Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization?

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

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

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.

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.

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.

In vivo imaging is a platform technology with the power to put function in its natural structural context. With the drive to translate stem cell therapies into pre-clinical and clinical trials, early selection of the right imaging techniques is paramount to success. There are many instances in regenerative medicine where the biological, biochemical, and biomechanical mechanisms behind the proposed function of stem cell therapies can be elucidated by appropriate imaging. Imaging techniques can be divided according to whether labels are used and as to whether the imaging can be done in vivo. In vivo human imaging places additional restrictions on the imaging tools that can be used. Microscopies and nanoscopies, especially those requiring fluorescent markers, have made an extraordinary impact on discovery at the molecular and cellular level, but due to their very limited ability to focus in the scattering tissues encountered for in vivo applications they are largely confined to superficial imaging applications in research laboratories. Nanoscopy, which has tremendous benefits in resolution, is limited to the near-field (e.g. near-field scanning optical microscope (NSNOM)) or to very high light intensity (e.g. stimulated emission depletion (STED)) or to slow stochastic events (photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)). In all cases, nanoscopy is limited to very superficial applications. Imaging depth may be increased using multiphoton or coherence gating tricks. Scattering dominates the limitation on imaging depth in most tissues and this can be mitigated by the application of optical clearing techniques that can impose mild (e.g. topical application of glycerol) or severe (e.g. CLARITY) changes to the tissue to be imaged. Progression of therapies through to clinical trials requires some thought as to the imaging and sensing modalities that should be used. Smoother progression is facilitated by the use of comparable imaging modalities throughout the discovery and trial phases, giving label-free techniques an advantage wherever they can be used, although this is seldom considered in the early stages. In this paper, we will explore the techniques that have found success in aiding discovery in stem cell therapies and try to predict the likely technologies best suited to translation and future directions.

Chapter 14 - Super-resolution Microscopy: A Comparison of Commercially Available Options

The field of optical nanoscopy , a paradigm referring to the recent cutting-edge developments aimed at surpassing the widely acknowledged 200nm-diffraction limit in traditional optical microscopy, has gained recent prominence & traction in the $21^{\mathrm {st}}$ century. Numerous optical implementations allowing for a new frontier in traditional confocal laser scanning fluorescence microscopy to be explored (termed super-resolution fluorescence microscopy ) have been realized through the development of techniques such as stimulated emission and depletion (STED) microscopy, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), amongst others. Nonetheless, it would be apt to mention at this juncture that optical nanoscopy has been explored since the mid-late $20^{\mathrm {th}}$ century, through several computational techniques such as deblurring and deconvolution algorithms. In this review, we take a step back in the field, evaluating the various in silico methods used to achieve optical nanoscopy today, ranging from traditional deconvolution algorithms (such as the Nearest Neighbors algorithm) to the latest developments in the field of computational nanoscopy, founded on artificial intelligence (AI). An insight is provided into some of the commercial applications of AI-based super-resolution imaging, prior to delving into the potentially promising future implications of computational nanoscopy. This is facilitated by recent advancements in the field of AI, deep learning (DL) and convolutional neural network (CNN) architectures, coupled with the growing size of data sources and rapid improvements in computing hardware, such as multi-core CPUs & GPUs, low-latency RAM and hard-drive capacities.

Nanoscopic imaging techniques provide a powerful set of tools for static and dynamic fluorescence microscopy below the diffraction limit of light. Among these super-resolution techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are based on the detection of single fluorophores, whose signals are localized with great precision. The strength of single-molecule approaches lies in their high localization accuracy and their molecular specificity and in the development of multicolor approaches, three-dimensional imaging, and the quantification of absolute molecule numbers. These capabilities make PALM and STORM imaging ideally suited to visualize the ultrastructures of synapses, to track the movements of synaptic components, and to quantify the molecular plasticity at synapses.Key wordsActive zone (AZ)Dendritic spineGluA1 (AMPA receptor subunit GluR1)GluN2B (NMDA receptor subunit NR2B)Photoactivated localization microscopy (PALM)Postsynaptic density (PSD)Stochastic optical reconstruction microscopy (STORM)

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.

Super-resolution Microscopy in Plant Cell Imaging.

Super-resolution microscopy enables imaging of structures smaller than the diffraction limit. Single-molecule localization microscopy methods, such as photoactivation localization microscopy and stochastic optical reconstruction microscopy, reconstruct images by plotting the centroids of fluorescent point sources from a series of frames in which only a few molecules are fluorescing at a time. These approaches require simpler instrumentation than methods that depend on structured illumination and thus are becoming widespread. The functionalized rhodamine derivative reported in this paper spontaneously converts between a bright and dark state due to pH-dependent cyclization. At pH 7, less than 0.5% of the dye molecules are fluorescent at any given time. Blinking occurs on time scales of seconds to minutes and can therefore be used for single-molecule localization microscopy without sample treatment or activation. The dye is bright and straightforward to use, and it is easy to synthesize and functionalize. Thus, it has potential to become a new and powerful addition to the toolset for super-resolution imaging.

Single Molecule Counting using STORM/PALM

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.

This short review begins with the theories of Airy, Rayleigh and Abbe on microscope resolution. Next, the principal developments in microscopy in the last half-century are examined for relevance to ophthalmology: confocal microscopy, photoactivation light microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), structured illumination (SI), 2-photon and multiphoton excitation microscopy with a focused beam. Except for confocal, these are difficult to apply to the eye in vivo, as are the interference methods available in microscopes. However, interferometry in the form of coherence tomography is now a major ophthalmic method which has diverged from microscopy. Multiphoton excitation microscopy with an unfocussed beam is a new, low-damage microscope method so-far not exploited in ophthalmoscopy. The Mesolens, which throws off the historic limitations in microscopy set by the human eye, is described as a possible future aid to ophthalmology of the anterior eye.