Characterization of a Hg2+-Selective Fluorescent Probe Based on Rhodamine B and Its Imaging in Living Cells.

A small organic molecule P was synthesized and characterized as a fluorometric and colorimetric dual-modal probe for Hg2+. The sensing characteristics of the proposed probe for Hg2+ were studied in detail. A fluorescent enhancing property at 583 nm (>30 fold) accompanied with a visible colorimetric change, from colorless to pink, was observed with the addition of Hg2+ to P in an ethanol-water solution (8:2, v/v, 20 mM HEPES, pH 7.0), which would be helpful to fabricate Hg2+-selective probes with "naked-eye" and fluorescent detection. Meanwhile, cellular experimental results demonstrated its low cytotoxicity and good biocompatibility, and the application of P for imaging of Hg2+ in living cells was satisfactory.

Hyperion Image Analysis Depicts a Preliminary Landscape of Tumor Immune Microenvironment in OSCC with Lymph Node Metastasis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) constitutes the most common types of oral cancer. Because its prognosis varies significantly, identification of a tumor immune microenvironment could be a critical tool for treatment planning and predicting a more accurate prognosis. This study is aimed at utilizing the Hyperion imaging system to depict a preliminary landscape of the tumor immune microenvironment in OSCC with lymph node metastasis. METHODS: We collected neoplasm samples from OSCC patients. Their formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained and stained utilizing a panel of 26 clinically relevant metal-conjugated antibodies. Detection and analysis were performed for these stained cells with the Hyperion imaging system. RESULTS: Four patients met our inclusion criteria. We depicted a preliminary landscape of their tumor immune microenvironment and identified 25 distinct immune cell subsets from these OSCC patients based on phenotypic similarity. All these patients had decreased expression of CD8+ T cells in tumor specimens. Variety in cell subsets was seen, and more immune activated cells were found in patient A and patient B than those in patient C and patient D. Such differences in tumor immune microenvironments can contribute to forecasting of individual prognoses. CONCLUSION: The Hyperion imaging system helped to delineate a preliminary and multidimensional landscape of the tumor immune microenvironment in OSCC with lymph node metastasis and provided insights into the influence of the immune microenvironment in determination of prognoses. These results reveal possible contributory factors behind different prognoses of OSCC patients with lymph node metastasis and provide reference for individual treatment planning.

Coefficient of variation as an image-intensity metric for cytoskeleton bundling.

The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. Although the skewness of the pixel intensity distribution derived from fluorescently-labeled cytoskeletons has been widely used as a metric to evaluate the degree of bundling in digital microscopy images, its versatility has not been fully validated. Here, we applied the coefficient of variation (CV) of intensity values as an alternative metric, and compared its performance with skewness. In synthetic images representing extremely bundled conditions, the CV successfully detected degrees of bundling that could not be distinguished by skewness. On actual microscopy images, CV was better than skewness, especially on variable-angle epifluorescence microscopic images or stimulated emission depletion and confocal microscopy images of very small areas of around 1 µm2. When blur or noise was added to synthetic images, CV was found to be robust to blur but deleteriously affected by noise, whereas skewness was robust to noise but deleteriously affected by blur. For confocal images, CV and skewness showed similar sensitivity to noise, possibly because optical blurring is often present in microscopy images. Therefore, in practical use with actual microscopy images, CV may be more appropriate than skewness, unless the image is extremely noisy.

Cyclometalated iridium(III) complexes containing an anthracene unit for sensing and imaging singlet oxygen in cellular mitochondria.

Singlet oxygen (1O2), as a highly reactive oxygen species, plays an important role in the physical, chemical and biomedical fields, especially during photodynamic therapy (PDT) process. In this work, two iridium(III) complexes containing an anthracene unit in their diimine ligand were designed and synthesized to monitor 1O2 in living cells. The complexes were weakly emissive owing to the photoinduced electron transfer process, but exhibited intense luminescence upon capturing 1O2, resulting from the formation of the corresponding endoperoxide analogues. The remarkable turn-on luminescence response was specific toward 1O2 and in preference to other reactive oxygen species. The utilization of one of the complexes for imaging 1O2 in living cells has also been demonstrated using three different cells lines. Cells incubated with the complexes were hardly emissive. Further light irradiation at 475 nm triggered intracellular emission turn on, indicative of the production of 1O2 photochemically. The emissive pattern was well colocalized with commercially available MitoTracker, suggesting the potential applications of the complexes for imaging mitochondria 1O2. The 1O2 capturing properties rendered the complexes low photocytotoxicity since 1O2-caused oxidative damage toward cellular molecules and structures was inhibited.

Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells.

We observed prominent effects of doxorubicin (Dox), an anthracycline widely used in anti-cancer therapy, on the aggregation and intracellular distribution of both partners of the H2A-H2B dimer, with marked differences between the two histones. Histone aggregation, assessed by Laser Scanning Cytometry via the retention of the aggregates in isolated nuclei, was observed in the case of H2A. The dominant effect of the anthracycline on H2B was its massive accumulation in the cytoplasm of the Jurkat leukemia cells concomitant with its disappearance from the nuclei, detected by confocal microscopy and mass spectrometry. A similar effect of the anthracycline was observed in primary human lymphoid cells, and also in monocyte-derived dendritic cells that harbor an unusually high amount of H2B in their cytoplasm even in the absence of Dox treatment. The nucleo-cytoplasmic translocation of H2B was not affected by inhibitors of major biochemical pathways or the nuclear export inhibitor leptomycin B, but it was completely diminished by PYR-41, an inhibitor with pleiotropic effects on protein degradation pathways. Dox and PYR-41 acted synergistically according to isobologram analyses of cytotoxicity. These large-scale effects were detected already at Dox concentrations that may be reached in the typical clinical settings, therefore they can contribute both to the anti-cancer mechanism and to the side-effects of this anthracycline.

A homogeneous SIRPα-CD47 cell-based, ligand-binding assay: Utility for small molecule drug development in immuno-oncology.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria.

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.

Application of laser scanning cytometry in vascular smooth muscle remodeling.

Pulmonary artery hyperplasia is the result of proliferation of the pulmonary arterial smooth muscles (PASM). Hypoxia-induced PASM proliferation in the fetus and the newborn is the primary cause of persistent pulmonary hypertension of the newborn (PPHN). This study was performed to characterize the utility of the Laser Scanning Cytometry (LSC) method in elucidating arterial cytoskeletal remodeling in an in vitro model of PPHN. The aim was to demonstrate the following: (a) LSC is a valid method for the analysis of nuclear and cytosolic fluorescence and (b) the cumulative effects of mechanical stretch together with hypoxia promote reactive oxygen species (ROS) formation. The molecular events in response to hypoxia and the mechanical overload of the pulmonary circuit were demonstrated in vitro by subjecting hypoxic cultured primary PASM or human airway smooth muscles (hASM) to repetitive stretch-relaxation cycles at rates comparable to dynamic stretch in vivo. The altered cytoskeleton in the form of filamentous to globular actin (F:G actin) ratio was imaged and quantified at the cellular level by LSC as an endpoint. LSC can remove the nuclear G-actin fluorescence from the total G-actin fluorescence. Pulsatile stretch was found to significantly increase the total endogenous ROS and superoxide anion release in normoxic and hypoxic conditions in primary PASM fibers. The effect of stretch was predominant in increasing superoxide anion release, only under hypoxic conditions. These findings, obtained by LSC in vitro are amenable to validation in any in vivo model of interest. The in vitro model is clinically relevant to human pulmonary vascular remodeling.

A stocked toolbox for understanding the role of astrocytes in disease.

Our understanding of astrocytes and their role in neurological diseases has increased considerably over the past two decades as the diverse roles of these cells have become recognized. Our evolving understanding of these cells suggests that they are more than support cells for neurons and that they play important roles in CNS homeostasis under normal conditions, in neuroprotection and in disease exacerbation. These multiple functions make them excellent candidates for targeted therapies to treat neurological disorders. New technological advances, including in vivo imaging, optogenetics and chemogenetics, have allowed us to examine astrocytic functions in ways that have uncovered new insights into the dynamic roles of these cells. Furthermore, the use of induced pluripotent stem cell-derived astrocytes from patients with a host of neurological disorders can help to tease out the contributions of astrocytes to human disease. In this Review, we explore some of the technological advances developed over the past decade that have aided our understanding of astrocyte function. We also highlight neurological disorders in which astrocyte function or dysfunction is believed to have a role in disease pathogenesis or propagation and discuss how the technological advances have been and could be used to study each of these diseases.

γH2AX is increased in peripheral blood lymphocytes of Alzheimer's disease patients in the South Australian Neurodegeneration, Nutrition and DNA Damage (SAND) study of aging.

An early cellular response to DNA double-strand breaks is the phosphorylation of histone H2AX to form γH2AX. Although increased levels of γH2AX have been reported in neuronal nuclei of Alzheimer's disease (AD) patients, γH2AX responses in the lymphocytes of individuals with mild cognitive impairment (MCI) and AD remain unexplored. In this study, the endogenous γH2AX level was measured, using laser scanning cytometry (LSC) and visual scoring, in lymphocyte nuclei from MCI (n = 18), or AD (n = 20) patients and healthy controls (n = 40). Levels were significantly elevated in nuclei of the AD group compared to the MCI and control groups, and there was a concomitant increase, with a significant trend, from the control group through MCI to the AD group. A significant negative correlation was seen between γH2AX and the mini mental state examination (MMSE) score, when the analysis included all subjects. Receiver Operation Characteristic curves were carried out for different γH2AX parameters; visually scored percent cells containing overlapping γH2AX foci displayed the best area under the curve value of 0.9081 with 85% sensitivity and 92% specificity for the identification of AD patients versus control. Plasma homocysteine, creatinine, and chitinase-3-like protein 1 (CHI3L1) were positively correlated with lymphocyte γH2AX signals, while glomerular filtration rate (GFR) was negatively correlated. Finally, there was a diminished γH2AX response to X-rays in lymphocytes of the MCI and AD groups compared to the control group. Our results indicate that lymphocyte γH2AX levels are a potential marker for identifying individuals at increased risk of developing AD. Prospective studies with normal healthy individuals are needed to test whether there is indeed a link between γH2AX levels and AD risk.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages.

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

A microfluidic oxygen gradient demonstrates differential activation of the hypoxia-regulated transcription factors HIF-1α and HIF-2α.

Gas-perfused microchannels generated a linear oxygen gradient via diffusion across a 100 µm polydimethylsiloxane (PDMS) membrane. The device enabled exposure of a single monolayer of cells sharing culture media to a heterogeneous oxygen landscape, thus reflecting the oxygen gradients found at the microscale in the physiological setting and allowing for the real-time exchange of paracrine factors and metabolites between cells exposed to varying oxygen levels. By tuning the distance between two gas supply channels, the slope of the oxygen gradient was controlled. We studied the hypoxic activation of the transcription factors HIF-1α and HIF-2α in human endothelial cells within a spatial linear gradient of oxygen. Quantification of the nuclear to cytosolic ratio of HIF immunofluorescent staining demonstrated that the threshold for HIF-1α activation was below 2.5% O2 while HIF-2α was activated throughout the entire linear gradient. We show for the first time HIF-2α is subject to hyproxya, hypoxia by proxy, wherein hypoxic cells activate HIF in close-proximity normoxic cells. These results underscore the differences between HIF-1α and HIF-2α regulation and suggest that a microfluidic oxygen gradient is a novel tool for identifying distinct hypoxic signaling activation and interactions between differentially oxygenated cells.

ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry.

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.

Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.

We developed appropriate surgical procedures for single and repetitive multi-photon imaging of spinal cord in vivo. By intravenous anesthesia, artificial ventilation and laminectomy, acute experiments were performed in the dorsal and lateral white matter. By volatile anesthesia and minimal-invasive surgery, chronic repetitive imaging up to 8 months were performed in the dorsal column through the window between two adjacent spines. Transgenic mouse technology enabled simultaneous imaging of labeled axons, astrocytes and microglia. Repetitive imaging showed positional shifts of microglia over time. These techniques serve for investigations of cellular dynamics and cell-cell interactions in intact and pathologically changed spinal tissue.

Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

High Content, Multi-Parameter Analyses in Buccal Cells to Identify Alzheimer's Disease.

Alzheimer's disease (AD) is a degenerative brain disorder and is the most common form of dementia. Minimally invasive approaches are required that combine biomarkers to identify individuals who are at risk of developing mild cognitive impairment (MCI) and AD, to appropriately target clinical trials for therapeutic discovery as well as lifestyle strategies aimed at prevention. Buccal mucosa cells from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing cohort (n=60) were investigated for cytological markers that could be used to identify both MCI and AD individuals. Visual scoring of the buccal cytome demonstrated a significantly lower frequency of basal and karyorrhectic cells in the MCI group compared with controls. A high content, automated assay was developed using laser scanning cytometry to simultaneously measure cell types, nuclear DNA content and aneuploidy, neutral lipid content, putative Tau and amyloid-ß (Aß) in buccal cells. DNA content, aneuploidy, neutral lipids and Tau were similar in all groups. However, there was significantly lower Tau protein in both basal and karyolytic buccal cell types compared with differentiated buccal cells. Aß, as measured by frequency of cells containing Aß signal, as well as area and integral of Aß signal, was significantly higher in the AD group compared with the control group. Buccal cell Aß was correlated with mini-mental state examination (MMSE) scores (r = -0.436, P=0.001) and several blood-based biomarkers. Combining newly identified biomarkers from buccal cells with those already established may offer a potential route for more specific biomarker panels which may substantially increase the likelihood of better predictive markers for earlier diagnosis of AD.

Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.