Serial Electron Microscopy Research Articles

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004.Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion,which are critical for a holistic understanding of the Leishmania life cycle.

A Convenient All-Cell Optical Imaging Method Compatible with Serial SEM for Brain Mapping.

The mammalian brain, with its complexity and intricacy, poses significant challenges for researchers aiming to understand its inner workings. Optical multilayer interference tomography (OMLIT) is a novel, promising imaging technique that enables the mapping and reconstruction of mesoscale all-cell brain atlases and is seamlessly compatible with tape-based serial scanning electron microscopy (SEM) for microscale mapping in the same tissue. However, currently, OMLIT suffers from imperfect coatings, leading to background noise and image contamination. In this study, we introduced a new imaging configuration using carbon spraying to eliminate the tape-coating step, resulting in reduced noise and enhanced imaging quality. We demonstrated the improved imaging quality and validated its applicability through a correlative light-electron imaging workflow. Our method successfully reconstructed all cells and vasculature within a large OMLIT dataset, enabling basic morphological classification and analysis. We also show that this approach can perform effectively on thicker sections, extending its applicability to sub-micron scale slices, saving sample preparation and imaging time, and increasing imaging throughput. Consequently, this method emerges as a promising candidate for high-speed, high-throughput brain tissue reconstruction and analysis. Our findings open new avenues for exploring the structure and function of the brain using OMLIT images.

Two subcompartments of the smooth endoplasmic reticulum in perisynaptic astrocytic processes: ultrastructure and distribution in hippocampal and neocortical synapses

Perisynaptic astrocytic processes involved in the tripartite synapse functioning respond to its activation by local depolarization with calcium release from the intracellular stores inside nodes of astrocytic processes and develop local and generalized calcium events. However, based on the first electron microscopy studies a point of view was formed that terminal astrocytic lamellae are devoid of any organelles, including the main astrocytic calcium store - the cisternae of the smooth endoplasmic reticulum. Indeed, analysis of smooth endoplasmic reticulum cisternae could be limited by their weak electron contrast, the studying of astrocytic processes on single sections, and insufficient optical resolution of the equipment used. Here, by using serial section transmission electron microscopy and 3D reconstructions, we analyzed astrocytic processes in murine hippocampal and cortical synapses. As a result of unit membranes contrast enhancement, it was shown for the first time that perisynaptic processes of astrocytes with a morphology of thin branchlets contain two types of smooth endoplasmic reticulum cisternae and microvesicles. Unlike branchlets, membrane organelles inside terminal lamellae were comprised by only short fragments of thin smooth endoplasmic reticulum cister-nae and microvesicles, whose groups tend to be located in close proximity to active zones of the most active synapses. We speculate both on reliability of the alternative methods in electron microscopy while studying astrocytic microenvironment of synapses and structure-function aspects of smooth endoplasmic reticulum cisternae compartmentalization inside the perisynaptic processes of astrocytes.

Direct association with the vascular basement membrane is a frequent feature of myelinating oligodendrocytes in the neocortex

BackgroundOligodendrocyte lineage cells interact with the vasculature in the gray matter. Physical and functional interactions between blood vessels and oligodendrocyte precursor cells play an essential role in both the developing and adult brain. Oligodendrocyte precursor cells have been shown to migrate along the vasculature and subsequently detach from it during their differentiation to oligodendrocytes. However, the association of mature oligodendrocytes with blood vessels has been noted since the discovery of this glial cell type almost a century ago, but this interaction remains poorly explored.ResultsHere, we systematically investigated the extent of mature oligodendrocyte interaction with the vasculature in mouse brain. We found that ~ 17% of oligodendrocytes were in contact with blood vessels in the neocortex, the hippocampal CA1 region and the cerebellar cortex. Contacts were made mainly with capillaries and sparsely with larger arterioles or venules. By combining light and serial electron microscopy, we demonstrated that oligodendrocytes are in direct contact with the vascular basement membrane, raising the possibility of direct signaling pathways and metabolite exchange with endothelial cells. During experimental remyelination in the adult, oligodendrocytes were regenerated and associated with blood vessels in the same proportion compared to control cortex, suggesting a homeostatic regulation of the vasculature-associated oligodendrocyte population.ConclusionsBased on their frequent and close association with blood vessels, we propose that vasculature-associated oligodendrocytes should be considered as an integral part of the brain vasculature microenvironment. This particular location could underlie specific functions of vasculature-associated oligodendrocytes, while contributing to the vulnerability of mature oligodendrocytes in neurological diseases.

Prolonged exposure of neonatal mice to sevoflurane leads to hyper-ramification in microglia, reduced contacts between microglia and synapses, and defects in adult behavior.

Prolonged exposure to general anesthetics during development is known to cause neurobehavioral abnormalities, but the cellular and molecular mechanisms involved are unclear. Microglia are the resident immune cells in the central nervous system and play essential roles in normal brain development. In the study, postnatal day 7 (P7) C57BL/6 mice were randomly assigned to two groups. In the sevoflurane (SEVO), mice were exposed to 2.5% sevoflurane for 4 h. In the control group, mice were exposed to carrier gas (30% O2/70% N2) for 4 h. Fixed brain slices from P14 to P21 mice were immunolabeled for ionized calcium-binding adapter molecule 1 (IBA-1) to visualize microglia. The morphological analysis of microglia in the somatosensory cortex was performed using ImageJ and Imaris software. Serial block face scanning electron microscopy (SBF-SEM) was performed to assess the ultrastructure of the microglia and the contacts between microglia and synapse in P14 and P21 mice. The confocal imaging of brain slices was performed to assess microglia surveillance in resting and activated states in P14 and P21 mice. Behavioral tests were used to assess the effect of microglia depletion and repopulation on neurobehavioral abnormalities caused by sevoflurane exposure. The prolonged exposure of neonatal mice to sevoflurane induced microglia hyper-ramification with an increase in total branch length, arborization area, and branch complexity 14 days after exposure. Prolonged neonatal sevoflurane exposure reduced contacts between microglia and synapses, without affecting the surveillance of microglia in the resting state or responding to laser-induced focal brain injury. These neonatal changes in microglia were associated with anxiety-like behaviors in adult mice. Furthermore, microglial depletion before sevoflurane exposure and subsequent repopulation in the neonatal brain mitigated anxiety-like behaviors caused by sevoflurane exposure. Our experiments indicate that general anesthetics may harm the developing brain, and microglia may be an essential target of general anesthetic-related developmental neurotoxicity.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 μm in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cN functions in mammals.

Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex

Astrocytes are increasingly understood to be important regulators of central nervous system (CNS) function in health and disease; yet, we have little quantitative understanding of their complex architecture. While broad categories of astrocytic structures are known, the discrete building blocks that compose them, along with their geometry and organizing principles, are poorly understood. Quantitative investigation of astrocytic complexity is impeded by the absence of high-resolution datasets and robust computational approaches to analyze these intricate cells. To address this, we produced four ultra-high-resolution datasets of mouse cerebral cortex using serial electron microscopy and developed astrocyte-tailored computer vision methods for accurate structural analysis. We unearthed specific anatomical building blocks, structural motifs, connectivity hubs, and hierarchical organizations of astrocytes. Furthermore, we found that astrocytes interact with discrete clusters of synapses and that astrocytic mitochondria are distributed to lie closer to larger clusters of synapses. Our findings provide a geometrically principled, quantitative understanding of astrocytic nanoarchitecture and point to an unexpected level of complexity in how astrocytes interact with CNS microanatomy.

3D reconstruction shows mouse skeletal muscle may decline in function across aging due to the MICOS complex

Mitochondria are active organelles, undergoing harmonized cycles of fission and fusion, driven by ‘mitochondrial dynamics,’ to retain their morphology, distribution, and size. Mitochondrial dynamics has emerged as a critical process in maintaining cellular homeostasis. Interestingly, it has been long respected that a decrease in mitochondrial function escorts aging in specific tissues. For example, skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. This decrease might, in turn, contribute to the observed age-dependent decline in organ function by mutations or alterations in mitochondrial fusion and fission proteins associated with several diseases. However, new players in regulating mitochondrial shape and cristae shape have been recently studied, such as the mitochondrial contact site and cristae organizing system (MICOS) complex. The MICOS complex is a multi-protein interaction hub that helps define cristate and mitochondria architecture. However, the MICOS complex has not been implicated in regulating organelle structure changes during aging. Thus, we hypothesized that loss of the MICOS complex during aging may increase mitochondrial fragmentation, decrease nanotunnels, and alter cristae morphology. To do this, we examined the three-dimensional morphology of mitochondria networks in young (3-month) and aged (2-year) murine gastrocnemius muscle via serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. We found differences in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, MICOS dynamics, and gene expression in skeletal muscle during aging. We also found an association between OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Furthermore, the loss of the MICOS complex was linked with decreased oxidative capacity and altered mitochondrial metabolism. Potentially, this suggests a novel relationship between the MICOS complex and aging in skeletal muscle.

Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Distinctive synaptic structural motifs link excitatory retinal interneurons to diverse postsynaptic partner types

Neurons make converging and diverging synaptic connections with distinct partner types. Whether synapses involving separate partners demonstrate similar or distinct structural motifs is not yet well understood. We thus used serial electron microscopy in mouse retina to map output synapses of cone bipolar cells (CBCs) and compare their structural arrangements across bipolar types and postsynaptic partners. Three presynaptic configurations emerge-single-ribbon, ribbonless, and multiribbon synapses. Each CBC type exploits these arrangements in a unique combination, a feature also found among rabbit ON CBCs. Though most synapses are dyads, monads and triads are also seen. Altogether, mouse CBCs exhibit at least six motifs, and each CBC type uses these in a stereotypic pattern. Moreover, synapses between CBCs and particular partner types appear biased toward certain motifs. Our observations reveal synaptic strategies that diversify the output within and across CBC types, potentially shaping the distinct functions of retinal microcircuits.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Improving the Neural Segmentation of Blurry Serial SEM Images by Blind Deblurring.

Serial scanning electron microscopy (sSEM) has recently been developed to reconstruct complex largescale neural connectomes, through learning-based instance segmentation. However, blurry images are inevitable amid prolonged automated data acquisition due to imprecision in autofocusing and autostigmation, which impose a great challenge to accurate segmentation of the massive sSEM image data. Recently, learning-based methods, such as adversarial learning and supervised learning, have been proven to be effective for blind EM image deblurring. However, in practice, these methods suffer from the limited training dataset and the underrepresentation of high-resolution decoded features. Here, we propose a semisupervised learning guided progressive decoding network (SGPN) to exploit unlabeled blurry images for training and progressively enrich high-resolution feature representation. The proposed method outperforms the latest deblurring models on real SEM images with much less ground truth input. The improvement of the PSNR and SSIM is 1.04 dB and 0.086, respectively. We then trained segmentation models with deblurred datasets and demonstrated significant improvement in segmentation accuracy. The A-rand (Bogovic et al. 2013) decreased by 0.119 and 0.026, respectively, for 2D and 3D segmentation.

Ultrastructural effects of sleep and wake on the parallel fiber synapses of the cerebellum.

Multiple evidence in rodents shows that the strength of excitatory synapses in the cerebral cortex and hippocampus is greater after wake than after sleep. The widespread synaptic weakening afforded by sleep is believed to keep the cost of synaptic activity under control, promote memory consolidation, and prevent synaptic saturation, thus preserving the brain's ability to learn day after day. The cerebellum is highly plastic and the Purkinje cells, the sole output neurons of the cerebellar cortex, are endowed with a staggering number of excitatory parallel fiber synapses. However, whether these synapses are affected by sleep and wake is unknown. Here, we used serial block face scanning electron microscopy to obtain the full 3D reconstruction of more than 7000 spines and their parallel fiber synapses in the mouse posterior vermis. This analysis was done in mice whose cortical and hippocampal synapses were previously measured, revealing that average synaptic size was lower after sleep compared to wake with no major changes in synapse number. Here, instead, we find that while the average size of parallel fiber synapses does not change, the number of branched synapses is reduced in half after sleep compared to after wake, corresponding to ~16% of all spines after wake and ~8% after sleep. Branched synapses are harbored by two or more spines sharing the same neck and, as also shown here, are almost always contacted by different parallel fibers. These findings suggest that during wake, coincidences of firing over parallel fibers may translate into the formation of synapses converging on the same branched spine, which may be especially effective in driving Purkinje cells to fire. By contrast, sleep may promote the off-line pruning of branched synapses that were formed due to spurious coincidences.

Immunohistochemical characterization of bipolar cells in four distantly related avian species.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

Methods to expose subsurface objects of interest identified from 3D imaging: The intermediate sample preparation stage in the correlative microscopy workflow.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows.