David E. Wolf

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

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.

Lipid domains in sperm plasma membranes

Mammalian sperm have unusual plasma membranes compared to those of somatic cells. After leaving the testes, sperm cease plasma membrane lipid and protein systhesis. A major fraction of mammalian sperm plasma membranes are lipid linked. A large fraction of their lipid chains are highly unsaturated. Biophysical studies reveal that lipids are regionalized on the sperm surface and are highly immobile. This immobile fraction evolves with sperm development. This non-diffusing fraction is also observed in bilayers reconstituted from lipid extracts of sperm head plasma membranes, suggesting the existence of gel phase domains in these membranes. This hypothesis is further supported by differential scanning calorimetry, which shares at least two relatively broad phase transitions with physiological temperature falling between these major transitions.

Influence of alcohols, temperature, and region on the mobility of lipids in neuronal membrane

Fluorescence recovery after photobleaching was used to examine lipid diffusibility in different regions of Aplysia neurons. Differences in diffusion of 1-acyl-2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)]aminohexanoyl)phosphatidylcholine (NBD-C6-PC) in the cell body, axon hillock, and axon were not apparent. Lipid diffusibility during temperature variations and exposure to alcohols was also examined by photobleaching techniques. For these studies, all measurements were made on the cell body. Alcohols were found to be selective in their effects upon the diffusibility of lipid probes. Neither ethanol nor butanol affected the diffusibility of NBD-PC. However, at the same concentrations, both of these alcohols caused a significant increase in the diffusion coefficient (D) for rhodamine-phosphatidylethanolamine (Rho-PE). The diffusion coefficient for NBD-PC in the cell body plasma membrane did not increase with warming, between 4 degrees C and 25 degrees C. The fraction of lipid probe free to diffuse (per cent recovery; %R) however, increased as temperature increased, within this range. The nonconventional relationship between temperature and D was even more pronounced for Rho-PE. As temperature increased, D became smaller for this probe, concurrent with an increase in %R. These results suggest that immobile viscous lipid is recruited into a mobile fraction as temperature increases, resulting in the maintenance of constant diffusibility. The effects of temperature on D and %R, and the selective effects of alcohols on lipid diffusibility suggest that the membrane is heterogeneously organized, on a submicroscopic scale, into domains. The implications of this organization for nerve function and responses of nervous systems to temperature and anesthetics are discussed.

Quantitative analysis of digital microscope images.

Publisher Summary The chapter discusses quantitative analysis of digital microscope images and presents several exercises to provide examples to explain the concept. The chapter also presents the basic concepts in quantitative analysis for imaging, but these concepts rest on a well-established foundation of signal theory and quantitative data analysis. The chapter presents several examples for understanding the imaging process as a transformation from sample to image and the limits and considerations of quantitative analysis. The chapter introduces to the concept of digitally correcting the images and also focuses on some of the more critical types of data transformation and some of the frequently encountered issues in quantization. Image processing represents a form of data processing. There are many examples of data processing such as fitting the data to a theoretical curve. In all these cases, it is critical that care is taken during all steps of transformation, processing, and quantization.

Fundamentals of fluorescence and fluorescence microscopy.

Publisher Summary This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. The chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence—boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. The chapter also examines how fluorescence is measured in the steady state and time-domain and how fluorescence is applied in the modern epifluorescence microscope.

Differential biological effects of K252 kinase inhibitors are related to membrane solubility but not to permeability

Abstract: K252a and K252b are related protein kinase inhibitors that, dependent on conditions, can either inhibit or potentiate the effects of neurotrophic factors. K252a, an ester, is more potent and more cytotoxic on intact cells than K252b, a carboxylic acid. To understand better why these drugs elicit different degrees of biological responses, we analyzed their hydrophobicity, cell permeability, and subcellular distribution. As judged by partitioning between organic and aqueous phases, both compounds are hydrophobic. The partition coefficients were 15.6:1 (organic/aqueous phases) for K252a and 4.4:1 for K252b. The ratio of fluorescence excitation at 352 nm to that at 340 nm for the K252 compounds in the organic alcohol 1‐decanol versus water provides a simple assay of binding of these compounds to phospholipid membranes. This ratio shifted for K252a, but not K252b, in the presence of phospholipid vesicles, indicating that K252a dissolved in the hydrophobic interior of the membrane. Using quantitative video fluorescence microscopy, we found that K252a strongly labeled both Sf9 insect cells and PC12 rat pheochromocytoma cells, probably staining intracellular membranes. The uptake of K252a was rapid and apparently irreversible. K252b also quickly entered Sf9 and PC12 cells, but staining was much weaker. Hence, K252a and K252b are similar in that they both rapidly enter cells but greatly differ in their membrane solubility.

Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells

FCS is an important technique for biophysicists, biochemists, and cell biologists. FCS represents an example of how one can make use of the microscope and electronics to extract information beyond the resolution limit of classical optics. It can be used to study single-molecules both in solution and in living cells and can be used to monitor a wide variety of macromolecular interactions. When used as an in vitro technique, FCS measurements are easy to conduct and can be made on simplified instrumentation. When used in vivo on living cells, many additional factors must be considered when evaluating experimental data. Despite these concerns, FCS represents a new approach that has broad applicability for the determination of molecular stoichiometry both in vivo and in vitro for a variety of membrane and soluble receptor systems.

Diffusion and the Control of Membrane Regionalization

The modern biologist has a concept of the physical structure and nature of biological membranes that is derived from the “fluid mosaic model.”’ This popular model envisages the membrane as being composed of a lipid bilayer. The membrane proteins are situated in this bilayer and in some cases span it. The lipid bilayer matrix is thought to act as a quasi two-dimensional fluid. Because of the fluid nature of the bilayer, the membrane lipids and proteins are thought to be free to diffuse within the membrane. At least three types of diffusional motion should be allowed: lateral diffusion within the plane of the membrane, transverse diffusion across the membrane, and rotational diffusion around axes perpendicular and parallel to the plane of the bilayer. Implicit in the wordfluid is the idea that the lipid bilayer has a bulk physical property called its “bulk membrane fluidity” or inversely its “bulk membrane viscosity,” which controls these diffusional motions. From a mathematical viewpoint this bulk fluidity enables the biophysicist to calculate the diffusion coefficients that define the rates of these motions. A corollary to the existence of such a property is that if a physiological transformation alters membrane fluidity, then, regardless of the probe or the technique that one uses to measure fluidity and regardless of whether one measures lateral, transverse, or rotational diffusion, the same change in fluidity will be observed. The rapid diffusion of constituent molecules is a fundamental, and defining, property of fluids. Indeed, one of the germinal experiments that led to the development of the “fluid mosaic model” was the heterokaryon fusion experiments of Frye and E d i d h 2 In these experiments, it was shown that on a heterokaryon formed by fusing a human and mouse cell, both the human and mouse surface antigens were rapidly intermixed by random lateral diffusion until they were both distributed uniformly over the entire cell surface. Diffusion is a random p r o c e ~ s . ~ It is best understood by describing it as a random walk of molecules, much like the aimless reelings of a drunken begga~.~*~ As in the case of the heterokaryon fusion experiments, diffusion should cause polarized distributions of surface molecules to rapidly randomize to homogeneity. (For a discussion of just how rapidly one can expect this to happen in biological membranes, the reader is referred to reference 5. ) Biological membranes, however, are capable of overcoming free random diffusion and localizing or polarizing the distribution of their surface components. Probably the clearest and most dramatic example of such surface regionalization occurs in the spermatozoa1 plasma membrane. Spermatozoa are, of course,

Imaging Membrane Organization and Dynamics

In this chapter, we will discuss how the conventional techniques of nonradiative fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) can be extended, by coupling these techniques to low-light-level video microscopy and digital image processing, to provide an additional dimension of information. That is, what new information can be gained when, rather than integrating the fluorescence signal over a photomultiplier tube, one instead retains the spatial information by using a two-dimensional video detector? In this chapter, we wish to emphasize the conceptual aspects of these experiments rather than focusing on specific issues of instrumentation and software. For a discussion of these issues the reader is referred to several comprehensive texts on this subject (Castleman, 1979; Inoue, 1986; Makovski, 1983; Taylor and Wang, 1989). In our discussion, we wish also to emphasize how certain empirical manipulations of experimental data can greatly simplify complicated analysis without sacrificing either validity or accuracy.

The optics of microscope image formation.

Abstract Although geometric optics gives a good understanding of how the microscope works, it fails in one critical area, which is explaining the origin of microscope resolution. To accomplish this, one must consider the microscope from the viewpoint of physical optics. This chapter describes the theory of the microscope-relating resolution to the highest spatial frequency that a microscope can collect. The chapter illustrates how Huygens’ principle or construction can be used to explain the propagation of a plane wave. It is shown that this limit increases with increasing numerical aperture (NA). As a corollary to this, resolution increases with decreasing wavelength because of how NA depends on wavelength. The resolution is higher for blue light than red light. Resolution is dependent on contrast, and the higher the contrast, the higher the resolution. This last point relates to issues of signal-to-noise and dynamic range. The use of video and new digital cameras has necessitated redefining classical limits such as those of Rayleigh’s criterion.