Single-molecule Super-resolution Microscopy Research Articles

Revealing operando surface defect–dependent electrocatalytic performance of Pt at the subparticle level

Understanding the operando defect-tuning performance of catalysts is critical to establish an accurate structure-activity relationship of a catalyst. Here, with the tool of single-molecule super-resolution fluorescence microscopy, by imaging intermediate CO formation/oxidation during the methanol oxidation reaction process on individual defective Pt nanotubes, we reveal that the fresh Pt ends with more defects are more active and anti-CO poisoning than fresh center areas with less defects, while such difference could be reversed after catalysis-induced step-by-step creation of more defects on the Pt surface. Further experimental results reveal an operando volcano relationship between the catalytic performance (activity and anti-CO ability) and the fine-tuned defect density. Systematic DFT calculations indicate that such an operando volcano relationship could be attributed to the defect-dependent transition state free energy and the accelerated surface reconstructing of defects or Pt-atom moving driven by the adsorption of the CO intermediate. These insights deepen our understanding to the operando defect-driven catalysis at single-molecule and subparticle level, which is able to help the design of highly efficient defect-based catalysts.

Visualizing DNA damage and repair using single molecule super resolution microscopy.

Single molecule super resolution microscopy overcomes the diffraction limit by separating individual fluorophore emissions over time, resulting in spatial resolutions that are far superior to epifluorescence microscopy. This allows for DNA damage response (DDR) events to be investigated in greater detail. A variety of DNA damaging drugs can be used on S-phase synchronized immortalized cell lines alongside 5-ethynyl-2'-deoxyuridine (EdU) pulse labelling to ultimately visualize DNA repair pathways at distinct time points and quantify colocalizations between nascent DNA and immunolabeled DDR proteins. This chapter will outline super resolution microscopy assays to interrogate the spatiotemporal organization of DNA repair proteins at damaged foci during DDR events within immortalized cell lines.

Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles

Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.

Polarization microscopy: from ensemble structural imaging to single-molecule 3D orientation and localization microscopy

Optical contrasts in microscopy are sensitive to light polarization, whose interaction with molecular dipoles provides an important lever for probing molecular orientation. Polarization microscopy has evolved considerably during the last decade, integrating strategies ranging from traditional linear dichroism to single-molecule orientation and localization imaging. This review aims to provide a summary of concepts and techniques behind orientation and structural imaging at the molecular level, from ensemble microscopy in 2D to single-molecule super-resolution microscopy in 3D.

Far-Field Polarization Optics Control the Nanometer-Scale Pattern of High-Fluorescence Dissymmetry Emission from Achiral Molecules near Plasmonic Nanodimers

Chiroptical spectroscopies like circular dichroism report on the handedness of molecules but are often limited to the high-concentration regime because of the mismatch between the wavelength of propagating visible light and the size of molecules. Recent work has demonstrated that the electromagnetic fields generated near chiral plasmonic nanostructures can have high optical chirality that bridges these length scales and can induce high-fluorescence dissymmetry signals from achiral dyes by redirecting their emission. As a step toward single-molecule chiral sources, the induced fluorescence dissymmetry and apparent emission pattern near chiral plasmonic nanoparticles must be quantified. Here, we measure the induced fluorescence dissymmetry from the achiral dye Cy5.5 near a chiral gold nanodimer structure, and we map out the apparent emission pattern of the dyes using super-resolution single-molecule microscopy. Our high-sensitivity approach quantifies the fluorescence dissymmetry from sampling only ∼100 zeptomoles of Cy5.5 and measures from Cy5.5 near gold nanodimers median fluorescence dissymmetry factors that are 2 orders of magnitude greater than those of synthesized chiral fluorescent molecules. We observe a spatial correlation between the simulated optical chirality about a plasmonic gold nanodimer and experimentally obtained super-resolution apparent emission patterns, and we use the incident polarization to control our detection bias for the brightest molecules to sample nanometer-scale regions of high electric flux.

Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms.

Single-molecule superresolution microscopy is a powerful tool for the study of biological structures on size scales smaller than the optical diffraction limit. Imaging samples at cryogenic temperatures (77 K) reduces the quantum yield of photobleaching for many fluorescent labels, yielding localization precisions below 10 nm. Cryogenic imaging further enables correlation with cryogenic electron tomography. A key limitation in applying methods such as PALM and STORM to samples maintained at 77 K is the limited number of fluorophores known to undergo efficient turn-on and turn-off mechanisms necessary to control the sparsity of active emitters. We find that mApple, a red-emitting fluorescent protein, undergoes a novel turn-off mechanism in response to simultaneous illumination with two colors of light. This turn-off mechanism enables localization of many individual molecules in initially bright samples, but the final density of localizable emitters is limited by relatively inefficient turn-on (photoactivation). Bulk excitation and emission spectroscopy shows that mApple has access to two distinct emissive states as well as dark states accessible optically or through changes in pH. The bright and stable emission of mApple enables widefield collection of single-molecule emission spectra, which highlight the complex nature and environmental sensitivity of states observed in red fluorescent proteins.

The role of O-GlcNAcylation in mRNA nuclear export studied by single-molecule super-resolution microscopy.

Characterizing the relationship between the nuclear export of mRNA and the O-GlcNAcylation of nuclear pore proteins is a critical process for understanding the dynamic effects of post-translational modifications on membrane protein structures. Specifically, it was reported that the concentration of O-GlcNAcylated nucleoporins (Nups) in the nuclear pore complexes (NPCs) can be significantly altered by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitor drugs. Combining two single-molecule super-resolution microscopy methods, STORM and SPEED microscopy, we found that the amount of messenger ribonucleoprotein particles (mRNPs) that successfully export through the NPC is approximately doubled when increasing the O-GlcNAcylation in the NPC. Reversely, five-fold less mRNPs can exit the nucleus to reach the cytoplasm with the decreased O-GlcNAcylation of Nups. Moreover, a virtual 3D imaging by SPEED microscopy revealed that the spatial distributions of these altered O-GlcNAcylated Nups colocalized well with the locations of Nups that are rich in phenylalanine-glycine (FG) repeats, suggesting the passage of mRNPs through the selective permeability barrier in the NPC is regulated by the amount of O-GlcNAcylated FG-Nups.

Dithered single-objective light sheet for improved super-resolution imaging throughout mammalian cells.

Single-molecule super-resolution fluorescence microscopy is a powerful method for imaging detailed biological structures at the nanoscale. However, imaging single molecules precisely in thick samples such as mammalian cells is notoriously difficult due to increased fluorescence background. Light sheet fluorescence microscopy in which the sample is illuminated with a thin plane of light mitigates the problem of increased fluorescence background in thick samples and results in increased signal-to-background ratio and improved localization precision. However, light sheet illumination is sensitive to shadowing artifacts from imperfections in the optical path and scattering throughout the sample. Additionally, most light sheet systems employ two objectives, which may suffer from steric hindrance and drift between the sample and illumination objective. To overcome these disadvantages, we have developed a single-objective tilted light sheet illumination system which employs dithering to reduce stripe artifacts and achieve homogeneous illumination of the sample. Our light sheet has been designed with a width, thickness, and confocal parameter specifically suited for cellular imaging. We further improve the imaging capabilities of our system with a microfluidic device enabling Exchange-PAINT for multi-target imaging without chromatic offsets, using engineered point spread functions for 3D imaging, and employing deep learning algorithms for high-density localization of single molecules. Our approach achieves improved localization precision, imaging speeds, and multi-target accuracy, enabling fast and precise multi-target 3D single-molecule super-resolution cellular imaging. Here, we focus on the design and construction of our single-objective dithered light sheet and its range of applications from nuclear protein investigation to whole cell imaging. We think the improved cellular imaging resulting from this system will impact our understanding of the relationship between organization and function of subcellular structures and help us and others answer relevant biomedical questions involved in disease pathogenesis.

Construction and characterization of a versatile multimodal single-molecule super-resolution microscope for 3D whole cell studies.

The cellular environment is dense and complex, and imaging with 3D single-molecule super-resolution microscopy requires careful consideration of appropriate illumination methods to use in relation to the goals of the study. There is, however, typically a tradeoff between performance and simplicity when selecting illumination schemes. Epi-illumination is a common and simple technique, but it generates high unwanted background fluorescence and causes photodamage and photobleaching throughout the imaged cell. Total internal reflection fluorescence (TIRF) is another technique which provides excellent contrast, but it is limited to imaging a thin volume at the bottom of a sample and is thus incompatible with whole-cell imaging. Light sheet illumination is an alternative, whole-cell compatible approach where a thin sheet of light is directed orthogonally to the detection axis to optically section a cell and reduce the fluorescence background, photobleaching, and photodamage. In this work we demonstrate a versatile microscopy platform which integrates the sectioning and background reducing capability of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. A flat-field intensity profile improves the control of fluorophore blinking kinetics and the resolution across the entire field of view when compared to a standard Gaussian intensity profile. Additionally, this microscope is designed to allow very fast switching between illumination modalities and laser wavelengths, using primarily commercially available parts. When combined with point spread function engineering, our system conveniently enables 3D single-molecule super-resolution imaging and molecular counting throughout whole cells with optimized and adjustable illumination modalities.

Super-resolution imaging of antibody-conjugated biodegradable periodic mesoporous organosilica nanoparticles for targeted chemotherapy of prostate cancer.

Biodegradable periodic mesoporous organosilica nanoparticles (nanoPMOs) are widely used as responsive drug delivery platforms for targeted chemotherapy of cancer. However, the evaluation of their properties such as surface functionality and biodegradability is still challenging, which has a significant impact on the efficiency of chemotherapy. In this study, we have applied direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule super-resolution microscopy technique, to quantify the degradation of nanoPMOs triggered by glutathione and the multivalency of antibody-conjugated nanoPMOs. Subsequently, the effect of these properties on cancer cell targeting, drug loading and release capability, and anticancer activity is also studied. Due to the higher spatial resolution at the nanoscale, dSTORM imaging is able to reveal the structural properties (i.e., size and shape) of fluorescent and biodegradable nanoPMOs. The quantification of nanoPMOs' biodegradation using dSTORM imaging demonstrates their excellent structure-dependent degradation behavior at a higher glutathione concentration. The surface functionality of anti-M6PR antibody-conjugated nanoPMOs as quantified by dSTORM imaging plays a key role in prostate cancer cell labeling: oriented antibody is more effective than random ones, while high multivalency is also effective. The higher biodegradability and cancer cell-targeting properties of nanorods conjugated with oriented antibody (EAB4H) effectively deliver the anticancer drug doxorubicin to cancer cells, exhibiting potent anticancer effects.

Selective Degradation and Quantification of Nucleoporins in the Nuclear Pore by Auxin-Inducible Degrons and Single-Molecule Microscopy.

There is a significant current question regarding the viable copy numbers of nucleoporins required for the function of the nuclear pore complex (NPC) in eukaryotic cells. The NPC consists of approximately 30 different nucleoporins in an eight-fold symmetry, meaning that there are multiple duplicates of each nucleoporin present within the nuclear pore. We recently developed a method that combines auxin-inducible degrons and single-molecule super-resolution microscopy to evaluate the copy number of nuclear basket nucleoporins required for the successful function of the NPC. Here, we describe the theory behind this auxin-inducible degron and single-molecule super-resolution microscopy method, and we detail a step-by-step process to selectively degrade nucleoporins either completely or in a stepwise manner. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Degradation of target nucleoporins Basic Protocol 2: Quantification of nucleoporin copy number via narrow-field fluorescence microscopy.

Fast DNA-PAINT imaging using a deep neural network

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we train the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-colour super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule imaging modality to enable fast single-molecule super-resolution microscopy.

Unraveling docking and initiation of mRNA export through the nuclear pore complex.

The nuclear export of mRNA through the nuclear pore complex (NPC) is a process required for the healthy functioning of human cells, making it a critical area of research. However, the geometries of mRNA and the NPC are well below the diffraction limit of light microscopy, thereby presenting significant challenges in evaluating the discrete interactions and dynamics involved in mRNA nuclear export through the native NPC. Recent advances in biotechnology and single-molecule super-resolution light microscopy have enabled researchers to gain granular insight into the specific contributions made by discrete nucleoporins in the nuclear basket of the NPC to the export of mRNA. Specifically, by expanding upon the docking step facilitated by the protein TPR in the nuclear basket as well as identifying NUP153 as being the primary nuclear basket protein initiating export through the central channel of the NPC.

The effect of stress on biophysical characteristics of misfolded protein aggregates in living Saccharomyces cerevisiae cells.

Aggregation of misfolded or damaged proteins is often attributed to numerous metabolic and neurodegenerative disorders. To reveal underlying mechanisms and cellular responses, it is crucial to investigate protein aggregate dynamics in cells. Here, we used super-resolution single-molecule microscopy to obtain biophysical characteristics of individual aggregates of a model misfolded protein ∆ssCPY* labelled with GFP. We demonstrated that oxidative and hyperosmotic stress lead to increased aggregate stoichiometries but not necessarily the total number of aggregates. Moreover, our data suggest the importance of the thioredoxin peroxidase Tsa1 for the controlled sequestering and clearance of aggregates upon both conditions. Our work provides novel insights into the understanding of the cellular response to stress via revealing the dynamical properties of stress-induced protein aggregates.

Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses.

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

Fourier Ring Correlation and Anisotropic Kernel Density Estimation Improve Deep Learning Based SMLM Reconstruction of Microtubules.

Single-molecule super-resolution microscopy (SMLM) techniques like dSTORM can reveal biological structures down to the nanometer scale. The achievable resolution is not only defined by the localization precision of individual fluorescent molecules, but also by their density, which becomes a limiting factor e.g., in expansion microscopy. Artificial deep neural networks can learn to reconstruct dense super-resolved structures such as microtubules from a sparse, noisy set of data points. This approach requires a robust method to assess the quality of a predicted density image and to quantitatively compare it to a ground truth image. Such a quality measure needs to be differentiable to be applied as loss function in deep learning. We developed a new trainable quality measure based on Fourier Ring Correlation (FRC) and used it to train deep neural networks to map a small number of sampling points to an underlying density. Smooth ground truth images of microtubules were generated from localization coordinates using an anisotropic Gaussian kernel density estimator. We show that the FRC criterion ideally complements the existing state-of-the-art multiscale structural similarity index, since both are interpretable and there is no trade-off between them during optimization. The TensorFlow implementation of our FRC metric can easily be integrated into existing deep learning workflows.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy.

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the “nano–bio interface”). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano–bio interface with 10–20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R (the reciprocal of the radius) membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano–bio interface using 3D SR microscopy.

Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution.

We demonstrate an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently gate images at 39 MHz, allowing fluorescence lifetime to be captured on standard camera sensors. Lifetime imaging of single molecules is enabled in wide field with exposure times of less than 100 ms. This capability allows combination of wide-field FLIM with single-molecule super-resolution localization microscopy. Fast single-molecule dynamics such as FRET and molecular binding events are captured from wide-field images without prior spatial knowledge. A lifetime sensitivity of 1.9 times the photon shot-noise limit is achieved, and high throughput is shown by acquiring wide-field FLIM images with millisecond exposure and >108 photons per frame. Resonant electro-optic FLIM allows lifetime contrast in any wide-field microscopy method.

Abstract 1032: Super-resolution imaging studies of zanidatamab: Providing insights into its bispecific mode of action

Abstract Zanidatamab (ZW25) is a biparatopic antibody that simultaneously binds two distinct epitopes of the oncogenic cell surface receptor HER2. Zanidatamab is currently being evaluated in global Phase 1, Phase 2 and registration-enabling clinical trials as a potential new treatment for patients with HER2-expressing cancers, including biliary tract, gastroesophageal adenocarcinomas, breast, and other tumor types. The extracellular domain of the HER2 receptor comprises four domains with zanidatamab binding to epitopes on domain 2 and the membrane proximal domain 4. Domain 2 and domain 4 of HER2 are also respectively targeted by the antibodies pertuzumab and trastuzumab, currently approved for clinical use in a subset of HER2 overexpressing cancers. The unique engineering and HER2-engagement of zanidatamab results in multiple mechanisms of action, including dual HER2 signal blockade, increased antibody binding, receptor clustering, and removal of HER2 from the cell surface, and potent effector function. Preclinical work shows that zanidatamab can exhibit differentiated activity over trastuzumab or combination of trastuzumab and pertuzumab but the precise molecular mechanism by which zanidatamab differentiates itself from these approved agents remains unclear. Structural modeling of zanidatamab in complex with HER2 suggests that unlike trastuzumab and pertuzumab, steric features of zanidatamab induce a complex of this antibody with HER2 comprised of alternating chains of the antibody and HER2 molecules, with each copy of zanidatamab bridging two HER2 molecules and vice-versa. In this study we report on the spatial distribution of cell surface HER2 molecules and the effect of antibody-induced receptor reorganization on prototypical cancer cells with high and low expression levels of this receptor. We employ super-resolution single-molecule microscopy to map the receptor distribution below the diffraction limit of traditional optical imaging techniques. The use of Direct Stochastic Optical Reconstruction Microscopy (dSTORM) allows us to quantitatively differentiate properties such as cluster size, frequency and receptor density induced by the antibodies. We demonstrate that the unique geometry of HER2 engagement achieved by zanidatamab results in the induction of strikingly aggregated HER2 receptor cluster we refer to as “capping” on the cell surface. This effect induced by zanidatamab is notably distinct relative to the receptor reorganization observed with trastuzumab or combination of trastuzumab and pertuzumab which typically induce multiple smaller microclusters on the cell surface. We observe differences in the lifetime and persistence of these capped configurations induced by zanidatamab, leading us to believe that these cell surface reorganization events are likely coupled to events such as internalization and receptor down regulation. Citation Format: Surjit Dixit, Libin Abraham, Nina Weiser, Michael R. Gold. Super-resolution imaging studies of zanidatamab: Providing insights into its bispecific mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1032.