Electron Microscopy at Scale.

The essential details of cellular interactions at synaptic level in the brain are still largely unknown. In this issue, Kasthuri et al. report new experimental and computational technologies for large-scale electron microscopy data collection and analysis, and through saturated reconstruction uncover synaptic connectional specificity that cannot be predicted by simple axonal-dendritic proximity.

Saturated Reconstruction of a Volume of Neocortex.

We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and mitochondria) are rendered and itemized in a database. We explore these data to study physical properties of brain tissue. For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters' rule). This online minable database provides general access to the intrinsic complexity of the neocortex and enables further data-driven inquiries.

High-definition imaging using line-illumination modulation microscopy.

The microscopic visualization of large-scale three-dimensional (3D) samples by optical microscopy requires overcoming challenges in imaging quality and speed and in big data acquisition and management. We report a line-illumination modulation (LiMo) technique for imaging thick tissues with high throughput and low background. Combining LiMo with thin tissue sectioning, we further develop a high-definition fluorescent micro-optical sectioning tomography (HD-fMOST) method that features an average signal-to-noise ratio of 110, leading to substantial improvement in neuronal morphology reconstruction. We achieve a >30-fold lossless data compression at a voxel resolution of 0.32 × 0.32 × 1.00 µm3, enabling online data storage to a USB drive or in the cloud, and high-precision (95% accuracy) brain-wide 3D cell counting in real time. These results highlight the potential of HD-fMOST to facilitate large-scale acquisition and analysis of whole-brain high-resolution datasets.

The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications.

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Best Practices and Progress in Precision-Cut Liver Slice Cultures.

Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell-cell and cell-matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.

Label-Free Multiphoton Microscopy: Much More Than Fancy Images.

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy.

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu's method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.

Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Moving human muscle physiology research forward: an evaluation of fiber type-specific protein research methodologies.

Human skeletal muscle is a heterogeneous tissue composed of multiple fiber types that express unique contractile and metabolic properties. While analysis of mixed fiber samples predominates and holds value, increasing attention has been directed toward studying proteins segregated by fiber type, a methodological distinction termed "fiber type-specific." Fiber type-specific protein studies have the advantage of uncovering key molecular effects that are often missed in mixed fiber homogenate studies but also require greater time and resource-intensive methods, particularly when applied to human muscle. This review summarizes and compares current methods used for fiber type-specific protein analysis, highlighting their advantages and disadvantages for human muscle studies, in addition to recent advances in these techniques. These methods can be grouped into three categories based on the initial processing of the tissue: 1) muscle-specific fiber homogenates, 2) cross sections of fiber bundles, and 3) isolated single fibers, with various subtechniques for performing fiber type identification and protein quantification. The relative implementation for each unique methodological approach is analyzed from 83 fiber type-specific studies of proteins in live human muscle found in the literature to date. These studies have investigated several proteins involved in a wide range of cellular functions that are important to muscle tissue. The second half of this review summarizes key findings from this ensemble of fiber type-specific human protein studies. We highlight examples of where this analytical approach has helped to improve understanding of important physiological topics such as insulin sensitivity, muscle hypertrophy, muscle fatigue, and adaptation to different exercise programs.

Method for Combined Observation of Serial Sections of Stented Arteries Embedded in Resin by Light Microscopy and Transmission Electron Microscopy.

We have developed a new method for obtaining information on whole tissues by light microscopy (LM) and ultrastructural features by transmission electron microscopy (TEM). This method uses serial sections of a stented artery embedded in resin. Stents were implanted in porcine coronary arteries in this study. The heart was perfusion fixed in a 2% paraformaldehyde and 1.25% glutaraldehyde mixed solution. The stented artery was then removed, fixed in 1% osmium, embedded in Quetol 651 resin, and sectioned serially. For LM, the black color of osmium was removed from the section by immersion in periodic acid and hydrogen peroxide after deplasticization. These sections were stained with hematoxylin and eosin and Elastica-Masson trichrome stain. For TEM, thin sections were re-embedded in Quetol 812 resin by the resupinate method and cut into ultrathin sections. A clear, fine structure was obtained, and organelles, microvilli, and cell junctions in the endothelium were easily observed. The combined observation of adjacent specimens by LM and TEM enabled us to relate histopathological changes in the millimeter scale to those in the nanometer scale.

High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation.

A longstanding limitation of imaging with serial block-face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block-face due to image jitter. Typically, variable-pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal-to-noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block-face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block-face ultramicrotome. This system enables the application of nitrogen gas precisely over the block-face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high-resolution block-face imaging of even the most charge prone of epoxy-embedded biological samples.

Evaluation of laser ablation microtomy for correlative microscopy of hard tissues.

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 µm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy.

Three-Dimensional Reconstruction of Erythrocytes Using the Novel Method For Corrective Realignment of the Transmission Electron Microscopy Cross-Section Images.

The detailed kinetics study of erythrocyte deformability is useful for the early diagnosis of blood diseases and for monitoring the blood rheology. Present solutions for a three-dimensional (3D) reconstruction of erythrocytes has a limited potential. This study aimed to use erythrocyte transmission electron images (ETIs) to evaluate the morphological relationship between adjacent ETIs and generate erythrocytes 3D model. First, ultrathin serial sections of skeletal muscle tissue were obtained using an ultramicrotome. Further, the set of ETIs in a capillary were captured by transmission electron microscopy. The images were aligned by translations and rotations using custom software to optimize the morphological relationship between adjacent ETIs. These coordinate transformations exploit the unique principal axis of inertia of each image to define the body coordinate system and hence provide the means to accurately reconnect the adjacent ETIs. The sum of the distances between the corresponding points on the boundary of adjacent ETIs was minimized and, further, was optimized by using physiological relationship between the adjacent ETIs. The analysis allowed to define precise virtual relationship between the adjacent erythrocytes. Finally, extracted erythrocytes' cross-section images allowed to generate 3D model of the erythrocytes.

Reliability of cartilage digestion and FDA-EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts.

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA-EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA-EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA-EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.

Large scale three-dimensional reconstruction of an entire Caenorhabditis elegans larva using AutoCUTS-SEM.

Large scale, high resolution three dimensional (3D) ultrastructural reconstruction of cells and tissues has become increasingly important to our understanding of complex biological systems. There have been a few partial 3D ultra-structures of Caenorhabditis elegans (C. elegans) reported, however 3D reconstruction of a whole nematode has never been achieved. Here, we independently developed a technique called automatic collector of ultrathin sections scanning electron microscopy and using this methodology, generated a 3D reconstruction of an entire C. elegans larva with 100 nm axial and 15 nm lateral resolution. Compared to the current available ATUM (automated tape-collecting ultramicrotome) technique, our work provides another alternative complete solution that can be applied to obtain large scale 3D ultrastructure of tissues. Our workflow includes an automated hardware system for high throughput serial section collection, a software package for automatic SEM imaging, and an image reconstruction program. These combined techniques can now be used together to rapidly provide access to understand the anatomy of the whole nematodes.

Raster-Mode Continuous-Flow Liquid Microjunction Mass Spectrometry Imaging of Proteins in Thin Tissue Sections.

Mass spectrometry imaging by use of continuous-flow liquid microjunction sampling at discrete locations (array mode) has previously been demonstrated. In this Letter, we demonstrate continuous-flow liquid microjunction mass spectrometry imaging of proteins from thin tissue sections in raster mode and discuss advantages (a 10-fold reduction in analysis time) and challenges (suitable solvent systems, data interpretation) of the approach. Visualization of data is nontrivial, requiring correlation of solvent-flow, mass spectral data acquisition rate, data quality, and liquid microjunction sampling area. The latter is particularly important for determining optimum pixel size. The minimum achievable pixel size is related to the scan time of the instrument used. Here we show a minimum achievable pixel size of 50 µm (x-dimension) when using an Orbitrap Elite; however a pixel size of 600 µm is recommended in order to minimize the effects of oversampling on image accuracy.

Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 µm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal ß-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 µm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.

Volume scanning electron microscopy for imaging biological ultrastructure.

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre-scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error-prone manual tasks of cutting and handling large numbers of sections, and imaging them one-by-one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z-resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block-face electron microscopy (SBEM), focused ion beam SEM (FIB-SEM) and automated tape-collecting ultramicrotome SEM (ATUM-SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.

Multiparametric Analysis of the Tumor Microenvironment: Hypoxia Markers and Beyond.

We have established a novel in situ protein analysis pipeline, which is built upon highly sensitive, multichannel immunofluorescent staining of paraffin sections of human and xenografted tumor tissue. Specimens are digitized using slide scanners equipped with suitable light sources and fluorescence filter combinations. Resulting digital images are subsequently subjected to quantitative image analysis using a primarily object-based approach, which comprises segmentation of single cells or higher-order structures (e.g., blood vessels), cell shape approximation, measurement of signal intensities in individual fluorescent channels and correlation of these data with positional information for each object. Our approach could be particularly useful for the study of the hypoxic tumor microenvironment as it can be utilized to systematically explore the influence of spatial factors on cell phenotypes, e.g., the distance of a given cell type from the nearest blood vessel on the cellular expression of hypoxia-associated biomarkers and other proteins reflecting their specific state of activation or function. In this report, we outline the basic methodology and provide an outlook on possible use cases.