Single-molecule Super-resolution Imaging Research Articles

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells.

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

Integrating microfluidics and a tilted single-objective light sheet for whole-cell multi-target 3D single-molecule super-resolution imaging

Integrating microfluidics and a tilted single-objective light sheet for whole-cell multi-target 3D single-molecule super-resolution imaging

One-Shot Dual-Detection-Based Single-Molecule Super-Resolution Imaging Method for Real-Time Observation of Spatiotemporal Catalytic Activity Variations on the Plasmonic Gold Nanoparticle Surface.

Understanding the relationship between the surface properties of a single plasmonic nanoparticle and its catalytic performance is critical for developing highly efficient nanocatalysts. In this study, a one-shot dual-detection-based single-molecule super-resolution imaging method in the evanescent field was developed to observe real-time spatiotemporal catalytic activity on a single plasmonic gold nanoparticle (AuNP) surface. The scattering intensity of AuNPs and the fluorescence of resorufin molecules produced on the AuNP surface were obtained simultaneously to investigate the relationship between nanoparticles and catalytic reactions at a single-molecule level. Chemisorbed adsorbates (i.e., catalytic product and resorufin) changed the electron density of individual AuNPs throughout the catalytic cycle, resulting in the fluctuation of the scattering intensity of individual AuNPs, which was attributed to the electron transfer between reactant resazurin molecules and AuNPs. The increase in the electron density of individual AuNPs affected the catalytic reaction rate. Furthermore, sequential mapping of individual catalytic events at the subdiffraction limit resolution was completed for real-time surface dynamics and spatiotemporal activity variations on the single AuNP surface. The developed method can aid in understanding surface-property-dependent catalytic kinetics and facilitate the development of nanoparticle-based heterogeneous catalysts at subdiffraction limit resolution.

Towards cloud-enabled fully automated end-to-end single molecule imaging.

Single Molecule Super-resolution imaging has revolutionised understanding biological processes on the sub-100 nm scale. Our group developed Fluorophore Localisation Imaging with Photobleaching (FLImP), a super-resolution imaging method that can achieve <4 nm resolution. In concert with molecular dynamics simulations, FLImP was used to reveal the in-situ atomic resolution structures of ligand-bound EGFR oligomers at the plasma membrane of cells (Needham et al., Nat Comms 2016), and those of ligand-unbound dimers and oligomers on cells (Zanetti-Domingues et al., Nat Comms 2018), shedding new light on the mechanisms of EGFR auto-inhibition, phosphorylation and signalling.

Investigating molecular mechanisms of key proteins underlying synapticplasticity using single-molecule tracking and super-resolution imaging.

Memory is encoded in networks formed by communicating neurons by the modification of synaptic contacts. In particular, dendritic spines are key structures in synaptic plasticity. These small protrusive structures can strengthen synaptic paths through morphological changes achieved by the remodeling of the actin cytoskeleton. One hypothesis regarding synaptic plasticity involves a particular actin-binding protein, the holoenzyme calcium/calmodulin-dependent kinase II (CaMKII) and its major function in actin network restructuring. Given the dodecameric structure of CaMKII, there is an intrinsic multivalency in the interaction modes of the holoenzyme that offers a unique potential for organizing the actin cytoskeleton. However, information about the binding kinetics, dynamics, and the nanoscale architecture of CaMKII-actin network organization at the single-molecule level is limited.In this work we characterize the binding kinetics and dynamics of individual fluorescently labeled CaMKII holoenzymes via single-molecule tracking (SMT) and provide nanoscale structural details of actin networks by implementing single-molecule super-resolution imaging (SRI) in a reconstituted CaMKII-actin assembly adhered to a coverslip. We employ a custom-built illumination scheme utilizing a flat-field total internal reflection fluorescence (TIRF) illumination approach. This method provides excellent contrast with even illumination across the sample, which effectively facilitates SMT for quantitative determination of binding kinetics and molecular dynamics. The flat-field illumination profile also improves the resolution over the entire field of view for SRI by homogenizing fluorophore blinking rates, since the fluorophore blinking kinetics is directly dependent on the effective illumination intensities. Our method closes an experimental gap present in the field and provides us with novel information of CaMKII-actin interactions at the nanoscale.

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.

Multi-target 3D single-molecule super resolution imaging throughout mammalian cells with a single-objective tilted light sheet.

Single-molecule based super-resolution fluorescence microscopy offers an approach to sub-diffraction-limited imaging of biological samples. While these approaches have proved significant to understanding subcellular structures, numerous super-resolution systems suffer from drawbacks such as decreased localization precision due to high background fluorescence, lack of control of the extracellular environment, decreased multi-target accuracy due to chromatic offsets in multicolor imaging, and the inability to image structures in three dimensions (3D). Light sheet fluorescence microscopy, which involves illuminating the sample with a thin sheet of light, offers a gentle illumination scheme that greatly reduces photobleaching, photodamage, and out of focus background fluorescence, thereby improving the localization precision. Here, we present a single-objective tilted light sheet super-resolution setup with five added innovations: (i) a microfluidic chip with reflective sidewalls and transparent top, (ii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iii) deep learning for high density single emitter localization, (iv) double helix point spread functions for 3D imaging, and (iv) galvanometric dithering of the illumination beam to reduce scattering artifacts and provide homogenous sample illumination. By utilizing light sheet illumination in conjunction with Exchange-PAINT and deep learning, this setup allows for the use of high concentrations of imager strands given that background fluorescence is drastically reduced and sparse emitters are no longer necessary for accurate localization. Furthermore, sequential washes of imager strands are enabled through the use of microfluidics, which allows for accurate, multi-target reconstructions that are not subject to chromatic offsets. Thus, we demonstrate a setup that offers improved localization precision, extracellular environment control, multi-target information, and 3D imaging that can easily be implemented for fast, accurate, and precise multi-target super-resolution whole cell imaging.

Three-dimensional single-molecule super-resolution imaging of proteins involved in primary cilia formation.

Primary cilia are organelles that protrude from the cell membrane of most human cells where they perform critical sensory and signaling functions. Defects and mutations in proteins in the primary cilium cause a range of diseases and syndromes collectively termed ciliopathies. Intracellular primary cilium formation begins with the recruitment of Golgi-derived pre-ciliary vesicles to the distal appendages of the mother centriole, but the molecular mechanisms behind the recruitment of the distal appendage proteins are not fully understood. The proteins Rab34 and MyosinVa were found through structured illumination microscopy (SIM) to be closely associated with these pre-ciliary vesicles during this step. However, because of the sub-diffraction size and three-dimensional (3D) structures of the centriole and of the involved proteins, higher resolution multi-target 3D microscopy is required to resolve their organization. To address this limitation, 3D two-color direct stochastic optical reconstruction microscopy (dSTORM) was implemented, providing a single-molecule localization precision of roughly 20 nm in all dimensions. A two-channel 4f emissions system was constructed using a 2-μm axial range double helix phase mask for single-molecule localization in 3D through point spread function engineering. This system allowed for 3D single-molecule super-resolution imaging of Rab34 and MyosinVa in two different color-channels together with imaging of the centriolar protein FOP in both color-channels for context and precise channel registration. The resulting 3D super-resolved reconstructions revealed the nanoscale structures formed by these proteins at the pre-ciliary vesicles, the structural relation between Rab34 and MyosinVa, and their position in relation to the mother centriole.

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.

Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.

Cell scientist to watch – Verena Ruprecht

ABSTRACT Verena Ruprecht received her PhD in Biophysics from the Johannes Kepler University in 2010 for her work on developing single-molecule super-resolution imaging tools in the lab of Gerhard Schütz. Following a research visit in Didier Marguet's lab at the Centre d'Immunologie de Marseille-Luminy (CIML) in France, she moved to the Institute of Science and Technology (IST) in Austria for a postdoc, working jointly with Carl-Philipp Heisenberg and Michael Sixt. There, she discovered a unique amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Verena started her independent laboratory at the Centre for Genomic Regulation (CRG) in Barcelona, Spain, in September 2016. Her group combines genetic and biophysical methods with multi-scale imaging and mathematical modelling to study cellular dynamics in embryo development. In 2020, Verena was selected as an EMBO Young Investigator and in the same year awarded an HFSP Young Investigator Grant for a collaborative project to study the biophysics of zebrafish fertilization.

Tracking the Coupling of Single Emitters to Plasmonic Nanoantennas with Single-Molecule Super-Resolution Imaging

Metal nanoantennas enable the manipulation of light emission and detection at the single photon level by confining light into very small volumes. Emitters coupled to these plasmonic structures are ...

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding.

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

Polarization-Encoded Colocalization Microscopy at Cryogenic Temperatures

Super-resolution localization microscopy is based on determining the positions of individual fluorescent markers in a sample. The major challenge in reaching an ever higher localization precision lies in the limited number of collected photons from single emitters. To tackle this issue, it has been shown that one can exploit the increased photostability at low temperatures, reaching localization precisions in the subnanometer range. Another crucial ingredient of single-molecule super-resolution imaging is the ability to activate an individual emitter within a diffraction-limited spot. Here, we report on the photoblinking behavior of organic dyes at low temperature and elaborate on the limitations of this ubiquitous phenomenon for selecting single molecules. We then show that recording the emission polarization not only provides access to the molecular orientation, but it also facilitates the assignment of photons to individual blinking molecules. Furthermore, we employ periodical modulation of the excitation polarization as a robust method to effectively switch fluorophores. We benchmark each approach by resolving two emitters on different DNA origami structures.

3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing.

That genome function may respond to its three-dimensional (3D) organization highlights the need for methods that can image genomes with superior coverage as well as greater genomic and optical resolution. Here, we push toward this goal by introducing OligoFISSEQ, a suite of three methods that leverage fluorescent in situ sequencing of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how only four rounds of sequencing are sufficient to produce 3D maps of 66 genomic targets across 6 chromosomes in hundreds to thousands of cells. We then use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.

Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry.

We report the design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit.

Addressing systematic errors in axial distance measurements in single-emitter localization microscopy.

Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.

Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left–right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein–protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein–protein interactions.

Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-point accumulation for imaging in nanoscale topography.

.In the brain, the strength of each individual synapse is defined by the complement of proteins present or the “local proteome.” Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline

Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.