Single-molecule Localization-based Super-resolution Microscopy Research Articles

Precise measurement of nanoscopic septin ring structures with deep learning-assisted quantitative superresolution microscopy.

The combination of image analysis and superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning (DL)-based object recognition enable the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, DL-based image recognition may introduce bias. This prohibits the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. Here we address this problem by using changes of septin ring structures in single molecule localization-based superresolution microscopy data as a paradigm. We identify potential sources of bias resulting from different training approaches by rigorous testing of trained models using real or simulated data covering a wide range of possible results. In a quantitative comparison of our models, we find that a trade-off exists between measurement accuracy and the range of recognized phenotypes. Using our thus verified models, we find that septin ring size can be explained by the number of subunits they are assembled from alone. Furthermore, we provide a new experimental system for the investigation of septin polymerization.

Probing Chromatin Compaction and Its Epigenetic States in situ With Single-Molecule Localization-Based Super-Resolution Microscopy.

Chromatin organization play a vital role in gene regulation and genome maintenance in normal biological processes and in response to environmental insults. Disruption of chromatin organization imposes a significant effect on many cellular processes and is often associated with a range of pathological processes such as aging and cancer. Extensive attention has been attracted to understand the structural and functional studies of chromatin architecture. Biochemical assays coupled with the state-of-the-art genomic technologies have been traditionally used to probe chromatin architecture. Recent advances in single molecule localization microscopy (SMLM) open up new opportunities to directly visualize higher-order chromatin architecture, its compaction status and its functional states at nanometer resolution in the intact cells or tissue. In this review, we will first discuss the recent technical advantages and challenges of using SMLM to image chromatin architecture. Next, we will focus on the recent applications of SMLM for structural and functional studies to probe chromatin architecture in key cellular processes. Finally, we will provide our perspectives on the recent development and potential applications of super-resolution imaging of chromatin architecture in improving our understanding in diseases.

Accelerating 3D single-molecule localization microscopy using blind sparse inpainting.

.Significance: Single-molecule localization-based super-resolution microscopy has enabled the imaging of microscopic objects beyond the diffraction limit. However, this technique is limited by the requirements of imaging an extremely large number of frames of biological samples to generate a super-resolution image, thus requiring a longer acquisition time. Additionally, the processing of such a large image sequence leads to longer data processing time. Therefore, accelerating image acquisition and processing in single-molecule localization microscopy (SMLM) has been of perennial interest.Aim: To accelerate three-dimensional (3D) SMLM imaging by leveraging a computational approach without compromising the resolution.Approach: We used blind sparse inpainting to reconstruct high-density 3D images from low-density ones. The low-density images are generated using much fewer frames than usually needed, thus requiring a shorter acquisition and processing time. Therefore, our technique will accelerate 3D SMLM without changing the existing standard SMLM hardware system and labeling protocol.Results: The performance of the blind sparse inpainting was evaluated on both simulation and experimental datasets. Superior reconstruction results of 3D SMLM images using up to 10-fold fewer frames in simulation and up to 50-fold fewer frames in experimental data were achieved.Conclusions: We demonstrate the feasibility of fast 3D SMLM imaging leveraging a computational approach to reduce the number of acquired frames. We anticipate our technique will enable future real-time live-cell 3D imaging to investigate complex nanoscopic biological structures and their functions.

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron-alkylated BODIPY probe for localization-based super-resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron-photodealkylation reaction with a single low-power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization-based microscopy of biological systems. The probe can be easily linked to nucleophile-containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super-resolution imaging of microtubules is demonstrated. This is the first example of single-molecule localization-based super-resolution microscopy using a visible-light-activated BODIPY compound as a fluorescent probe.

Improved split fluorescent proteins for endogenous protein labeling

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell–cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow–green split-mNeonGreen21–10/11 that improves the ratio of complemented signal to the background of FP1–10-expressing cells compared to the commonly used split GFP1–10/11; as well as a 10-fold brighter red-colored split-sfCherry21–10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.

Photostable and photoswitching fluorescent dyes for super-resolution imaging.

Super-resolution fluorescence microscopy is a recently developed imaging tool for biological researches. Several methods have been developed for detection of fluorescence signals from molecules in a subdiffraction-limited area, breaking the diffraction limit of the conventional optical microscopies and allowing visualization of detailed macromolecular structures in cells. As objectives are exposed to intense laser in the optical systems, fluorophores for super-resolution microscopy must be tolerated even under severe light irradiation conditions. The fluorophores must also be photoactivatable and photoswitchable for single-molecule localization-based super-resolution microscopy, because the number of active fluorophores must be controlled by light irradiation. This has led to growing interest in these properties in the development of fluorophores. In this mini-review, we focus on the development of photostable and photoswitching fluorescent dyes for super-resolution microscopy. We introduce recent efforts, including improvement of fluorophore photostability and control of photoswitching behaviors of fluorophores based on photochemical and photophysical processes. Understanding and manipulation of chemical reactions in excited fluorophores can develop highly photostable and efficiently photoswitchable fluorophores that are suitable for super-resolution imaging applications.

Single-molecule analysis of fluorescent carbon dots towards localization-based super-resolution microscopy

The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature. In this study, we have systematically characterized the single-molecule fluorescence of three carbon dots and compared them with a standard fluorescent probe. Each of these carbon dots showed a long-lived dark state in the presence of an electron acceptor. The electron transfer mechanism was investigated in single-molecule as well as in ensemble experiments. The average on–off rate between the fluorescent bright and dark states, which is one of the important parameters for single-molecule localization-based super-resolution microscopy, was measured by changing the laser power. We report that the photon budget and on–off rate of these carbon dots were good enough to achieve single-molecule localization with a precision of ~35 nm.

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

Bayesian cluster identification in single-molecule localization microscopy data.

Single-molecule localization-based super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. Although many algorithms exist for the identification and localization of molecules from raw image data, methods for analyzing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here we present a model-based Bayesian approach to evaluate molecular cluster assignment proposals, generated in this study by analysis based on Ripley's K function. The method takes full account of the individual localization precisions calculated for each emitter. We validate the approach using simulated data, as well as experimental data on the clustering behavior of CD3ζ, a subunit of the CD3 T cell receptor complex, in resting and activated primary human T cells.

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain.

The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.

Spectral demixing avoids registration errors and reduces noise in multicolor localization-based super-resolution microscopy

Multicolor single molecule localization-based super-resolution microscopy (SMLM) approaches are challenged by channel crosstalk and errors in multi-channel registration. We recently introduced a spectral demixing-based variant of direct stochastic optical reconstruction microscopy (SD-dSTORM) to perform multicolor SMLM with minimal color crosstalk. Here, we demonstrate that the spectral demixing procedure is inherently free of errors in multicolor registration and therefore does not require multicolor channel alignment. Furthermore, spectral demixing significantly reduces single molecule noise and is applicable to astigmatism-based 3D multicolor imaging achieving 25 nm lateral and 66 nm axial resolution on cellular nanostructures.

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast.

Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2-4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.

Direct stochastic optical reconstruction microscopy (dSTORM).

Single-molecule localization-based super-resolution microscopy can be performed with regular, bright, and photostable organic fluorophores. We review a concept termed direct stochastic optical reconstruction microscopy (dSTORM), which operates conventional fluorophores as photoswitches and provides an optical resolution of ~20 nm. We introduce the principle of dSTORM, illustrate experimental schemes, and discuss approaches for data analysis.

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C–C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

Photoactivation and Saturated Emission in Blended Conjugated Polymer Nanoparticles

Blended poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT)/poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) conjugated polymer nanoparticles were prepared and characterized by conventional and single-particle fluorescence spectroscopy. The particles exhibit red emission and improved quantum efficiency resulting from highly efficient energy transfer from donor PFBT to acceptor MEH-PPV as well as suppression of MEH-PPV aggregation. Photobleaching results indicate better photostability in the blended sample compared to undoped MEH-PPV nanoparticles and photoactivation of donor emission, which could be useful for single-molecule localization-based super-resolution microscopy. Single blended nanoparticles exhibit bright fluorescence as well as saturation behavior at very low excitation intensities. These and other properties of the blended conjugated polymer nanoparticles could provide substantial improvements in resolution when employed in super-resolution microscopy.

Novel Labeling Schemes for Single-Molecule Localization Microscopy

Single molecule localization-based superresolution microscopy methods, such as PALM or STORM, have been breakthrough techniques of the last years. Until now however, they require special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific labeling. On the other hand, many laboratories will have most of their constructs in GFP form and entire genomes are available as functional GFP-fusion proteins.Here, we report a method that makes all these constructs available for superresolution microscopy by targeting GFP with tiny, high-affinity antibodies coupled to blinking dyes. It thus combines the molecular specificity of genetic tagging with the high photon yield of organic dyes and minimal linkage error, as demonstrated on microtubules, living neurons and yeast cells. We show that in combination with GFP-libraries, virtually any known protein can immediately be used in superresolution microscopy and that high-throughput superresolution imaging using simplified labeling schemes is possible.The labeling density in superresolution microscopy based on photoactivatable fluorophores is limited by the fact that a small, but significant fraction is always in the bright state. To overcome this limitation we implemented binding-activated localization microscopy (BALM), which is based on the localization of individual binding events of fluorophores that show a fluorescence enhancement upon binding to their target structures. Using nucleic acid stains on double-stranded DNA we yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm in vitro and could visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy can be extended to other dyes and targets such as protein structures.

Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.

A Simple, Versatile Method for GFP-Based Single Molecule Localization Microscopy

Single-molecule localization-based superresolution microscopy methods such as PALM or STORM, have been breakthrough techniques of the last years. Until now however, they require special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific labeling. On the other hand, many laboratories will have most of their constructs in GFP form and entire genomes are available as functional GFP-fusion proteins.Here, we report a method that makes all these constructs available for superresolution microscopy by targeting GFP with tiny, high-affinity antibodies coupled to blinking dyes. It thus combines the molecular specificity of genetic tagging with the high photon yield of organic dyes and minimal linkage error.Direct STORM on microtubules labeled with our novel antibodies showed that indeed the linkage error was minimal, whereas the large size of standard antibodies resulted in an additional error of >10 nm in immunolabeling. The high brightness of our labels enabled us to perform rapid time-lapse dSTORM and sptPALM on living neurons expressing the outer membrane protein GPI-GFP. Three-dimensional dSTORM on microtubules using the bi-plane approach allowed us to distinguish overlapping microtubules with an axial separation of ∼100 nm. Using a budding yeast GFP-tag genomic library we could readily image several GFP-tagged proteins targeted to specific intracellular locations.In summary, targeting of GFP-labeled constructs with tiny antibodies provides fast and simple access to superresolution microscopy of virtually any known protein in cells. Since for several organisms the entire genome is available as GFP-tagged constructs, all these proteins are immediately accessible without the requirement for cloning or the generation of antibodies. Finally, due to a simple one-step labeling protocol, our technique opens the door to high-throughput localization analysis of entire genomes at the nanoscopic level in cells.