Single-molecule Microscopy Techniques Research Articles

Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.

Extracellular vesicles (EVs) play a key role in cell-cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells.

Single molecule microscopy to profile the effect of zinc status on transcription factor dynamics

The regulation of transcription is a complex process that involves binding of transcription factors (TFs) to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. Here we employ multifocus microscopy and highly inclined laminated optical sheet microscopy to track TF dynamics in response to perturbations in labile zinc inside cells. We sought to define whether zinc-dependent TFs sense changes in the labile zinc pool by determining whether their dynamics and DNA binding can be modulated by zinc. We used fluorescently tagged versions of the glucocorticoid receptor (GR), with two C4 zinc finger domains, and CCCTC-binding factor (CTCF), with eleven C2H2 zinc finger domains. We found that GR was largely insensitive to perturbations of zinc, whereas CTCF was significantly affected by zinc depletion and its dwell time was affected by zinc elevation. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

First person – Radoslaw Gora

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Radoslaw Gora is first author on ‘ Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue’, published in DMM. Radoslaw is a PhD student in the lab of Dr Marcel Schaaf at Leiden University, Leiden, The Netherlands, investigating protein mobility patterns by use of single-molecule microscopy techniques.

Speed Microscopy: High-Speed Single Molecule Tracking and Mapping of Nucleocytoplasmic Transport.

The nuclear pore complex (NPC) functions as a gateway through which molecules translocate into and out of the nucleus. Understanding the transport dynamics of these transiting molecules and how they interact with the NPC has great potentials in the discovery of clinical targets. Single-molecule microscopy techniques are powerful tools to provide sub-diffraction limit information about the dynamic and structural details of nucleocytoplasmic transport. Here we detail single-point edge-excitation subdiffraction (SPEED) microscopy, a high-speed superresolution microscopy technique designed to track and map proteins and RNAs as they cross native NPCs.

Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII – LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Single-Molecule Biophysical Techniques to Study Actomyosin Force Transduction.

Inside the cellular environment, molecular motors can work in concert to conduct a variety of important physiological functions and processes that are vital for the survival of a cell. However, in order to decipher the mechanism of how these molecular motors work, single-molecule microscopy techniques have been popular methods to understand the molecular basis of the emerging ensemble behavior of these motor proteins.In this chapter, we discuss various single-molecule biophysical imaging techniques that have been used to expose the mechanics and kinetics of myosins. The chapter should be taken as a general overview and introductory guide to the many existing techniques; however, since other chapters will discuss some of these techniques more thoroughly, the readership should refer to those chapters for further details and discussions. In particular, we will focus on scattering-based single-molecule microscopy methods, some of which have become more popular in the recent years and around which the work in our laboratories has been centered.

Protein adsorption measurements on low fouling and ultralow fouling surfaces: A critical comparison of surface characterization techniques

Ultralow protein fouling behavior is a common target for new high-performance materials. Ultralow fouling is often defined based on the amount of irreversibly adsorbed protein (< 5 ng cm−2) measured by a surface ensemble averaging method. However, protein adsorption at solid interfaces is a dynamic process involving multiple steps, which may include adsorption, desorption, and irreversible protein denaturation. In order to better optimize the performance of antifouling surfaces, it is imperative to fully understand how proteins interact with surfaces, including kinetics of adsorption and desorption, conformation, stability, and amount of adsorbed proteins. Defining ultralow fouling surfaces based on a measurement at or near the limit of detection of a surface-averaged measurement may not capture all of this behavior. Single-molecule microscopy techniques can resolve individual protein-surface interactions with high temporal and spatial resolution. This information can be used to tune the properties of surfaces to better resist protein adsorption. In this work, we demonstrate how combining surface plasmon resonance, X-ray photoelectron spectroscopy, atomic force microscopy, and single-molecule localization microscopy provides a more complete picture of protein adsorption on low fouling and ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In this case, comparing the surfaces using surface plasmon resonance alone is insufficient to rank their resistance to protein adsorption. Our results suggest a revision of the accepted definition of ultralow fouling surfaces is timely: with the advent of time-resolved studies of protein adsorption kinetics at the single-molecule level, it is neither necessary nor sufficient to rely on a surface averaging techniques to qualify ultralow fouling surfaces. Since protein adsorption is a dynamic process, understanding how surface properties affect the kinetics of protein adsorption will enable the design of future generations of advanced antifouling materials. Statement of significanceThe design of ultralow fouling surfaces is often optimized based on a single surface-averaging technique measuring the amount of irreversibly adsorbed protein. This work provides a critical comparison of alternative techniques for evaluating protein adsorption on low fouling and ultralow fouling surfaces, and demonstrates how additional information about the dynamics of protein-surface interactions at the interface can be obtained by application of single-molecule microscopy. This approach could be used to better elucidate mechanisms of protein resistance and design principles for advanced ultralow fouling materials.

Accurate Investigation on the Fluorescence Resonance Energy Transfer between Single Organic Molecules and Monolayer WSe2 by Quantum Coherent Modulation-Enhanced Single-Molecule Imaging Microscopy.

Hybrid organic two-dimensional (2D) materials heterostructures are attracting tremendous attention for optoelectronic applications due to their low-cost processing and complementary advantages. However, accurate understanding of the fundamental physics on the interface of the hybrid heterostructures at the single-molecule level remains largely unexplored. Here, we investigated the fluorescence resonance energy transfer (FRET) between the single organic molecules and monolayer WSe2 through a newly developed single molecule microscopy technique, quantum coherent modulation-enhanced single-molecule imaging microscopy (QCME-SMIM). It is shown that the extremely weak energy transfer signal was successfully extracted from the huge fluorescence background, originating from the emission of monolayer WSe2. The observed energy transfer efficiency is in agreement with a d-4 distance dependence, with a Förster radius of ∼6 nm for the hybrid structures. Our work not only provides valuable insight into the FRET process at the single-molecule level across such hybrid organic-2D interfaces, but also demonstrates the feasibility of the newly developed technique for investigating the fundamental physics of electron transfer kinetics.

Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Single Molecule Microscopy Is Useful Technique For DNA Hairpin FRET Systems.

Nanometre sized semiconductor quantum dots as fluorescent materials are now becoming as ubiquitous as the organic dyes that preceded them. By virtue of their tuneable optical properties, narrow emission profiles and photostability, QDs have proved themselves as resilient and multifarious in both their applications and their underlying photophysics compared to their organic dye counterparts. Single Molecule Microscopy techniques (SM) are especially relevant to macromolecular biochemistry as chemical processes occurring within large molecules that are often too complicated to study in the ensemble. Using both total internal reflection (TIR) and Confocal Microscopy (CM) and both image processing and photon counting to achieve understanding of the binding kinetics and dynamic properties exhibited by the molecule via FRET studies. Several versions of the system have been examined to optimize the FRET pairing of donor/acceptor tags, achieve quantitative and ratiometric bioconjugation to nanocrystals and perfect techniques of microscopy and image processing.

Structural evolution and membrane interactions of Alzheimer's amyloid-beta peptide oligomers: new knowledge from single-molecule fluorescence studies.

Amyloid-β peptide (Aβ) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single-molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid-β, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar-to-picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aβ40 to Aβ42. Here, we discuss several types of single-molecule imaging that have been applied to the study of Aβ oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aβ-induced toxicity, is included in hopes of inspiring future development in this area of research.

Single-molecule imagingin vivo: the dancing building blocks of the cell

A cell can be viewed as a dynamic puzzle, where single pieces shuffle in space, change their conformation to fit different partners, and new pieces are generated while old ones are destroyed. Microscopy has become capable of directly observing the pieces of the puzzle, which are single molecules. Single-molecule microscopy in vivo provides new insights into the molecular processes underlying the physiology of a cell, allowing not only for visualizing how molecules distribute with nanometer resolution in the cellular environment, but also for characterizing their movement with high temporal precision. This approach reveals molecular behaviors normally invisible in ensemble measurements. Depending on the molecule, the process, and the cellular region studied, single molecules can be followed by conventional epifluorescence microscopy, or by illuminating only a thin region of the cell, as in Total Internal Reflection Fluorescence (TIRF) and Selective Plane Illumination Microscopy (SPIM), and by limiting the amount of detectable molecules, as in Fluorescence Speckle Microscopy (FSM) and Photo-Activation (PA). High spatial resolution can be obtained by imaging only a fraction of the molecules at a time, as in Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), or by de-exciting molecules in the periphery of the detection region as in Stimulated Emission-Depletion (STED) microscopy. Single-molecule techniques in vivo are becoming widespread; however, it is important to choose the most suited technique for each biological question or sample. Here we review single-molecule microscopy techniques, describe their basic principles, advantages for in vivo application, and discuss the lessons that can be learned from live single-molecule imaging.

Dynamic and Ordered Assembly of Single Spliceosomes

Spliceosome assembly is a highly dynamic and complex process involving the addition of 5 snRNAs and >100 proteins to a pre-mRNA transcript. This process has largely been analyzed by discontinuous assays that provide limited kinetic information. By combining yeast genetics, chemical biology, and a multi-wavelength single molecule microscopy technique (Colocalization Single Molecule Spectroscopy, CoSMoS), we monitored the formation of single spliceosomes. This approach revealed that individual spliceosome subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes. No single subcomplex binding event disproportionately limits the speed of the assembly reaction. While not all pre-mRNAs acquire a functional spliceosome, commitment of pre-mRNAs to splicing increases as assembly progresses. Therefore, spliceosome assembly is not a one-way, unbranched process as some models depict. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in complex environments.

The complexity of mesoporous silica nanomaterials unravelled by single molecule microscopy

Mesoporous silica nanomaterials are a novel class of materials that offer a highly complex porous network with nanometre-sized channels into which a wide amount of differently sized guests can be incorporated. This makes them an ideal host for various applications for example in catalysis, chromatography and nanomedicine. For these applications, analyzing the host properties and understanding the complicated host-guest interactions is of pivotal importance. In this perspective we review some of our recent work that demonstrates that single molecule microscopy techniques can be utilized to characterize the porous silica host with unprecedented detail. Furthermore, the single molecule studies reveal sample heterogeneities and are a highly efficient tool to gain direct mechanistic insights into the host-guest interactions. Single molecule microscopy thus contributes to a thorough understanding of these nanomaterials enabling the development of novel tailor-made materials and hence optimizing their applicability significantly.

Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by Förster/fluorescence resonance energy transfer (FRET) spectroscopy.

A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy

Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato ( Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated number-average and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk.

Structure-activity Relationships In Synthetic Systems Of Coupled Motor Proteins

Motor proteins play an essential role in regulating the internal organization of the cytoplasm by maneuvering a wide variety of vesicles and organelles. Many of these transport processes depend on the ability of motors to function collectively, in groups that contain more than one motor molecule. Utilizing protein engineering and DNA-self-assembly techniques, we have created experimental models of these multi-motor systems. Our synthetic assemblies afford precise control over the number of coupled motors in a construct, as well as their relative positions and the mechanical compliance of the linkage between each motor and the solid support to which it is anchored. Furthermore, the molecular architecture of these assemblies has been characterized using both bulk methods and single-molecule microscopy techniques. Our initial optical trapping experiments revealed rich behavior in a system of two coupled kinesin motors; the force-velocity relationship for individual constructs reveals that coupled kinesin motors can move at higher-than-predicted velocities when under high loads. Additionally, the system's stepping dynamics appears to vary significantly with applied load. Here, we discuss our efforts to characterize the effect of mechanical compliance and inter-motor separation upon these behaviors. Understanding these structure-activity relationships is critical to our broader goal of a comprehensive, mechanistic model of collective motor protein transport. Such a model would provide important insight into the process by which a cell controls its internal order.