Thick Tissue Sections Research Articles

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.

INSIHGT: A scalable and homogeneous 3D multiplexed molecular mapping platform for spatial biology and precision medicine.

e15180 Background: The dynamic interplay between cancer cells and their microenvironment is a determinant of disease evolution and response to treatment. However, the current state of three-dimensional (3D) tumor microenvironment analysis is hindered by inadequate penetration of antibodies into thick tissue sections, leading to a substantial gap in acquiring unbiased, precise 3D multiplexed immunohistochemistry (IHC) images. Methods: We developed INSIHGT to surmount these limitations by enabling multi-round, multiplexed 3D IHC in standard formalin-fixed tissues. This fully automatable, fast-track method requires a simple series of chemical cocktail exchanges, translating to a tissue-to-image time of 6 days for 1 cm³ samples and as little as 6 hours for biopsy specimens. INSIHGT offers a scalable and efficient solution for detailed 3D spatial mapping of cancerous cells and their interactions within their native context. Results: 3D IHC with INSIHGT shows ideal penetration homogeneity, which quantitatively matches reference signals in 1cm3 samples. We further validated INSIHGT with 309 commercially available primary antibodies and is compatible with simultaneous fluorescent in situ hybridization (FISH) to detect RNA transcripts or DNA chromosomal abnormalities. Notably, the quality of post-INSIHGT traditional histological analyses, such as H&E and standard IHC, was not altered. We demonstrated a 3D mapping of over 20 markers in a 1 mm-thick tumor sample across seven INSIHGT cycles, and an atlas of multiple tumour samples with 3D IHC & H&E. The large datasets seamlessly integrates with single-cell segmentation and analysis workflows, yielding comprehensive maps that elucidate protein expression, cell morphology, spatial positioning, and potential cell-cell interactions. Conclusions: INSIHGT provides an unparalleled level of visualization and analytical detail concerning the spatial dynamics within the tumor microenvironment at a single-cell resolution. As a transformative next generation histopathology platform, it can uncover new structural and molecular details within clinical specimens, accelerating biomarker discovery and novel diagnostic assays development, thereby enriching the field of tumor biology and propelling the progress of personalized oncology treatments.

Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

Cryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Optimizing Ideal Thickness of Sections for Epidermis in Intra-Operative Frozen Sections.

To estimate and evaluate the optimal thickness of tissue section for skin and dermal appendages intra-operatively and to compare the morphology and architecture of the epidermis and its appendages at 7 and 10 microns. After obtaining clearance from the institutional human ethical committee, 101 skin margins were prospectively analyzed using a two-step embedding technique. After multiple trials and errors, 7- and 10-micron thicknesses were selected for the present study. Artefacts, staining characteristics, cellular morphology, cellular outline, and nuclear outline were assessed and scored as unacceptable or acceptable. The data were entered in a Microsoft Excel spreadsheet and analyzed using SPSS software. There was a statistically significant difference between the sections obtained at 7 microns and 10 microns (p-value: <0.001), the latter were better in all the parameters analyzed. However, no difference was noted in the characteristics of the dermal appendages (p-value: >0.05). While mucosal margins can be obtained at the usual 5-7 microns, the same thickness is not optimal for skin margins intra-operatively. Frozen sections for the skin margins may be set at 10 microns, to save time, minimize artefacts, and for better readability.

Phenotypic characterization of liver tissue heterogeneity through a next-generation 3D single-cell atlas

Three-dimensional (3D) geometrical models are potent tools for quantifying complex tissue features and exploring structure–function relationships. However, these models are generally incomplete due to experimental limitations in acquiring multiple (> 4) fluorescent channels in thick tissue sections simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we combined deep-tissue immunostaining, multiphoton microscopy, deep-learning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatial single-cell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the effect on the liver's overall structure when changing the hepatic environment after removing KCs. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs results in morphological changes in hepatocytes and HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, opening up avenues for in-depth investigations into tissue structure across both normal and pathological conditions.

Clinico-pathological evaluation of gastric adeno carcinoma with reference to ki-67 LI immuno-staining

Gastric cancer is a life-threatening disease accounting for the one of the most common types of disease and second most that cause the cancer which lead to death. Many molecular markers were established as a possible prognostic factor apart from routine grading and TNM staging. The variable prognosis of gastric cancer within a pathological grade necessitates a newer marker which can be correlated with the prognosis and aggressiveness of the disease. ki-67 is a nuclear protein that is known as a marker of cellular proliferation and ribosomal RNA transcription. According to analysis, the ki-67 is a nuclear proliferation antigen affecting the health of the people as it cannot be cycle in the resting phase. Moreover, the fraction of ki-67 is clinical course of various cancers. To evaluate the expression of ki-67 LI in gastric cancer and correlate the findings with various clinico-pathological features.Prospective study was conducted from October 2018 to September 2021 at the department of Pathology, MKCG Medical College, Berhampur, Odisha. Two sets of 3–4-micron thickness of tissue sections prepared from blocks of histologically confirmed cases of gastric adenocarcinoma were taken, one for routine H&amp;E and other for ki-67 LI IHC study. IHC protocol of DAKO was followed along with DAB visualisation. The results were correlated with different clinico-pathological features of Gastric cancer. Higher ki-67 LI was correlated significantly with tumour location, histological grade and type but not with age and sex.In our study, IHC assessment of ki-67 LI correlates well with histological grade and type of tumour. ki-67 LI can be taken as a useful method in identifying aggressiveness of the tumour with an indication of adjuvant chemotherapy. Post-operative follow-up and large sample size could throw more light.

The Expression of Mucin-4 (MUC4) in Sarcomas Apart From Sclerosing Epithelioid Fibrosarcoma and Low-Grade Fibromyxoid Sarcoma.

Background Low-grade fibromyxoid sarcoma (LGFMS) and sclerosing epithelioid fibrosarcoma (SEF) are two rare but aggressive soft tissue sarcomas that can be difficult to distinguish due to histopathological similarities. The present study examines the diagnostic capacities of mucin-4 (MUC4), a transmembrane mucin, in identifying different types of sarcomas and broadens its evaluation to include a wide range of sarcomas. Methods Immunohistochemical (IHC) examination of tissue samples from various sarcomas was performed using a mouse anti-MUC4 monoclonal antibody. IHC was conducted on 4-mm thick formalin-fixed paraffin-embedded tissue sections after pressure cooker antigen retrieval with a mouse anti-MUC4 monoclonal antibody. Results MUC4 was shown to be highly expressed in SEF (n=13) and LGFMS (n=10), while focal positivity in synovial sarcoma (n=1). Other sarcomas, such as malignant peripheral nerve sheath tumors, fibrosarcoma, leiomyosarcoma, liposarcoma, and myxofibrosarcoma, exhibited no expression (n=0). These findings are consistent with previous research and support MUC4 specificity as a SEF and LGFMS marker.This study provides information on the diagnostic efficacy of MUC4, particularly in the context of certain subtypes. It not only helps our understanding of these unique instances, but it also provides context for histopathological and IHC findings in soft tissue sarcoma. Furthermore, this study investigates the influence of age and gender on MUC4 expression in a range of sarcomas, which was typically understudied in the literature and found no relation with expression of MUC4. Conclusion In conclusion, this study adds to our understanding of soft tissue sarcomas by emphasizing the crucial role of MUC4 in certain sarcoma subtypes while acknowledging the complex variety of the sarcoma landscape. Further research is needed to understand the molecular mechanism that governs marker expression patterns, as well as the therapeutic implications.

Spatial Multiomics Profiling of Angioimmunoblastic T-Cell Lymphoma

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of non-Hodgkin lymphoma (NHL) characterized primarily by a T follicular helper cell (TFH) phenotype. AITL presents with a highly complex tumor microenvironment with the tumor cells on a scaffold of highly arborized CD21+ follicular dendritic cells found in proximity to high endothelial venules (HEVs). The organized structure of the lymph node with distinct B cell and T cell zones is disrupted in the lymph node samples from patients with AITL. Lymphoid follicles in most AITL cases tend to be depleted, not hyperplastic as in other lymphomas. AITL tumor cells express CD10, BCL6, PD1, CXCL13, C-X-C motif chemokine receptor 5 (CXCR5), ICOS, and signaling lymphocytic activation molecule-associated protein (SAP) with diagnosis based on the expression of at least 2 of these markers. Our study uses novel spatial omics technology (Deterministic Barcoding in Tissue (DBiT-seq) and multiplexed immunofluorescence staining (CODEX)) to fully characterize the transcriptional and proteomic landscape of AITL. Methods: In this study, initial FFPE biopsy specimens from 22 patients with AITL were collected from archives. 10 µm thick tissue sections on poly-L-lysine coated slides were prepared for the performance of multiplexed immunofluorescence imaging (CODEX) for 12 immune-related markers: Podoplanin, CD20, CD4, CD8, CD68, CD45RO, CD3e, GranzymeB, PD1, Ki67, CD21 and CD34 using the PhenoCycler-Fusion system. We also validated anti-EBV Latent Membrane Protein 1 antibody. Cell segmentation was performed using a Stardist-based model in QuPath and downstream analysis done using the Seurat package. On an adjacent tissue section, we used Spatial CITE-seq to co-map the whole transcriptome and ~160 proteins. Results: From the multiplexed immunofluorescence staining, we observed CD21+ Follicular dendritic cells in close proximity to HEVs as expected from the spatial architecture of AITL. We also found 11 spatially distinct clusters differentially expressing the marker proteins. We observed a lower number of CD8+ cytotoxic T cells relative to CD4+ Helper T cells. We also identified cellular neighborhoods and their composition found a population of FDCs that directly interact with the tumor cells. From spatial CITE-seq, we average of ~670 genes were found in each spot. We also found the expression of CXCL13, TET2, DNMT3A and PDCP1 which are markers associated with AITL. There was a strong correlation between the transcript signals detected in Spatial CITE-seq and the protein expression from CODEX. Conclusions: Our study revealed the spatial organization of the tumor microenvironment of AITL. We envision that combining multiplexed immunofluorescence and Spatial CITE-seq will further our understanding of the AITL tumor environment and could lead to the discovery of novel clinical targets.

Age dynamics of strain differences in the morphofunctional state of pancreatic beta- and amylin-producing cells in SHR and Wistar rats

Large-scale epidemiological studies have shown that cardiac pathology and progressive atherosclerosis in patients with diabetes mellitus occurred already at the stage of prediabetes. Obesity and insulin resistance affect cardiometabolic health due to pleiotropic effects of insulin. Despite the vast range of research, some aspects remain hidden links in the overall pathogenesis of metabolic and hemodynamic disorders. The aim of the work was to study the morphofunctional state of pancreatic islets (PIs), beta- and amylin-producing cells in male rats of Wistar strain (normotensive) and SHR (with spontaneous development of hypertension) in age dynamics. Materials and methods. The study was carried out using 38 male Wistar rats and SHRs aged 7 and 24 months. Non-invasive blood pressure (BP) detection procedures were done using the BP-2000 Blood Pressure Analysis System. The morphofunctional state of PIs was examined in serial 5-μm thick pancreatic tissue sections. Beta- and amylin-producing cells were detected after histological preprocessing and the use of monoclonal FITC-conjugated antibodies. Image file processing was done via ImageJ software (National Institutes of Health, USA). Levels of glycemia were monitored with a SUPER GLUCOCARD-II glucometer. Results. SHRs were hyperglycemic both at 7 and at 24 months, 8.41 ± 0.15 mmol/l and 8.90 ± 0.14 mmol/l, respectively, with elevated BP, 155 ± 5 / 80 ± 5 mm Hg and 165 ± 5 / 90 ± 5 mm, respectively. Old SHRs developed PI hypertrophy mainly associated with the increased number and percentage of beta-cells, apparently in response to hyperglycemia. Both in the PIs of adult and old SHRs, the number of amylin-producing cells was lower while the content of amylin was higher than those in the age-matched Wistar rats. Conclusions. Male SHRs are characterized by a persistent increase in blood pressure and abnormalities of carbohydrate metabolism already at adult age, one of the manifestations of which is hyperglycemia worsening with age. Chronic hyperglycemia in SHRs due to the higher insulin requirement finds its expression in low content of this hormone in the islets at adult age and decreased its content in beta-cells in old animals.

Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.

Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection. The two-step procedure also hinders the further improvement of spatial resolution to <10 μm as needed for subcellular proteomics. Herein, we developed a high-throughput and high-resolution spatial proteomics platform by seamlessly coupling deep ultraviolet (DUV) laser ablation (LA) with nanoPOTS (Nanodroplet Processing in One pot for Trace Samples)-based sample preparation. We demonstrated the DUV-LA system can quickly isolate and collect tissue samples at a throughput of ∼30 spots/min and a spatial resolution down to 2 μm from a 10 μm thick human pancreas tissue section. To improve sample recovery, we developed a proximity aerosol collection approach by placing DMSO droplets close to LA spots. We demonstrated the DUV-LA-nanoPOTS platform can detect an average of 1312, 1533, and 1966 proteins from ablation spots with diameters of 7, 13, and 19 μm, respectively. In a proof-of-concept study, we isolated and profiled two distinct subcellular regions of the pancreas tissue revealed by hematoxylin and eosin (H&E) staining. Quantitative proteomics revealed proteins specifically enriched to subcellular compartments.

An intelligent workflow for sub-nanoscale 3D reconstruction of intact synapses from serial section electron tomography

BackgroundAs an extension of electron tomography (ET), serial section electron tomography (serial section ET) aims to align the tomographic images of multiple thick tissue sections together, to break through the volume limitation of the single section and preserve the sub-nanoscale voxel size. It could be applied to reconstruct the intact synapse, which expands about one micrometer and contains nanoscale vesicles. However, there are several drawbacks of the existing serial section ET methods. First, locating and imaging regions of interest (ROIs) in serial sections during the shooting process is time-consuming. Second, the alignment of ET volumes is difficult due to the missing information caused by section cutting and imaging. Here we report a workflow to simplify the acquisition of ROIs in serial sections, automatically align the volume of serial section ET, and semi-automatically reconstruct the target synaptic structure.ResultsWe propose an intelligent workflow to reconstruct the intact synapse with sub-nanometer voxel size. Our workflow includes rapid localization of ROIs in serial sections, automatic alignment, restoration, assembly of serial ET volumes, and semi-automatic target structure segmentation. For the localization and acquisition of ROIs in serial sections, we use affine transformations to calculate their approximate position based on their relative location in orderly placed sections. For the alignment of consecutive ET volumes with significantly distinct appearances, we use multi-scale image feature matching and the elastic with belief propagation (BP-Elastic) algorithm to align them from coarse to fine. For the restoration of the missing information in ET, we first estimate the number of lost images based on the pixel changes of adjacent volumes after alignment. Then, we present a missing information generation network that is appropriate for small-sample of ET volume using pre-training interpolation network and distillation learning. And we use it to generate the missing information to achieve the whole volume reconstruction. For the reconstruction of synaptic ultrastructures, we use a 3D neural network to obtain them quickly. In summary, our workflow can quickly locate and acquire ROIs in serial sections, automatically align, restore, assemble serial sections, and obtain the complete segmentation result of the target structure with minimal manual manipulation. Multiple intact synapses in wild-type rat were reconstructed at a voxel size of 0.664 nm/voxel to demonstrate the effectiveness of our workflow.ConclusionsOur workflow contributes to obtaining intact synaptic structures at the sub-nanometer scale through serial section ET, which contains rapid ROI locating, automatic alignment, volume reconstruction, and semi-automatic synapse reconstruction. We have open-sourced the relevant code in our workflow, so it is easy to apply it to other labs and obtain complete 3D ultrastructures which size is similar to intact synapses with sub-nanometer voxel size.

Protective effect of paeoniflorin in diabetic nephropathy: A preclinical systematic review revealing the mechanism of action.

Paeoniflorin (PF), the main active glucoside of Paeonia Lactiflora, has many pharmacological activities, such as inhibition of vasodilation, hypoglycemia, and immunomodulation. Although the current evidence has suggested the therapeutic effects of PF on diabetic nephropathy (DN), its potential mechanism of action is still unclear. A systematic review and meta-analysis of the existing literature on paeoniflorin treatment in DN animal models was performed to evaluate the efficacy and mechanism of PF in DN animal models. The risk of bias in each study was judged using the CAMARADES 10-item quality checklist with the number of criteria met varying from 4 / 10 to 7 / 10, with an average of 5.44. From inception to July 2022, We searched eight databases. We used the Cochrane Collaboration's 10-item checklist and RevMan 5.3 software to assess the risk of bias and analyze the data. Three-dimensional dose/time-effect analyses were conducted to examine the dosage/time-response relations between PF and DN. Nine animal studies were systematically reviewed to evaluate the effectiveness of PF in improving animal models of DN. Meta-analysis data and intergroup comparisons indicated that PF slowed the index of mesangial expansion and tubulointerstitial injury, 24-h urinary protein excretion rate, expression of anti-inflammatory mediators (mRNA of MCP-1, TNF-α, iNOS, and IL-1 β), and expression of immune downstream factors (P-IRAK1, TIRF, P-IRF3, MyD88, and NF-κBp-p65). Furthermore, modeling methods, animal species, treatment duration, thickness of tissue sections during the experiment, and experimental procedures were subjected to subgroup analyses. The present study demonstrated that the reno-protective effects of PF were associated with its inhibition on macrophage infiltration, reduction of inflammatory mediators, and immunomodulatory effects. In conclusion, PF can effectively slow down the progression of DN and hold promise as a protective drug for the treatment of DN. Due to the low bioavailability of PF, further studies on renal histology in animals are urgently needed. We therefore recommend an active exploration of the dose and therapeutic time frame of PF in the clinic and in animals. Moreover, it is suggested to actively explore methods to improve the bioavailability of PF to expand the application of PF in the clinic.

Evaluating the optimal tissue thickness for mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 μm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated. Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy. Analyzing sectioned tissues at higher thickness (>10μm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7μm) does not result in a significant increase in lipid annotations. This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.

Study of the cell proliferation index (Ki67) in inflammatory odontogenic cysts

IntroductionInflammatory odontogenic cysts are lesions that develop from the cellular remains of odontogenesis. They are lesions that may recur after incomplete surgical enucleation with a variable clinical appearance depending on the clinical form. These clinical variations could be explained by the biological behaviour of the lesion cells which can be explored by the study of the cell proliferation index (Ki67). This justifies the initiation of this work whose objective was to study the level of expression of Ki67 and to measure the index of proliferation in odontogenic inflammatory cysts. Material and methodThis was a cross-sectional study with an analytical aim on surgical parts of odontogenic inflammatory cysts over a period of 31 months in the odontostomatology department of the Hôpital Général Idrissa Pouye. The Ki67 immunohistochemistry study was performed on 3μ thick histological sections of paraffin-fixed tissue. The inclusion criteria were any surgical specimen of odontogenic inflammatory cyst that we could collect. The variables studied were sociodemographic, clinical and histopathological. ResultsRadiculo-dental cysts represented 80.5% of the surgical specimens collected and residual cysts 12.2%. The percentage of Ki67-labelled cells was greater than 15% in 80.5% of the odontogenic inflammatory cysts and less than 7% in 19.5% of the cases. In addition, intense labelling was noted in radiculo-dental cysts and weak labelling in the other clinical forms of inflammatory odontogenic cysts obtained. An association was noted between the percentage of Ki67 labelled cells and histological types (p-value <0.001); but also between the intensity of Ki67 labelling and histological types (p-value <0.001). ConclusionThe cell proliferation index (Ki67) could be used as a prognostic biomarker in odontogenic inflammatory cysts.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Infrared laser ablation dynamics using light scattering

The dynamics of infrared laser ablation at atmospheric pressure was studied employing continuous visible laser light scattering in the expanding plume. A pulsed infrared optical parametric oscillator at 2940 nm wavelength and 5 ns pulse width was used to irradiate samples comprising liquid glycerol or 50 µm thick sections of rat liver tissue. The scattered light from the expanding laser ablation plume was measured using a 532 nm continuous laser parallel to the target and several millimeters above the ablated spot. The scattered light was recorded using a photomultiplier detector and the signal used to estimate the time at which the plume front passed through the continuous laser beam. The velocity of the ablation plume was obtained from consecutive time and distance measurements and the fraction of the laser energy transferred to the expanding shock wave was determined using the Taylor shock wave model. Plume modeling calculations indicate that the ablation is driven by phase explosion that is thermally confined and near the stress confinement regime.

High-throughput single-molecule localization microscopy: Potential clinical applications.

High-throughput single-molecule localization microscopy: Potential clinical applications.

Nontoxic Fluorescent Nanoprobes for Multiplexed Detection and 3D Imaging of Tumor Markers in Breast Cancer

Multiplexed fluorescent immunohistochemical analysis of breast cancer (BC) markers and high-resolution 3D immunofluorescence imaging of the tumor and its microenvironment not only facilitate making the disease prognosis and selecting effective anticancer therapy (including photodynamic therapy), but also provides information on signaling and metabolic mechanisms of carcinogenesis and helps in the search for new therapeutic targets and drugs. The characteristics of imaging nanoprobe efficiency, such as sensitivity, target affinity, depth of tissue penetration, and photostability, are determined by the properties of their components, fluorophores and capture molecules, and by the method of their conjugation. Regarding individual nanoprobe components, fluorescent nanocrystals (NCs) are widely used for optical imaging in vitro and in vivo, and single-domain antibodies (sdAbs) are well established as highly specific capture molecules in diagnostic and therapeutic applications. Moreover, the technologies of obtaining functionally active sdAb-NC conjugates with the highest possible avidity, with all sdAb molecules bound to the NC in a strictly oriented manner, provide 3D-imaging nanoprobes with strong comparative advantages. This review is aimed at highlighting the importance of an integrated approach to BC diagnosis, including the detection of biomarkers of the tumor and its microenvironment, as well as the need for their quantitative profiling and imaging of their mutual location, using advanced approaches to 3D detection in thick tissue sections. The existing approaches to 3D imaging of tumors and their microenvironment using fluorescent NCs are described, and the main comparative advantages and disadvantages of nontoxic fluorescent sdAb-NC conjugates as nanoprobes for multiplexed detection and 3D imaging of BC markers are discussed.

Tissue libraries enable rapid determination of conditions that preserve antibody labeling in cleared mouse and human tissue.

Difficulty achieving complete, specific, and homogenous staining is a major bottleneck preventing the widespread use of tissue clearing techniques to image large volumes of human tissue. In this manuscript, we describe a procedure to rapidly design immunostaining protocols for antibody labeling of cleared brain tissue. We prepared libraries of 0.5-1.0 mm thick tissue sections that are fixed, pre-treated, and cleared via similar, but different procedures to optimize staining conditions for a panel of antibodies. Results from a library of mouse tissue correlate well with results from a similarly prepared library of human brain tissue, suggesting mouse tissue is an adequate substitute for protocol optimization. These data show that procedural differences do not influence every antibody-antigen pair in the same way, and minor changes can have deleterious effects, therefore, optimization should be conducted for each target. The approach outlined here will help guide researchers to successfully label a variety of targets, thus removing a major hurdle to accessing the rich 3D information available in large, cleared human tissue volumes.