Visualizing the Ultrastructures and Dynamics of Synapses by Single-Molecule Nanoscopy

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.

The combination of optical sectioning provided by a two-photon temporal focusing with lateral super resolution provided by PALM enables imaging of protein distributions in cells with a lateral localization precision better than 20 nm at multi ple imaging planes over an axial range of 10μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells. The present super-resolution techniques, such as photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), do not allow imaging of whole cells and organelles that are typically up to 15 μm deep into the cell, because they are limited to an imaging depth of order to of a fraction of the wavelength. The two photon temporal focusing provides the optical sectioning necessary for a 3D super resolution technique.

Single Molecule Counting using STORM/PALM

New technologies are ready to revolutionize glomerular imaging and significantly improve or replace immunofluorescence and electron microscopy, which have driven research and diagnosis of glomerular diseases for over 50 years. Advanced forms of transmission and scanning electron microscopy have revealed the detailed spatial relationships of the glomerular basement membrane, podocytes, and endothelial cells. These may be overshadowed by super resolution microscopy (SRM), which combines the advantages of immunofluorescence and electron microscopy, offers high resolution identification of specific molecules, and images large, physiologically relevant volumes of the glomerulus. Rapidity, ease of use and low cost with some types of SRM make them potentially suitable for routine diagnosis. SRM visualizes structures below the classical diffraction limit of conventional light microscopy by adding a time variable to either the illumination of the specimen, or to the fluorescence signal emitted by it. Ensemble techniques vary illumination and include Structured Illumination Microscopy (SIM) and Stimulation Emission Depletion Microscopy (STED). Single molecule localization techniques vary the light emission by fluorescence labels in the specimen, and include Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM). Technologies such as expansion microscopy and genetic labeling can also create effective super resolution imaging by non-optical, specialized preparation techniques. All technologies require dark field fluorescence and some require computer image analysis and reconstruction. Replicating successful application in other areas of biology, SIM, STED, and STORM have visualized normal and nephrotic disease podocytes, and have confirmed their appearances to be similar to those seen by electron microscopy, but with added new information on cell configuration and protein localization. STORM has also localized podocyte cytoskeleton and adhesion proteins, and glomerular basement membrane proteins at a resolution never before possible. These pioneering efforts show the promise of super resolution microscopy, and lay the groundwork for future study and new diagnostic tools for glomerular diseases.

Advanced microscopy techniques (such as STORM, STED, andSIM) have recently allowed the visualization of biological samples beyond the diffraction limit of light. Thanks to this breakthrough, the organization of molecules can be revealed within single cells as never before.Here, we describe the application of STochastic Optical Reconstruction Microscopy (STORM) for the study of polycomb group of proteins (PcG) in the context of chromatin organization. We present a clustering algorithm to quantitatively analyze the spatial distribution of nuclear molecules (e.g., EZH2 or its associated chromatin mark H3K27me3) imaged by 2D STORM. This distance-based analysis uses x-y coordinates of STORM localizations to group them into "clusters." Clusters are classified as singles if isolated or into islands if they form a group of closely associated clusters. For each cluster, the algorithm calculates the number of localizations, the area, and the distance to the closest cluster.This approach can be used for every type of adherent cell lineand allows the imaging of every protein for which an antibody is available. It represents a comprehensive strategy to visualize and quantify how PcG proteins and related histone marks organize in the nucleus at nanometric resolution.

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (tThresh and dThresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.

Expression of the AMPA receptor subunit GluR1 is associated with granule cell maturation in the dentate gyrus

Genistein inhibits amyloid peptide 25-35-induced neuronal death by modulating estrogen receptors, choline acetyltransferase and glutamate receptors

This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of superresolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate superresolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.Mark Bates, Christian Eggeling

PALM-Based Super-Resolution Imaging and its Applications

ABSTRACTMycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and synthetic biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic to both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300- to 800-nm range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed superresolution imaging techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have, however, not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform single-molecule localization microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photoconvertible fluorescent protein mEos3.2, enabling photo-activated localization microscopy (PALM) in most Mycoplasma species. We also describe the application of direct stochastic optical reconstruction microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development of SMLM strategies to study these organisms in the future.IMPORTANCE Mycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small sizes of these bacteria, well below a micron, limits the usefulness of traditional fluorescence imaging methods, as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art superresolution microscopy techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photoconvertible fluorescent protein, and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nm. Our work paves the way toward a better understanding of mycoplasma biology through imaging of subcellular structures at the nanometer scale.

Conventional microscopy techniques, namely, the confocal microscope or deconvolution processes, are resolution limited to approximately 200-250 nm by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20-50 nm in diameter. In the mid-2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular co-localization questions. This "breaking" of the diffraction barrier, affording resolution beyond 200 nm, is termed super-resolution microscopy. More recent approaches include single-molecule localization (such as photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) and point spread function engineering (such as stimulated emission depletion (STED) microscopy). In this review, we explain basic principles behind currently commercialized super-resolution setups and address advantages and considerations in applying these techniques to protein co-localization in biological systems.

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy

Fluorescent proteins are promising toolsfor studying intracellular signaling processes in lymphocytes. This brief review summarizes fluorescence-based imaging techniques developed in recent years and discusses new methodological advances, such as fluorescent photoswitches, fluorescence recovery after photobleaching (FRAP), fluorescent resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), total internal reflection fluorescence (TIRF) and other techiques. This survey also highlights recent advances in vitro imaging of live tissues, novel applications of flow cytometry with genetically modifed fluorescent proteins, and future prospects for the development of new immunological test systems based on fluorescent protein technology.