Jan Slavík

Use of FITC as a Fluorescent Probe for Intracellular pH Measurement

Fluorescein Isothiocyanate (FITC) is widely used in biology and medicine as a fluorescent marker for labeling various proteins. Particularly fluorescence marking of antibodies could not be imagined without FITC. However, at the same time FITC displays pH-indicative properties. This paper evaluates the limits of the use of FITC as a pH indicator in biological material, namely, for intracellular and intraorganellar pH measurement.

2′,7′-Bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy

The widely used fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell ofSaccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

Applications of fluorescent probes in cellular biology measurement of intracellular pH

Abstract Fluorescent dyes are capable of giving in vivo information on intracellular ionic composition, membrane potential, membrane fluidity, polarity, localisation of receptors, enzymes and enzymatic activity, and they can selectively label various intracellular compartments and cellular constituents. The tremendous potential inherent in fluorescent techniques is illustrated by the example of in vivo intracellular pH measurement.

Fusion of macrophages on an implant surface is associated with down-regulated expression of ligands for galectin-1 and -3 in the rat.

Galectins have a wide range of biological activities which are elicited by binding to appropriate glycoligands. Besides regulation of the expression of the galectins the extent of the presence of suitable binding sites will be relevant to infer the cellular responsiveness to this class of sugar receptors. Thus ligand presentation requires monitoring by the tissue lectin. We demonstrate the expression of galectin-3 by macrophages and foreign-body giant multinucleate cells colonizing a cellophane implant in the rat by the A1D6 monoclonal antibody. The extents of ligand presence are visualized in the same cells by biotinylated galectin-3 and also by galectin-1 which is produced by diverse mammalian cell types and widely distributed. Labeled mistletoe (VAA) and tomato (LEA) lectins are used as tools to assess the degree of similarity of the binding profile between endogenous and exogenous proteins. The presentation of alpha-galactosides is monitored with a natural immunoglobulin G subfraction obtained by two consecutive affinity chromatography steps. The binding of labeled galectins and plant lectins was significantly lower to foreign-body giant multinucleate cells than to mononuclear macrophages. The application of the alpha-galactoside-specific probe yielded no significant staining. The potential problem of epitope accessibility could be excluded by the concomitant positivity obtained with an IgG subfraction with selectivity to beta-galactosides also obtained by affinity chromatography. These results provide no evidence for a role of alpha-galactosides for the binding of galectins in the rat macrophages colonizing the implant. The reduced level of expression of glycoligands for galectin-1 and -3 in foreign-body giant multinucleate cells in contrast with the mononuclear macrophages suggests an inhibitory influence of macrophage fusion on the expression of galectin-reactive molecules.

Fluorescent Probes in Biology and Medicine. Measurement of Intracellular pH Values in Individual Cells.

The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H+, Ca2+, Mg2+, K+, Na+, Fe3+, Cl-, Zn2+, Cd2+, Hg2+, Pb2+, Ba2+, La3+. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth dyes gives chance for the separated detection of another six peak pairs. The literature data on simultaneous applications of several fluorescent dyes are rare, usually it is only pH and calcium, pH and membrane potential or pH and cytoskeleton changes that are mentioned. Nevertheless, I am sure that in the near future it will be quite common to employ several fluorescent dyes simultaneously. So, in a few years, you may expect to be comfortably seated in an armchair in front of the monitor screen, sip your coffee and follow simultaneously several physiological parameters trying to find out new relations among them. In this respect the potential of fluorescent probes is unsurpassed if you just recall only the discovery of calcium waves and calcium spikes during the past years.

The Effect of Lysosomal pH on Lactoferrin-Dependent Iron Uptake in Tritrichomonas foetus

Tritrichomonas foetus is a parasitic protozoan which causes a sexually transmitted disease of cattle. The establishment of infection depends on the ability of T. foetus to acquire iron from the host as was demonstrated in experimentally infected mice1. Most of the iron available in mucosal secretions, the environment colonised by this parasite, is rather firmly bound to host iron-binding proteins such as lactoferrin and transferrin. Trichomonads as well as other pathogens therefore evolved specific mechanisms which allow them to withdraw iron from these proteins2,3,4. It was shown recently that lactoferrin is specifically bound to the surface of T. foetus, endocytosed and transported into hydrolase containing lysosome-like organelles5. Although there is no direct evidence for release of iron within this cell compartment, the low pH in the lysosomes might provide a suitable environment for such process. Since information about the intracellular pH of protozoa is rather limited6,7 and since no data are available on the pH of the endo/lysosomal compartment in trichomonads, we attempted (1) to determine the pH of the cytoplasm and the lysosome-like organelles of T. foetus, (2) to monitor pH changes in these organelles in trichomonads treated with agents inhibiting endo/lysosomal acidification, and (3) to investigate whether a pH increase in the lysosomal-like organelles influences the iron uptake from lactoferrin by T. foetus.

ARTIFACTS IN FLUORESCENCE RATIO IMAGING

Successful development of reliable fluorescent ratio indicators for ion concentration, membrane fluidity and membrane potential and steady improvement of the imaging equipment (microscopes, detectors, computers) have made fluorescence ratio imaging microscopy a widely used method in biomedicai research (Slavik, 1994). An increasing number of papers deal especially with Ca2+ and pH measurements. The proper interpretation of experimental data requires not only a profound knowledge of the fluorescent probe used and a reliable calibration standard curve but one must be also aware of artifacts inherent in the opto-electronical level of the technique. These artifacts can arise from the following causes: blurring of the image due to the involvement of out-of-focus light, background fluorescence, improper adjustment of the detection part of experimental setup, movement of the sample, and bleaching of the dye.

Distribution of Individual Cytoplasmic pH Values in a Cell Suspension

The application of pH-sensitive fluorescent probes represents both a progressive and an easy way of in vivo determination of cytoplasmic pH values. The ratio microscopy can deliver pH values of a large number of cells simultaneously. This illustrates the tremendous progress of the pH-measuring techniques, as 31P NMR, ion-selective microelectrodes, distribution of weak acids or destruction (permeabilization) of cells yielded only one single value from each measurement procedure.