Fluorophores for single-molecule localization microscopy

Super-resolution fluorescence microscopy allows for obtaining images with a resolution of 10–20 nm, far exceeding the diffraction limit of conventional optical microscopy (200–350 nm), and provides an opportunity to study in detail the subcellular structures and individual proteins in both living and fixed cells. Among these methods, single-molecule localization microscopy (SMLM) has become widespread. SMLM techniques are based on special fluorophores capable of photoswitching. The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications. We discuss recent progress and prospects for the development of new effective labels suitable for SMLM.     

Study of single-molecule dynamics and reactions with classic light microscopy.

Single-molecule studies in the life sciences often deal with observation or spectroscopy. Studies of reactions are rare, and the light microscope has been used for such experiments only occasionally. In an experimental environment, for example, as is required for most nearfield scanning or electron microscopies, it is difficult to study single-molecule reactions of biological relevance. Therefore, we have developed techniques to study single-molecule reactions with classic (nonscanning) farfield light microscopy. The conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH (a reduced form of NAD+), pyruvate, and H+ catalyzed by a few LDH-1 enzyme molecules has been studied in substrate solutions with different viscosity using the NADH autofluorescence. It is even possible to monitor the progress of the reaction by phase-contrast microscopy via scattering or absorption by product molecules. As an example for a single-molecule reaction with a macromolecule as substrate, the handling and enzymatic cutting of fluorescently stained lambda-DNA is studied. In solutions containing 10 mM magnesium and 66 mM potassium ions at pH 7.9, an individual DNA molecule tends to collapse into a globular structure. When moved through an aqueous solution, it becomes stretched by viscosity drag. After stopping the motion, the molecule collapses and the dynamics of this process can be quantified. When a restriction enzyme is present, sequence-specific cutting can be directly observed in the light microscope. The theoretical restriction pattern, as predicted from the sequence of the molecule, can be generated directly under visual inspection.     

Biophysical nanotools for single-molecule dynamics

The focus of the cell biology field is now shifting from characterizing cellular activities to organelle and molecular behaviors. This process accompanies the development of new biophysical visualization techniques that offer high spatial and temporal resolutions with ultra-sensitivity and low cell toxicity. They allow the biology research community to observe dynamic behaviors from scales of single molecules, organelles, cells to organoids, and even live animal tissues. In this review, we summarize these biophysical techniques into two major classes: the mechanical nanotools like dynamic force spectroscopy (DFS) and the optical nanotools like single-molecule and super-resolution microscopy. We also discuss their applications in elucidating molecular dynamics and functionally mapping of interactions between inter-cellular networks and intra-cellular components, which is key to understanding cellular processes such as adhesion, trafficking, inheritance, and division.     

Localization accuracy in single-molecule microscopy

One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.     

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.     

Deconvolving single-molecule intensity distributions for quantitative microscopy measurements

In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.     

A new technique for visualizing proanthocyanidins by light microscopy

We describe a new technique for visualizing proanthocyanidin-containing elements in plant tissues. Our innovation is the fixation of condensed tannins with an exogenous protein prior to alcohol dehydration. In this way, tannins do not undergo partial solubilization during the dehydration sequence and appear as sharply contoured globules of various diameters.     

A method for visualizing the acrosome by light microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol:acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.     

Fluorescence microscopy

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.     

Eight years of single-molecule localization microscopy

Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.     

Progress in quantitative single-molecule localization microscopy

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).     

Bayesian estimation for species identification in single-molecule fluorescence microscopy

In this article we describe a recursive Bayesian estimator for the identification of diffusing fluorophores using photon arrival-time data from a single spectral channel. We present derivations for all relevant diffusion and fluorescence models, and we use simulated diffusion trajectories and photon streams to evaluate the estimator's performance. We consider simplified estimation schemes that bin the photon counts within time intervals of fixed duration, and show that they can perform well in realistic parameter regimes. The latter results indicate the feasibility of performing identification experiments in real time. It will be straightforward to generalize our approach for use in more complicated scenarios, e.g., with multiple spectral channels or fast photophysical dynamics.     

Fingerprinting polysaccharides with single-molecule atomic force microscopy

We report the use of an atomic force microscopy (AFM)-based force spectroscopy technique to identify, at the single-molecule level, the components of mixtures of polysaccharides. Previously, we showed that the elasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. These transitions produce atomic fingerprints in the force-extension spectrum that are characteristic of the ground-energy conformation of the pyranose ring and the type of glycosidic linkages. Using this approach we find that commercially available agarose and lambda-carrageenan contain molecules that, when stretched in an atomic force microscope, produce a force spectrum characteristic of alpha-(1-->4) d-glucans. We have identified these molecules as amylopectin or floridean starch, a storage polysaccharide in algae. Our methodology can identify individual polysaccharide molecules in solution, which is not possible by any other spectroscopic technique, and therefore is an important addition to the arsenal of analytical techniques used in carbohydrate research.     

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.     

Virus-derived platforms for visualizing protein associations inside cells

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein muNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the muNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent muNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.     

Rapid microscale procedure for visualizing intracellular plasmid DNA by electron microscopy

A rapid microscale procedure is described that releases plasmid DNA in situ from the bacterial cell and that allows selective observation of the plasmid bound to cellular components. The released plasmid DNA was adsorbed preferentially on mica in a divalent cation-free medium then processed for electron microscopy. The plasmid DNAs studied were pAO3 (1683 base pairs (bp²)), λdv021 (3505 bp), pTSO118 (4000 bp), pAO65 (4786 bp), ColE1 (6500 bp), and RSF2124 (11,400 bp). These DNAs were seen as supercoiled circles or as relaxed circles of corresponding length. Occasionally an internal loop of replicating DNA was present. One micrometer of measured length corresponded to 3100 bp.     

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.