Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

High data output and automated 3D correlative light-electron microscopy method

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.     

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.     

有机荧光团及荧光标记细胞的阴极荧光成像研究

目的 阴极荧光（CL）成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法 本研究应用扫描电镜（SEM）和阴极荧光谱仪系统（SEM-CL）,研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果 实验结果显示,在低能量（2.5～5 keV）和低束流（～10 pA）电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到～30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论 在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联（CCLEM）成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。     

Find your coat: Using correlative light and electron microscopy to study intracellular protein coats

Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale.     

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)     

Correlative video-light–electron microscopy: development,impact and perspectives

Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the ‘history,’ or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light–electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular ‘context’ to the fluorescent dynamic structures of interest. CLEM ‘multiplies’ the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.     

光电关联显微镜技术及其在植物学研究中的应用

光电关联显微镜技术(correlative light and electron microscopy,CLEM)将光学显微镜的高灵敏度和大视场与电子显微镜的高分辨率相结合,弥补了各自成像的局限,可在原位获得更全面、更精细的定位及结构信息,近年来受到广泛关注.目前,该技术广泛应用于亚细胞结构与特定结构的观察、蛋白质的精...     

An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy

Fluorescence recovery after photobleaching (FRAP) provides an important quantitative readout of the mobility of fluorescently tagged structures in live tissue. Here we present a protocol for visualizing FRAP signal at the ultrastructural level, permitting the nature of recovered fluorescence signal to be studied at greater resolution than afforded by conventional light microscopy. Specifically we use FRAP, fixation, photoconversion and correlative light and electron microscopy (CLEM) to examine the ultrastructural organization of mobile FM1-43-labeled vesicles in synapses of cultured hippocampal neurons. At photobleached synapses, the FRAP signal can be visualized as photoconverted electron-dense vesicles. The combination of FRAP and CLEM provides a powerful tool for examining the specific localization of imported vesicles in relation to synaptic architecture. Moreover, with the increasing availability of photoconvertible fluorophores, this approach should be readily applicable to other systems where an ultrastructural characterization of FRAP signal is desirable. After cultures are prepared and ready to use, this protocol takes 2-3 days.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

Monitoring lysosomal fusion in electrofused hybridoma cells

Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

The use of markers for correlative light electron microscopy

Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a “warning” that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.     

Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.     

Imaging single virus particles on the surface of cell membranes by high-resolution scanning surface confocal microscopy

We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 ± 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell.     

Four-color, 4-D time-lapse confocal imaging of chick embryos

Our understanding of the molecular mechanisms that direct cell motility, cell division, and cell shaping has benefited from innovations in cell labeling and the ability to resolve intracellular dynamics with multispectral, high-resolution imaging. However, due to difficulties with in vivo cell marking and monitoring, most studies have been restricted to fixed tissue or cells in culture. Here, we report the delivery of multiple (up to four), multicolor fluorescent protein (FP) constructs and four-dimensional (4-D), multispectral time-lapse confocal imaging of cell movements in living chick embryos. Cell cytoskeletal components are fluorescently tagged after microinjection and electroporation of a cocktail of FP constructs into specific regions of chick embryos. We tested 11 different FP constructs in various two-, three-, and four-color combinations using multispectral imaging and linear unmixing to limit the crosstalk between different emission spectra. We monitored intracellular dynamics in individual multicolored migrating cells in vivo and developed a set of advantageous imaging parameters for 4-D time-lapse confocal microscopy. We find that the number of four-color labeled cells in a typical embryo is approximately 10% of the total number of fluorescently labeled cells; this value consistently increases showing that approximately 50% of the total labeled cells have only one-color. We find that multicolored cells are photostable for time-lapses of approximately 2-3 h. Thus, cell labeling with up to four FP color schemes combined with multispectral, 4-D confocal time-lapse imaging offers a powerful tool to simultaneously analyze cellular and molecular dynamics during chick embryogenesis.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.