Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

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 (t_Thresh and d_Thresh) 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.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.     

Optical techniques for imaging membrane topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.     

Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color.     

Applications of immunogold and lectin-gold labeling in tumor research and diagnosis

Immunohistochemistry and carbohydrate histochemistry have had an enormous impact on both tumor research and diagnosis. In particular, immunogold labeling has provided significant advantages over classical fluorescence and enzyme-based techniques. In light microscopy, the silver-intensified gold labeling has proven highly sensitive and precise in localization. In electron microscopy, the gold particle marker was a prerequisite for succesful and unequivocal antigen detection in electron-dense cellular structures such as secretory granules. In this review we demonstrate the usefulness of light and electron microscopiaal gold labeling techniques as applied in tumor research and diagnosis. The examples include expression of β-1,6 branches and specific sialoglycoconjugates in colon carcinoma, b-12 carbohydrate epitope in breast carcinoma, polysialic acid in neuroendocrine tumors of lung, adrenal and thyroid, as well as studies on proinsulin to insulin conversion in insulinomas. In addition, practical hints for prevention of background taining, tissue fixation, and silver intensification of gold labeling are given.     

Dissecting tripartite synapses with STED microscopy

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.     

3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.     

Translocation through the nuclear pore: Kaps pave the way

Transport through the nuclear pore complex (NPC), a keystone of the eukaryotic building plan, is known to involve a large channel and an abundance of phenylalanine–glycine (FG) protein domains serving as binding sites for soluble nuclear transport receptors and their cargo complexes. However, the conformation of the FG domains in vivo, their arrangement in relation to the transport channel and their function(s) in transport are still vividly debated. Here, we revisit a number of representative transport models—specifically Brownian affinity gating, selective phase gating, reversible FG domain collapse, and reduction of dimensionality (ROD)—in the light of new data obtained by optical single transporter recording, optical superresolution microscopy, artificial nanopores, and many other techniques. The analysis suggests that a properly adapted, simplified version of the ROD model accounts well for the available data. This has implications for nucleocytoplasmic transport in general.     

Optical imaging of nanoscale cellular structures

Visualization of subcellular structures and their temporal evolution is of utmost importance to understand a vast range of biological processes. Optical microscopy is the method of choice for imaging live cells and tissues; it is minimally invasive, so processes can be observed over extended periods of time without generating artifacts due to intense light irradiation. The use of fluorescence microscopy is advantageous because biomolecules or supramolecular structures of interest can be labeled specifically with fluorophores, so the images reveal information on processes involving only the labeled molecules. The key restriction of optical microscopy is its moderate resolution, which is limited to about half the wavelength of light (～200 nm) due to fundamental physical laws governing wave optics. Consequently, molecular processes taking place at spatial scales between 1 and 100 nm cannot be studied by regular optical microscopy. In recent years, however, a variety of super-resolution fluorescence microscopy techniques have been developed that circumvent the resolution limitation. Here, we present a brief overview of these techniques and their application to cellular biophysics.     

Intravital microscopy: Imaging host–parasite interactions in the brain

Intravital fluorescence microscopy (IVM) is a powerful technique for imaging multiple organs, including the brain of living mice and rats. It enables the direct visualisation of cells in situ providing a real‐life view of biological processes that in vitro systems cannot. In addition, to the technological advances in microscopy over the last decade, there have been supporting innovations in data storage and analytical packages that enable the visualisation and analysis of large data sets. Here, we review the advantages and limitations of techniques predominantly used for brain IVM, including thinned skull windows, open skull cortical windows, and a miniaturised optical system based on microendoscopic probes that can be inserted into deep tissues. Further, we explore the relevance of these techniques for the field of parasitology. Several protozoan infections are associated with neurological symptoms including Plasmodium spp., Toxoplasma spp., and Trypanosoma spp. IVM has led to crucial findings on these parasite species, which are discussed in detail in this review.     

A Multi-layered Protein Network Stabilizes the Escherichia coli FtsZ-ring and Modulates Constriction Dynamics

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.     

Precise Time Superresolution by Event Correlation Microscopy

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.     

Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.     

High-resolution confocal imaging and three-dimensional rendering

Intrinsic opacity and inhomeogeniety of most biological tissues have prevented the efficient light penetration and signal detection for high-resolution confocal imaging of thick tissues. Here, we summarize recent technical advances in high-resolution confocal imaging for visualization of cellular structures and gene expression within intact whole-mount thick tissues. First, we introduce features of the FocusClear technology that render biological tissue transparent and thus improve the light penetration and signal detection. Next, a universal fluorescence staining method that labels all nuclei and membranes is described. We then demonstrate the postrecording image processing techniques for 3D visualization. From these images, regions of interest in the whole-mount brain can be segmented and volume rendered. Together, these technical advances in confocal microscopy allow visualization of structures within whole-mount tissues up to 1mm thick at a resolution similar to that of the observation of single cells in culture. Practical uses and limitations of these techniques are discussed.     

Profilin connects actin assembly with microtubule dynamics

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.     

STED nanoscopy of actin dynamics in synapses deep inside living brain slices

It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.