Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Building a super-resolution fluorescence cryomicroscope.

Correlated super-resolution fluorescence microscopy and cryo-electron microscopy enables imaging with both high labeling specificity and high resolution. Naturally, combining two sophisticated imaging techniques within one workflow also introduces new requirements on hardware, such as the need for a super-resolution fluorescence capable microscope that can be used to image cryogenic samples. In this chapter, we describe the design and use of the "cryoscope"; a microscope designed for single-molecule localization microscopy (SMLM) of cryoEM samples that fits right into established cryoEM workflows. We demonstrate the results that can be achieved with our microscope by imaging fluorescently labeled vimentin, an intermediate filament, within U2OS cells grown on EM grids, and we provide detailed 3d models that encompass the entire design of the microscope.

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy.

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

Single-Cell Single-Molecule RNA-FISH Combined with Immunofluorescence and High-Speed and High-Resolution Scanning Analysis to Visualize the Reactivation of Latent HIV-1.

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.

Single molecule delivery into living cells.

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

Label-free detection and profiling of individual solution-phase molecules.

Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques1 are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method2-16 capable of revealing details of molecular conformation in solution17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light-molecule interactions in high-finesse fibre-based Fabry-Pérot microcavities19-21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics22,23 and Pound-Drever-Hall (PDH) cavity locking24 to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.

Single-particle Cryo-EM and molecular dynamics simulations: A perfect match.

Knowledge of the structure and dynamics of biomolecules is key to understanding the mechanisms underlying their biological functions. Single-particle cryo-electron microscopy (cryo-EM) is a powerful structural biology technique to characterize complex biomolecular systems. Here, we review recent advances of how Molecular Dynamics (MD) simulations are being used to increase and enhance the information extracted from cryo-EM experiments. We will particularly focus on the physics underlying these experiments, how MD facilitates structure refinement, in particular for heterogeneous and non-isotropic resolution, and how thermodynamic and kinetic information can be extracted from cryo-EM data.

From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

Trapping and recapturing single DNA molecules with pore-cavity-pore device.

Single-molecule detection technology is a technique capable of detecting molecules at the single-molecule level, characterized by high sensitivity, high resolution, and high specificity. Nanopore technology, as one of the single-molecule detection tools, is widely used to study the structure and function of biomolecules. In this study, we constructed a small-sized nanopore with a pore-cavity-pore structure, which can achieve a higher reverse capture rate. Through simulation, we investigated the electrical potential distribution of the nanopore with a pore-cavity-pore structure and analyzed the influence of pore size on the potential distribution. Accordingly, different pore sizes can be designed based on the radius of gyration of the target biomolecules, restricting their escape paths inside the chamber. In the future, nanopores with a pore-cavity-pore structure based on two-dimensional thin film materials are expected to be applied in single-molecule detection research, which provides new insights for various detection needs.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

Single-Molecule Fluorescence Probes Interactions between Photoactive Protein-Silver Nanowire Conjugate and Monolayer Graphene.

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.

Single-Molecule FRET Analyses of NMDA Receptors.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Accurate single-molecule spot detection for image-based spatial transcriptomics with weakly supervised deep learning.

Image-based spatial transcriptomics methods enable transcriptome-scale gene expression measurements with spatial information but require complex, manually tuned analysis pipelines. We present Polaris, an analysis pipeline for image-based spatial transcriptomics that combines deep-learning models for cell segmentation and spot detection with a probabilistic gene decoder to quantify single-cell gene expression accurately. Polaris offers a unifying, turnkey solution for analyzing spatial transcriptomics data from multiplexed error-robust FISH (MERFISH), sequential fluorescence in situ hybridization (seqFISH), or in situ RNA sequencing (ISS) experiments. Polaris is available through the DeepCell software library (https://github.com/vanvalenlab/deepcell-spots) and https://www.deepcell.org.

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

Deep learning-based spectroscopic single-molecule localization microscopy.

Significance: Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale. Aim: Develop a novel computational approach leveraging deep learning to reconstruct both label-free and fluorescence-labeled sSMLM imaging data. Approach: We developed a two-network-model based deep learning algorithm, termed DsSMLM, to reconstruct sSMLM data. The effectiveness of DsSMLM was assessed by conducting imaging experiments on diverse samples, including label-free single-stranded DNA (ssDNA) fiber, fluorescence-labeled histone markers on COS-7 and U2OS cells, and simultaneous multicolor imaging of synthetic DNA origami nanoruler. Results: For label-free imaging, a spatial resolution of 6.22 nm was achieved on ssDNA fiber; for fluorescence-labeled imaging, DsSMLM revealed the distribution of chromatin-rich and chromatin-poor regions defined by histone markers on the cell nucleus and also offered simultaneous multicolor imaging of nanoruler samples, distinguishing two dyes labeled in three emitting points with a separation distance of 40 nm. With DsSMLM, we observed enhanced spectral profiles with 8.8% higher localization detection for single-color imaging and up to 5.05% higher localization detection for simultaneous two-color imaging. Conclusions: We demonstrate the feasibility of deep learning-based reconstruction for sSMLM imaging applicable to label-free and fluorescence-labeled sSMLM imaging data. We anticipate our technique will be a valuable tool for high-quality super-resolution imaging for a deeper understanding of DNA molecules' photophysics and will facilitate the investigation of multiple nanoscopic cellular structures and their interactions.

Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

In Situ Imaging of Proteins Using DNA-PAINT Super-Resolution Microscopy.

The spatial resolution of conventional light microscopy is restricted by the diffraction limit to hundreds of nanometers. Super-resolution microscopy enables single digit nanometer resolution by circumventing the diffraction limit of conventional light microscopy. DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) belongs to the family of single-molecule localization super-resolution approaches. Unique features of DNA-PAINT are that it allows for sub-nanometer resolution, spectrally unlimited multiplexing, proximity detection, and quantitative counting of target molecules. Here, we describe prerequisites for efficient DNA-PAINT microscopy.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.