http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_1 A novel approach for detecting nucleic acid in solution has been adopted for real-time imaging of native mRNAs in living cells. This method utilizes hybridization probes, called “molecular beacons,” that generate fluorescent signals only when they are hybridized to a complementary target sequence. Nuclease-resistant molecular beacons are designed to efficiently hybridize to accessible regions within RNAs and then be detected via fluorescence microscopy. The target regions chosen for probe binding are selected using two computer algorithms, mfold and OligoWalk, that predict the secondary structure of RNAs and help narrow down sequence stretches to which the probes should bind with high affinity in vivo. As an example, molecular beacons were designed against regions of oskar mRNA, microinjected into living Drosophila melanogaster oocytes and imaged via confocal microscopy. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_10 Laser capture microdissection (LCM) is a technique for isolating pure cell populations from a heterogeneous tissue section or cytological preparation via direct visualization of the cells. This technique is applicable to molecular profiling of diseased and disease-free tissue, permitting correlation of cellular molecular signatures with specific cell populations. DNA, RNA, or protein analysis can be performed with the microdissected tissue by any method with adequate sensitivity. The principle components of LCM technology are (1) visualization of the cells of interest via microscopy, (2) transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods, and tissuepreservation protocols that allow microdissection of fresh or archival specimens. LCM platforms are available as a manual system (PixCell; Arcturus Bioscience) or as an automated system (AutoPix™). 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_11 Laser capture microdissection (LCM) enables the removal of discrete microstructures or cell types from properly prepared histological sections. Extraction of RNA from microdissected tissue followed by quantitative reverse transcriptase-polymerase chain (QRT-PCR) reaction permits the analysis of cell-type or microstructure-specific gene expression changes that occur in response to various stimuli in the environment. In our lab, the combination of LCM and QRT-PCR has proven very useful in the determination of the in vivo gene expression changes that occur in bronchiolar epithelium in response to inhalation of crocidolite asbestos. A detailed description of the preparation of cDNA from bronchiolar epithelial cells obtained by LCM is described in this work. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_12 Fluorescence in situ hybridization (FISH) is a nonisotopic labeling and detection method that provides a direct way to determine the relative location or copy number of specific DNA sequences in nuclei or chromosomes. With recent advancements, this technique has found increased application in a number of research areas, including cytogenetics, prenatal diagnosis, cancer research and diagnosis, nuclear organization, gene loss and/or amplification, and gene mapping. The availability of different types of probe and the increasing number of FISH techniques has made it a widespread and diversely applied technology. Multicolor karyotyping by multicolor FISH and spectral karyotyping interphase FISH and comparative genomic hybridization allow genetic analysis of previously intractable targets. We present a brief overview of FISH technology and describe in detail methods of probe labeling and detection for different types of tissue sample, including microdissected nuclei from formalin-fixed paraffin-embedded tissue sections. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_13 Of the technologies available for measuring gene expression, microarrays using cDNA targets is one of the most common and well-developed high-throughput techniques. With this technique, the expression levels of thousands of genes are measured simultaneously. DNA probes are immobilized on solid surfaces, either membrane-based or chemically coated glass surfaces. On glass arrays, the probes are hybridized with fluorescent-labeled target samples. Fluorescence intensities, which reflect gene expression levels, are detected by imaging the array using a laser or white-light source and capturing the image using photomultiplier tube detection or a charge-coupled device camera. Different laser-based scanners are used in laboratories to scan microarray images. This chapter discusses the imaging process and the protocols being developed. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_14 Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_15 Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neuro-transmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a buildup of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes and vesicle swelling have also been resolved. These findings reveal the molecular machinery and mechanism of cell secretion. In this chapter, the discovery of the porosome, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane are discussed. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_16 The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remained unknown. Earlier studies from our laboratory demonstrated the association of the α-subunit of heterotrimeric GTP-binding protein G±i3 with zymogen granule membrane and implicated its involvement in vesicle swelling. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and found to be KCl sensitive. However, Ca2+ had no effect on zymogen granule size. Taken together, these results indicated that zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G±i3 protein. Subsequently, our studies demonstrated that the water channel aquaporin-1 (AQP1) is also present at the zymogen granule membrane and participates in rapid GTP-induced and G±i3-mediated vesicular water gating and swelling. Isolated zymogen granules exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of zymogen granules with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxy-terminal domain of AQP1 blocked GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the zymogen granule membrane is involved in basal GTP-induced and G±i3-mediated rapid gating of water into zymogen granules of the exocrine pancreas. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_17 This chapter describes the use of the atomic force microscope (AFM) to probe and map out regional variations in apparent elastic properties of living cells. The importance of mechanics in the field of cell biology is becoming more widely appreciated, and the AFM has unique advantages for cell mechanics applications. However, care must be taken in the acquisition, analysis, and interpretation of AFM indentation data. To help make this powerful technique accessible to a broad range of investigators, detailed procedures are provided for all stages of the AFM experiment from sample preparation through data analysis and visualization. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_18 Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_19 Electron microscopy of single particles has recently become a very popular field in both biological and material sciences. It might be difficult for a novice researcher new to this field to know how to start tackling a new project. This chapter is designed to serve as a guideline for anyone starting a new project to determine a three-dimensional structure using single-particle techniques. The chapter describes the basic techniques necessary to prepare the samples and acquire the data to calculate a three-dimensional reconstruction in easy-to-understand, step-by-step instructions. It starts with the basic preparation of support films and the usage of a variety of staining techniques needed to assess the quality of the sample and the viability of the project. It ends with a detailed description of vitreous ice preparations designed to acquire high-resolution structural information. Guidelines and tips are given on how to record the best images with an electron microscope. Although this chapter is geared to researchers new to the field, experts might find it not only useful as a reference but also valuable because of the number of practical tips included. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_2 Molecules that have no center of symmetry are able to convert light to its second harmonic, at twice the frequency and half the wavelength. This only happens with any efficiency at very high light intensities such as are given by a pulsed laser, and because the efficiency of the process depends on the square of the intensity, it will be focal plane selective in exactly the same way as two-photon excitation of fluorescence. Because of its unusual molecular structure and its high degree of crystallinity, collagen is, by far, the strongest source of second harmonics in animal tissue. Because collagen is also the most important structural protein in the mammalian body, this provides a very useful imaging tool for studying its distribution. No energy is lost in second-harmonic imaging, so the image will not fade, and because it is at a shorter wavelength than can be excited by two-photon fluorescence, it can be separated easily from multiple fluorescent probes. It is already proving useful in imaging collagen with high sensitivity in various tissues, including cirrhotic liver, normal and carious teeth, and surgical repair of tendons. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_20 Three-dimensional electron microscopy of single macromolecular assemblies has made large strides forward over the last decade. A large number of image processing techniques have been developed and many have found general distribution. For the proper usage of the wide range of available techniques, a clear concept of all processing steps is essential. This chapter provides step-by-step instruction for the three-dimensional reconstruction of an unknown macromolecule. Where possible, the limitations of the techniques are explained. The chapter attempts to be sufficiently general such so as not to adhere to a single image processing system. Described are alignment techniques for two and three dimensions, classification procedures, and the usage of three-dimensional reconstruction algorithms. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_21 A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm k 1.2 mm × 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_3 Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ ([Ca2+]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca2+] is essential to understanding the role of Ca2+ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca2+ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca2+] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca2+ in cells with high temporal and spatial resolution. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_4 Lung tissue consists of more than 40 individual cell types that might interact to produce adverse pathologies. After injury, a number of signaling proteins expressed in various epithelial and other cell types have been linked to the advent of apoptosis, compensatory proliferation, and adaptation to stress. We describe here the use of immunochemistry and multifluorescence approaches using confocal laser scanning microscopy to define the signaling pathways (protein kinases C and mitogen-activated protein kinases) activated by asbestos fibers after inhalation. Using these approaches, we are able to localize signaling events in distinct cell types of the lung and determine their status in the cell cycle (resting or nonresting). Moreover, we are able to determine whether various signaling proteins colocalize in cells and the sites affected by asbestos fibers. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_5 The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a “pretty picture.” Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate the subjective nature of assessing the CLSM and allow different machines to be compared. These tests are essential if one is to make intensity measurements. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_6 Comparative quantitation has become an increasingly desirable tool in determining compositional differences of aortic plaque lesion in transgenically altered mice. To this end, methodology has been developed to identify lipid, cellularity, collagen, and elastin components using traditional bright-field microscopy, fluorescence, and polarized light microscopy, employing both confocal and wide-field imaging systems. Subsequent imaging processing and analysis on the digitally captured images reveals differences in compositional components as influenced by diet, age, and gender. This method can be expanded to employ a rich variety of histochemical and immunohistochemical staining protocols. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_7 Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_8 The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF-κB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59259-993-6_9 Biological samples from human tissues are characterized by complexity and heterogeneity. The ability to make rapid, reliable, quantitative fluorochromatic measurements on clinical samples allows the development of new and practical assays that could influence diagnosis and treatment in a variety of clinical applications. Laser scanning cytometry (LSC) is a very versatile and adaptable technology that allows for the quantitative analysis of cell samples that are unsuitable for flow cytometry by virtue of their presentation and context. Crucially, it allows the direct visualization of cells and rare events and the correlation of imagery with fluorochromatic measurements. In this chapter, we describe early experiments in the study of cytotoxic drug uptake and resistance in human tumor cells and in the study of sputum cells from asthmatic patients, which harness the specific capabilities of LSC to practical clinical problems. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1385/1-59745-010-3:103 The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, with both MRI physicists and biologists starting to use MRI to identify mouse models of human disease. The purpose of this chapter is to provide details of the animal handling necessary for routine and robust in vivo imaging with particular emphasis on multiple-mouse imaging. In addition, techniques for perfusion-fixation for postmortem imaging of specimens and whole mice are given. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1385/1-59745-010-3:129 Magnetic resonance imaging (MRI) is increasingly becoming an important tool to study anatomy of rodent brains. Compared with histology, it has clear advantages because the entire 3D object can be captured as an image nondestructively. However, low imaging resolution and a small number of available contrast mechanisms are two critical disadvantages. In this article, the future potential of magnetic resonance (MR) microimaging is discussed, with special emphasis on diffusion tensor microimaging as an effective contrast mechanism for the developing central nervous system. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1385/1-59745-010-3:151 MRI-based perfusion imaging techniques can be classified into those that use exogenously administered contrast agents and those that use an endogenous material that reflects blood flow. This chapter focuses on the technique of arterial spin labeling (ASL), in which endogenous water is made a freely diffusible perfusion tracer by perturbing the magnetization of blood water in arteries prior to their entry into tissue of interest. The technique is totally noninvasive and allows repeated quantitative blood flow measurements in a time scale limited only by the spin lattice relaxation time (T1). Absolute quantitation requires measurement of T1, transit time, and labeling efficiency, as well as careful control for magnetization transfer effects. Two main variants of the ASL technique are in use: continuous ASL (CASL) and pulsed ASL (PASL). This chapter describes basic theory for CASL, and experimental and computational procedures for obtaining quantitative perfusion maps of the brain. Extension of the technique for renal perfusion imaging is outlined. 2005-11-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1385/1-59745-010-3:175 Quantitative magnetic resonance spectroscopy (MRS) and imaging (MRI) measurements of energy metabolism (i.e., cerebral metabolic rate of oxygen consumption, CMRo2), blood circulation (i.e., cerebral blood flow, CBF; and cerebral blood volume, CBV), and functional MRI (fMRI) were used to interpret the physiological basis of blood oxygenation level-dependent (BOLD) image contrast at 7 T in rat brain. Multimodal signals over a wide range of activity, for primarily glutamatergic neurons, were measured. Because each parameter that can influence the BOLD image contrast was measured quantitatively and separately, multimodal measurements of changes in CMRo2, CBF, CBV, and BOLD signal allowed calibration as well as validation of fMRI. Good agreement between changes in CMRo2 calculated from BOLD theory and measured by 13C MRS reveal that BOLD signal changes at 7 T are closely linked with alterations in neuronal glucose oxidation of glutamatergic neurons, both for activation and de-activation paradigms. Additional neurochemical and neurophysiological studies with fMRI suggest that the BOLD response from the cerebral cortex is closely linked to neurotransmitter release and energetic demand of glutamatergic neurons. Thus, calibrated fMRI may be used to reflect energetic changes of ensembles of glutamatergic neurons in the cerebral cortex. 2005-11-01T05:00:00Z