Single molecule microscopy

Hampered by their susceptibility to nucleophilic attack and chemical bleaching, electron-deficient squaraine dyes have long been considered unsuitable for biological imaging. This study unveils a surprising twist: in aqueous environments, bleaching is not irreversible but rather a reversible spontaneous quenching process. Leveraging this new discovery, we introduce a novel deep-red squaraine probe tailored for live-cell super-resolution imaging. This probe enables single-molecule localization microscopy (SMLM) under physiological conditions without harmful additives or intense lasers and exhibits spontaneous blinking orchestrated by biological nucleophiles, such as glutathione or hydroxide anion...

Azobenzenes are fascinating molecular machines that can reversibly transform between two isomeric forms by an external stimulus. Diazocine, a type of bridged azobenzene, has been shown to possess enhanced photoexcitation properties. Due to the distortion caused by the ethyl bridge in the E-isomer, the Z-form becomes the thermodynamically stable configuration. Despite a comprehensive understanding of its photophysical properties, there is still much to learn about the behavior of diazocine on a metal surface...

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces...

Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics across many timescales. With TCSPC, Fluorescence correlation spectroscopy (FCS) and pulsed interleaved excitation-Förster resonance energy transfer (PIE-FRET) are collected simultaneously on diffusing molecules to extract diffusion characteristics and proximity information. This article is a guide to calibrating FCS and PIE-FRET measurements with several biological samples including liposomes, streptavidin-coated quantum dots, proteins, and nucleic acids for reliable determination of diffusion coefficients and FRET efficiency...

UNLABELLED: Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood...

Amyloid-beta (Aβ42) aggregates are characteristic signatures of Alzheimer's disease, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aβ42 fibrils. We quantify correlations between fibril architectures, measured by SMOLM, and their growth and decay visualized by single-molecule localization microscopy (SMLM)...

Phenotypic profiling by high throughput microscopy has become one of the leading tools for screening large sets of perturbations in cellular models. Of the numerous methods used over the years, the flexible and economical Cell Painting (CP) assay has been central in the field, allowing for large screening campaigns leading to a vast number of data-rich images. Currently, to analyze data of this scale, available open-source software ( i.e. , CellProfiler) requires computational resources that are not available to most laboratories worldwide...

UNLABELLED: Subgenomic flavivirus RNAs (sfRNAs) are structured RNA elements encoded in the 3'-UTR of flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze the production of sfRNAs using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) and super-resolution microscopy during West Nile virus, Zika virus, or Dengue virus serotype 2 infection. We show that sfRNAs are initially localized diffusely in the cytosol or in processing bodies (P-bodies)...

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells...

The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex...

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis...

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site...

Denoising diffusion models embody a type of generative artificial intelligence that can be applied in computer vision, natural language processing and bioinformatics. In this Review, we introduce the key concepts and theoretical foundations of three diffusion modelling frameworks (denoising diffusion probabilistic models, noise-conditioned scoring networks and score stochastic differential equations). We then explore their applications in bioinformatics and computational biology, including protein design and generation, drug and small-molecule design, protein-ligand interaction modelling, cryo-electron microscopy image data analysis and single-cell data analysis...

Recent microscopy and nuclear magnetic resonance (NMR) studies have noticed substantial suppression of intracellular diffusion for positively charged proteins, suggesting an overlooked role of electrostatic attraction in nonspecific protein interactions in a predominantly negatively charged intracellular environment. Utilizing single-molecule detection and statistics, here, we quantify in aqueous solutions how protein diffusion, in the limit of low diffuser concentration to avoid aggregate/coacervate formation, is modulated by differently charged interactor proteins over wide concentration ranges...

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes...

Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy technique method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-impermeable dye for quantifying BBB extravasation. A deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction...

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay...

Small molecule drugs play a pivotal role in the arsenal of anticancer pharmacological agents. Nonetheless, their small size poses a challenge when directly visualizing their localization, distribution, mechanism of action (MOA), and target engagement at the subcellular level in real time. We propose a strategy for developing triple-functioning drug beacons that seamlessly integrate therapeutically relevant bioactivity, precise subcellular localization, and direct visualization capabilities within a single molecular entity...

The deployment of small-molecule fluorescent agents plays an ever-growing role in medicine and drug development. Herein, we complement the portfolio of powerful fluorophores, reporting the serendipitous discovery and development of a novel class with an imidazo[1,2-a]pyridinium triflate core, which we term PyrAtes. These fluorophores are synthesized in a single step from readily available materials (>60 examples) and display Stokes shifts as large as 240 nm, while also reaching NIR-I emissions at λmax as long as 720 nm...

Interferometric scattering microscopy (iSCAT) has rapidly developed as a quantitative tool for the label-free detection of single macromolecules and nanoparticles. In practice, this measurement records the interferometric scattering signal of individual nanoparticles in solution as they land and stick on a coverslip, exhibiting an intensity that varies linearly with particle volume and an adsorption rate that reflects the solution-phase transport kinetics of the system. Together, such measurements provide a multidimensional gauge of the particle size and concentration in solution over time...