Resection status and margin control in intraoperative frozen sectioning analysis of oral squamous cell carcinoma.

PURPOSE: Intraoperative frozen section analysis (IFSA) is a well-established procedure for determining the intraoperative soft tissue resection status in patients with oral squamous cell carcinoma (OSCC). Margin status is a major predictor of the patient´s outcome, histologically free margins of ≥ 5 mm are demanded. This study evaluates the accuracy of IFSA, the impact of margin status and the impact of intraoperative margin revision on disease-free survival (DFS) and overall survival (OS). METHODS: This retrospective study included 213 patients with OSCC. IFSA results were compared with definitive histopathological reports, Kaplan-Meier analysis was performed. Cut-off values were calculated for resection margins considering known risk factors. RESULTS: IFSA showed positive margins in 8 cases (3.8%). Kaplan-Meier analysis revealed no significant differences for OS or DFS if R0-status was achieved by initial resection or immediate re-resection. Final histopathological evaluation revealed false-positive IFSA in 3/8 cases (37.5%) and false-negative IFSA in 1/205 cases (0.5%). Sensitivity was 83.3% and specificity was 98.6%. Analysis of optimal cut-off values showed no general need for larger resection margins in patients with risk factors. Cut-off values were slightly higher for patients with the risk factor alcohol consumption (7 mm for OS and DFS) or pN + ECS- disease (7 mm for DFS). Optimal cut-off values for tumour-margin-distance were around 6 mm. CONCLUSION: IFSA provides a valuable assessment method for intraoperative soft tissue resection margins. Risk factors seemingly do not significantly influence the extent of tumour resection.

GAUSS-EM, guided accumulation of ultrathin serial sections with a static magnetic field for volume electron microscopy.

Serial sectioning electron microscopy (EM) of millimeter-scale three-dimensional (3D) anatomical volumes requires the collection of thousands of ultrathin sections. Here, we report a high-throughput automated approach, GAUSS-EM (guided accumulation of ultrathin serial sections-EM), utilizing a static magnetic field to collect and densely pack thousands of sections onto individual silicon wafers. The method is capable of sectioning hundreds of microns of tissue per day at section thicknesses down to 35 nm. Relative to other automated volume EM approaches, GAUSS-EM democratizes the ability to collect large 3D EM volumes because it is simple and inexpensive to implement. We present two exemplar EM volumes of a zebrafish eye and mouse olfactory bulb collected with the method.

Dental sectioning for intraoral equine cheek teeth extractions: 29 cases.

The objectives of this retrospective study were to describe cheek teeth extraction by the sectioning technique, the decision making to use this technique and its potentially associated complications. Sectioning for dental extraction purpose was used in 29/461 (6.3%) of cases. Oro-sinusal fistula was the main post-operative complication, with 4/29 (13.7%) cases developing a macroscopic communication between the alveolus of the tooth extracted and the adjacent sinus compartment. All teeth where sectioning was attempted were successfully extracted. Sectioning for dental extraction appears to be a safe technique that can be used instead of or in addition too other minimal invasive cheek teeth extraction techniques. Thorough preoperative planning including oroscopic examination and medical imaging modalities are required to help in decision making, as well as excellent sedation and analgesia and horse compliance.

In Vivo Intelligent Fluorescence Endo-Microscopy by Varifocal Meta-Device and Deep Learning.

Endo-microscopy is crucial for real-time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta-device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo-microscope is developed based on varifocal meta-lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta-lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field-of-view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high-resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc.

Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Unusual Atrophic Nervus Intermedius in a Patient with Refractory Nervus Intermedius Neuralgia and History of Ipsilateral Sudden-Onset Central Facial Palsy and Sensorineural Hearing Loss: Cadaveric-Clinical Images with Surgical Video.

Nervus intermedius (NI) arises from the superior salivary nucleus, solitary nucleus, and trigeminal tract. It leaves the pons as 1 to 5 roots and travels between the facial and vestibulocochlear nerves before merging with the facial nerve within the internal auditory canal. The mastoid segment of the facial nerve then gives rise to a sensory branch that supplies the posteroinferior wall of the external auditory meatus and inferior pina. This complex pathway renders the nerve susceptible to various pathologies, leading to NI neuralgia. Here, the authors present an unusual intraoperative finding of an atrophic NI in a patient with refractory NI neuralgia and a history of ipsilateral sudden-onset central facial palsy and microvascular decompression for trigeminal neuralgia. The patient underwent NI sectioning via the previous retrosigmoid window and achieved partial ear pain improvement. The gross size of the NI is compared with a cadaveric specimen through stepwise dissection. This case highlights the potential significance of subtle central ischemic events and subsequent atrophy of NI in the pathogenesis of NI neuralgia, as well as the ongoing need to investigate the therapeutic efficacy of nerve sectioning.

Blueprints from plane to space: outlook of next-generation three-dimensional histopathology.

Here, we summarize the literature relevant to recent advances in three-dimensional (3D) histopathology in relation to clinical oncology, highlighting serial sectioning, tissue clearing, light-sheet microscopy, and digital image analysis with artificial intelligence. We look forward to a future where 3D histopathology expands our understanding of human pathophysiology and improves patient care through cross-disciplinary collaboration and innovation.

Ultrathin H-MXM as An "Ion Freeway" for High-Performance Osmotic Energy Conversion.

Nanofluidic membranes are currently being explored as potential candidates for osmotic energy harvesting. However, the development of high-performance nanofluidic membranes remains a challenge. In this study, the ultrathin MXene membrane (H-MXM) is prepared by ultrathin slicing and realize the ion horizontal transportation. The H-MXM membrane, with a thickness of only 3 µm and straight subnanometer channels, exhibits ultrafast ion transport capabilities resembling an "ion freeway". By mixing artificial seawater and river water, a power output of 93.6 W m-2 is obtained. Just as cell membranes have an ultrathin thickness that allows for excellent penetration, this straight nanofluidic membrane also possesses an ultrathin structure. This unique feature helps to shorten the ion transport path, leading to an increased ion transport rate and improveS performance in osmotic energy conversion.

Depth sectioning using environmental and atomic-resolution STEM.

(Scanning) transmission electron microscopy (TEM) images of samples in gas and liquid media are acquired with an environmental cell (EC) via silicon nitride membranes. The ratio of sample signal against the background is a significant factor for resolution. Depth-sectioning scanning TEM (STEM) is a promising technique that enhances the signal for a sample embedded in a matrix. It can increase the resolution to the atomic level, thereby enabling EC-STEM applications in important areas. This review introduces depth-sectioning STEM and its applications to high-resolution EC-STEM imaging of samples in gases and in liquids.

Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice.

Histological analysis is a morphological technique and an effective method for understanding the pathology of rheumatoid arthritis (RA). RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These pathologies can be observed in a collagen-induced arthritis model mouse using formaldehyde-fixated paraffin-embedded (FFPE) samples. For the preparation of FFPE samples, the conditions of the fixation and decalcification process significantly affect tissue staining results. Since the lesion sites include bone tissue, a decalcification process is necessary when preparing an FFPE sample. Therefore, selecting an optimal condition for the fixating and decalcifying solution is important. In this chapter, we describe the procedures of preparing paraffin samples, including fixation, decalcification, embedding, and sectioning from the RA model mouse, as well as different staining methods (hematoxylin and eosin, tartrate-resistant acid phosphatase).

Otolith sectioning reveals higher maximum age in greater weever (Trachinus draco).

Growth and maximum age are two key parameters that inform resilience of fish populations to exploitation. Existing information on those for greater weever inhabiting the eastern North Sea is based on the analysis of whole otoliths. Here, we present a reanalysis using sectioned otoliths. The results reveal a different growth pattern and a higher maximum age than that previously reported. The higher maximum age makes greater weever populations more vulnerable to exploitation. Such information can serve as a basis for the estimation of the growth curve that can be used for future assessment of the species.

A mechanical and three-dimensional finite element study of the optimum tooth sectioning depth during the extraction of low-level horizontally impacted mandibular third molar.

The present study aims to determine the optimum sectioning depth for the extraction of low-level horizontally impacted mandibular third molar (LHIM3M) using mechanical and finite element analysis. One hundred and fifty extracted mandibular third molars were randomly divided into three groups: 1, 2 or 3 mm of tooth tissue was retained at the bottom of the crown. The breaking force of teeth was tested in a universal strength testing machine. The fracture surface was observed and the type of tooth breakage was recorded. According to the three groups, corresponding 3D finite element models were created. The breaking force obtained in the mechanical study was, respectively, applied and the stress and strain of the teeth and surrounding tissues were analysed. Breaking force decreased as sectioning depth increased. The 2 mm group produced the lowest rate of incomplete breakage (10%). In the 2 mm model, the stresses were evenly distributed in the tooth tissue at the bottom of the fissure, and the maximal stress was located in the tissue close to the root segment. The maximum values of stresses in the bone and of strains in the periodontal ligament of the second molar and bone were lower in the 1 mm model than in other models. Their distribution was similar in the three models. A sectioning depth of 1 mm group saves labour during the extraction of LHIM3M, compared to 2 and 3 mm; 2 mm might be the appropriate sectioning depth in terms of breakage shapes.

The views of psychiatrists on proposed changes to the England and Wales Mental Health Act 1983 legislation for people with intellectual disability: A national study.

BACKGROUND: The Draft Mental Health Bill proposes removal of both intellectual disability and autism from Section 3 of the Mental Health Act for England and Wales (MHA). This would lead to people with intellectual disability (PwID) and/or autism could not be detained beyond 28 days, in the absence of diagnosed co-occurring mental illness. AIM: To obtain views of psychiatrists working with PwID in England and Wales regarding the proposed MHA changes. This study focusses specifically on the impact on PwID. METHODS: A cross-sectional online mixed methodology survey of Likert and free-text response questions was developed, to ascertain perceptions of proposed legislative changes to the MHA. A non-discriminatory exponential snowballing technique leading to non-probability sampling was used to disseminate the survey. Quantitative data was analysed using descriptive statistics, Mann-Whitney and Fisher's exact tests. Thematic analysis was conducted on free text responses. RESULTS: A total of 82 psychiatrists (33%) from approximately 250 eligible completed the survey. Nearly two-thirds (64%) reported good awareness of the proposed changes, with over half (55%) reporting disagreement with the changes. Psychiatrists working in inpatient settings for PwID reported increased awareness of the changes, less agreement with the reforms, and increased expectations of the reforms having negative unintended consequences, compared to their peers working exclusively in the community. Consultants reported greater disagreement with the changes compared to their non-consultant peers. Qualitative analysis identified five main themes: impact on diagnosis and treatment, seeking alternative options, introducing inequities, resources, and meeting holistic care goals through the Care, Education and Treatment Reviews (CETR) process. CONCLUSION: Psychiatrists working with PwID report widespread disagreement with the proposed changes to the MHA for PwID, with greater levels of disagreement among those working in inpatient services. Caution with respect to the proposed changes, and monitoring of the impact of the changes if implemented, is advised.

The effect of visuo-haptic exploration on the development of the geometric cross-sectioning ability.

Cross-sectioning is a shape understanding task where the participants must infer and interpret the spatial features of three-dimensional (3D) solids by depicting their internal two-dimensional (2D) arrangement. An increasing body of research provides evidence of the crucial role of sensorimotor experience in acquiring these complex geometrical concepts. Here, we focused on how cross-sectioning ability emerges in young children and the influence of multisensory visuo-haptic experience in geometrical learning through two experiments. In Experiment 1, we compared the 3D printed version of the Santa Barbara Solids Test (SBST) with its classical paper version; in Experiment 2, we contrasted the children's performance in the SBST before and after the visual or visuo-haptic experience. In Experiment 1, we did not identify an advantage in visualizing 3D shapes over the classical 2D paper test. In contrast, in Experiment 2, we found that children who had the experience of a combination of visual and tactile information during the exploration phase improved their performance in the SBST compared with children who were limited to visual exploration. Our study demonstrates how practicing novel multisensory strategies improves children's understanding of complex geometrical concepts. This outcome highlights the importance of introducing multisensory experience in educational training and the need to make way for developing new technologies that could improve learning abilities in children.

Exploration of fs-laser ablation parameter space for 2D/3D imaging of soft and hard materials by tri-beam microscopy.

Tri-beam microscopes comprising a fs-laser beam, a Xe+ plasma focused ion beam (PFIB) and an electron beam all in one chamber open up exciting opportunities for site-specific correlative microscopy. They offer the possibility of rapid ablation and material removal by fs-laser, subsequent polishing by Xe-PFIB milling and electron imaging of the same area. While tri-beam systems are capable of probing large (mm) volumes providing high resolution microscopical characterisation of 2D and 3D images across exceptionally wide range of materials and biomaterials applications, presenting high quality/low damage surfaces to the electron beam can present a significant challenge, especially given the large parameter space for optimisation. Here the optimal conditions and artefacts associated with large scale volume milling, mini test piece manufacture, serial sectioning and surface polishing are investigated, both in terms of surface roughness and surface quality for metallic, ceramic, mixed complex phase, carbonaceous, and biological materials. This provides a good starting place for those wishing to examine large areas or volumes by tri-beam microscopy across a range of materials.

Multiplane HiLo microscopy with speckle illumination and non-local means denoising.

Significance: HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Aim: Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Approach: Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. Results: We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. Conclusions: Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

Serial-section Electron Tomography and Quantitative Analysis of Microtubule Organization in 3D-reconstructed Mitotic Spindles.

For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/). Key features • High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM). • Serial-section electron tomography of selected cells. • Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization.

High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.

Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed. One of them is the milling of thin crystal lamella using focused ion beam (FIB), which was successfully applied to several protein crystals. Here, we present a new approach that uses cryo-sectioning after high pressure freezing of dextran embedded protein crystals. 150 to 200 nm thick cryo-sections of hen egg white lysozyme tetragonal crystals where used for electron diffraction experiments. Complete diffraction data up to 2.9 Å resolution have been collected and the lysozyme structure has been solved by molecular replacement and refined against these data. Our data demonstrate that cryo-sectioning can preserve protein structure at high resolution and can be used as a new sample preparation technique for 3D electron diffraction experiments of protein crystals. The different orientations found in the crystal chips and their large number resulting from the cryo-sectioning make the latter an attractive approach as it combines advantages from both blotting approaches (number of crystals) and FIB-milling (controlled thickness and absence of solvent around the crystal).

High precision vibration sectioning for 3D imaging of the whole central nervous system.

BACKGROUND: Imaging and reconstruction of the morphology of neurons within the entire central nervous system (CNS) is important for deciphering the neural circuitry and related brain functions. With combination of tissue clearing and light sheet microscopy, previous studies have imaged the mouse CNS at cellular resolution, while remaining single axons unresolvable due to the tradeoff between sample size and imaging resolution. This could be improved by sectioning the sample into thick slices and imaged with high resolution light sheet microscopy as described in our previous study. However, the achievable quality for 3D imaging of serial thick slices is often hindered by surface undulation and other artifacts introduced by sectioning and handling limitations. NEW METHODS: In order to improve the imaging quality for mouse CNS, we develop a high-performance vibratome system for sample sectioning and handling automation. The sectioning mechanism of the system was modeled theoretically and verified experimentally. The effects of process parameters and sample properties on sectioning accuracy were studied to optimize the sectioning outcome. The resultant imaging outcome was demonstrated on mouse samples. RESULTS: Our theoretical model of vibratome effectively depicts the relationship between the sample surface undulation errors and the sectioning parameters. With the guidance of the theoretical model, the vibratome is able to achieve a local surface undulation error of ±0.5 µm and a surface arithmetic mean deviation (Sa) of 220 nm for 300-µm-thick tissue slices. Imaging results of mouse CNS show the continuous sectioning capability of the vibratome. COMPARISON WITH EXISTING METHOD: Our automatic sectioning and handling system is able to process serial thick slices for 3D imaging of the whole CNS at a single-axon resolution, superior to the commercially available vibratome devices. CONCLUSION: Our automatic sectioning and handling system can be optimized to prepare thick sample slices with minimal surface undulation and manual manipulation in support of 3D brain mapping with high-throughput and high-accuracy.

Whole-brain Optical Imaging: A Powerful Tool for Precise Brain Mapping at the Mesoscopic Level.

The mammalian brain is a highly complex network that consists of millions to billions of densely-interconnected neurons. Precise dissection of neural circuits at the mesoscopic level can provide important structural information for understanding the brain. Optical approaches can achieve submicron lateral resolution and achieve "optical sectioning" by a variety of means, which has the natural advantage of allowing the observation of neural circuits at the mesoscopic level. Automated whole-brain optical imaging methods based on tissue clearing or histological sectioning surpass the limitation of optical imaging depth in biological tissues and can provide delicate structural information in a large volume of tissues. Combined with various fluorescent labeling techniques, whole-brain optical imaging methods have shown great potential in the brain-wide quantitative profiling of cells, circuits, and blood vessels. In this review, we summarize the principles and implementations of various whole-brain optical imaging methods and provide some concepts regarding their future development.