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.

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.

Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems.

Serial block face imaging with the scanning electron microscope has been developed as an alternative to serial sectioning and transmission electron microscopy for the ultrastructural analysis of the three-dimensional organization of cells and tissues. An ultramicrotome within the microscope specimen chamber permits sectioning and imaging to a depth of many microns within resin-embedded specimens. The technology has only recently been adopted by plant microscopists and here we describe some specimen preparation procedures suitable for plant tissue, suggested microscope imaging parameters and discuss the software required for image reconstruction and analysis.

A multicentre study of the precision and accuracy of the TruSlice and TruSlice Digital histological dissection devices.

BACKGROUND: Five key factors enabling a good surgical grossing technique include a flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. TruSlice and TruSlice Digital are new innovative tools based on a guillotine configuration. The TruSlice has plastic inserts whilst the TruSlice Digital has an electronic micrometre attached: both features enable these dissection factors to be controlled. The devices were assessed in five hospitals in the UK. MATERIAL AND METHODS: A total of 267 fixed tissue samples from 23 tissue types were analysed, principally the breast (n = 32) skin (30), rectum (28), colon (27) and cervix (17). Precision and accuracy were evaluated by measuring the defined thickness, and the consistency of achieving the defined thickness of tissue samples taken respectively. Both parameters were expressed as a total percentage of compliance for the cohort of samples accessed. RESULTS: Overall, the mean (standard deviation) score for precision was 81 (11) % whilst the accuracy score was 82 (11) % (both p < 0.05, chi-squared test), although this varied with type of tissue. Accuracy and precision were strongly correlated (rp = 0.83, p < 0.001). CONCLUSION: The TruSlice Digital devices offer an assured precision and accuracy performance which is reproducible across an assortment of tissue types. The use of a micrometre to set tissue slice thickness is innovative and should comply with laboratory accreditation requirements, alleviating concerns of how to tackle issues such as the 'measurement of uncertainty' at the grossing bench.

Elastic volume reconstruction from series of ultra-thin microscopy sections.

Anatomy of large biological specimens is often reconstructed from serially sectioned volumes imaged by high-resolution microscopy. We developed a method to reassemble a continuous volume from such large section series that explicitly minimizes artificial deformation by applying a global elastic constraint. We demonstrate our method on a series of transmission electron microscopy sections covering the entire 558-cell Caenorhabditis elegans embryo and a segment of the Drosophila melanogaster larval ventral nerve cord.

Towards the lean lab: the industry challenge.

In anatomic pathology, the current state encompassing the pre-analytic processes of tissue collection, handling, examination, preparation, processing, and storage are largely uncontrolled, inconsistently performed, and/or not standardized according to the sound scientific data. Pre-analytic defects result in nearly three-quarters of the problems in laboratory diagnostics. This is evident in quality surveys from well-respected institutions that document high miss rates in the required basics of information related to patient and tissue identity, let alone parameters documenting quality aspects related to the surgical specimen and its preservation. This talk will describe the historical approach to tissue processing and identify gaps from worldwide observations in current laboratory practices. It will also offer potential methodological and technological solutions and process improvements that laboratories may consider in serving the ultimate users of pathology information: the clinician and the patient. It illustrates the need for scientifically validated specimen guidelines and a performance based, standardized and documented "chain of custody" of the pre-analytical steps from the patient's body through fixation. For thought leaders and professional standard setters, opportunities for optimizing molecular studies exist in specimen collection, transfer, grossing, fixation, and decalcification protocols. In this evolving era of molecular profiling and personalized therapeutic decision-making, a well-reasoned and coordinated focus on pre-analytic processes that optimizes specimens for subsequent testing will result in: Improved specimen quality for molecular testing Improved accuracy of diagnostic and molecular test results Reduced Turnaroundtimes for same-day diagnosis Enhanced satisfaction of clinicians and patients.

Development of new and accurate measurement devices (TruSlice and TruSlice Digital) for use in histological dissection: an attempt to improve specimen dissection precision.

Histological dissection of human tissue has relied on conventional procedures, which have largely remained unchanged for decades. Practices to determine measurement parameters employed in these procedures have largely relied on the use of rulers and weighing scales. It is well documented in the scientific literature that both fixation and processing of tissue can significantly affect the viability of the of tissue sections both for tinctorial and immunocytochemical investigations. Both of these factors can be compounded in their negative effects by inappropriate sampling of tissue at histological cut up. There are five key factors to ensure good surgical grossing technique, flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. Meeting these factors implies the devices are fit for purpose. Here we describe an innovative approach to designing cut up devices to improve accuracy and precision, which take these five key requirements into consideration. The devices showed accuracy and precision, enabling tissue slices to be produced in a uniformly perpendicular fashion to within 2 mm in thickness and to enable consistency and reproducibility of performance across a series of tissue types. The application of a digital rule on one of these devices ensures accuracy and also enables quality control issues to be clearly assessed. As cellular pathology laboratories conform to ever increasing standards of compliance and performance in practice, the advent of assured precision and accuracy at cut up is awaited. Recommendations from accreditation bodies such as the United Kingdom Accreditation Service (UKAS) continue to push for improvements in this area of histological investigation. These newly designed devices may give the answers to these requirements and provide the impetus for a new generation of innovative equipment for histological dissection.

Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography.

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells - especially those in tissue - are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.

Evaluation of coils for imaging histological slides: signal-to-noise ratio and filling factor.

PURPOSE: To investigate the relative gain in sensitivity of five histology coils designed in-house to accommodate tissue sections of various sizes and compare with commercial mouse head coils. METHODS: The coil set was tailored to house tissue sections ranging from 5 to1000 µm encased in either glass slides or coverslips. RESULTS: Our simulations and experimental measurements demonstrated that although the sensitivity of this flat structure consistently underperforms relative to a birdcage head coil based on the gain expected from their respective filling factor ratios, our results demonstrate that it can still provide a remarkable gain in sensitivity. Our study also describes preparation protocols for freshly excised sections, as well as premounted tissue slides of both mouse and human specimens. Examples of the exceptional level of tissue detail and the near-perfect magnetic resonance imaging to light microscopic image coregistration are provided. CONCLUSION: The increase in filling factor achieved by the histology radiofrequency (RF) probe overcomes the losses associated with electric leaks inherent to this structure, leading to a 6.7-fold improvement in performance for the smallest coil implemented. Alternatively, the largest histology coil design exhibited equal sensitivity to the mouse head coil while nearly doubling the RF planar area coverage.

Development of a live tissue microtome: reflections of an amateur machinist.

Live circular tissue slices of nearly identical diameter and thickness can be generated from most tissues by the use of two instruments; a coring tool to cut cylindrical tissue cores which are subsequently sliced by a microtome into thin circular sections 5-8 mm in diameter and 50-300 microns thick. The sections are of very similar geometry permitting direct comparisons without normalization. Both instruments operate submerged in a cold isotonic medium that caries the cut slices outside the microtome. The slices are cut by a rapidly oscillating disposable Gillette blade mounted at an angle of 20 degrees to the vertical main axis of the tissue core. The latter is advanced against the oscillating blade by a weighted plunger pushing it against a screw adjustable limit plate that defines the desired slice thickness. Both instruments can be sterilized and slices can be obtained at a rate of approximately one every ten seconds.

Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing.

We describe a technique suitable for routine three-dimensional (3-D) analysis of mouse embryos that is based on episcopic fluorescence images captured during serial sectioning of wax-embedded specimens. We have used this procedure to describe the cardiac phenotype and associated blood vessels of trisomic 16 (Ts16) and Cited2-null mutant mice, as well as the expression pattern of an Myf5 enhancer/beta-galactosidase transgene. The consistency of the images and their precise alignment are ideally suited for 3-D analysis using video animations, virtual resectioning or commercial 3-D reconstruction software packages. Episcopic fluorescence image capturing (EFIC) provides a simple and powerful tool for analyzing embryo and organ morphology in normal and transgenic embryos.

High-throughput analysis of gene expression on tissue sections by in situ hybridization.

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.

Vertical optical sectioning using a magnetically driven confocal microscanner aimed for in vivo clinical imaging.

This paper presents a confocal microscanner for direct vertical optical sectioning of biological samples. Confocal imaging is performed by transverse (X-axis) and axial (Z-axis) scanning of a focused laser beam using an optical fiber and a microlens respectively. The actuators are fabricated by laser micromachining techniques and are driven by electromagnetic forces. Optical and mechanical performance of the system is predicted by simulation software packages and characterized by experimental measurements. The scanner has lateral resolution of 3.87 µm and axial resolution of 10.68 µm with a field of view of 145 µm in X and 190 µm in Z directions. Confocal imaging of a polymer layer deposited on a silicon wafer and onion epidermal cells is demonstrated.

Observation of three-dimensional internal structure of steel materials by means of serial sectioning with ultrasonic elliptical vibration cutting.

A three-dimensional (3D) internal structure observation system based on serial sectioning was developed from an ultrasonic elliptical vibration cutting device and an optical microscope combined with a high-precision positioning device. For bearing steel samples, the cutting device created mirrored surfaces suitable for optical metallography, even for long-cutting distances during serial sectioning of these ferrous materials. Serial sectioning progressed automatically by means of numerical control. The system was used to observe inclusions in steel materials on a scale of several tens of micrometers. Three specimens containing inclusions were prepared from bearing steels. These inclusions could be detected as two-dimensional (2D) sectional images with resolution better than 1 mum. A three-dimensional (3D) model of each inclusion was reconstructed from the 2D serial images. The microscopic 3D models had sharp edges and complicated surfaces.

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.

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).

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.

Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver.

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160 degrees C up to about -30 degrees C. The microtome is set for a cutting thickness of 540-1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method.

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca2+, and 10(-4) M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of less than 1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15 degrees K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5 - 10 micron, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was approximately 3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released. Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.