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.

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.

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.

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.

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.

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.

Laser-scanning cytometry can quantify human adipocyte browning and proves effectiveness of irisin.

Laser-scanning cytometry is presented as a tool allowing population scale analysis of ex vivo human brown adipogenic differentiation. It combines texture analysis and detection of Ucp1 protein content in single brown adipocytes of mixed cell populations with gene expression pattern and functional characteristics of browning. Using this method we could validate mouse data in human samples demonstrating the effectiveness of irisin to induce "beige" differentiation of subcutaneous white adipocytes.

Cross-Linking Cellulosic Fibers with Photoreactive Polymers: Visualization with Confocal Raman and Fluorescence Microscopy.

The properties of paper sheets can be tuned by adjusting the surface or bulk chemistry using functional polymers that are applied during (online) or after (offline) papermaking processes. In particular, polymers are widely used to enhance the mechanical strength of the wet state of paper sheets. However, the mechanical strength depends not only on the chemical nature of the polymeric additives but also on the distribution of the polymer on and in the lignocellulosic paper. Here, we analyze the photochemical attachment and distribution of hydrophilic polydimethylacrylamide-co-methacrylate-benzophenone P(DMAA-co-MABP) copolymers with defined amounts of photoreactive benzophenone moieties in model paper sheets. Raman microscopy was used for the unambiguous identification of P(DMAA-co-MABP) and cellulose specific bands and thus the copolymer distribution within the cellulose matrix. Two-dimensional Raman spectral maps at the intersections of overlapping cellulose fibers document that the macromolecules only partially surround the cellulose fibers, favor to attach to the fiber surface, and connect the cellulose fibers at crossings. Moreover, the copolymer appears to accumulate preferentially in holes, vacancies, and dips on the cellulose fiber surface. Correlative brightfield, Raman, and confocal laser scanning microscopy finally reveal a reticular three-dimensional distribution of the polymer and show that the polymer is predominately deposited in regions of high capillarity (i.e., in proximity to fine cellulose fibrils). These data provide deeper insights into the effects of paper functionalization with a copolymer and aid in understanding how these agents ultimately influence the local and overall properties of paper.

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

Quantification of fluorescent reporters in plant cells.

Fluorescent reporters are powerful tools for plant research. Many studies require accurate determination of fluorescence intensity and localization. Here, we describe protocols for the quantification of fluorescence intensity in plant cells from confocal laser scanning microscope images using semiautomated software and image analysis techniques.

Live cell imaging of the cytoskeleton and cell wall enzymes in plant cells.

The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis.

γH2AX responses in human buccal cells exposed to ionizing radiation.

DNA double strand breaks are induced by ionizing radiation (IR), leading to the phosphorylation of the core histone protein H2AX (termed γH2AX). The understanding of the γH2AX responses in irradiated human buccal cells is still very limited. We used visual scoring and laser scanning cytometry (LSC) methods to investigate γH2AX signaling following exposure of human buccal cells (from six individuals) to ionizing radiation at 0-4 Gy. The frequency of nuclei containing 15-30 γH2AX foci was significantly elevated 30 min post-IR exposure (by visual scoring). Concomitantly, there was a significant decrease in the frequency of cells without foci following exposure to IR. IR-induced γH2AX signal as determined by laser scanning cytometry (which included γH2AX integral and MaxPixel value) increased significantly in all individual's 2N nuclei 30 min post-IR and was similar for all three nuclear shapes identified. Individuals with the lowest baseline γH2AX integral (i.e., in nonirradiated cells) showed the greatest fold stimulation of γH2AX and significant dose-responses to IR doses of 1, 2, and 4 Gy. In 5 out of 6 individuals, the frequency of visually scored γH2AX in nuclei showed a strong correlation (up to r = 0.999) with LSC scored γH2AX integrals. The γH2AX response and subsequent decline varied between individuals but remained elevated above baseline levels 24 h post IR exposure. γH2AX response in irradiated human buccal cells has potential to be used as an index of baseline DNA damage in population studies. The variable response to IR exposure between individuals should be taken into consideration when using the γH2AX assay for radiation biodosimetry.

Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and its potential application in radiation biodosimetry.

Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

Bringing CLARITY to gray matter atrophy.

Gray matter atrophy has been shown to be a strong correlate to clinical disability in multiple sclerosis (MS) and its most commonly used animal model, experimental autoimmune encephalomyelitis (EAE). However, the relationship between gray mater atrophy and the spinal cord pathology often observed in EAE has never been established. Here EAE was induced in Thy1.1-YFP mice and their brains imaged using in vivo magnetic resonance imaging (MRI). The brains and spinal cords were subsequently optically cleared using Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY). Axons were followed 5mm longitudinally in three dimensions in intact spinal cords revealing that 61% of the axons exhibited a mean of 22 axonal ovoids and 8% of the axons terminating in axonal end bulbs. In the cerebral cortex, we observed a decrease in the mean number of layer V pyramidal neurons and a decrease in the mean length of the apical dendrites of the remaining neurons, compared to healthy controls. MRI analysis demonstrated decreased cortical volumes in EAE. Cross-modality correlations revealed a direct relationship between cortical volume loss and axonal end bulb number in the spinal cord, but not ovoid number. This is the first report of the use of CLARITY in an animal model of disease and the first report of the use of both CLARITY and MRI.

Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other kinetic events that can be measured during S-phase.