Multiparametric Fluorescence Microscopy Research Articles

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Orthogonal cell polarity imaging by multiparametric fluorescence microscopy

The cellular microenvironment is a complex medium due to high concentrations of proteins and an intertwined framework of cellular organelles. In particular, cellular micro-polarity controls several biological processes, since it modulates hydrophobic/hydrophilic interactions and, hence, recognition, signalling and binding events.In this work, we have developed an unprecedented methodology to construct accurate environment polarity images using multiparametric fluorescence microscopy, via a multi-linear calibration of orthogonal parameters: the fluorescence lifetime and the spectral shift of a series of solvatochromic dyes. For this approach, we have synthesized and fully characterized N-substituted 2-methoxy-9-acridone dyes as suitable bioimaging polarity probes. However, to fully comprehend the complex links between microenvironment polarity and the dyes’ properties, we have endeavoured a multidisciplinary approach, in which we have studied the photophysics of our fluorophores using spectroscopic tools and state-of-the-art computational chemistry. This profound knowledge permitted to use these dyes as intracellular polarity probes, quantitatively and robustly probing the microenvironment of different cellular compartments.Our new methodology may pave the way to further developments in accurate sensing of cellular microenvironment parameters.

Spatiotemporal dynamics of host cell modification caused by herpesvirus infection

Herpesviruses are large and complex DNA viruses that are composed of an icosahedral capsid, a proteinaceous layer termed the tegument, and a glycoprotein rich lipid envelope. One important area of host-pathogen interaction that is still poorly understood is the extensive change to intracellular organelles and cellular morphology that occur within the infected cell during active virus replication. In order to characterise the spatiotemporal dynamics of host cell remodelling caused by herpesvirus infection, we use novel multiparametric fluorescence microscopy methods compatible with live-cell imaging. In addition, we apply expansion microscopy to map 3D rearrangement in great detail. The remodelling of the host cell is correlated to the stage of virus replication which can highly vary between individual cells. Therefore, we have constructed a recombinant reporter virus that expresses eYFP-tagged ICP0, a multifunctional immediate early tegument protein, as well as mCherry-tagged glycoprotein C (gC), a late protein that is a major component of the viral envelope. The sequential expression of these two viral proteins provides us with an intrinsic time stamp for the stage of virus infection in each cell. With this fluorescent reporter virus, we are able to describe the remodeling of- the three-dimensional architecture of microtubules and the actin network,- compartments of the secretory and endocytic pathways which are intimately linked to viral envelope protein synthesis, maturation and transport,- and key antiviral and inflammatory signalling platforms (mitochondria and peroxisomes).

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.

Interactive, Graph-based Visual Analysis of High-dimensional, Multi-parameter Fluorescence Microscopy Data in Toponomics

In Toponomics, the function protein pattern in cells or tissue (the toponome) is imaged and analyzed for applications in toxicology, new drug development and patient-drug-interaction. The most advanced imaging technique is robot-driven multi-parameter fluorescence microscopy. This technique is capable of co-mapping hundreds of proteins and their distribution and assembly in protein clusters across a cell or tissue sample by running cycles of fluorescence tagging with monoclonal antibodies or other affinity reagents, imaging, and bleaching in situ. The imaging results in complex multi-parameter data composed of one slice or a 3D volume per affinity reagent. Biologists are particularly interested in the localization of co-occurring proteins, the frequency of co-occurrence and the distribution of co-occurring proteins across the cell. We present an interactive visual analysis approach for the evaluation of multi-parameter fluorescence microscopy data in toponomics. Multiple, linked views facilitate the definition of features by brushing multiple dimensions. The feature specification result is linked to all views establishing a focus+context visualization in 3D. In a new attribute view, we integrate techniques from graph visualization. Each node in the graph represents an affinity reagent while each edge represents two co-occurring affinity reagent bindings. The graph visualization is enhanced by glyphs which encode specific properties of the binding. The graph view is equipped with brushing facilities. By brushing in the spatial and attribute domain, the biologist achieves a better understanding of the function protein patterns of a cell. Furthermore, an interactive table view is integrated which summarizes unique fluorescence patterns. We discuss our approach with respect to a cell probe containing lymphocytes and a prostate tissue section.

Abstract 2154: Simultaneous analysis of cell death mechanisms and oxidative stress using live cell fluorescence microscopy

Abstract Reactive oxygen species (ROS) can directly damage DNA, proteins and lipids, while increased oxidative stress has been reported to be associated with apoptotic and autophagic cell death. Importantly, increased oxidative stress and dysregulation of cell death mechanisms have been linked with cancer development. To better understand the connection between increased oxidative stress and cell death pathways, it is important to be able to monitor both of these events simultaneously in the same cell and amongst mixed populations of cells. The goal of this study was to utilize multi-parametric fluorescence microscopy to examine the temporal progression of oxidative stress relative to cell death. By using a fluorogenic caspase substrate in combination with a near infrared fluorescent probe for ROS, we observed that increased oxidative stress was followed by activation of caspase-3/7 after induction by several agonists. We examined the effect of various agonists in both primary cells as well as cancer cell lines. Loss of mitochondrial membrane potential, nuclear condensation, and cell proliferation were also measured to establish a more comprehensive assessment of the cellular response to stress in these models. By simultaneously examining multiple parameters over time, we were able to define the temporal progression of apoptosis relative to the onset of oxidative stress. Furthermore, we were able to characterize the mechanism of cell death by discriminating between cells which were apoptotic (active caspase-3/7), autophagic (LC3B-positive autophagosomes), or both. This multi-parametric approach provided detailed spatial information at the cellular level so that correlations and temporal resolution could be determined between oxidative stress and cell death mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2154. doi:10.1158/1538-7445.AM2011-2154

Human vs. machine: evaluation of fluorescence micrographs

To enable high-throughput screening of molecular phenotypes, multi-parameter fluorescence microscopy is applied. Object of our study is lymphocytes which invade human tissue. One important basis for our collaborative project is the development of methods for automatic and accurate evaluation of fluorescence micrographs. As a part of this, we focus on the question of how to measure the accuracy of microscope image interpretation, by human experts or a computer system. Following standard practice we use methods motivated by receiver operator characteristics to discuss the accuracies of human experts and of neural network-based algorithms. For images of good quality the algorithms achieve the accuracy of the medium-skilled experts. In images with increased noise, the classifiers are outperformed by some of the experts. Furthermore, the neural network-based cell detection is much faster than the human experts.

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three‐dimensional (3‐D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3‐D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross‐talk, fluorescence photobleaching and cell movements. To identify true fluorescence co‐localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co‐localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.