whole-cell-super-resolution-imaging-via-dna-paint-on-spinning-disk

Whole-cell Super-Resolution Imaging <em>via</em> DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Single-molecule localization microscopy (SMLM) enables nanoscale imaging of cellular structures but is typically restricted to imaging near the coverslip ...

University College London

Quantifying the extent to which points are clustered in single-molecule localization microscopy data is vital to understanding the spatial relationships between molecules in the underlying sample. Many existing computational approaches are limited in their ability to process large-scale data sets, to deal effectively with sample heterogeneity, or require subjective user-defined analysis parameters. Here, we develop a supervised machine-learning approach to cluster analysis which is fast and accurate. Trained on a variety of simulated clustered data, the neural network can classify millions of points from a typical single-molecule localization microscopy data set, with the potential to include additional classifiers to describe different subtypes of clusters. The output can be further refined for the measurement of cluster area, shape, and point-density. We demonstrate this approach on simulated data and experimental data of the kinase Csk and the adaptor PAG in primary human T cell immunological synapses.

Machine learning for cluster analysis of localization microscopy data.

Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points. Unexpectedly, these clusters do not co-localize with PAG at the membrane but instead provide a ready pool of monomers to downregulate signaling. By generating CRISPR-Cas9 knockout T cells, our data also identify that a major risk factor for autoimmune diseases, the protein tyrosine phosphatase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.

Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

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 (P2Y), 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 P2Y 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 P2Y oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y mechanistic action and therapeutic targeting.

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

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Pancreatic ductal adenocarcinoma (PDAC) continues to show no improvement in survival rates. One aspect of PDAC is elevated ATP levels, pointing to the purinergic axis as a potential attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, this axis plays essential roles in fibrosis, inflammation response, and immune function. Analyzing the main members of the PDAC extracellular purinome using publicly available databases discerned which members may impact patient survival.  presents as the purinergic gene with the strongest association with hypoxia, the highest cancer cell-specific expression, and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification and pharmacological strategies, we demonstrate mechanistically that this ATP-driven invasion requires direct protein-protein interactions between P2Y and αV integrins. DNA-PAINT super-resolution fluorescence microscopy reveals that P2Y regulates the amount and distribution of integrin αV in the plasma membrane. Moreover, receptor-integrin interactions were required for effective downstream signaling, leading to cancer cell invasion. This work elucidates a novel GPCR-integrin interaction in cancer invasion, highlighting its potential for therapeutic targeting.

Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion.

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.

Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function.

Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein-protein association () and dissociation () constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.

Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy.

Single-Molecule Localization Microscopy (SMLM) has traditionally faced challenges to optimize signal-to-noise ratio, penetration depth, field-of-view (FOV), and spatial resolution simultaneously. Here, we show that DNA-PAINT imaging on a Spinning Disk Confocal with Optical Photon Reassignment (SDC-OPR) system overcomes these trade-offs, enabling high-resolution imaging across multiple cellular layers and large FOVs. We demonstrate the system's capability with DNA origami constructs and biological samples, including nuclear pore complexes, mitochondria, and microtubules, achieving a spatial resolution of 6 nm in the basal plane and sub-10 nm localization precision at depths of 9 µm within a 53 × 53 µm² FOV. Additionally, imaging of the developing Drosophila eye epithelium at depths up to 9 µm with sub-13 nm average localization precision, reveals distinct E-cadherin populations in adherens junctions. Quantitative analysis of Collagen IV deposition in this epithelium indicated an average of 46 ± 27 molecules per secretory vesicle. These results underscore the versatility of DNA-PAINT on an SDC-OPR for advancing super-resolution imaging in complex biological systems.

Super-resolution imaging in whole cells and tissues via DNA-PAINT on a spinning disk confocal with optical photon reassignment.

Understanding how viruses enter and fuse with host cells is crucial for developing effective antiviral therapies. The process of viral entry and fusion involves a series of complex steps that allow the virus to breach the host cell membrane and deliver its genetic material inside, with viral fusogens often co-operating to attain the required energy for successful membrane fusion. This co-operative clustering of fusogens in viral envelopes is similar to receptor clustering in cellular systems, where receptors aggregate to initiate signalling cascades. Single-molecule localisation microscopy (SMLM) approaches have emerged as powerful tools to study these intricate mechanisms, allowing the observation of proteins with unprecedented levels of detail. These technologies provide unparalleled insights into the dynamics of viral entry and fusion at a molecular level, revealing how the co-ordinated action of fusogens facilitates membrane fusion. By employing the newest advances in SMLM techniques, such as DNA-PAINT and MINFLUX, we anticipate that precise information on the key steps of viral fusion can be revealed with high spatial and temporal resolutions, identifying critical points in the process that can be targeted by antiviral strategies.

Article	Year
Machine learning for cluster analysis of localization microscopy data. Nature communications\| PubMed ID: 32198352	2020
Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation. Cell reports\| PubMed ID: 33357425	2020
Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules. Nature communications\| PubMed ID: 33483489	2021
Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization. Biomolecules\| PubMed ID: 34680136	2021
Determination of association equilibrium constant from single molecule fluorescence localization microscopy. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology\| PubMed ID: 35749053	2022
Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion. eLife\| PubMed ID: 36942939	2023
Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function. Open biology\| PubMed ID: 37161290	2023
Quantitative Analysis of Protein-Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy. Nano letters\| PubMed ID: 39432814	2024
Super-resolution imaging in whole cells and tissues via DNA-PAINT on a spinning disk confocal with optical photon reassignment. Nature communications\| PubMed ID: 40442066	2025
Single-molecule localisation microscopy approaches reveal envelope glycoprotein clusters in single-enveloped viruses: a potential functional role? Biochemical Society transactions\| PubMed ID: 40495469	2025

Sabrina Simoncelli

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