Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Quantitative in vivo imaging of embryonic development: opportunities and challenges

Animal models are critically important for a mechanistic understanding of embryonic morphogenesis. For decades, visualizing these rapid and complex multidimensional events has relied on projection images and thin section reconstructions. While much insight has been gained, fixed tissue specimens offer limited information on dynamic processes that are essential for tissue assembly and organ patterning. Quantitative imaging is required to unlock the important basic science and clinically relevant secrets that remain hidden. Recent advances in live imaging technology have enabled quantitative longitudinal analysis of embryonic morphogenesis at multiple length and time scales. Four different imaging modalities are currently being used to monitor embryonic morphogenesis: optical, ultrasound, magnetic resonance imaging (MRI), and micro-computed tomography (micro-CT). Each has its advantages and limitations with respect to spatial resolution, depth of field, scanning speed, and tissue contrast. In addition, new processing tools have been developed to enhance live imaging capabilities. In this review, we analyze each type of imaging source and its use in quantitative study of embryonic morphogenesis in small animal models. We describe the physics behind their function, identify some examples in which the modality has revealed new quantitative insights, and then conclude with a discussion of new research directions with live imaging.     

A genetic in vivo system to detect asymmetrically distributed RNA

Many RNAs show polarized or otherwise non-random subcellular distributions. To create a method for genome-wide genetic screens for RNAs with asymmetric subcellular distributions, we have combined methods for gene tagging and live imaging of messenger RNA (mRNA). A pilot screen in a highly polarized, differentiated cell in the Drosophila larva, the branched terminal cell of the tracheal system, demonstrates the feasibility of the method for identifying new asymmetrically localized mRNAs in vivo.     

Imaging the living plant cell: From probes to quantification

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

Specific examples are used to illustrate some of the challenges of live cell imaging, from designing genetically encoded probes to choosing a pipeline for image analysis and quantification.     

Visualizing new dimensions in Drosophila myoblast fusion

Over several years, genetic studies in the model system, Drosophila melanogastor, have uncovered genes that when mutated, lead to a block in myoblast fusion. Analyses of these gene products have suggested that Arp2/3-mediated regulation of the actin cytoskeleton is crucial to myoblast fusion in the fly. Recent advances in imaging in Drosophila embryos, both in fixed and live preparations, have led to a new appreciation of both the three-dimensional organization of the somatic mesoderm and the cell biology underlying myoblast fusion.     

Molecular mechanisms of membrane polarity in renal epithelial cells

Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.     

Live imaging early immune cell ontogeny and function in zebrafish Danio rerio

The success of a robust vertebrate inflammatory response is in part because of the migratory potential of its haematopoietic components. Once these cells converge at an inflammatory site, they interact with each other as well as non‐immune tissues and infectious agents to help manage both the scale and the duration of any ensuing response. Exactly how these blood cells, that constitute the innate and adaptive immune systems, contribute to such immune responses remain largely unknown. Traditionally, assessing these contributions relied upon histological analysis of fixed tissue sections complemented with in vitro dynamic data. Although informative, translating results from these studies into the multicellular whole‐animal setting remain difficult. Recently, non‐invasive live imaging of the immune system in animal models is providing significant insights into how immune cells function within their intact natural environment. Although the majority of these studies have been conducted within mice, another vertebrate, the zebrafish Danio rerio is being recognized as an ideal platform for non‐invasive live imaging applications. The optical transparency, rapid development, genetic tractability and highly conserved innate and adaptive immune systems of this well‐established developmental model have been exploited in a number of recent studies evaluating the immunocompetence of fluorescently tagged blood cells. In addition, similar live imaging studies are helping to dissect the ontogeny of blood‐cell development by tracking various haematopoietic precursor cells to assess their contribution to different blood lineages. This review will examine some recent advances that have helped D. rerio emerge as a live imaging platform as well as its potential to offer valuable insights into the genetics behind diseases associated with immune cell dysfunction.     

Imaging and characterisation of the surface of live cells

Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.     

Highly effective detection of inflamed cells using a modified bradykinin ligand labeled with FITC fluorescence

Detection of inflammation in live cells is important because long-lasting inflammation is considered to be a primary cause of several diseases. However, few reports have been published on imaging analysis of inflammation in live cells. In this study, we developed an effective imaging system for detection of inflamed cells using a bradykinin ligand (BK) or a modified BK (mBK), which has specific affinity with the cellular B1R receptor. Synthetic BK or mBK labeled with FITC at the N-terminus was employed for discriminating between inflamed and normal cells; this method was found to be effective for detection of inflammation in live cells. In addition, using the mBK-based cell imaging system, we successfully performed flow-based analysis of live cell inflammation on a micro-chip channel, composed of a Starna flow cell and PDMS (Polydimethylsiloxane) walls. The BK-based cell imaging methods designed here would be a useful platform for development of a high-throughput live cell analysis system for investigating the factors underlying inflammation or for screening of anti-inflammation candidate drugs.     

CRISPR/dCas9系统在活细胞成像领域的研究进展

研究不同基因、染色体以及基因与染色体之间的时空关系在遗传学、发育生物学和生物医学等领域具有重要意义。CRISPR/Cas9基因编辑技术具有优异的靶向性,已经成为应用最广泛的基因编辑工具。近年来,研究人员基于Cas9的核酸酶失活突变体dCas9发展了一系列先进的活细胞成像技术,为染色质、基因组特定位点的高分辨成像提供了快速、方便的研究工具。文中从细胞递送方式、荧光信号优化以及正交多色成像3个方面对CRISPR/dCas9系统在活细胞成像中的研究进展进行了综述,并对该领域的发展趋势进行了展望。     

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

Multicolor protein labeling in living cells using mutant β-lactamase-tag technology

Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools.     

Confocal laser scanning microscopy

Many technological advancements of the past decade have contributed to improvements in the photon efficiency of the confocal laser scanning microscope (CLSM). The resolution of images from the new generation of CLSMs is approaching that achieved by the microscope itself because of continued development in digital imaging methods, laser technology and the availability of brighter and more photostable fluorescent probes. Such advances have made possible novel experimental approaches for multiple label fluorescence, live cell imaging and multidimensional microscopy.     

Chemical probes shed light on protein function

Site-specific protein labeling with synthetic dyes is an emerging technique for live cell imaging. A protein or peptide tag fused to the protein of interest provides the means for attachment of a fluorophore or other small molecule probe, to allow non-invasive imaging of the dynamics of protein localization. The past two years have seen significant advances in such methods, the publication of a number of new tags for labeling, and the imaginative application of established techniques to tackle previously intractable biological questions.     

Single Cell Analysis of Drug Distribution by Intravital Imaging

Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level.     

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine–biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and β-mercaptoethanol proved to be effective. We therefore established a staining protocol using β-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.M.F. Langhorst and S. Genisyuerek contributed equally to this work.     

Intravital microscopy: a novel tool to study cell biology in living animals

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.