DNA-based ForceChrono probes for deciphering single-molecule force dynamics in living cells.

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

Unraveling Burst Selection Bias in Single-Molecule FRET of Species with Unequal Brightness and Diffusivity.

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.

Single-Molecule Maps of Membrane Insertion by Amyloid-ß Oligomers Predict Their Toxicity.

The interaction of small Amyloid-ß (Aß) oligomers with the lipid membrane is an important component of the pathomechanism of Alzheimer's disease (AD). However, oligomers are heterogeneous in size. How each type of oligomer incorporates into the membrane, and how that relates to their toxicity, is unknown. Here, we employ a single molecule technique called Q-SLIP (Quencher-induced Step Length Increase in Photobleaching) to measure the membrane insertion of each monomeric unit of individual oligomers of Aß42, Aß40, and Aß40-F19-Cyclohexyl alanine (Aß40-F19Cha), and correlate it with their toxicity. We observe that the N-terminus of Aß42 inserts close to the center of the bilayer, the less toxic Aß40 inserts to a shallower depth, and the least toxic Aß40-F19Cha has no specific distribution. This oligomer-specific map provides a mechanistic representation of membrane-mediated Aß toxicity and should be a valuable tool for AD research.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Universal inverse modeling of point spread functions for SMLM localization and microscope characterization.

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single-molecule localization, aberration correction and deconvolution. Here we present universal inverse modeling of point spread functions (uiPSF), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single-molecule localization microscopy (SMLM). Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system- or sample-specific characteristics, for example, the bead size, field- and depth- dependent aberrations, and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single-molecule blinking data.

DNA mismatch and damage patterns revealed by single-molecule sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically. This publication introduces the freestanding bilayer microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase-separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.

Visualization of Individual RNA Molecules by Proximity Ligation-Based Chromogenic In Situ Hybridization Assay.

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.

Single-Cell Imaging of mRNA by Target RNA-Initiated RCA.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

Single-Molecule RNA Imaging in Live Cells with an Avidity-Based Fluorescent Light-Up Aptamer biRhoBAST.

Observing individual RNA molecules provides valuable insights into their regulation, interactions with other cellular components, organization, and functions. Although fluorescent light-up aptamers (FLAPs) have recently shown promise for RNA imaging, their wider applications have been mostly hindered by poor brightness and photostability. We recently developed an avidity-based FLAP known as biRhoBAST that allows for single-molecule RNA imaging in live or fixed cells and tracking individual mRNA molecules in living cells due to its excellent photostability and high brightness. Here, we present step-by-step detailed protocols starting from cloning biRhoBAST repeats into the target RNA sequence, to imaging dynamics of single mRNA molecules. Additionally, we address the validation of single-molecule imaging experiments through single-molecule fluorescence in situ hybridization (smFISH) and colocalization studies.

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.

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.

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.

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.

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.

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.