Single molecule fluorescence image patterns linked to dipole orientation and axial position: application to myosin cross-bridges in muscle fibers

Background

Photoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.

Methods and Findings

Fluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ² minimization.

Conclusions

Results indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

单分子荧光检测技术的发展及其在植物生物学研究中的应用

单分子荧光检测技术是利用荧光基团对目的分子标记后,在单分子水平成像并追踪分子的构象变化、动力学特征以及分子之间相互作用的研究方法.相较于传统分子生物学和遗传学的研究手段,单分子检测技术可以对单个分子的动态和特性进行分析,特别是瞬时或偶发性的事件,从而更加深入地挖掘在群体测量中被掩盖的信息.该技术已广泛应用于动物细胞生物...     

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.Since its advent 20 years ago, single-molecule fluorescence imaging has given rise to a host of exciting experiments (Ambrose and Moerner 1991). Beyond enabling fundamental investigations of the physics of emissive molecules, one main advantage of this technique is its use in biologically relevant, live-cell experiments. Optical fluorescence microscopy is an important instrument for cell biology, as light can be used to noninvasively probe a sample with relatively small perturbation of the specimen, enabling dynamical observation of the motions of internal structures in living cells. Single-molecule epifluorescence microscopy extends these capabilities by achieving nanometer-scale resolution, taking advantage of the fact that one can precisely characterize the point spread function (PSF) of a microscope, allowing the center of a distribution, and thus the exact position of an emitter, to be localized with accuracy much better than the diffraction limit itself. This localization accuracy improves beyond the diffraction limit roughly as one over the square root of the number of detected photons (Thompson et al. 2002). Detecting 100 photons from a single, isolated molecule can therefore improve the resolution of an optical measurement from the ∼250-nm diffraction limit down to 25 nm.Single-molecule imaging has been used in the investigation of a number of live-cell samples. In 2000, the lateral heterogeneity of the plasma membrane was investigated by tracing the motion of single dye-labeled lipids in native human airway smooth muscle (HASM) cells (Schütz et al. 2000), and epidermal growth factor (EGF) receptor signaling was explored with a fluorescent protein fusion and a labeled ligand (Sako et al. 2000). Single fluorophore-labeled molecules have subsequently been used in many ways (Moerner 2003), for instance to investigate the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane of Chinese hamster ovary (CHO) cells (Vrljic et al. 2002; Vrljic et al. 2005) and to explore the real-time dynamic behavior of cell-penetrating-peptide (CPP) molecular transporters on the plasma membrane of CHO cells (Lee et al. 2008). Furthermore, in 2001, Harms et al. characterized the emission of fluorescent proteins in biocompatible environments and noted that the yellow fluorescent protein EYFP was well-suited to single-molecule imaging in cells (Harms et al. 2001). Such fluorescent proteins can be genetically encoded as tags for native proteins in cells; these fusions have been used in many live-cell single-molecule experiments.More recently, single-molecule epifluorescence microscopy has been used to probe the inner workings of live bacteria. The small size of prokaryotic cells makes the optical diffraction limit particularly noticeable, which has stimulated the push toward superlocalization and superresolution to overcome this obstacle. As a result, the nascent field of bacterial structural biology has benefited greatly from single-molecule investigations of proteins in live cells. The overall shapes of such cells can be seen in a standard light microscope, but those interested in probing subcellular details, such as protein structure and localization, have typically had to resort to in vitro characterization combined with extrapolation to the cellular environment, as well as to indirect methods such as biochemical assays. Although cryo-electron microscopy can provide extremely high spatial resolution, fixation or plunge-freezing is essential, and methods for identifying specific proteins out of many are still lacking. As a consequence, bacterial cell biology is an area of study ripe for investigation with direct, noninvasive optical methods of probing position, coupling and structure, with resolution below the standard diffraction limit.Several groups have extended single-molecule imaging techniques to live bacterial samples. In 2004, single PleC proteins were visualized in Caulobacter crescentus cells (Deich et al. 2004), and the behavior of this system is described in more detail later. More recently, Xie and coauthors have used single-molecule fluorescence techniques to study DNA-binding proteins, mRNA, and membrane proteins to provide much insight into the mechanisms of bacterial gene expression; these efforts have been documented in a recent review (Xie et al. 2008). As well, Conley et al. used covalently linked Cy3-Cy5-thiol switchable fluorophores to illuminate the stalks of C. crescentus cells with high resolution (Conley et al. 2008). In this article, we focus on the application of single-molecule imaging and single-molecule-based superresolution imaging to investigate the localization, movement, and structure of three important proteins, PleC, PopZ, and MreB, in live C. crescentus cells.     

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

Artifacts in single-molecule localization microscopy

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.     

Identifying and characterizing the biological targets of metallotherapeutics: Two approaches using Au(I)-protein interactions as model systems

A significant challenge in the field of medicinal inorganic chemistry is the identification of biological targets of metal-based drugs and the characterization of the metal–biomolecule adducts. A classic example is Au(I), which has long been used to treat rheumatoid arthritis despite a poor understanding of its biological targets due to the lability, reactivity, and “spectroscopic silence” that are characteristic of Au(I). Here, we report two qualitative methods for characterizing Au(I)–protein adducts: a thiol-reactive probe that facilitates the identification of biological cysteine–Au(I) adducts and a photoreactive Au(I) complex that produces a covalent bond between the Au(I) complex and the biomolecule.     

Characterization of physical properties of supported phospholipid membranes using imaging ellipsometry at optical wavelengths

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.     

Raman ChemLighter: Fiber optic Raman probe imaging in combination with augmented chemical reality

Raman spectroscopy using fiber optic probe combines non‐contacted and label‐free molecular fingerprinting with high mechanical flexibility for biomedical, clinical and industrial applications. Inherently, fiber optic Raman probes provide information from a single point only, and the acquisition of images is not straightforward. For many applications, it is highly crucial to determine the molecular distribution and provide imaging information of the sample. Here, we propose an approach for Raman imaging using a handheld fiber optic probe, which is built around computer vision–based assessment of positional information and simultaneous acquisition of spectroscopic information. By combining this implementation with real‐time data processing and analysis, it is possible to create not only fiber‐based Raman imaging but also an augmented chemical reality image of the molecular distribution of the sample surface in real‐time. We experimentally demonstrated that using our approach, it is possible to determine and to distinguish borders of different bimolecular compounds in a short time. Because the method can be transferred to other optical probes and other spectroscopic techniques, it is expected that the implementation will have a large impact for clinical, biomedical and industrial applications.      

Colorful Protein-Based Fluorescent Probes for Collagen Imaging

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni²⁺-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.     

Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology

Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus–cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.     

Recent advances in exploiting ionic liquids for biomolecules: Solubility,stability and applications

The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications.     

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.     

Quantifying and Optimizing Single-Molecule Switching Nanoscopy at High Speeds

Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5–25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.     

Single-molecule fluorescence studies from a bioinorganic perspective

In recent years, single-molecule methods have enabled many innovative studies in the life sciences, which generated unprecedented insights into the workings of many macromolecular machineries. Single-molecule studies of bioinorganic systems have been limited, however, even though bioinorganic chemistry represents one of the frontiers in the life sciences. With the hope to stimulate more interest in applying existing and developing new single-molecule methods to address compelling bioinorganic problems, this review discusses a few single-molecule fluorescence approaches that have been or can be employed to study the functions and dynamics of metalloproteins. We focus on their principles, features and generality, possible further bioinorganic applications, and experimental challenges. The fluorescence quenching via energy transfer approach has been used to study the O₂-binding of hemocyanin, the redox states of azurin, and the folding dynamics of cytochrome c at the single-molecule level. Possible future applications of this approach to single-molecule studies of metalloenzyme catalysis and metalloprotein folding are discussed. The fluorescence quenching via electron transfer approach can probe the subtle conformational dynamics of proteins, and its possible application to probe metalloprotein structural dynamics is discussed. More examples are presented in using single-molecule fluorescence resonance energy transfer to probe metallochaperone protein interactions and metalloregulator-DNA interactions on a single-molecule basis.     

Chemistry in living systems

Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. Although genetically encoded tags such as GFP are widely used to monitor discrete proteins, they can cause significant perturbations to a protein's structure and have no direct extension to other classes of biomolecules such as glycans, lipids, nucleic acids and secondary metabolites. In recent years, an alternative tool for tagging biomolecules has emerged from the chemical biology community--the bioorthogonal chemical reporter. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. This review highlights the development of bioorthogonal chemical reporters and reactions and their application in living systems.     

Translational diffusion of individual class II MHC membrane proteins in cells

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.     

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and assembly of native myosin on muscle polyribosomes

We have used the overload-induced growth of avian muscles to study the assembly of the newly synthesized myosins which were separated by non-denaturing pyrophosphate-polyacrylamide gel electrophoresis. Using this model, we have observed the appearance of fast-like isomyosins in polyribosomes prepared from slow anterior latissimus dorsi muscle after 72 h of overload. These new isoforms comigrating with native myosin from fast posterior latissimus dorsi muscle were not yet present in cellular extracts from the same muscle. The in vitro translation system utilizing muscle specific polyribosomes directs the synthesis of the corresponding myosin isoforms. Under denaturing conditions, myosin heavy chains and light chains dissociate to the expected subunit composition of each specific isoform. The synthesis and assembly of native myosin on polyribosomes indicate that myosin exists as a single mature protein prior to the incorporation in the thick filament.     

DNA origami as biocompatible surface to match single-molecule and ensemble experiments

Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements.