Total internal reflection fluorescence microscopy for single-molecule imaging in living cells

Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

Imaging molecular interactions in living cells

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.     

Focusing light on infection in four dimensions

The fusion of cell biology with microbiology has bred a new discipline, cellular microbiology, in which the primary aim is to understand host-pathogen interactions at a tissue, cellular and molecular level. In this context, we require techniques allowing us to probe infection in situ and extrapolate quantitative information on its spatiotemporal dynamics. To these ends, fluorescent light-based imaging techniques offer a powerful tool, and the state-of-the-art is defined by paradigms using so-called multidimensional (multi-D) imaging microscopy. Multi-D imaging aims to visualize and quantify biological events through time and space and, more specifically, refers to combinations of: three (3D, volume), four (4D, time) and five (5D, multiwavelength)-dimensional recordings. Successful multi-D imaging depends upon understanding the available technologies and their limitations. This is especially true in the field of microbiology where visualization of infectious/pathogenic activities inside living host systems presents particular technical challenges. Thus, as multi-D imaging rapidly becomes a common bench tool to the cellular microbiologist, this review provides the new user with some of the necessary technical insight required to get the best from these methods.     

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.     

Imaging the boundaries—innovative tools for microscopy of living cells and real-time imaging

Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes “at work” and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein–protein interaction designed for use in living cells.     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

FRET-FLIM applications in plant systems

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.     

Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic. Furthermore, interaction with other cell types, including stromal fibroblasts and immune cells, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo. Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo. As such, they can serve an important role in reducing the need for experiments on living animals.     

Fluorescence complementation: an emerging tool for biological research

Numerous technologies based on utilizing fluorescent proteins have been developed for biological research, and fluorescence complementation (FC) is a recent application for visualization of molecular events in living cells and organisms. Currently, ten fluorescent proteins have been demonstrated to support FC. Over the past five years, FC-based technologies have been developed to visualize a variety of molecular events, such as protein-protein interactions, post-translational modifications, protein folding, conformational changes, RNA-protein interactions, mRNA localization and DNA hybridization. In addition, FC has also been used for drug discovery. These applications are providing fascinating insights into many biological processes. Here, we review the principles and applications of FC technologies, discuss their current challenges and examine prospects for future advances.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

Computational imaging in cell biology

Microscopy of cells has changed dramatically since its early days in the mid-seventeenth century. Image analysis has concurrently evolved from measurements of hand drawings and still photographs to computational methods that (semi-) automatically quantify objects, distances, concentrations, and velocities of cells and subcellular structures. Today's imaging technologies generate a wealth of data that requires visualization and multi-dimensional and quantitative image analysis as prerequisites to turning qualitative data into quantitative values. Such quantitative data provide the basis for mathematical modeling of protein kinetics and biochemical signaling networks that, in turn, open the way toward a quantitative view of cell biology. Here, we will review technologies for analyzing and reconstructing dynamic structures and processes in the living cell. We will present live-cell studies that would have been impossible without computational imaging. These applications illustrate the potential of computational imaging to enhance our knowledge of the dynamics of cellular structures and processes.     

Localization of protein-protein interactions in live cells using confocal and spectral imaging FRET microscopy

Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy. The specific aims of the investigation of protein dynamics in live specimens dictate the selection of the microscope methodology. In this article confocal and spectral imaging methods to monitor the dimerization of alpha enhancer binding protein (C/EBPalpha) in the pituitary GHFT1-5 living cell nucleus have been described. Also outline are issues involved in protein imaging using light microscopy techniques and the advantages of lifetime imaging of protein-protein interactions.     

Imaging dynamic cell signaling in vivo with new classes of fluorescent reporters

Dynamical features of cell signaling are the essence of living organisms. To understand animal development, it is fundamental to investigate signaling dynamics in vivo. Robust reporters are required to visualize spatial and temporal dynamics of enzyme activities and protein–protein interactions involved in signaling pathways. In this review, we summarize recent development in the design of new classes of fluorescent reporters for imaging dynamic activities of proteases, kinases, and protein–protein interactions. These reporters operate on new physical and/or chemical principles; achieve large dynamic range, high brightness, and fast kinetics; and reveal spatiotemporal dynamics of signaling that is correlated with developmental events such as embryonic morphogenesis in live animals including Drosophila and zebrafish. Therefore, many of these reporters are great tools for biological discovery and mechanistic understanding of animal development and disease progression.     

Illuminating intracellular signaling and molecules for single cell analysis

Fluorescent and bioluminescent proteins are now widely used for detection of small molecules and various intracellular events ranging from protein conformational change to cell death in living cells. To analyze the dynamics of molecular processes in real time at the level of single cells, engineered protein-based probes with higher sensitivity and selectivity are required. The probes can be entirely genetically encoded and can comprise fusions of different proteins or domains. This review specifically examines basic concepts of designing genetically encoded fluorescent and bioluminescent probes developed in the past decade, highlighting some potential applications for basic research and for drug discovery.     

Four-color, 4-D time-lapse confocal imaging of chick embryos

Our understanding of the molecular mechanisms that direct cell motility, cell division, and cell shaping has benefited from innovations in cell labeling and the ability to resolve intracellular dynamics with multispectral, high-resolution imaging. However, due to difficulties with in vivo cell marking and monitoring, most studies have been restricted to fixed tissue or cells in culture. Here, we report the delivery of multiple (up to four), multicolor fluorescent protein (FP) constructs and four-dimensional (4-D), multispectral time-lapse confocal imaging of cell movements in living chick embryos. Cell cytoskeletal components are fluorescently tagged after microinjection and electroporation of a cocktail of FP constructs into specific regions of chick embryos. We tested 11 different FP constructs in various two-, three-, and four-color combinations using multispectral imaging and linear unmixing to limit the crosstalk between different emission spectra. We monitored intracellular dynamics in individual multicolored migrating cells in vivo and developed a set of advantageous imaging parameters for 4-D time-lapse confocal microscopy. We find that the number of four-color labeled cells in a typical embryo is approximately 10% of the total number of fluorescently labeled cells; this value consistently increases showing that approximately 50% of the total labeled cells have only one-color. We find that multicolored cells are photostable for time-lapses of approximately 2-3 h. Thus, cell labeling with up to four FP color schemes combined with multispectral, 4-D confocal time-lapse imaging offers a powerful tool to simultaneously analyze cellular and molecular dynamics during chick embryogenesis.