Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 angstrom using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents pro-tein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Some living eukaryotes during and after scanning electron microscopy

Electron microscopy (EM) is an essential imaging method in biological sciences. Since biological specimens are exposed to radiation and vacuum conditions during EM observations, they die due to chemical bond breakage and desiccation. However, some organisms belonging to the taxa of bacteria, fungi, plants, and animals (including beetles, ticks, and tardigrades) have been reported to survive hostile scanning EM (SEM) conditions since the onset of EM. The surviving organisms were observed (i) without chemical fixation, (ii) after mounting to a precooled cold stage, (iii) using cryo-SEM, or (iv) after coating with a thin polymer layer, respectively. Combined use of these techniques may provide a better condition for preservation and live imaging of multicellular organisms for a long time beyond live-cell EM.

Immuno-correlative light and electron microscopy (iCLEM) using STEM v1

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy and the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-STEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-STEM involves the examination of ultrathin tissue sections (62-70 nm) using immunofluorescence labelling and subsequent SEM imaging to obtain a high resolution overview of these sections with greater morphological detail compared to iCLEM-SEM. This protocol should be of particular interest to EM facilities with SEM, but not TEM access.

High-precision targeting workflow for volume electron microscopy

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

Invaginating Structures in Synapses – Perspective

Invaginating structures are common in the synapses of most animals. However, the details of these invaginating structures remain understudied in part because they are not well resolved in light microscopy and were often misidentified in early electron microscope (EM) studies. Utilizing experimental techniques along with the latest advances in microscopy, such as focused ion beam-scanning EM (FIB-SEM), evidence is gradually building to suggest that the synaptic invaginating structures contribute to synapse development, maintenance, and plasticity. These invaginating structures are most elaborate in synapses mediating rapid integration of signals, such as muscle contraction, mechanoreception, and vision. Here we argue that the synaptic invaginations should be considered in future studies seeking to understand their role in sensory integration and coordination, learning, and memory. We review the various types of invaginating structures in the synapses and discuss their potential functions. We also present several new examples of invaginating structures from a variety of animals including Drosophila and mice, mainly using FIB-SEM, with which we trace the form and arrangement of these structures.

Immuno-correlative light and electron microscopy (iCLEM) using SEM v1

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-SEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-SEM provides a large quantifiable overview of 200 nm tissue sections without the presence of grid bars, and thicker sections enhance the immunofluorescent labelling.

Immuno-correlative light and electron microscopy (iCLEM) using TEM v1

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-TEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-TEM provides high resolution ultrastructural detail of cells, and consecutive ultrathin (62-70 nm) tissue sections can be examined using different labelling techniques involving the use of immunofluorescent and immunogold markers.

Preparation of large-scale digitization samples for automated electron microscopy of tissue and cell ultrastructure

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time- consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by automated acquisition of virtually all structures in a given EM section. Resulting datasets allow convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We therefore established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared to other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.

Characterization of Subcellular Organelles in Cortical Perisynaptic Astrocytes

Perisynaptic astrocytic processes (PAPs) carry out several different functions, from metabolite clearing to control of neuronal excitability and synaptic plasticity. All these functions are likely orchestrated by complex cellular machinery that resides within the PAPs and relies on a fine interplay between multiple subcellular components. However, traditional transmission electron microscopy (EM) studies have found that PAPs are remarkably poor of intracellular organelles, failing to explain how such a variety of PAP functions are achieved in the absence of a proportional complex network of intracellular structures. Here, we use serial block-face scanning EM to reconstruct and describe in three dimensions PAPs and their intracellular organelles in two different mouse cortical regions. We described five distinct organelles, which included empty and full endosomes, phagosomes, mitochondria, and endoplasmic reticulum (ER) cisternae, distributed within three PAPs categories (branches, branchlets, and leaflets). The majority of PAPs belonged to the leaflets category (~60%), with branchlets representing a minority (~37%). Branches were rarely in contact with synapses (<3%). Branches had a higher density of mitochondria and ER cisternae than branchlets and leaflets. Also, branches and branchlets displayed organelles more frequently than leaflets. Endosomes and phagosomes, which accounted for more than 60% of all the organelles detected, were often associated with the same PAP. Likewise, mitochondria and ER cisternae, representing ~40% of all organelles were usually associated. No differences were noted between the organelle distribution of the somatosensory and the anterior cingulate cortex. Finally, the organelle distribution in PAPs did not largely depend on the presence of a spine apparatus or a pre-synaptic mitochondrion in the synapse that PAPs were enwrapping, with some exceptions regarding the presence of phagosomes and ER cisternae, which were slightly more represented around synapses lacking a spine apparatus and a presynaptic mitochondrion, respectively. Thus, PAPs contain several subcellular organelles that could underlie the diverse astrocytic functions carried out at central synapses.

TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.