which we perturbed using the small-molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion.

Interactive simulation and visualization of point spread functions in single molecule imaging

The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. Optical aberrations and fixed fluorophore dipoles often result in non-isotropic and distorted PSFs, impairing and biasing conventional fitting approaches. Further, PSF shapes are deliberately modified in PSF engineering approaches for providing improved sensitivity, e.g., for 3D localization or determination of dipole orientation. As this can lead to highly complex PSF shapes, a tool for visualizing expected PSFs would facilitate the interpretation of obtained data and the design of experimental approaches. To this end, we introduce a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. Our tool incorporates a wide range of microscope and fluorophore parameters, including orientationally constrained fluorophores, as well as custom aberrations, transmission and phase masks, thus enabling an accurate representation of various imaging conditions. An additional feature is the simulation of crowded molecular environments with overlapping PSFs. Further, our app directly provides the Cramér–Rao bound for assessing the best achievable localization precision under given conditions. Finally, our software allows for the fitting of custom aberrations directly from experimental data, as well as the generation of a large dataset with randomized simulation parameters, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.

Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging.

Imaging neuronal architecture has been a recurrent challenge over the years, and the localization of synaptic proteins is a frequent challenge in neuroscience. To quantitatively detect and analyze the structure of synapses, we recently developed free SODA software to detect the association of pre and postsynaptic proteins. To fully take advantage of spatial distribution analysis in complex cells, such as neurons, we also selected some new dyes for plasma membrane labeling. Using Icy SODA plugin, we could detect and analyze synaptic association in both conventional and single molecule localization microscopy, giving access to a molecular map at the nanoscale level. To replace those molecular distributions within the neuronal three-dimensional (3D) shape, we used MemBright probes and 3D STORM analysis to decipher the entire 3D shape of various dendritic spine types at the single-molecule resolution level. We report here the example of synaptic proteins within neuronal mask, but these tools have a broader spectrum of interest since they can be used whatever the proteins or the cellular type. Altogether with SODA plugin, MemBright probes thus provide the perfect toolkit to decipher a nanometric molecular map of proteins within a 3D cellular context.

Large-FOV 3D localization microscopy by spatially variant point spread function generation.

Accurate characterization of the microscopic point spread function (PSF) is crucial for achieving high-performance localization microscopy (LM). Traditionally, LM assumes a spatially invariant PSF to simplify the modeling of the imaging system. However, for large fields of view (FOV) imaging, it becomes important to account for the spatially variant nature of the PSF. Here, we propose an accurate and fast principal components analysis-based field-dependent 3D PSF generator (PPG3D) and localizer for LM. Through simulations and experimental three-dimensional (3D) single-molecule localization microscopy (SMLM), we demonstrate the effectiveness of PPG3D, enabling super-resolution imaging of mitochondria and microtubules with high fidelity over a large FOV. A comparison of PPG3D with a shift-variant PSF generator for 3D LM reveals a threefold improvement in accuracy. Moreover, PPG3D is approximately 100 times faster than existing PSF generators, when used in image plane-based interpolation mode. Given its user-friendliness, we believe that PPG3D holds great potential for widespread application in SMLM and other imaging modalities.

Near-Infrared Perylenecarboximide Fluorophores for Live-Cell Super-Resolution Imaging.

Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N3) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.

Comparison of the SMLM technique and the MSSR algorithm in confocal microscopy for super-resolved imaging of cellulose fibres.

Nowadays, the use of super-resolution microscopy (SRM) is increasing globally due to its potential application in several fields of life sciences. However, a detailed and comprehensive guide is necessary for understanding a single-frame image's resolution limit. This study was performed to provide information about the structural organisation of isolated cellulose fibres from garlic and agave wastes through fluorophore-based techniques and image analysis algorithms. Confocal microscopy provided overall information on the cellulose fibres' microstructure, while techniques such as total internal reflection fluorescence microscopy facilitated the study of the plant fibres' surface structures at a sub-micrometric scale. Furthermore, SIM and single-molecule localisation microscopy (SMLM) using the PALM reconstruction wizard can resolve the network of cellulose fibres at the nanometric level. In contrast, the mean shift super-resolution (MSSR) algorithm successfully determined nanometric structures from confocal microscopy images. Atomic force microscopy was used as a microscopy technique for measuring the size of the fibres. Similar fibre sizes to those evaluated with SIM and SMLM were found using the MSSR algorithm and AFM. However, the MSSR algorithm must be cautiously applied because the selection of thresholding parameters still depends on human visual perception. Therefore, this contribution provides a comparative study of SRM techniques and MSSR algorithm using cellulose fibres as reference material to evaluate the performance of a mathematical algorithm for image processing of bioimages at a nanometric scale. In addition, this work could act as a simple guide for improving the lateral resolution of single-frame fluorescence bioimages when SRM facilities are unavailable.

SEMORE: SEgmentation and MORphological fingErprinting by machine learning automates super-resolution data analysis

The morphology of protein assemblies impacts their behaviour and contributes to beneficial and aberrant cellular responses. While single-molecule localization microscopy provides the required spatial resolution to investigate these assemblies, the lack of universal robust analytical tools to extract and quantify underlying structures limits this powerful technique. Here we present SEMORE, a semi-automatic machine learning framework for universal, system- and input-dependent, analysis of super-resolution data. SEMORE implements a multi-layered density-based clustering module to dissect biological assemblies and a morphology fingerprinting module for quantification by multiple geometric and kinetics-based descriptors. We demonstrate SEMORE on simulations and diverse raw super-resolution data: time-resolved insulin aggregates, and published data of dSTORM imaging of nuclear pore complexes, fibroblast growth receptor 1, sptPALM of Syntaxin 1a and dynamic live-cell PALM of ryanodine receptors. SEMORE extracts and quantifies all protein assemblies, their temporal morphology evolution and provides quantitative insights, e.g. classification of heterogeneous insulin aggregation pathways and NPC geometry in minutes. SEMORE is a general analysis platform for super-resolution data, and being a time-aware framework can also support the rise of 4D super-resolution data.

Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

Bayesian optimized unbiased complete clustering for single molecule localization microscopy

Bayesian optimized unbiased complete clustering for single molecule localization microscopy

Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag

Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag

A probabilistic approach to detect intermolecular interactions in single-molecule localization microscopy

A probabilistic approach to detect intermolecular interactions in single-molecule localization microscopy

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

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

Revealing the 3D nanoscale organization of MyosinH in the apical complex of toxoplasma gondii through single-molecule localization microscopy with the double-helix point spread function

Revealing the 3D nanoscale organization of MyosinH in the apical complex of toxoplasma gondii through single-molecule localization microscopy with the double-helix point spread function

Investigating connections between caveolae and the membrane phase transition using single-molecule localization microscopy

Investigating connections between caveolae and the membrane phase transition using single-molecule localization microscopy

Local Water Content in Polymer Gels Measured with Super-Resolved Fluorescence Lifetime Imaging.

Water molecules play an important role in the structure, function, and dynamics of (bio-) materials. A direct access to the number of water molecules in nanoscopic volumes can thus give new molecular insights into materials and allow for fine-tuning their properties in sophisticated applications. The determination of the local water content has become possible by the finding that H2 O quenches the fluorescence of red-emitting dyes. Since deuterated water, D2 O, does not induce significant fluorescence quenching, fluorescence lifetime measurements performed in different H2 O/D2 O-ratios yield the local water concentration. We combined this effect with the recently developed fluorescence lifetime single molecule localization microscopy imaging (FL-SMLM) in order to nanoscopically determine the local water content in microgels, i.e. soft hydrogel particles consisting of a cross-linked polymer swollen in water. The change in water content of thermo-responsive microgels when changing from their swollen state at room temperature to a collapsed state at elevated temperature could be analyzed. A clear decrease in water content was found that was, to our surprise, rather uniform throughout the entire microgel volume. Only a slightly higher water content around the dye was found in the periphery with respect to the center of the swollen microgels.

Lokaler Wassergehalt in Polymergelen bestimmt über superauflösende Fluoreszenzlebensdauer‐Bildgebung

AbstractWassermoleküle spielen eine wichtige Rolle für die Struktur, Funktion und Dynamik von (Bio‐) Materialien. Ein direkter Zugang zur Anzahl der Wassermoleküle in nanoskopischen Volumina kann daher neue molekulare Einblicke in Materialien geben und eine Feinabstimmung ihrer Eigenschaften in anspruchsvollen Anwendungen ermöglichen. Die Bestimmung des lokalen Wassergehalts wurde durch die Entdeckung möglich, dass H2O die Fluoreszenz von rot‐emittierenden Farbstoffen löscht. Da deuteriertes Wasser, D2O, keine signifikante Fluoreszenzlöschung hervorruft, lässt sich über Fluoreszenzlebensdauermessungen bei unterschiedlichen H2O/D2O‐Verhältnissen die lokale Wasserkonzentration bestimmen. Wir kombinierten diesen Effekt mit der vor Kurzem entwickelten Fluoreszenzlebensdauer‐Einzelmolekül‐Lokalisierungsmikroskopie (FL‐SMLM), um die lokale Wasserkonzentration in Mikrogelen, d. h. weichen Hydrogelpartikeln, die aus einem in Wasser gequollenen vernetzten Polymer bestehen, nanoskopisch zu bestimmen. Die Änderung des Wassergehalts von thermoresponsiven Mikrogelen beim Übergang von ihrem gequollenen Zustand bei Raumtemperatur zu einem kollabierten Zustand bei erhöhter Temperatur konnte analysiert werden. Es wurde eine deutliche Abnahme des Wassergehalts festgestellt, die zu unserer Überraschung über das gesamte Mikrogelvolumen recht gleichmäßig war. In der Peripherie der gequollenen Mikrogele war die Wasserkonzentration um den Farbstoff herum nur geringfügig größer als in deren Zentrum.