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Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri‑infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation. [ABSTRACT FROM AUTHOR]

Abstract Background The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. Methods Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. Results On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. Conclusions Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

• A sub-100 nm perovskite-based fluorescent probe modified with the survivin antibody was developed. • Polyvinyl pyrrolidone (PVP) coated perovskite crystals can keep the probe with great fluorescence intensity and resistance of water and oxygen. • The survivin protein can be an ideal fluorescence target for rapid diagnosis of colorectal cancer. Due to non-specific signs in the early stage of colorectal cancers, most patients result in a low early diagnosis rate. Survivin protein is upregulated in many malignant tumors, making it an ideal diagnostic and therapeutic target for colorectal cancers. However, the survivin protein is located in the cell nucleus, which needs the probe to penetrate the complex nuclear membrane structure for targeting and recognizing. Here, we have developed a sub-100 nm perovskite-based fluorescent probe modified with the survivin antibody, which can freely target survivin protein through the nuclear membrane. Polyvinyl pyrrolidone (PVP) coated perovskite crystals are obtained by the supersaturated crystallization, which keep the probe with great fluorescence intensity and resistance of water and oxygen. We have established colorectal cancer cell lines and colorectal cancer tissues to test the feasibility and efficacy of the probe. After investigating the fluorescence intensity values of 20 probing samples, the average fluorescence intensity of colorectal cancer sections is 8.7 times higher than that of paracancerous tissues. Our work demonstrates that the survivin can be an ideal fluorescence target for rapid diagnosis of colorectal cancer, which also provides a research foundation for fluorescence-guided surgery and nano-therapy for targeting the survivin protein. [ABSTRACT FROM AUTHOR]

Background: The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification.Methods: Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures.Results: On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment.Conclusions: Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4′,6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes.

Isolation of extremely rare circulating tumor cell (CTC) clusters from the bloodstream of patients enables minimally invasive diagnosis and prognosis while providing information on their role in metastasis. A few technologies specifically developed for the enrichment of CTC clusters fail to achieve a high enough processing throughput to be practical in clinical settings or risk damaging large clusters owing to their structural design producing high shear forces. Here, we outline a methodology developed for rapid and effective enrichment of CTC clusters from cancer patients, independent of the cluster size and cell surface markers. Minimally invasive access to tumor cells in hematogenous circulation will be an integral part of cancer screening and personalized medicine., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Autoimmune diseases are strictly connected with the presence of autoantibodies in patient serum. Detection of Antinucleolar Antibodies (ANAs) in patient serum is performed using a laboratory technique named Indirect Immunofluorescence (IIF) followed by manual evaluation on the acquired slides from specialized personnel. In this procedure, several limitations appear and several automatic techniques have been proposed for the task of ANA detection. In this work we present a method achieving state-of-the-art performance on a publicly available dataset. More precisely, two powerful and rotation invariant descriptors are incorporated into a two stage classification scheme where the feature vectors are represented and fused in the dissimilarity space. Then, in a second level dissimilarity vectors are classified using a linear SVM classifier. Evaluation on the HEp-2 cell contest dataset yields a 70.16% performance on cell-level classification. Furthermore we provide results in Image Level Classification where a 78.57% classification rate was achieved. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) has been widely applied to study cellular functions;however, the relationship between cellular elasticity and ultrastructure density of a live cell remains to be discovered. The objective of this study was thus to extend our previous method of integrating AFM and immunofluorescence imaging to measure the ultrastructure distribution-related local mechanical properties of live cells. First, the morphology of a live cell was obtained by AFM. Second, the indentation sites were selected and flexible force volume indentation was performed. Third, the immunofluorescence image of the cell was obtained. The last was the mapping of the indentation site to the immunofluorescence image and obtaining the relationship between the local elastic properties and cytoskeleton density. The results on differentiated rat Schwann cells (RSCs) showed that the elastic modulus of stress fibers is higher than those of the nucleus and cytosol. The local elastic modulus of the live RSCs is correlated to the actin density, and the stress fiber that behaves like a pretension beam can give RSCs enough strength to envelop axons during myelination. In particular, the elastic properties of the live RSCs were twofold lower than those of the fixed. The results demonstrated the integrated method's applicability for a live cell. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood-derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single-cell suspension generation from different tissue Alternate Protocol 1: Single-cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan-MDSCs or PMN-MDSC and M-MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN-MDSC and M-MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples., (© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.)

A cost-effective method that integrates high-resolution morphology using an atomic force microscope and immunofluorescence imaging for measuring the local mechanical properties of a cell was developed. By considering the normal indentation conditions and the distribution of the underlying cytoskeleton, a criterion for selecting indentation sites was proposed. PC-12 cells cultivated under normal and high D-glucose medium are employed to demonstrate the applicability of the proposed method. The apparent Young's modulus for each indentation site was estimated by fitting the data with a pyramidal punch contact mechanics model. The results showed that the cell bodies cultivated in the high D-glucose medium were higher but their growth cones were shorter than those cultivated in a normal medium. The Young's moduli of the growth cones were positively correlated with the density of the actin filament in the cytoskeleton. The Young's moduli at the growth cone and the nucleus region of cells cultivated in the high D-glucose medium were higher and lower, respectively, than those of the control group. The results demonstrated the integrated method could correlate local mechanical properties and distribution of actin filament of the growth cone of PC-12 cell. [ABSTRACT FROM AUTHOR]

Abstract: Immunofluorescence imaging on polymeric biomaterials is often inhibited by autofluorescence and other optical phenomena. This often limits the analysis that can be performed on cells that are in contact with these materials. This study outlines a method that will quench these inhibitive optical phenomena on a variety of polymeric materials, including poly(glycerol sebacate), poly(urethane), poly(l-lactide–co-ε-caprolactone), and poly(lactic acid–co-glycolic acid). The method uses a simple material treatment method utilizing Sudan Black B (SB), which is commonly used as an autofluorescence quenching molecule in tissue histology, but has not yet been used in biomaterials analysis. The quenching mechanism in the selected polymers is investigated using attenuated total reflectance Fourier transform infrared spectroscoy, ultraviolet–visible light absorbance and fluorescence analysis, and scanning electron microscopyobservation of the material morphology prior to and after SB treatment. The results point to SB eliminating the inhibitive light phenomena of these materials by two methods: (i) chemical interaction between SB and the polymer molecules and (ii) physical interaction whereby SB forms a physical barrier that can absorb scattered light and quench autofluorescence interference during fluorescence microscopy. The studies show that the treatment of polymers with SB is robust across the polymers tested, in both porous and non-porous formats. The method does not interfere with immunofluorescent imaging of fluorescently labeled biological cells cultured on these polymers. This quick, simple, and affordable method enables a variety of analyses to be conducted that may otherwise have been impractical or impossible. [Copyright &y& Elsevier]

Inflammasomes are multiprotein complexes that assemble in host cells upon recognition of infection or danger via pattern recognition receptors and/or danger recognition receptors. The assembly of inflammasomes results in the activation of caspase-1 and is followed by the enzymatic maturation and secretion of inflammatory cytokines like interleukin 1β (IL-1β) and IL-18.In the oral cavity, gingival epithelial cells (GECs) line the mucosa and have a barrier role for invading pathogens. In these cells, the NLRP3 inflammasome is the best studied and has been shown to play a role in the inflammatory immune response against a variety of oral pathogens. As such, in order to study gingivitis and other oral pathologic inflammatory conditions, studying the activation of inflammasomes is important. The simplest way to detect inflammasome activation is to detect the activated caspase-1, as well as the secretion of mature IL-1β and IL-18, via ELISA, Western blot, and immunofluorescence techniques.Here we describe the detection of NLRP3 inflammasome activation by the oral pathogen Porphyromonas gingivalis in human GECs via these three methods and mention other methods and techniques that we have successfully used together with these in our quest to understand the host-pathogen interaction., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4′,6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes. [ABSTRACT FROM AUTHOR]

Understanding the morphogenesis and differentiation of tissues and organs from progenitor fields requires methods to visualize this process. Despite an ever-growing recognition that ECM plays an important role in tissue development, studies of ECM movement, and patterns in live tissue are scarce. Here, we describe a method in which a living limb bud is immunolabeled prior to fixation using fluorescent antibodies that recognize two ECM constituents, fibronectin and fibrillin 2. The results show that undifferentiated mesenchyme in quail embryos can be distinguished from prechondrogenic cellular condensations, in situ, via ECM antibodies-indicating the developmental transition from naïve mesenchyme to committed skeletal tissue. We conclude that our live tissue injection method is a general approach that allows visualization of the structural characteristics and the distribution pattern of ECM scaffolds, in situ. With slight modifications, the method will produce robust fluorescence images of ECM scaffolds in any suitable tissue mass and allow multiple kinds of optical analyses including virtual 3D reconstructions., (© 2018 Elsevier Inc. All rights reserved.)

Inflammatory conditions induce redistribution of junctional adhesion receptors toward the apical regions of endothelial cells promoting lymphocyte TEM. Much of the molecular structures of TEM have been revealed; however, the biophysical mechanisms underlying this process remain to be fully elucidated. Here, we used immunofluorescence microscopy and AFM to study endothelial distribution of adhesion molecules upon lymphocyte activation and transmigration. Our immunofluorescence results revealed redistribution of JAM-A and PECAM-1 but not ICAM-1 or VCAM-1 toward the apical junctional regions of HUVECs following a 6-h stimulation with TNF-α and IFN-γ. Consistently, our SCFS studies revealed that Jurkat cell adhesion to stimulated HUVEC monolayers was significantly greater in junctional regions. Enhanced adhesion was mediated mostly by JAM-A receptors. Further AFM adhesion mapping of the homophilic JAM-A/JAM-A interaction on the surfaces of HUVECs revealed a greater number of JAM-A receptors available for binding along junctional regions after TNF-α and IFN-γ stimulation. Our data reveal for the first time that adhesion "hot spots" of JAM-A receptors are involved in initiating lymphocyte TEM under inflammatory conditions.