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Cryostat sectioning is a popular but labor-intensive method for preparing histological brain sections. We have developed a modification of the commercially available CryoJane tape collection method that significantly improves the ease of collection and the final quality of the tissue sections. The key modification involves an array of UVLEDs to achieve uniform polymerization of the glass slide and robust adhesion between the section and slide. This report presents system components and detailed procedural steps, and provides examples of end results; that is, 20 μm mouse brain sections that have been successfully processed for routine Nissl, myelin staining, DAB histochemistry, and fluorescence. The method is also suitable for larger brains, such as rat and monkey.

Geo-seq combines laser capture microdissection and single-cell RNA-seq technology to enable transcriptome analysis of small quantities of cells from defined geographical locations. Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use geographical position sequencing (Geo-seq), a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously. The Geo-seq protocol allows the profiling of transcriptome information from only a small number cells and retains their native spatial information. This protocol has wide potential applications to address biological and pathological questions of cellular properties such as prospective cell fates, biological function and the gene regulatory network. Geo-seq has been applied to investigate the spatial transcriptome of mouse early embryo, mouse brain, and pathological liver and sperm tissues. The entire protocol from tissue collection and microdissection to sequencing requires ∼5 d, Data analysis takes another 1 or 2 weeks, depending on the amount of data and the speed of the processor.

Background Glioma is the most common malignant tumor of the central nervous system, and intraoperative accurate diagnosis is critical for formulating rational surgical strategies. Traditional intraoperative frozen section pathology has certain limitations, while molecular diagnosis provides a new direction for precise glioma diagnosis and treatment. This study aimed to explore the clinical value of intraoperative frozen section pathology alone and combined with molecular diagnosis to improve diagnostic accuracy and optimize surgical strategies for gliomas. Methods A total of 230 patients preoperatively diagnosed with glioma via MRI at Qilu Hospital of Shandong University from January 2021 to December 2023 were enrolled. All patients underwent intraoperative frozen section pathology (traditional diagnosis group, n = 113), while a subset also underwent molecular diagnosis (molecular diagnosis group, n = 117). Using postoperative histopathological diagnosis as the \"gold standard\", we evaluated the accuracy of intraoperative frozen section pathology alone and combined with molecular diagnosis, as well as the impact of molecular diagnostic techniques on surgical strategies. Results The accuracy of intraoperative frozen section pathology was 77.88% (88/113). When combined with molecular diagnosis, the accuracy of intraoperative integrated diagnosis improved to 94.87% (111/117). Among the molecular diagnosis group, 20 cases showed discrepancies between intraoperative frozen section pathology and postoperative histopathological diagnosis. Molecular diagnosis corrected 14 of these errors, resulting in only 6 cases with unresolved discrepancies, achieving a correction rate of 70% (14/20). Additionally, 86.32% (101/117) of patients had their surgical strategies optimized based on positive molecular markers, including extended tumor resection (58 cases, 57.43%) and molecular total resection (43 cases, 42.57%). Conclusions The combination of intraoperative frozen section pathology with molecular diagnosis significantly improve the accuracy of intraoperative diagnosis, optimize surgical strategies, and provide critical support for personalized treatment and precision medicine. This study also validates the clinical value of molecular diagnostic techniques in intraoperative diagnosis of glioma, laying a foundation for future advancements in diagnostic and therapeutic paradigms.

Molecular analysis of postmortem samples of brain tissue obtained from 11 children with autism showed that the prefrontal and temporal cortexes in 10 of these children had patches of neuronal disorganization. Autism is, in part, a heritable developmental disorder involving macroscopic early brain overgrowth in the majority of cases 1 – 7 and dysfunction 8 that affects several cortical and subcortical regions mediating autistic symptoms, including prefrontal and temporal cortexes. 4 , 9 – 11 The underlying cortical defects remain uncertain. Despite the early diagnosable onset, in more than 40 studies, the average age of patients with autism in postmortem analyses was 22 years. 4 Three previous case studies that evaluated Nissl-stained sections of brains obtained from patients with autism ranging in age from 4 to 60 years described individual instances of heterotopias, slight focal laminar disorganization, 12 , . . .

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

Cryosectioning is known as a common and well-established histological method, due to its easy accessibility, speed, and cost efficiency. However, the creation of bone cryosections is especially difficult. In this study, a cryosectioning protocol for trabecular bone that offers a relatively cheap and undemanding alternative to paraffin or resin embedded sectioning was developed. Sections are stainable with common histological dying methods while maintaining sufficient quality to answer a variety of scientific questions. Furthermore, this study introduces an automated protocol for analysing such sections, enabling users to rapidly access a wide range of different stainings. Therefore, an automated ‘QuPath’ neural network-based image analysis protocol for histochemical analysis of trabecular bone samples was established, and compared to other automated approaches as well as manual analysis regarding scattering, quality, and reliability. This highly automated protocol can handle enormous amounts of image data with no significant differences in its results when compared with a manual method. Even though this method was applied specifically for bone tissue, it works for a wide variety of different tissues and scientific questions.

Abstract Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin–stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.

Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue’s highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.

Histology is the gold standard for analyzing tissue structure and cell morphology. Immunostaining on thin tissue sections enables precise visualization of antigens and proteins. However, for cryosectioning small tissues such as organoids, spheroids, and tumoroids there is a lack of standardized, time- and cost-effective methods, limiting the throughput of analysis. Here, we have adapted to cryosectioning our previously developed HistoBrick approach, in which small tissue arrangement is spatially controlled within arrayed mini-wells. By testing various embedding matrices, we show that an 8% PEGDA and 2.5% gelatine mixture is optimal, providing essential structural support to maintain sample integrity during cryosectioning. This embedding matrix preserves fragile substructures of human retinal organoids, which are particularly susceptible to damage during sample preparation. Using PEGDA-gelatine HistoBricks for the simultaneous embedding of 16 retinal organoids, we analyzed a time course of retinal organoid development. We observed the maintenance of photoreceptors cell bodies up to week 98 in culture, while photoreceptor outer segments were gradually lost. Further, we observed displaced photoreceptors in the region of outer segments. The PEGDA-gelatine HistoBrick is a cost-efficient tool that can be implemented for small tissue studies to increase throughput in experiments such as large-scale screenings or toxicology research.

Cryosection pathology is essential for intraoperative diagnosis of diffuse midline gliomas, yet it often leads to diagnostic errors and may prompt unnecessary re-biopsies before completion of the formal molecular assessment. In this study, we propose an AI-augmented framework, CryoAID, for rapid molecular outcome prediction during surgery for patients with diffuse midline glioma. CryoAID integrates a generative model to correct cryosection artefacts and a pathology foundation model to predict molecular statuses directly from cryosection images. We validate CryoAID across multiple cohorts to predict tumoural molecular statuses in the internal ( n  = 326), external multi-centre ( n  = 52), and consecutive ( n  = 68) datasets. In particular, CryoAID accurately predicts major molecular statuses (e.g., ATRX, H3K27M, and TP53) using cryosection images that were previously deemed disqualified for molecular examinations. Beyond tumour cells, CryoAID reveals highly differential clinical features, including glial cell proliferation, abundant cytoplasm, and localised endothelial proliferation. In the retrospective analyses, CryoAID reduces re-biopsy rates by 26.4% and 26.6% in the internal and consecutive datasets, respectively. Our findings demonstrate that the AI-augmented pathology workflow can extract diagnostic value from specimens previously considered non-viable by traditional histopathology. This approach represents a shift towards real-time molecular pathology, potentially reducing re-biopsies and improving diagnostic precision for patients with diffuse midline glioma. Cryosection pathology is essential for intraoperative diagnosis of diffuse midline gliomas (DMGs), but often requires re-sampling for an accurate assessment. Here, the authors develop CryoAID, an AI-augmented framework for rapid and accurate molecular outcome prediction during surgery for DMG, reducing re-biopsy rates in clinical cohorts.