ATUM-FIB microscopy for targeting and multiscale imaging of rare events in mouse cortex.

Here, we describe a detailed workflow for ATUM-FIB microscopy, a hybrid method that combines serial-sectioning scanning electron microscopy (SEM) with focused ion beam SEM (FIB-SEM). This detailed protocol is optimized for mouse cortex samples. The main processing steps include the generation of semi-thick sections from sequentially cured resin blocks using a heated microtomy approach. We demonstrate the different imaging modalities, including serial light and electron microscopy for target recognition and FIB-SEM for isotropic imaging of regions of interest. For complete details on the use and execution of this protocol, please refer to Kislinger et al. (2020).

Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy.

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays.

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

Ultra-thin sectioning and grinding of epoxy plastinated tissue.

With classical sheet plastination techniques such as E12, the level and thickness of the freeze-cut sections decide on what is visible in the final sheet plastinated sections. However, there are other plastination techniques available where we can look for specific anatomical structures through the thickness of the tissue. These techniques include sectioning and grinding of plastinated tissue blocks or thick slices. The ultra-thin E12 technique, unlike the classic E12 technique, starts with the plastination of a large tissue block. High temperatures (30-60°C) facilitate the vacuum-forced impregnation by decreasing the viscosity of the E12 and increasing the vapour pressure of the intermediary solvent. By sectioning the cured tissue block with a diamond band saw plastinated sections with a thickness of <300 µm can be obtained. The thickness of plastinated sections can be further reduced by grinding. Resulting sections of <100 µm are suitable for histological staining and microscopic studies. Anatomical structures of interest in thick plastinate slices can be followed by variable manual grinding in a method referred to as Tissue Tracing Technique (TTT). In addition, the tissue thickness can be adapted to the transparency or darkness of tissue types in different regions of the same plastinated section. The aim of this study was to evaluate the advantages of techniques based on sectioning and grinding of plastinated tissue (E12 ultra-thin and TTT) compared to conventional sheet-forming techniques (E12).

Histological comparison between laser microtome sections and ground specimens of implant-containing tissues.

Evaluation of bone regeneration and peri-implant bone apposition can only be accomplished using laboratory techniques that allow assessment of decalcified hard tissue. It is known that 5-15µm thick sections can be prepared with the cutting-grinding technique, but their production causes a high material loss (≥0.5mm) between two sections and requires years of training and experience. With the development of the laser microtome it has become possible to cut decalcified bone without high sample material loss. Many scientific publications deal with the application possibilities of the individual methods So far, there is no comparison work between the cutting-grinding technique and laser microtomy. For this reason, new tissue sections were prepared by laser microtome and analyzed histologically from samples that had been previously been prepared by the cutting-grinding technique. Using both methods, it could be demonstrated that the different implants were completely surrounded by a connective tissue layer. In sections (50-100µm) produced by the routine cutting-grinding technique, magnifications up to 20× revealed no detailed histological information because cell structures could not be clearly identified. By contrast, laser microtome sections (10µm) revealed these information as e.g. osteocytes are already clearly visible at 10× magnification. Furthermore, the interface between implant and the surrounding bone could be clearly demonstrated due to visible demarcation between a capsule and connective tissue. At the histological level, laser microtome sections were clearly superior at thicknesses ≥30µm compared to sections produced by the cutting-grinding technique. In addition, laser microtomy has the advantages of time saving and markedly reduced sample loss, especially in cases of the production of serial sections.

Large Volume Electron Microscopy and Neural Microcircuit Analysis.

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

Morphological description of male genital organs of Marca's marmoset (Mico marcai).

Morphological characterisation of the genital organs of primates may bring significant contributions to the understanding of different reproductive behaviours and support new conservation strategies. However, relevant or detailed descriptions of genital morphology of several primate species are still lacking. This study describes the gross and microscopic anatomy of the internal and external genitalia of Marca's marmoset (Mico marcai). The same organs described in other primate species were identified here, but some anatomical particularities were detected, such as absence of a dartos tunic, presence of a vas deferens ampulla, absence of spongious erectile tissue in the pelvic urethra, separation of prostate gland lobes by a longitudinal sulcus and lack of septation in the corpus cavernosus and spongiosus at the level of the shaft and free portion of the penis. Keratinised type 1 spicules arising from epidermal or dermal projections were found in the free portion of the penis. Microscopic analysis revealed a small bone (baculum) consisting of peripheral compact bone and a central, non-ossified area filled with vascular tissue at the distal end of this portion of the penis. Results of this study may support further comparative studies of primates' reproductive ecology.

Application of Tissue Microarray in Esophageal Adenocarcinoma.

Tissue microarray technology could allow immunohistochemical staining or in situ hybridization on hundreds of different tissue samples simultaneously. It allows faster analysis and considerably reducing costs incurred in staining. The technique also provides a high-throughput analysis of multiple tissues for the different types of research. In the literature, many researches of esophageal adenocarcinoma use tissue microarray to enhance the output. In this chapter, we have a brief overview of tissue microarray technologies, the advantages and disadvantages of tissue microarray, and related troubleshootings.

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.

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

Preparation of Scaffolds from Decellularized Testicular Matrix.

Biological scaffolds composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Here we describe a simple and robust methodology for the preparation of decellularized testicular matrix (DTM) scaffolds with minimal damage to the native three-dimensional structure and tissue-specific ECM components. Such DTM scaffolds can help to gain a better insight into the molecular composition and function of testicular ECM and to develop new tissue engineering approaches to treat various types of male fertility disorders.

A Novel Mohs Precision Tool.

BACKGROUND: Effective treatment by Mohs micrographic surgery requires preparation of high-quality slides. OBJECTIVE: To examine a novel tissue alignment device designed to address variability in tissue processing because of excessive sample trimming. MATERIALS AND METHODS: A device was designed to account for angular errors and unparalleled tissue embedding. A retrospective chart review was performed both with and without the use of the device over the course of a 4-year period (2012-2015). RESULTS: Between January 1, 2012, and June 10, 2014, before device implementation, mean number of stages per case was 1.65 (n = 3,680) and mean number of surgeries per day was 6.34 (n = 640). Between June 11, 2014, and October 02, 2015, with device implemented, the average number of stages per case between decreased to 1.58 (n = 2,562) and the number of daily surgeries increased to 7.05 (n = 358). This represents a significant decrease in number of stages per case by 0.07 stages (95% CI: -0.01 to -0.13, p = .02), as well as an increase in the number of cases per day by 0.71 cases (95% CI: 0.12-1.3, p < .01). CONCLUSION: Slide preparation using the novel alignment device may result in less tissue waste and more cases being performed daily.

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.

Preparation of viable adult ventricular myocardial slices from large and small mammals.

This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-µm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation-contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in ∼4 h.

Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration.

High-resolution images obtained from plant tissues processed for light microscopy, transmission electron microscopy, and immunohistochemistry have provided crucial links between plant subcellular structure and physiology during photorespiration as well as the impact of photorespiration on plant evolution and development. This chapter presents established protocols to guide researchers in the preparation of plant tissues for high-resolution imaging with a light and transmission electron microscope and detection of proteins using immunohistochemistry. Discussion of concepts and theory behind each step in the process from tissue preservation to staining of resin-embedded tissues is included to enhance the understanding of all steps in the procedure. We also include a brief protocol for quantification of cellular parameters from high-resolution images to help researchers rigorously test hypotheses.

3D printed pathological sectioning boxes to facilitate radiological-pathological correlation in hepatectomy cases.

Radiogenomics promises to identify tumour imaging features indicative of genomic or proteomic aberrations that can be therapeutically targeted allowing precision personalised therapy. An accurate radiological-pathological correlation is critical to the process of radiogenomic characterisation of tumours. An accurate correlation, however, is difficult to achieve with current pathological sectioning techniques which result in sectioning in non-standard planes. The purpose of this work is to present a technique to standardise hepatic sectioning to facilitateradiological-pathological correlation. We describe a process in which three-dimensional (3D)-printed specimen boxes based on preoperative cross-sectional imaging (CT and MRI) can be used to facilitate pathological sectioning in standard planes immediately on hepatic resection enabling improved tumour mapping. We have applied this process in 13 patients undergoing hepatectomy and have observed close correlation between imaging and gross pathology in patients with both unifocal and multifocal tumours.

Custom-built tools for the study of deer antler biology.

Deer antlers can be developed into multiple novel models to study growth and development of tissues for biomedical research. To facilitate this process, we have invented and further refined five custom-built tools through three decades of antler research. These are: 1. Pedicle growth detector to pinpoint the timing when pedicle growth is initiated, thus stimuli for pedicle and first antler formation can be investigated and identified. 2. Thin periosteum slice cutter to thinly slice (0.2mm or 0.7 mm thick) a whole piece of antlerogenic periosteum (AP) or pedicle periosteum (PP), which facilitates gene delivery into cells resident in these tissues, thus making transgenic antlers possible. 3. The porous periosteum multi-needle punch to effectively loosen the dense AP or PP tissue. This allows most cells of the periosteum to come into direct contact with treating solutions, thus making artificial manipulation of antler development possible. 4. The intra-dermal pocket maker to cut the thin dermal tissue (less than 2 mm in thickness) of a male deer calf horizontally into two layers to make an intra-dermal pocket. This allows loading of AP tissue intra-dermally to test the theory of "antler stem cell niche" in vivo. 5. The sterile periosteum sampling system to allow aseptic collection of the AP, PP or the antler growth centre tissue on farm, thus allowing antler generation, regeneration or rapid growth to be investigated in vitro. Overall, we believe the application of contemporary cellular and molecular biological techniques coupled with these custom-built tools would greatly promote the establishment of this unique and novel model for the benefits of biomedical research, and hence human health.

RNA isolation from precision-cut lung slices (PCLS) from different species.

BACKGROUND: Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures. RESULTS: We established an optimized protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis. CONCLUSIONS: In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.

Jan Evangelista Purkyne (1787-1869) and his instruments for microscopic research in the field of neuroscience.

The findings obtained by the famous nineteenth-century Czech scientist Jan Evangelista Purkyne (1787-1869) in the field of microscopic structure of animal and human tissues, including the brain, spinal cord, and nerves, have already been described in depth in a number of older and newer publications. The present article contains an overview of the instruments and tools that Purkyne and his assistants used for microscopic research of tissue histology. Some of these instruments were developed either by Purkyne alone, such as the microtomic compressor, or together with his assistant Adolph Oschatz, such as the microtome. A brief overview of the development of the cutting engines suggests that the first microtome, a prototype of modern sliding microtomes, was designed and constructed under the supervision of Purkyne at the Institute of Physiology in Wroclaw. Purkyne and his assistants, thus, not only obtained important findings of animal and human nervous and other tissues but also substantially contributed to the development of instruments and tools for their study, a fact often forgotten today.

The Primary Root of Sorghum bicolor (L. Moench) as a Model System to Study Brassinosteroid Signaling in Crops.

Roots anchor plants to the soil and are essential for a successful plant growth and adaptation to the environment. Research on the primary root in the plant model system Arabidopsis thaliana has yielded important advances in the molecular and cellular understanding of root growth and development. Several studies have uncovered how the hormones brassinosteroids (BRs) control cell cycle and differentiation programs through different cell-specific signaling pathways that are key for root growth and development. Currently, an important challenge resides in the translation of the current knowledge on Arabidopsis roots into agronomically valuable species to improve the agricultural production and to meet the food security goals of the millennium. In this chapter, we characterize the primary root apex of the cereal Sorghum bicolor (L. Moench) (sorghum), analyze the physiological response of sorghum roots to BRs, and examine the phylogeny of the BRASSINOSTEROID INSENSITIVE1-like receptor family in Arabidopsis and its orthologous genes in sorghum. Overall, we support the use of sorghum as a suitable crop model species for the study of BR signaling in root growth and development. The methods presented enable any laboratory worldwide to use sorghum primary roots as a favorite organ for the study of growth and development in crops.