Thin Tissue Sections Research Articles

Preparation and Sectioning of Paraffin-Embedded Tissue for Histology and Histochemistry.

Cutting thin sections of plant tissues enables observation of internal structures and inner cell layers, and it facilitates various histochemical staining methods. Paraffin embedding gives the tissues a uniform stiffness that is sufficient for sectioning with a microtome. Here, we describe the procedures for preparation of paraffin-embedded tissues and the techniques for sectioning with a microtome. This method is applicable to various types of tissues, including rice shoot apex and immature inflorescence.

Fast interactive simulations of cardiac electrical activity in anatomically accurate heart structures by compressing sparse uniform cartesian grids

Background and Objective:Numerical simulations are valuable tools for studying cardiac arrhythmias. Not only do they complement experimental studies, but there is also an increasing expectation for their use in clinical applications to guide patient-specific procedures. However, numerical studies that solve the reaction–diffusion equations describing cardiac electrical activity remain challenging to set up, are time-consuming, and in many cases, are prohibitively computationally expensive for long studies. The computational cost of cardiac simulations of complex models on anatomically accurate structures necessitates parallel computing. Graphics processing units (GPUs), which have thousands of cores, have been introduced as a viable technology for carrying out fast cardiac simulations, sometimes including real-time interactivity. Our main objective is to increase the performance and accuracy of such GPU implementations while conserving computational resources. Methods:In this work, we present a compression algorithm that can be used to conserve GPU memory and improve efficiency by managing the sparsity that is inherent in using Cartesian grids to represent cardiac structures directly obtained from high-resolution MRI and mCT scans. Furthermore, we present a discretization scheme that includes the cross-diagonal terms in the computational cell to increase numerical accuracy, which is especially important for simulating thin tissue sections without the need for costly mesh refinement. Results:Interactive WebGL simulations of atrial/ventricular structures (on PCs, laptops, tablets, and phones) demonstrate the algorithm’s ability to reduce memory demand by an order of magnitude and achieve calculations up to 20x faster. We further showcase its superiority in slender tissues and validate results against experiments performed in live explanted human hearts. Conclusions:In this work, we present a compression algorithm that accelerates electrical activity simulations on realistic anatomies by an order of magnitude (up to 20x), thereby allowing the use of finer grid resolutions while conserving GPU memory. Additionally, improved accuracy is achieved through cross-diagonal terms, which are essential for thin tissues, often found in heart structures such as pectinate muscles and trabeculae, as well as Purkinje fibers. Our method enables interactive simulations with even interactive domain boundary manipulation (unlike finite element/volume methods). Finally, agreement with experiments and ease of mesh import into WebGL paves the way for virtual cohorts and digital twins, aiding arrhythmia analysis and personalized therapies.

Progress on three-dimensional visualizing skin architecture with multiple immunofluorescence staining and tissue-clearing approaches.

The skin forms the external covering of the body and is its largest organ, comprising many different cell types. Although the diversity of these cells has been widely studied with various histological methods, our understanding of skin architecture is mainly established on thin tissue sections, which restricted the information available to two dimensions. The development of innovative techniques to induce optical transparency ("clearing") in biological tissues has enabled researchers to visualize the three-dimensional reconstruction of intact organs and thick tissue sections at a cellular resolution. With the aid of tissue-clearing treatment, the labeled cutaneous nerve fibers and blood vessels can be followed for a longer distance on the thicker skin section or the whole mount skin under a fluorescence microscopy or a confocal microscopy. It is beneficial for demonstrating the morphological characteristics of nerve fibers and blood vessels themselves, as well as their spatial interconnection. In this review, we provide a brief summary of the literature on the use of tissue optical clearing methods and describe our experience of multiple fluorescent staining and tissue clearing approaches on thicker skin sections and whole-mount skin in our laboratory. Given the existing conventional methods, we expected to provide a more effective approach to comprehensively study skin architecture.

Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.

Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 μm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 200-μm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring, and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.

Evaluation of femtosecond-LA-ICP-TOFMS for multi-elemental mapping at cellular resolution of human-tissue from cancer patients

The characterization of the multi-elemental distributions within a thin tissue section of a liver metastasis from colorectal cancer patient after oxaliplatin-based chemotherapy, is critically evaluated using Ultraviolet Femtosecond Laser Ablation Inductively Coupled Plasma-Time of Flight Mass Spectrometry (UV-fs-LA-ICP-TOFMS). Different femtosecond laser ablation conditions, in terms of spot size and energy per pulse, were investigated to achieve proper ablation of the formalin-fixed paraffin-embedded tumor sections, while also trying to minimize the ablation of the glass substrate. Moreover, histological images of the tissues before and after the ablation processes were combined with UV-fs-LA-ICP-TOFMS elemental mapping to achieve multi-elemental spatial distributions at cellular level. For instance, spatial distributions of endogenous 31P, 56Fe, 63Cu and 64Zn analytes were determined within the thin tissue section. Furthermore, spatial distribution of elements such as 24Mg, 28Si, and 40Ca were employed to study glass substrate ablation during tissue analysis. Additional analytes, which might be partially associated to compounds employed on hematoxylin and eosin tissue staining processes, such as 23Na, 27Al, 32S, 39K, 79Br and 127I, were also detected. Finally, the accumulation of the oxaliplatin-based chemotherapy products in the stroma was confirmed from the spatial distribution of 196Pt ion signals within the tissue.

Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.

Simultaneous deep ultraviolet transmission and scattering microscopy for virtual histology.

In recent years, the emergence of a variety of novel optical microscopy techniques has enabled the generation of virtual optical stains of unlabeled tissue specimens, which have the potential to transform existing clinical histopathology workflows. In this work, we present a simultaneous deep ultraviolet transmission and scattering microscopy system that can produce virtual histology images that show concordance to conventional gold-standard histological processing techniques. The results of this work demonstrate the system's diagnostic potential for characterizing unlabeled thin tissue sections and streamlining histological workflows.

First Report of Alternaria gossypina Causing Stem Base Rot on Passilora edulis in Guangxi, China.

Passion fruit (Passilora edulis), known as the "king of fruit juices", is popular in southern China (Yuan et al. 2019). Stem base rot is a devastating disease of passion fruit commonly caused by several Fusarium spp. (Zakaria, 2022). In July 2022, typical symptoms of stem base rot were observed in a poorly managed "Qinmi No. 9" Golden passion fruit orchard in Jingxi (23°13'10"N, 106°5'23"E). The disease incidence had reached 40% (n=200) in the survey. Symptoms included ulceration and mutilation at the stem base, making the plants prone to breakage when pulled, wilting and drooping leaves above ground, and severe cases leading to the entire plant withering and dying. Fourteen plants with obvious symptoms were collected. Thin sections of plant tissue were cut from junction of sickness and health of stem, sterilized with ethanol and sodium hypochlorite, and placed on PDA medium at 28°C. Sixty fungal strains were obtained, 90% of which was identified as Fusarium based on morphology. 80% of Fusarium were F. oxysporum species complex (FOSC), but pathogenicity experiment showed all FOSC were weakly pathogenic. However, two severely pathogenic fungi with similar morphology but distinct from Fusarium were identified from the same plant. The representative strain C11 was selected for further study. C11 demonstrated a rapid growth rate, reaching a 90 mm diameter colony on PDA cultured at 25°C for 7 days. The colony displaced a round, flat shape with an overall light brown front, and cottony gray or light brown mycelium, while the reverse side was dark brown. Conidia production was observed as typically occurred in multiple chains after 14 days culture on OA medium, with round, oval or straight rod-like brown conidia ranging in size from 5.74-23.42×14.67-67.22, featuring 1-8 transverse septa and 0-3 mediastinum (Figure S1). For molecular identification, the internal transcribed spacer (ITS, OR616614), translation elongation factor 1-alpha (TEF, OR633298), alternaria major allergen (Alt a1, OR633294) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, OR633295), RNA polymerase subunit II gene (RPB2, OR633297), 18S Small subunit rDNA (SSU, OR616608) , 28S Large Subunit rDNA (LSU, OR616615), the KOG1058 gene regions (OR633296) and an approximately segment of the anonymous noncoding region (OPA10-2, OR633299) were amplified from C11 (Liu et al. 1999, Li et al. 2023, Andrew et al. 2009), and deposited in GenBank with accession number shown in the brackets. Phylogenetic trees were constructed in MEGA11 after splicing by BioEdit (Figure S3). Combining morphology and molecular analyses, C11 was identified as Alternaria gossypina (Woudenberg et al. 2015). To test the pathogenicity, the base of the seedling stem (20cm in height) of 50 healthy "Tainong No. 1" variety of purple passion fruit, which was more susceptible to stem-base rot, was puncture wound with needles, inoculated with 6 mm diameter colonies of fungi, and then wrapped in wet cotton (Ángel et al. 2018). Ten healthy seedlings inoculated with PDA were used as controls. These plants were cultured in an artificial greenhouse at 30±5℃with 80±5% humidity. After 15 days, the plants inoculated with C11 exhibited symptoms similar to those in the field, whereas the controls remained healthy. A. gossypina was reisolated from the diseased plant stems, with the morphology and GAPDH sequence consistent with the inoculated (Figure S1, S2). This is the first report of passion fruit stem rot caused by A. gossypina. This finding will aid in the prevention and control of stem rot in passion fruit.

Ce3D-IBEX: Achieving Multiplex 3-dimensional Imaging for Deep Phenotyping of Cells in Tissues

Abstract 2D multiplexed antibody-based imaging provides a framework to study cell-cell interactions in thin tissue sections but lacks the ability to interrogate spatial relationships in larger 3D micro-anatomical structures. Several methods for 3D imaging of optically cleared tissue exist, but current approaches do not provide a deep phenotyping capability as afforded by high content 2D imaging. Here,we developed a method to overcome this constraint by combining a fast hydrophilic tissue clearing technique, clearing-enhanced 3D (Ce3D), and the recently developed Iterative Bleaching Extends Multiplexity (IBEX) technique together with a robust image registration algorithm suite to create a highly multiplex 3D imaging method: Ce3D-IBEX. It has been used to analyze murine lung, lymph nodes, small intestine, retina, tumor, and cornea as well as human retina, kidney, liver, tonsil, and jejunum, yielding seamless views of large-scale tissue structures while enabling visualization of high dimensional phenotypic markers at single-cell resolution, permitting the identification of discrete cell subsets, quantification of protein expression level and their mutual spatial association within tissue structures. To date, 25+ markers have been visualized in a single 3D sample. This represents a major advance in the emerging field of highly multiplexed 3D imaging. With aims to open new avenues for characterization of spatial dynamics of immune cells in healthy and diseased tissue.

Abstract 2309: 3D spatial quantification of lymphocyte infiltration and collagen features in the tumor microenvironment using a novel assay: 3D I/O Pro™

Abstract Background: The composition of the tumor microenvironment (TME) is a major determinant of response to therapy in many solid tumors. To-date, characterization of the TME has been based on limited analysis of thin tissue sections. Here we demonstrate the utility of an end-to-end 3D spatial biology workflow, the 3D I/O Pro™, based on whole tissue imaging, to identify and quantify tumor cells, lymphocytes, and collagen features in human FFPE tissue samples. Methods: Five human colorectal cancer FFPE blocks were deparaffinized, stained with nuclear (TO-PRO-3) and general protein (eosin) dyes, and cleared using a modified iDISCO protocol. Entire samples were imaged at 2 µm/pixel resolution with a hybrid open-top light-sheet microscope, the 3Di™. Regions of interest (ROIs) with a volume of 0.5 mm3 were re-imaged at 0.33 µm/pixel. Cell nuclei and collagen were segmented and 3D spatial relationships between tumor cells, lymphocytes, and collagen fibers were quantified. Analyses were performed within select ROIs using 3Dai™ tools including U-Net, CytoMAP, CT-FIRE, CurveAlign, and custom Python scripts. Results: The ratio of stromal to tumor parenchymal lymphocytes (lymphocyte infiltration ratio) varied from 1.4 up to 9.1 in 3D volumes and 1 to 25 in 2D virtual sections taken throughout all 5 CRC samples. The ratio of the collagen fibers within 100 µm of the tumor border-oriented perpendicular to the tumor-stromal boundary compared to those oriented parallel to the tumor-stromal boundary was 0.37 in a proof-of-concept evaluation of a 2D virtual section with a lymphocyte infiltration ratio of 12. Qualitatively, areas with perpendicular collagen had more lymphocyte infiltration into the tumor parenchyma than areas with parallel collagen orientation. Conclusions: We demonstrated that the 3D I/O Pro™ pipeline can quantify lymphocyte density in tumor parenchyma and stroma and analyze collagen features, including orientation, within 3D ROIs in the TME. This workflow allows us to characterize tumors based on many complex spatial relationships and could have broad applicability in research and development of novel cancer therapies that target tumor fibrosis or other features of the TME. In the future, we plan to correlate features from the 3D TME with response to immunotherapy and use these features to refine histologic definitions of immune spatial phenotypes. Citation Format: Caleb Stoltzfus, Jasmine Wilson, Nathan Grant, Alexandra Alvarsson, Anna DeWitt, David Simmons, Brandy Olin, Bonnie Phillips, Laura A. Dillon, G. Travis Clifton, Nicholas P. Reder. 3D spatial quantification of lymphocyte infiltration and collagen features in the tumor microenvironment using a novel assay: 3D I/O Pro™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2309.

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Evaluation of Inlet Temperature with Three Sprayer Designs for Desorption Electrospray Ionization Mass Spectrometry Tissue Analysis.

Mass spectrometry imaging (MSI) allows for the spatially resolved detection of endogenous and exogenous molecules and atoms in biological samples, typically prepared as thin tissue sections. Desorption electrospray ionization (DESI) is one of the most commonly utilized MSI modalities in preclinical research. DESI ion source technology is still rapidly evolving, with new sprayer designs and heated inlet capillaries having recently been incorporated in commercially available systems. In this study, three iterations of DESI sprayer designs are evaluated: (1) the first, and until recently only, commercially available Waters sprayer; (2) a developmental desorption electro-flow focusing ionization (DEFFI)-type sprayer; and (3) a prototype of the newly released Waters commercial sprayer. A heated inlet capillary is also employed, allowing for controlled inlet temperatures up to 500 °C. These three sprayers are evaluated by comparative tissue imaging analyses of murine testes across this temperature range. Single ion intensity versus temperature trends are evaluated as exemplar cases for putatively identified species of interest, such as lactate and glutamine. A range of trends are observed, where intensities follow either increasing, decreasing, bell-shaped, or other trends with temperature. Data for all sprayers show approximately similar trends for the ions studied, with the commercial prototype sprayer (sprayer version 3) matching or outperforming the other sprayers for the ions investigated. Finally, the mass spectra acquired using sprayer version 3 are evaluated by uniform manifold approximation and projection (UMAP) and k-means clustering. This approach is shown to provide valuable insight that is complementary to the presented univariate evaluation for reviewing the parameter space in this study. Full spectral temperature optimization data are provided as supporting data to enable other researchers to design experiments that are optimal for specific ions.

Laser capture microdissection and native mass spectrometry for spatially-resolved analysis of intact protein assemblies in tissue.

Previously, we have shown that native ambient mass spectrometry imaging allows the spatial mapping of folded proteins and their complexes in thin tissue sections. Subsequent top-down native ambient mass spectrometry of adjacent tissue section enables protein identification. The challenges associated with protein identification by this approach are (i) the low abundance of proteins in tissue and associated long data acquisition timescales and (ii) irregular spatial distributions which hamper targeted sampling of the relevant tissue location. Here, we demonstrate that these challenges may be overcome through integration of laser capture microdissection in the workflow. We show identification of intact protein assemblies in rat liver tissue and apply the approach to identification of proteins in the granular layer of rat cerebellum.

Infrared Laser Ablation and Capture of Biological Tissue.

Sampling thin tissue sections with cellular precision can be accomplished using laser ablation microsampling for mass spectrometry analysis. In this work, the use of a pulsed mid-infrared (IR) laser for selecting small regions of interest (ROI) in tissue sections for offline liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The laser is focused onto the tissue section, which is rastered as the laser is fired. The ablated tissue is captured in a microwell array and processed in situ through reduction, alkylation, and digestion with a low liquid volume workflow. The resulting peptides from areas as small as 0.01mm2 containing 5 ng of protein are analyzed for protein identification and quantification using offline LC-MS/MS.

STalign: Alignment of spatial transcriptomics data using diffeomorphic metric mapping

Spatial transcriptomics (ST) technologies enable high throughput gene expression characterization within thin tissue sections. However, comparing spatial observations across sections, samples, and technologies remains challenging. To address this challenge, we develop STalign to align ST datasets in a manner that accounts for partially matched tissue sections and other local non-linear distortions using diffeomorphic metric mapping. We apply STalign to align ST datasets within and across technologies as well as to align ST datasets to a 3D common coordinate framework. We show that STalign achieves high gene expression and cell-type correspondence across matched spatial locations that is significantly improved over landmark-based affine alignments. Applying STalign to align ST datasets of the mouse brain to the 3D common coordinate framework from the Allen Brain Atlas, we highlight how STalign can be used to lift over brain region annotations and enable the interrogation of compositional heterogeneity across anatomical structures. STalign is available as an open-source Python toolkit at https://github.com/JEFworks-Lab/STalign and as Supplementary Software with additional documentation and tutorials available at https://jef.works/STalign.

Affordable optical clearing and immunolabelling in mouse brain slices

Traditional histological analysis is conducted on thin tissue sections, limiting the data capture from large tissue volumes to 2D profiles, and requiring stereological methods for 3D assessment. Recent advances in microscopical and tissue clearing methods have facilitated 3D reconstructions of tissue structure. However, staining of large tissue blocks remains a challenge, often requiring specialised and expensive equipment to clear and immunolabel tissue. Here, we present the Affordable Brain Slice Optical Clearing (ABSOC) method: a modified iDISCO protocol which enables clearing and immunolabeling of mouse brain slices up to 1 mm thick using inexpensive reagents and equipment, with no intensive expert training required. We illustrate the use of ABSOC in 1 mm C57BL/6J mouse coronal brain slices sectioned through the dorsal hippocampus and immunolabelled with an anti-calretinin antibody. The ABSOC method can be readily used for histological studies of mouse brain in order to move from the use of very thin tissue sections to large volumes of tissue – giving more representative analysis of biological samples, without the need for sampling of small regions only.

Impact of corpus callosum fiber tract crossing on polarimetric images of human brain histological sections: ex vivo studies in transmission configuration.

Imaging Mueller polarimetry is capable to trace in-plane orientation of brain fiber tracts by detecting the optical anisotropy of white matter of healthy brain. Brain tumor cells grow chaotically and destroy this anisotropy. Hence, the drop in scalar retardance values and randomization of the azimuth of the optical axis could serve as the optical marker for brain tumor zone delineation. The presence of underlying crossing fibers can also affect the values of scalar retardance and the azimuth of the optical axis. We studied and analyzed the impact of fiber crossing on the polarimetric images of thin histological sections of brain corpus callosum. We used the transmission Mueller microscope for imaging of two-layered stacks of thin sections of corpus callosum tissue to mimic the overlapping brain fiber tracts with different fiber orientations. The decomposition of the measured Mueller matrices was performed with differential and Lu-Chipman algorithms and completed by the statistical analysis of the maps of scalar retardance, azimuth of the optical axis, and depolarization. Our results indicate the sensitivity of Mueller polarimetry to different spatial arrangement of brain fiber tracts as seen in the maps of scalar retardance and azimuth of optical axis of two-layered stacks of corpus callosum sections The depolarization varies slightly () with the orientation of the optical axes in both corpus callosum stripes, but its value increases by 2.5 to 3 times with the stack thickness. The crossing brain fiber tracts measured in transmission induce the drop in values of scalar retardance and randomization of the azimuth of the optical axis at optical path length of . It suggests that the presence of nerve fibers crossing within the depth of few microns will be also detected in polarimetric maps of brain white matter measured in reflection configuration.

Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling.

Label-free molecular imaging techniques such as matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enable the direct and simultaneous mapping of hundreds of different metabolites in thin sections of biological tissues. However, in host-microbe interactions it remains challenging to localize microbes and to assign metabolites to the host versus members of the microbiome. We therefore developed a correlative imaging approach combining MALDI-MSI with fluorescence in situ hybridization (FISH) on the same section to identify and localize microbial cells. Here, we detail metaFISH as a robust and easy method for assigning the spatial distribution of metabolites to microbiome members based on imaging of nucleic acid probes, down to single-cell resolution. We describe the steps required for tissue preparation, on-tissue hybridization, fluorescence microscopy, data integration into a correlative image dataset, matrix application and MSI data acquisition. Using metaFISH, we map hundreds of metabolites and several microbial species to the micrometer scale on a single tissue section. For example, intra- and extracellular bacteria, host cells and their associated metabolites can be localized in animal tissues, revealing their complex metabolic interactions. We explain how we identify low-abundance bacterial infection sites as regions of interest for high-resolution MSI analysis, guiding the user to a trade-off between metabolite signal intensities and fluorescence signals. MetaFISH is suitable for a broad range of users from environmental microbiologists to clinical scientists. The protocol requires ~2 work days.

Convolutional neural networks applied to microtomy: Identifying the trimming-end cutting routine on paraffin-embedded tissue blocks

In the field of histopathology, the microtomy procedure yields thin sections of tissue embedded in paraffin blocks, then to be further processed for diagnostic purposes. Within microtomy, trimming is an initial but critical process in which the excess paraffin covering the tissue of interest is removed by continuous cutting routines, until the tissue is suitably exposed and ready for sectioning. Trimming is currently a time-consuming process that is manually held by technicians. In this paper, we present a method to automatize this process, by analyzing tissue block surface images resulting from each cyclic cutting routine. Two types of Convolutional Neural Networks (CNNs) were fine-tuned: one for binary segmentation, the other for multi-class classification tasks, by exploring and optimizing lightweight architectures to provide fast analytical results on cost-effective edge computing. Two sequential online conditions followed the CNNs to output the current stage of the block surface and rule if the trimming-end cutting routine was reached. We compared the results obtained through our method with the ones obtained by three skilled technicians processing 75 tissue blocks. The proposed method identified the trimming-end cutting routine approximately as accurate as the technicians did, yielding up to 90% of trimmed blocks of optimal quality and evidencing the potential of this tool in future automated trimming instruments. We deployed our method to an Edge TPU hardware accelerator to showcase its capability to provide immediate and objective results at every microtomy station applied with an ad hoc hardware, potentially guaranteeing a throughput 50% higher than manual trimming.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.