Microscopy Research and Technique virtual issue: "Correlative light and electron microscopy".

Simultaneous Correlative Light and Electron Microscopy of Samples in Liquid

Systems biology in 3D space--enter the morphome.

Correlative light and electron microscopy (CLEM) methods integrate light and electron microscopy on a single sample, literally bridging the gap between these two microscopy techniques. Most methods use fluorescent microscopy of thin or semi‐thin sections to define a region‐of‐interest, which then is traced back in the EM to provide subcellular context information (e.g. membrane organization, non‐labeled surroundings of the fluorescent structure). The most powerful application of CLEM is correlative live cell imaging‐EM, by which dynamic information is inferred to structures seen in static EM pictures. By merging the strengths of the two techniques a novel and integrated type of image is created that combines parameters that cannot ‐ or not easily ‐ be obtained when using separate images of related events. However, because imaging requirements are intrinsically different between light and electron microscopy, creating conditions that are ideal for both modalities is a challenging process. Moreover, correlating fluorescent labeling to the cellular architecture seen in the EM is not always straightforward. The most pressing challenges in CLEM are currently therefore 1. development of sample preparation methods that are appropriate for both LM and EM imaging. 2. development of bi‐modal probes that are visible in both LM and EM and 3. development of software that allows for rapid and accurate correlation of LM and EM images. The focus of our research is to develop new probes and CLEM pipelines that allow us to efficiently and with high accuracy define the three‐dimensional (3D) ultrastructural context of fluorescently‐tagged proteins previously localized in fixed or living cells. We apply our technologies to study the cellular pathways and mechanisms that control the cell's digestive system – i.e. the endo‐lysosomal system ‐ in health and disease conditions. The main CLEM technology that we use in the lab is based on the use of immunogold labeling of ultrathin cryosections (the Tokuyasu technique) [1]. Most CLEM approaches, however, are restricted in their EM approach by the lack of 3D structural information. To overcome this limitation, we apply Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM) as 3D‐EM approach in a live cell‐CLEM set up. To visualize endo‐lysosomes in live cells we combine fluorescent tagged endo‐lysosomal proteins (such as LAMP1‐mGFP) with endocytic tracers (such as fluorescently labeled dextran). This approach enables live‐cell tracking of specific endo‐lysosomal compartments, after which the samples are fixed, stained and resin‐embedded for FIB‐SEM imaging. Figure 1 presents an example of Dextran‐Alexa646 and/or LAMP1‐mGFP labeled endo‐lysosomal compartments in live cells (A) and in 3D‐EM (D), providing the cellular context at ultrastructural resolution. In my presentation I will show various examples of both immunoEM and FIB.SEM‐based CLEM approaches, which are designed to optimally image individual, membrane‐bounded compartments.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

Electron microscopy (EM) reveals cellular ultrastructure at high definition but faces the challenges of identification of specific subcellular structures and localization of specific macromolecules, whereas fluorescence microscopy (FM) can label and localize specific molecules in cells. Correlative light and electron microscopy (CLEM) combines the advantages of both microscopic techniques. Imaging vitreous hydrated samples at cryogenic temperatures using CLEM enables observations of cellular components of interest and their cellular context in a near-native state. This cryo-CLEM approach is further strengthened by incorporation of superresolution fluorescence microscopy, which can precisely pinpoint targets on electron micrographs. Cryogenic superresolution correlative light and electron microscopy (csCLEM) is an emerging and promising imaging technique that is expected to unveil its full power in ultrastructural studies. The present review describes the logic and principles behind this technique, how the method is implemented, the prospects, and the challenges.

SummaryFiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye‐labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold‐core rhodamine B‐labelled silica particles, 15 nm CdSe/CdS/ZnS core–shell–shell quantum dots (QDs) and 230 nm Y2O3:Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold‐core rhodamine B‐labelled silica NPs and QDs are quenched after a single exposure to 60 ke− nm–2 with an energy of 120 keV, while Y2O3:Eu3+ NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2O3:Eu3+ NPs are promising as robust fiducial marker in CLEM.Lay DescriptionLuminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye‐labelled silica particles, quantum dots and lanthanide‐doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye‐labelled silica NPs and QDs are quenched after a single exposure to 60 ke− nm–2 with an energy of 120 keV, while lanthanide‐doped inorganic NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of lanthanide‐doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide‐doped NPs are promising as robust fiducial marker in CLEM.

Correlative light and electron microscopy (CLEM) combines the strengths of light microscopy (LM) and electron microscopy (EM) to form a more complete picture of a cellular process. LM allows for a multitude of fluorescent tags and a wide field of view to select rare events, and EM allows for increased resolution and visualization of cellular ultrastructure [1]. We used three methods to examine external and internal morphologies of cells: cell cultures grown on coverslips, resin sections on coverslips, and resin sections on coated slot grids. Coverslip methods used indium-tin-oxide (ITO) coverslips with fiducial markers. The coverslips were coated with a 0.1% (w/v) Poly-L-lysine solution for 30 min, rinsed in water, and dried on filter paper overnight. Cells were grown and labeled on the coverslips depending on the requirements of the projects and imaged in a laser scanning confocal microscope (LSCM). All samples were imaged on a ZEISS Sigma HD VP Scanning Electron Microscope (SEM). Areas of interest on coverslip samples noted in LM were found in the SEM using ZEISS Shuttle and Find software. Slot grids were placed in a STEM holder and imaged using a backscatter detector. Correlation and overlay of images was performed using multiple software packages.

Correlative Light and Electron MIcroscopy

Correlative light and electron microscopy (CLEM) is a powerful tool for investigating cellular structure and function at the molecular level. However, while electron microscopy is often performed to great advantage at cryogenic temperatures, this is not always the case for light microscopy. One key challenge is the lack of cryo-compatible immersion objectives. In recent years, multiple cryoimmersion light microscopy (cryo-iLM) approaches have been described, but these techniques have never been used in correlative approaches. Here we present a novel workflow for correlative cryoimmersion light microscopy and electron cryomicroscopy (cryo-iCLEM). Cryo-electron tomography conducted before and after cryo-iLM reveals that cryo-iCLEM maintains ultra-thin, electron-transparent samples mechanically intact and does not degrade the ultrastructural preservation achieved through plunge-freezing. For cryo-iLM, the sample is first embedded in a viscous immersion medium at cryogenic temperatures and examined with a custom cryo-immersion objective. After cryo-iLM, the immersion medium is dissolved in liquid ethane, allowing for subsequent cryo-EM imaging. We further show that cryo-iCLEM can be used on FIB-lamellae, demonstrating that mechanically sensitive samples remain undamaged. Embedding the sample in the immersion fluid reduces contamination and thus allows data acquisition over many hours. Samples can therefore be examined in detail with the advantage of low bleaching rates of fluorophores at cryogenic temperatures. In the future, we hope that our approach can help improve the performance of many advanced light microscopy techniques when they are applied in the context of cryo-CLEM.

Correlative light and electron microscopy (CLEM) is an advanced imaging approach that faces critical challenges in the analysis of both materials and biological specimens. CLEM integrates the strengths of both light and electron microscopy, in a hardware and software correlative environment, to produce a composite image that combines the high resolution of the electron microscope with the large field of view of the light microscope. It enables a more comprehensive understanding of a sample’s microstructure, texture, morphology, and elemental distribution, thereby facilitating the interpretation of its properties and characteristics. CLEM has diverse applications in the geoscience field, including mineralogy, petrography, and geochemistry. Despite its many advantages, CLEM has some limitations that need to be considered. One of its major limitations is the complexity of the imaging process. CLEM requires specialized equipment and expertise, and it can be challenging to obtain high-quality images that are suitable for analysis. In this study, we present a CLEM workflow based on an innovative sample holder design specially dedicated to the examination of thin sections and three-dimensional samples, with a particular emphasis on geosciences.

A picture is worth a thousand words. Correlative light and electron microscopy (CLEM) is a valuable imaging tool as it bridges the resolution gap between the two modalities. Fluorescent markers are applied routinely to capture a broad field of view in two-dimensional space (2D), 3D, or 4D. In diffraction-limited light microscopy (LM), resolution approaches 200 nm. Alternatively, the physical properties of an electron beam allow an improvement in resolution approaching 1 nm for the visualization of ultrastructural features, including the surrounding architecture of some feature of interest. It follows that CLEM is especially powerful. Correlative Light and Electron Microscopy II, Volume 124 (Methods in Cell Biology) expands on techniques and applications introduced in Correlative Light and Electron Microscopy, Volume 111 (Methods in Cell Biology). The use of CLEM has wide applicability and can benefit many studies. For example, it can be used to directly identify a small organelle, thus bridging the resolution gap between LM and electron microscopy (EM). It can also be used to identify rare events or a region of interest for subsequent ultrastructural analysis, such as the dynamics of a vesicle being trafficked throughout the cell.

Correlative microscopy is a sophisticated imaging technique that combines optical and electron microscopes, with the most common approach being the integration of light microscopy and electron microscopy, known as correlative light and electron microscopy (CLEM). While CLEM provides a comprehensive view of biological samples, it presents a significant challenge in sample preparation due to the distinct processes involved in each technique. Striking a balance between these methods is crucial. Despite numerous approaches, achieving seamless imaging with CLEM remains a complex task. Exosomes, nanovesicles ranging from 30 to 150 nm in size, are enclosed by a lipid bilayer and released by various cell types. Visualizing exosomes poses difficulties due to their small size and minimal electric charge. However, imaging exosomes at high resolution offers a direct method to understand their morphology and functions. In this study, we evaluated exosome imaging with CLEM using a combination of confocal, transmission electron microscope, and scanning electron microscope (SEM). In addition, we conducted a comparative analysis of these two techniques, evaluating their suitability and efficiency in imaging nanoscale structures. In this study, we found that confocal-SEM correlation is more applicable for imaging exosomes. Moreover, we observed that exosomes were found in clusters in confocal-SEM correlation.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Light microscopy (LM) covers a relatively wide area and is suitable for observing the entire neuronal network. However, resolution of LM is insufficient to identify synapses and determine whether neighboring neurons are connected via synapses. In contrast, the resolution of electron microscopy (EM) is sufficiently high to detect synapses and is useful for identifying neuronal connectivity; however, serial images cannot easily show the entire morphology of neurons, as EM covers a relatively narrow region. Thus, covering a large area requires a large dataset. Furthermore, the three-dimensional (3D) reconstruction of neurons by EM requires considerable time and effort, and the segmentation of neurons is laborious. Correlative light and electron microscopy (CLEM) is an approach for correlating images obtained via LM and EM. Because LM and EM are complementary in terms of compensating for their shortcomings, CLEM is a powerful technique for the comprehensive analysis of neural circuits. This review provides an overview of recent advances in CLEM tools and methods, particularly the fluorescent probes available for CLEM and near-infrared branding technique to match LM and EM images. We also discuss the challenges and limitations associated with contemporary CLEM technologies.