http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_1 Unlike immunofluorescence confocal microscopy of fixed samples or microscopic surface analysis in material sciences that both involve largely indestructible samples, life-cell imaging focuses on live cells. Imaging live specimen is by definition minimally invasive imaging, and photon efficiency is the primordial concern, even before issues of spatial, temporal or, spectral resolution, of acquisition speed and image contrast come in. Beyond alerting the reader that good live-cell images are often not the crisp showcase images that you know from the front page, this chapter is concerned with providing a fresh look on one of the routine instruments in modern biological research. Irrespective of whether you are a young researcher setting up your own lab or a senior investigator choosing equipment for a new project, at some stage you will most likely face decision making on what (fluorescence) imaging set-up to buy. In as much as this choice is about a long-lived and often relatively costly piece of equipment and, more importantly, impacts on your future experimental program, this choice can be a tricky one. It involves considering a multitude of parameters, some of which are discussed here. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_2 Fluorescence imaging is an important tool for molecular biology research. There is a wide array of fluorescent labels and activatable probes available for investigation of biochemical processes at a molecular level in living cells. Given the large number of potential imaging agents and numerous variables that can impact the utility of these fluorescent materials for imaging, selection of the appropriate probes can be a difficult task. In this report an overview of fluorescent imaging agents and details on their optical and physical properties that can impact their function are presented. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_3 The analysis of live cells using automated fluorescence microscopy systems on an industrial scale is known as high content screening/analysis (HCS/A). Its development has been driven both by the demands of compound screening in the drug discovery industry and by the promise of whole genome functional analyses using siRNA knockouts. This chapter outlines the primary applications of HCS/A within the drug discovery process and in systems cell biology. It discusses specific issues which must be addressed when undertaking HCS/A, such as choice of cells, probes, labels, and assay type. Drawing from information gathered from surveys of key users of HCS/A in industry and academia, it then provides a detailed description of HCS/A user issues and requirements, before concluding with a summary of the imaging instrumentation currently available for live cell HCS/A. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_10 For more than a decade, fluorescence resonance energy transfer (FRET) imaging methods have been developed to study dynamic interactions between molecules at the nanometer scale in live cells. Here, we describe a protocol to measure FRET by the acceptor-sensitized emission method as detected by total internal reflection fluorescence (TIRF) imaging to study the interaction of appropriately labeled plasma membrane-associated molecules that regulate the earliest stages of antigen-mediated signaling in live B lymphocytes. This protocol can be adapted and applied to many cell types where there is an interest in understanding signal transduction mechanisms in live cells. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_11 Since the discovery of the diffraction barrier in the late nineteenth century, it has been commonly accepted that with far-field optical microscopy it is not possible to resolve structural details considerably finer than half the wavelength of light. The emergence of STED microscopy showed that, at least for fluorescence imaging, these limits can be overcome. Since STED microscopy is a far-field technique, in principle, the same sample preparation as for conventional confocal microscopy may be utilized. The increased resolution, however, requires additional precautions to ensure the structural preservation of the specimen. We present robust protocols to generate test samples for STED microscopy. These protocols for bead samples and immunolabeled mammalian cells may be used as starting points to adapt existing labeling strategies for the requirements of sub-diffraction resolution microscopy. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_12 Inositol trisphosphate and cyclic ADP-ribose, main intracellular Ca2+ messengers, induce release from the intracellular Ca2+ stores via inositol trisphosphate and ryanodine receptors, respectively. Recently, studies using novel messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releasing Ca2+ from calcium stores in organelles other than endoplasmic reticulum (ER) have been conducted. However, technical difficulties of Ca2+ measurements in relatively small Ca2+ stores prompted us to develop a new, more sensitive, and less damaging two-photon permeabilization technique. Applied to pancreatic acinar cells, this technique allowed us to show that all three messengers – IP3, cADPR, and NAADP – release Ca2+ from two intracellular stores: the endoplasmic reticulum and an acidic store in the granular region. This chapter describes a detailed procedure of using this technique with pancreatic acinar cells. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_13 Three-dimensional (3D) cell cultures are important tools in cell biology research and tissue engineering because they more closely resemble the architectural microenvironment of natural tissue, compared to standard two-dimensional cultures. Microscopy techniques that function well for thin, optically transparent cultures, however, are poorly suited for imaging 3D cell cultures. Three-dimensional cultures may be thick and highly scattering, preventing light from penetrating without significant distortion. Techniques that can image thicker biological specimens at high resolution include confocal microscopy, multiphoton microscopy, and optical coherence tomography. In this chapter, these three imaging modalities are described and demonstrated in the assessment of functional and structural features of 3D chitosin scaffolds, 3D micro-topographic substrates from poly-dimethyl siloxane molds, and 3D Matrigel cultures. Using these techniques, dynamic changes to cells in 3D microenvironments can be non-destructively assessed repeatedly over time. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_14 During the past 10 years, major developments in live-cell imaging methods have accompanied growing interest in the application of microfluidic techniques to biological imaging. The broad design possibilities of microfabrication and its relative ease of implementation have led to the development of a number of powerful imaging assays. Specifically, there has been great interest in the development of devices in which single cells can be followed in real-time over the course of several generations while the growth environment is changed. With standard perfusion chambers, the duration of a typical experiment is limited to one cell generation time. Using microfluidics, however, long-term imaging setups have been developed which can measure the effects of temporally controlled gene expression or pathway activation while tracking individual cells over the course of many generations. In this paper, we describe the details of fabricating such a microfluidic device for the purpose of long-term imaging of proliferating cells, the assembly of its individual components into a complete device, and then we give an example of how to use such a device to monitor real-time changes in gene expression in budding yeast. Our goal is to make this technique accessible to cell biology researchers without prior experience with microfluidic systems. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_15 Oxygen is essential for survival of aerobic organisms. Sensing changes in the environmental oxygen concentration and appropriate adaptation to such changes are essential for organisms to survive. Hypoxia inducible factor 1 (HIF-1) is the key transcription factor in controlling the expression of oxygen-dependent genes required for this adaptation. HIF-1 is a heterodimer of an oxygen dependent a-subunit and constitutive ß-subunit. Abundance and activity of HIF-1 is controlled by post-translational hydroxylation. Microscopic analysis of the assembly and activation process of HIF-1 has become an important tool to better understand HIF-1 regulation. Confocal laser microscopy provides exact images of HIF-1a that is translocated into the nucleus under hypoxia and its disappearance upon reoxygenation. To exactly follow the protein–protein interaction during the assembly process of HIF-1, both subunits were labeled by fusing them to fluorescent proteins. Fluorescence resonance energy transfer (FRET) was used to determine the interaction of both subunits in living cells by confocal microscopy. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_16 Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca2+, H+), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite – cellular oxygen – is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O2 in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O2 concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_17 Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na+, and briefly also aspects related to mitochondrial Ca2+ measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_18 Cell surface receptors are crucial in the regulation of a wide variety of signalling responses to extracellular stimuli such as soluble growth factors or matrix proteins. To respond effectively to rapidly changing environmental cues, many receptors are rapidly endo- or exo-cytosed to either subcellular or membrane compartments or they recruit specific intracellular binding partners. Recent advances in microscopy techniques have made it possible to study receptor behaviour in live cells to gain a better understanding of dynamics, binding partners and sub-cellular localisation. Here we describe several common currently used techniques to study receptor behaviour in living cells. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_19 Protein functions rely on their ability to engage in specific protein–protein interactions and form complexes that are dynamically regulated by stimuli. Bioluminescence resonance energy transfer (BRET) is a highly sensitive technique, which allows monitoring of interaction between two proteins: one tagged with the luminescent donor Renilla luciferase, the other with a fluorescent acceptor such as YFP. We adapted this method to single-cell imaging. To this aim, we tag proteins of interest, transfect cells with these fusions, and use the high-sensitivity microscopy, combined with electron multiplying cooled charge-coupled device (EMCCD) cameras and improved bioluminescence probes. We thus achieve rapid acquisition of high-resolution BRET images and study the localization and dynamics of protein–protein interactions in individual live cells. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_20 The changes that occur in electrochemical gradients across biological membranes provide us with invaluable information on physiological responses, pathophysiological processes and drug actions/toxicity. This chapter aims to provide researchers with sufficient information to carry out a quantitative assessment of mitochondrial energetics at a single-cell level thereby providing output on changes in the mitochondrial membrane potential (??m) through the utilization of potentiometric fluorescent probes (TMRM, TMRE, Rhodamine 123). As these cationic probes behave in a Nernstian fashion, changes at the plasma membrane potential (??p) need also to be accounted for in order to validate the responses obtained with ??m-sensitive fluorescent probes. To this end techniques that utilize ??p-sensitive anionic fluorescent probes to monitor changes in the plasma membrane potential will also be discussed. In many biological systems multiple changes occur at both a ??m and ??p level that often makes the interpretation of the cationic fluorescent responses much more difficult. This problem has driven the development of computational modelling techniques that utilize the redistribution properties of the cationic and anionic fluorescent probes within the cell to provide output on changes in ??m and ??p. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_21 Fluorescent imaging techniques are powerful tools that aid in studying protein dynamics and membrane domains and allow for the visualization and data collection of such structures as caveolae and clathrin-coated pits, key players in the regulation of cell communication and signaling. The family of image correlation spectroscopy (FICS) provides a unique way to determine details about aggregation, clustering, and dynamics of proteins on the plasma membrane. FICS consists of many imaging techniques which we will focus on including image correlation spectroscopy, image cross-correlation spectroscopy and dynamic image correlation spectroscopy. Image correlation spectroscopy is a tool used to calculate the cluster density, which is the average number of clusters per unit area along with data to determine the degree of aggregation of plasma membrane proteins. Image cross-correlation spectroscopy measures the colocalization of proteins of interest. Dynamic image correlation spectroscopy can be used to analyze protein aggregate dynamics on the cell surface during live-cell imaging in the millisecond to second range. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_4 Many fluorescent probes depend on the fluorescence resonance energy transfer (FRET) between fluorescent protein pairs. The efficiency of energy transfer becomes altered by conformational changes of a fused sensory protein in response to a cellular event. A structure-based approach can be taken to design probes better with improved dynamic ranges by computationally modeling conformational changes and predicting FRET efficiency changes of candidate biosensor constructs. FRET biosensors consist of at least three domains fused together: the donor protein, the sensory domain, and the acceptor protein. To more efficiently subclone fusion proteins containing multiple domains, a cassette-based system can be used. Generating a cassette library of commonly used domains facilitates the rapid subcloning of future fusion biosensor proteins. FRET biosensors can then be used with fluorescence microscopy for real-time monitoring of cellular events within live cells by tracking changes in FRET efficiency. Stimulants can be used to trigger a range of cellular events including Ca2+ signaling, apoptosis, and subcellular translocations. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_5 Peroxynitrite (ONOO–) and hypochlorous acid (HOCl) are two highly reactive oxygen species generated in biological systems. The overproduction of peroxynitrite or hypochlorous acid is implicated in a broad array of human pathologies including vascular, immunological, and neurodegenerative diseases. However, unambiguous detection of these reactive oxygen species has been relatively difficult due to their short biological half-lives and multiple reaction pathways. Based on their specific chemical reactions, we have developed fluorescent probes HKGreen-1 and HKOCl-1 for highly sensitive detection of peroxynitrite and hypochlorous acid, respectively. Both probes have been demonstrated to be able to discriminate corresponding reactive species from other reactive oxygen and nitrogen species (ROS and RNS) in not only chemical systems but also biological systems. The endogenous production of peroxynitrite in neuronal cells under oxygen-glucose deprivation (OGD) conditions has been visualized for the first time by utilizing HKGreen-1 probe, whilst the endogenous production of hypochlorous acid in macrophage cells upon stimulation with LPS, IFN-?, and PMA has been imaged by utilizing HKOCl-1 probe. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_6 Photo-activatable (caged) probes are powerful research tools for biological investigation. The superb maneuverability of a light beam allows researchers to activate caged probes with pinpoint accuracy. Recent developments in caging chemistry and two-photon excitation technique further enhance our capability to perform photo-uncaging with even higher spatial and temporal resolution, offering new photonic approaches to study cell signaling dynamics in greater detail. Here we present a sample method that combines the techniques of photo-activation and digital fluorescence microscopy to assay an important class of intracellular receptors for the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3). The imaging assay is performed in fully intact living cells using a caged and cell membrane permeable ester derivative of IP3, cm-IP3/PM. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_7 Phagocytosis is the process whereby specialized leukocytes ingest large particles. This is an extremely dynamic and localized process that requires the recruitment to the sites of ingestion of numerous effector proteins, together with extensive lipid remodelling. To investigate such a dynamic series of events in living cells, non-invasive methods are required. The use of fluorescent probes in conjunction with spectroscopic analysis is optimally suited for this purpose. Here we describe a method to express in RAW264.7 murine macrophages genetically encoded probes that allow for the spatio-temporal analysis of lipid distribution and metabolism during phagocytosis of immunoglobulin-opsonized beads. The fluorescence of the probes is best analysed by laser scanning or spinning disc confocal microscopy. While the focus of this chapter is on phagocytic events, this general method can be employed for the analysis of lipid distribution and dynamics during a variety of biological processes in the cell type of the investigator’s choice. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_8 To follow the cell division cycle in the living state, certain biological activity or morphological changes must be monitored keeping the cells intact. Mitotic events from prophase to telophase are well defined by morphology or movement of chromatin, nuclear envelope, centrosomes, and/or spindles. To paint or simultaneously visualize these mitotic subcellular structures, we have been using ECFP-histone H3 for chromatin and chromosomes, EGFP-Aurora-A for centrosomes and kinetochore spindles and DsRed-fused truncated peptide of importin alpha for the outer surface of nuclear envelope as living cell markers. Time-lapse images from prophase through to early G1 phase can be obtained by constructing a triple-fluorescent cell line (Sugimoto et al., Cell Struct. Funct. 27, 457–467, 2002). Here, we describe the multicolor imaging of mitosis of a human breast cancer cell line, MDA435, and a further application to characterizing the apoptotic chromatin condensation process in isolated nuclei by simultaneously visualizing kinetochores with EGFP and chromatin with a fluorescent dye, SYTO 59. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-404-3_9 Many cells undergo directed cell migration in response to external cues in a process known as chemotaxis. This ability is essential for many single-celled organisms to hunt and mate, the development of multicellular organisms, and the functioning of the immune system. Because of their relative ease of manipulation and their robust chemotactic abilities, the neutrophil-like cell line (HL-60) has been a powerful system to analyze directed cell migration. In this chapter, we describe the maintenance and transient transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays (micropipette and EZ-TAXIS). Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging. 2009-10-30T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-569-9_1 Piezoelectric sensors have become a versatile tool in biosensorics to study protein—protein and protein—small molecule interactions. Here we present theoretical background on piezoelectric sensors and instructions, how to modify their surface with various recognition elements for cholinesterases. These recognition elements comprise an organophosphate (paraoxon), a cocaine derivative (BZE-DADOO), and a tricyclic, aromatic compound (propidium). Additionally, a guide to the kinetic evaluation of the obtained binding curves is given in this chapter. 2008-12-11T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-569-9_10 A model immunosensor based on a labeling method using gold nanoparticles (AuNPs) and electrochemical detection is developed. Microparamagnetic beads (MB) as primary antibody immobilization platforms and AuNPs modified with a secondary antibody as high sensible electrochemical labels have been used. The carbon electrode used as transducer incorporates a magnet that allows the collection/ immobilization on its surface of the immunological sandwich attached to the MB. 2008-12-11T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-569-9_11 Methods for the construction of electrochemical composite biosensors using gold nanoparticles and Teflon as nonconducting-binding material are described in detail. The advantages of the incorporation of gold nanoparticles to the composite electrode matrices are highlighted, giving rise to bioelectrodes with improved analytical performance in terms of stability and sensitivity with respect to other biosensor designs. Three different biosensors have been considered: a tyrosinase biosensor in which the enzyme and gold nanoparticles are incorporated into graphite—Teflon composite electrode matrices by simple physical inclusion, a progesterone immunusensor in which the antibody is directly attached to the electrode surface and amperometric transduction is carried out at a colloidal gold—graphite—Teflon—tyrosinase composite biosensor, and a mediator-less glucose oxidase biosensor constructed by bulk incorporation of the enzyme into colloidal gold-multiwall carbon nanotubes—Teflon composite electrodes. 2008-12-11T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-569-9_12 The immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of various analytes at the point of care. We have developed lateral flow strip tests for the specific qualitative or semiquantitative detection of antigens, antibodies, and haptens, such as drug residues. Here, we describe in detail the preparation of three examples of the strip tests for detection of (a) the infectious bursal disease virus; (b) Trichinella specific antibodies, and (c) Clenbuterol residues in urine samples. 2008-12-11T05:00:00Z