Showing papers in "Methods in Cell Biology in 1989"

Fluorescence microscopy in three dimensions.

Abstract: The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity High-quality, cooled charge-coupled devices have recently become available Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information The impact of modern video microscopy techniques (Allen, 1985; Inoue, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated Cells are intrinsically three-dimensional This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied In either case, image processing techniques can be used to rapidly provide the desired representation of the data In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information The most straightforward of these methods uses only information from adjacent focal planes to correct the central one This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus A variation on this method allows efficient calculation of stereo pairs(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescence microscopy methods for yeast.

Abstract: Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Fluorescent indicators of ion concentrations.

Abstract: Publisher Summary Ion-concentration gradients across cell membranes are central to any understanding of biological energetics and signal transduction. The proton electrochemical gradient is generally accepted to be the key intermediary linking electron transport to adenosine triphosphate (ATP) synthesis in bacteria, chloroplasts, and mitochondria. Cytoplasmic pH is a powerful regulator of enzyme activities and is known to alkalinize in many types of cells undergoing mitogenic activation. The characterization of ion gradients requires knowing both extracellular and intracellular ion activities and free concentrations. The Na+ gradient is the energy source for secondary active transport of nutrients into animal cells and many co- or counter-transport systems for other ions. Cl- gradients are vital to the function of epithelia, the control of cell volume, and to the actions of many inhibitory neurotransmitters. Determination of extracellular ions usually poses much less of a problem than intracellular, because the extracellular medium is much more accessible. Extracellular concentrations of all the simple inorganic ions except K+ are comparable to or higher than intracellular, so detection is easier extracellularly.

Total internal reflection fluorescence microscopy.

Abstract: Total internal reflection fluorescence (TIRF) microscopy provides a means to selectively excite fluorophores in an aqueous or cellular environment that is very near a solid surface (within ≤100 nm: less than one-fifth the thickness of a confocal microscopy section) without exciting fluorescence from regions further from the surface. Fluorescence excitation by this thin zone of electromagnetic energy (called an evanescent field) results in images with very low background fluorescence, virtually no out-of-focus fluorescence, and minimal exposure of cells to light in any other planes in the sample. Figure 21-1 shows an example of TIRF on intact living cells in a culture compared with standard epifluorescence. The above features have led to numerous applications, including the following:

Fluorescence ratio imaging microscopy.

Abstract: Publisher Summary Rapid advancements in low-light-level imaging technology have made it possible to apply the fundamental principles of fluorescence spectroscopic analyses to living cells. The quantitation of an optical signal from living cells attached to a substrate is very difficult. Living cells move, change shape, and vary the local number density of membrane-bound organelles. Fluorescence ratio imaging provides a means of overcoming many of the problems associated with quantifying the complex temporal and spatial dynamics of molecules and organelles in living cells. This chapter describes the issues of fluorescence ratio imaging microscopy, including basic requirements of the instrumentation, validation of the basic tenets of ratioing as applied to microscopy, and some methods and issues in the application of ratio imaging. With continued development and improvement in fluorescent probes for ion measurements as well as the development of probes for other important molecules, the potential applicability of ratio imaging is enormous. Fluorescence ratio imaging is a spectroscopic technique and is usable for any technique that can be expressed by the relationship of signals at two or more wavelengths.

Fluorescence labeling and microscopy of DNA

Abstract: Publisher Summary This chapter discusses the in vivo labeling of macromolecules and other cellular constituents and their measurement by fluorescence microscopy. An inherent pitfall in this approach is that either the labeling and/or observation technique may perturb the cell such that the information one derives is misleading or outright erroneous. The problem certainly applies to the determination of DNA content and cell cycle progression in living cells. The chapter describes the techniques for in vivo measurements; an implicit requirement is that all experiments must be carefully controlled in the sense of demonstrating that the conditions for labeling and measurement do not impair cell viability. In some cases, this condition cannot be met and there is no satisfactory alternative but to use techniques based on fixation and labeling protocols that help to take a snapshot in time of the DNA metabolism in the living cell. The chapter also presents the use of fluorescence digital imaging microscopy (FDIM) for measuring DNA, its metabolism, and its structure.

Fluorescence polarization microscopy.

Abstract: Publisher Summary Fluorescence polarization techniques have long been used to measure the orientational distributions and rotational diffusion of molecules in solution or suspension. A commercial spectrofluorimeter is used for such studies. Fluorescence polarization through a microscope offers the possibility of analogous studies on the surface or interior of very small subvolumes of cells. However, fluorescence polarization with a microscope presents some unique experimental problems and opportunities not generally present with a spectrofluorimeter. The chief experimental problem is that the microscope objective often has a rather high aperture and can thereby look around the sample effectively integrating fluorescence over a range of emission solid angles. A second problem is that the perfectly polarized excitation illumination may suffer some depolarization as it is concentrated by that same objective. A third problem is that the nearby substrate may influence the polarization of rays emitted at high incidence angles but still captured by the objective. This chapter discusses the polarization from fluorophores in membranes-oriented flat on the microscope stage.

Low temperature-induced transport blocks as tools to manipulate membrane traffic.

Abstract: Publisher Summary This chapter discusses the use of temperature experiments in studies of protein transport in the exocytic pathway with emphasis on the description of immunocytochemical techniques used in protein detection in light and electron microscropy. The availability of well-characterized soluble and membrane-bound markers made it possible to discover thermosensitive steps in the endocytic pathway. Studies of virus glycoprotein transport have initiated the analysis of the function of distinct intracellular sites in the biosynthetic pathway at which protein movement is arrested at reduced temperatures. Temperature experiments with cultured cells grown on plastic or on glass coverslips require simple equipment. Incubation is most conveniently carried out in water baths adjusted to the appropriate temperature. Bath temperature can be easily monitored with a thermometer punched through the Styrofoam cover, and adjusted, if necessary, by adding a few chucks of ice. At 15°C proteins accumulate in a compartment between the endoplasmic reticulum (ER) and the Golgi complex, whereas at 20°C accumulation occurs at the level of a late-Golgi compartment. The reversible temperature-induced transport blocks provide valuable tools for the morphological and biochemical analysis of membrane traffic. Distinct threshold temperatures exist for different steps in transport pathways, but temperature reduction can also be used to slow down events among different sites and to increase resolution.

Poration by alpha-toxin and streptolysin O: an approach to analyze intracellular processes.

Abstract: Publisher Summary This chapter describes the purification and handling of α-toxin and streptolysin O (SLO) for the poration of cells. In contrast to other permeabilizing procedures, the pores inserted into the plasma membrane with the aid of bacterial toxins are stabilized by a proteinaceous ring like structure. Depending on the aim of an experiment, the cells can be made permeable either for large or for small molecules by selection of a suitable bacterial pore-forming protein. This allows permanent access to the cells' interior to investigate intracellular processes as diverse as fusion of secretory vesicles with the inner surface of the cell membrane, contraction, metabolism of hormones, fluxes of ions, or glucose metabolism. Besides the different secretory cells, poration by α-toxin or SLO was carried out with hepatocytes, rat basophilic leukemia cells, fibroblasts, and smooth muscle cells. This indicates that the novel approach of permeabilization of cells by channel-forming toxins has already become a widely used tool for the investigation of a variety of intracellular processes in situ .

Chapter 4 Using Temperature-Sensitive Mutants of VSV to Study Membrane Protein Biogenesis

Abstract: Publisher Summary This chapter describes experimental protocols that are illustrative of the method in which temperature-sensitive mutants of vesicular stomatitis virus (VSV) can be used. The discovery of these mutant viral proteins has created an opportunity to synchronize the transport of the totality of an integral membrane protein, not just a subpopulation that has been radiochemically tagged. Once synchronized, this transport has proved amenable to morphological characterization. These mutants also prove useful in determining the mechanisms, underlying the retention of proteins in the ER or at other specific steps along the “secretory” pathway. The rapid reversibility of the temperature-induced transport block indicates that the mechanism of retention of these mutants may involve subtle interactions with specific cellular machinery. The discovery that the G protein in some mutant VSV strains is temperature-sensitive for exit from the ER allows synchronizing its transport between the ER and the cell surface. To determine independently whether the G protein has been correctly transported to the Golgi apparatus and not to an inappropriate destination, it can be investigated whether its asparagine-linked oligosaccharides have been appropriately remodeled.

Resonance energy transfer microscopy.

Abstract: Publisher Summary Scientists have long sought technical approaches that would allow them to study the real-time behavior of cellular functions at the subcellular level. Fluorescence, which is the emission of photons that occur as the electrons in a chromophore decay from an excited state back to the ground state, has provided a tool by which scientists can examine various cellular properties. This chapter discusses how fluorescence can be used as a spectroscopic ruler to measure distances among cellular components and gain information about the interactions of these components on the molecular level. This method, known as “resonance energy transfer (RET),” is founded on the fact that a fluorophore (donor) in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor) nonradiatively through dipole–dipole interactions. This process requires some spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and, for a given donor-acceptor pair, the efficiency of the transfer process is dependent on their relative orientation and on the distance between them.

Immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy.

Abstract: We have presented our detailed methods for localizing antigens in cultured cells and tissue sections by IP at the EM level. Immunoperoxidase cytochemistry is particularly well suited for the study of sparse antigens as a result of the enzymatic amplification afforded by the method, and of molecules confined within a membrane-enclosed compartment wherein the DAB reaction produce can accumulate. Although IP is commonly used to localize membrane-compartmentalized molecules, reliable qualitative information can also be obtained on cytoplasmic antigens as well (Anderson et al., 1978; Merisko et al., 1986; Rodman et al., 1984). For these and other reasons, it is likely that IP cytochemistry will continue to be an important tool for the cell biologist especially in the study of membrane traffic. Other inventive combinations of immunocytochemical methods will likely be forthcoming, for example, combining IP localization with postembedding labeling by colloidal-gold conjugates to provide triple EM labeling.

Chapter 5 Enzymatic Approaches for Studying the Structure, Synthesis, and Processing of Glycoproteins

Abstract: Publisher Summary This chapter highlights the enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Enzymes that hydrolyze specifically at the inner-core di- N -acetylchitobiose moiety comprise a broad group of glycosidic enzymes known as “endo- β -N-acetylglucosaminidases,” or more commonly, “endoglycosidases.” Among these, the bacterial enzymes Endo H and Endo F are widely used because of their different substrate specificities and their commercial availability. Protein conformational effects can influence greatly the course and extent of deglycosylation by rendering the susceptible oligosaccharides at certain glycosylation sites partially or completely “inaccessible” to endoglycosidase action. Various denaturants have been employed to expose otherwise inaccessible oligosaccharide cores, including ionic, nonionic, and zwitterionic detergents as well as chaotropic salts and thiols. For the oligosaccharide cleaving enzymes to be effective, they must be used in conjunction with highly sensitive analytical techniques for characterizing the carbohydrate or protein moieties. The simplest method of determining whether deglycosylation has occurred is by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of control and enzyme-treated material. A significant decrease in molecular weight (i.e., increase in electrophoretic mobility) is a good indication that Asn-linked oligosaccharides have been enzymatically released.

Postembedding labeling on Lowicryl K4M tissue sections: detection and modification of cellular components.

Abstract: Publisher Summary This chapter discusses the procedures for low-temperature embedding in Lowicryl K4M together with various protocols for the light and electron microscopic localization of cellular constituents as they have been used in laboratory. The development of the Lowicryl resins, particularly the hydrophilic Lowicryl K4M, for low-temperature embedding by Carlemalm er al. (1982) and their successful introduction for postembedding immunolabeling can be considered a major achievement. This low-temperature embedding technique has not only provided superior preservation of antigens, but has also resulted in improved preservation of fine-structural details of mildly aldehyde-fixed tissues and cells, and a drastically reduced background staining. In addition, it dramatically improved the detectability of glycoconjugates with lectins, monoclonal antibodies, and glycosyltransferases. The basic principles of sectioning resin-embedded materials apply to Lowicryl K4M. Lowicryl K4M blocks can be sectioned with glass or diamond knives. The angle of the pyramids should be in the range of 55˚-60˚. It is recommended to trim the final pyramids with glass knives or on a trimming machine. For sectioning, precautions have to be taken to prevent wetting of the pyramid because Lowicryl K4M is hydrophilic.

Multiple spectral parameter imaging

Abstract: Publisher Summary This chapter discusses the requirements for performing multiple spectral parameter imaging on living cells. Many of these considerations also apply to fixed preparations. Cell functions, such as cell division, endocytosis, and cell migration involve a complex temporal and spatial interplay of multiple organelles, macromolecules, ions, and metabolites. Goal of cell biology is to define the role of each cellular constituent and to define the molecular interplay of multiple constituents that are required for the completion of specific cell functions. Therefore, a technique is required that will permit the quantitation of multiple physiological parameters in time and space within the same cells. Quantitative fluorescence microscopy, when combined with the increasing number of sensitive fluorescent and other luminescent probes available, offers a powerful approach for defining the chemical and molecular dynamics within living cells in time and space. An approach is developed that allows the analysis and correlation of up to five separate parameters based on the spectral isolation of distinct fluorescent probes. The use of a properly designed imaging workstation helps to employ these methods in both two and three dimensions and to harness the power of video-enhanced contrast microscopy.

A fluorescent derivative of ceramide: physical properties and use in studying the Golgi apparatus of animal cells.

Abstract: Publisher Summary This chapter discusses the synthesis and purification of C 6 -NBD ceramide and its application in the study of the Golgi apparatus of animal cells When cells are treated with c,-NBD-ceramide at low temperature, washed, and warmed to 37°C, the Golgi apparatus and later the plasma membrane becomes intensely fluorescent During the redistribution of intracellular fluorescence, the C 6 -NBD-ceramide is metabolized to fluorescent sphingomyelin and glucosylceramide Consistent with the increasing fluorescence at the plasma membrane over time, increasing amounts of each fluorescent metabolite can be removed from the cell surface by back-exchange to nonfluorescent acceptor liposomes Commercial sources of D - erythro -sphingosine can be contaminated with significant amounts of L -threo-sphingosine, resulting in two isomers of C 6 -NBD-ceramide being produced during synthesis Resonance energy transfer (RET) between C,-NBD-labeled lipids and Rhodamine B-labeled phosphatidylethanol-amine (N-Rh-PE) can be used to monitor the rate of transfer of C,-NBD-labeled lipids between two populations of lipid vesicles It is found that RET can also be used to test the ability of a C,-NBD-lipid to undergo transbilayer movement in unilamellar vesicles

Analysis of the synthesis, intracellular sorting, and function of glycoproteins using a mammalian cell mutant with reversible glycosylation defects.

Abstract: Publisher Summary This chapter summarizes the properties of the mammalian cell mutant ldl D. It reviews the recent experiments that exploit its novel characteristics and provide suggestions for the experimental manipulation of these cells. Classic biochemical genetics combined with recombinant-DNA technology provides a powerful approach for identifying and characterizing the genes, gene products, and biochemical and cellular functions that underlie the multicompartmental pathways. Seven recessive complementation groups ( ldl A– ldl G) are identified, which define genes required for the normal expression of LDL receptor activity. The ldl B, ldl C, and ldl D mutants exhibit global, pleiotropic glycosylation defects affecting protein- and lipid-linked oligosaccharides. ldl D cells were initially isolated in selections and screens were designed to identify Chinese hamster ovary (CHO) mutants expressing defects in the endocytosis of LDL. The defective synthesis of N-linked, 0-linked, and lipid-linked glycoconjugates and the LDL receptor deficiency of ldl D cells are because of a virtual absence of UDP-Gal/UDP-GalNAc 4-epimerase enzymatic activity. This enzyme catalyzes the reversible isomerizations of UDP-glucose to UDP-galactose (Gal) and UDP- N -acetylglucosamine to UDP- N -acetylgalactosamine (GalNAc).

Expression and analysis of the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells using retroviral vectors.

Abstract: Publisher Summary This chapter describes methods for studying the expression and transport of the polymeric immunoglobulin receptor (poly-IgR) in Madin–Darby canine kidney (MDCK) cells. This system has been very useful in analyzing protein traffic and transport in polarized cells. A variety of methods have been used to express cloned cDNAs in MDCK cells, both transiently and in stable cell lines. The retroviral system consists of an expression vector and a packaging cell line. In pDOL vector, the gene of interest (the rabbit poly-IgR) can be cloned into either a Bam HI or Sal I site, both of which are driven by the viral long terminal repeat (LTR) promoter. Secretory component (SC) is a large proteolytic fragment of the poly-IgR and is easily purified from rabbit bile. Growth of MDCK cells on filters leads to increased cell polarity and allows separate access to the apical and basolateral surfaces. Cells are maintained on 10-cm tissue culture plates in medium containing 5% FBS. It is not easy to monitor directly the growth of cells on filters and formation of a confluent monolayer. A simple qualitative test relies on hydrostatic pressure.

A hitchhiker's guide to analysis of the secretory pathway in yeast.

Abstract: Publisher Summary This chapter presents the analysis of the secretory pathway in yeast. The highly compartmentalized organization of cellular activities in the budding yeast, Saccharomyces cerevisiae, requires a mechanism that ensures the correct localization of protein and lipid. All cellular protein biosynthesis operates in the cytoplasm except for a small number of mitochondrial DNA-encoded proteins. Secretory proteins are synthesized on polyribosomes that preferentially associate with the endoplasmic reticulum (ER) shortly after initiation and discharge the nascent polypeptides into the ER. In yeast secretory proteins, the N-linked core oligosaccharides are trimmed to Man8GlcNAc2 and are extensively modified with additional mannose residues during their transit through the Golgi apparatus, producing a carbohydrate structure referred to as the “outer chain.” Protein secretion and plasma membrane assembly in S .cerevisiae are directed topologically to the bud portion of a growing cell, this region being the region of active cell surface growth. For 35S or 32P radiolabeling, the appropriate growth conditions depend largely on the proteins to be examined. Expression of a variety of regulated secretory and membrane proteins require growth conditions that starve yeast of a particular catabolite. Two commonly studied yeast proteins—the α-cell-specific mating pheromone ( α-factor) and the soluble vacuolar protease (CPY)—are produced constitutively.

Three-dimensional confocal fluorescence microscopy.

Abstract: Publisher Summary Biological material is organized in four dimensions: three spatial ones and a temporal one. Light microscopy is able to visualize biological objects in their natural watery condition and during their temporal development. Improved imaging is the optical sectioning property by which the contributions from out-of-focus areas in the specimen are effectively suppressed. In normal microscopy, these contributions lead to a strong reduction in the available image contrast. A three-dimensional microscope is obtained where each data point as collected represents the quantity of the specific contrast parameter used at a certain point in space. Deconvolution techniques have been developed for eliminating the out-of-focus information from conventional fluorescence microscopy. Confocal microscopy can deliver directly clear optical sections without the use of time-consuming image reconstruction algorithms. Image processing can also be used to enhance the confocal images. Computer-generated stereoscopic images are also used for the visualization of the three-dimensional biological information. This chapter discusses the number of aspects of confocal imaging, especially the advantages and drawbacks of the various scanning approaches.

Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopy.

Abstract: Publisher Summary This chapter discusses the utility of video microscopy to visualize, resolve, and measure widths and distances to precisions that are conventionally considered to be below the limit of the resolution of the light microscope. In an image-forming system, resolution is generally expressed as a measure of the ability to separate the images of two neighboring object points. In the case of a light microscope, producing diffraction-limited images, the resolution limit is defined as the minimum distance between two self-luminous or incoherently illuminated objects or structures whose diffraction images can visually be distinguished as coming from two points. When the diffraction images of the two points overlap to an extent that they can no longer be distinguished from that of an individual object, the two are said not to be resolved or that the distance is less than the limit of resolution. For microscopy, the contrast boosting ability of video allows the use of the best corrected objective lenses not only for bright field and fluorescence but also for polarized light and differential interference contrast (DIC) microscopy.

Designing, building, and using a fluorescence recovery after photobleaching instrument.

Abstract: Publisher Summary This chapter discusses the design, construction, and proper use of a fluorescence photobleaching recovery (FPR) instrument, the design of spot FPR instruments, and the development of intensified video imaging FPR instruments. The design possibilities of such instruments are essentially infinite. The chapter describes the instruments that are interfaced to personal computers. Such interfacing brings, at reasonable cost to the individual laboratory, the ability to perform both nonlinear least squared data fitting and image processing. Such interfacing requires compromise. However, the rapid evolution of the personal computer in the past 10 years promises continued growth of power and flexibility in the future. FPR is a technique for measuring the lateral diffusibility of macromolecules in membranes and aqueous phases. Many membrane proteins are not completely free to diffuse, and their diffusion rates are too slow to be controlled by lipid fluidity.

Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy

Abstract: Publisher Summary Immunocytochemistry provides a powerful technique for analysis of molecular organization within cells. Using specific antibody probes together with electron microscopy (EM), the localization of a protein within individual cells can be analyzed. During the electronmicroscopic analysis of yeast cells, it is found that samples prepared for routine EM could be labeled quite successfully with affinity-purified antiserum against the membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase), and colloidal gold reagents. This chapter describes the detailed protocols of these methods. Generation of reagent antibodies of high specificity is of utmost importance for accurate immunolocalization at the EM level. The electron micrographs presented in the chapter compare several fixation protocols and resins, and the effect of these variables on immunoreactivity of HMG-CoA reductase with affinity-purified antiserum.

Image fidelity: characterizing the imaging transfer function

Abstract: Publisher Summary This chapter discusses the various limitations and distortions inherent and introduced in quantitative fluorescence microscopy and the ways to compensate and/or eliminate them. It presents the basic ideas that govern the system approach for describing and analyzing quantitative imaging systems for fluorescence microscopy. Use of the linear, shift-invariant (LSI) model for describing the complete system has given an elegant tool, the optical transfer function (OW), to describe the imaging system's resolution capabilities. It has also enabled to understand how an object much smaller than a wavelength of light can be visualized. Using simple test patterns it is seen how it is possible to measure the contrast modulation transfer function (CMTF). The chapter describes how every acquired fluorescence image is a compromise between the actual distribution of light and the constraints of physics and engineering that prevent us from observing it. This distortion is inherent in the observation process. At the very best, the data is obtained through quantitative fluorescence microscopy that will be a distortion of reality. The chapter also discusses the tools for understanding that distortion.

Fluorescent indicators of membrane potential: microspectrofluorometry and imaging

Abstract: Publisher Summary The electric potential difference between the interior of a cell and the extracellular medium has long known to play an important role in the mediation, regulation, and delivery of many transmembrane signals. Fluorescent indicators of membrane potential have been developed over the past 15 years to achieve this end. Dyes have been broadly classified as either fast or slow, depending on whether they are suitable for the measurements of millisecond changes in excitable cells or smaller cell physiological changes occurring in the minute time scale. The fast dyes are applied to cases where AC coupling and/or signal averaging can be employed to extract a small potential-dependent optical response from a large light background. The slow dyes are much more sensitive but are applicable mainly to experiments with large populations of cells in a fluorometer. This chapter discusses the application of a fast dye in the measurements of both the spatial and temporal distribution of membrane potential induced by an external electric field. It also describes a new series of slow dyes that allow the measurement of membrane potential in individual cells through the microscope.

Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca2+ with fura-2.

Abstract: Publisher Summary The fluorescent reporter dyes quin2 and fura-2 are used by many investigators to quantitate the cytosolic free-calcium concentration [Ca2+]i in different types of cells. For the dyes to measure accurately [Ca2+]i, they must be selectively and uniformly distributed within the cytoplasmic matrix of the cells. However, in a number of cell types fura-2 does not remain within the cytoplasmic matrix; it accumulates within intracellular compartments, is secreted from the cells entirely, or both. This chapter reviews the evidence for fura-2 sequestration and secretion by cells and describes the use of organic-anion transport inhibitors to ameliorate problems caused by these processes. Mouse macrophages possess organic-anion transporters that remove fluorescent dyes, including fura-2, from the cytoplasmic matrix of these cells. The dyes are sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. The transporters that promote dye sequestration and secretion are inhibited by the drugs probenecid and sulfinpyrazone that block transport of organic anions in polarized epithelial cells.

Preservation of biological specimens for observation in a confocal fluorescence microscope and operational principles of confocal fluorescence microscopy.

Abstract: Publisher Summary Confocal scanning-laser microscopy combined with sophisticated image-processing techniques allow the cell biologist to explore the three-dimensional infrastructure of the cell, thus conferring a high degree of precision in localization experiments This chapter discusses some of the inherent problems in preservation of biological specimens so that reliable data can be acquired with a confocal fluorescence microscope The methodology is the result of an effort to study the genesis of epithelial polarity in the Madin-Darby canine kidney (MDCK) cell line While specific methods have been developed to study these particular cells, the lessons learned about their preservation should be widely applicable A description of the confocal principle introduces basic concepts of confocal fluorescence microscopy One can then understand how images are recorded and how the information may be interpreted