Note: multiplexed multiple-tau auto- and cross-correlators on a single field programmable gate array.

We introduce a new multiple-tau hardware correlator design for computing fluorescence correlation functions (CFs) in real time. Use of hardware resources is minimized by scheduling the computation of different segments of the CFs on a single correlator block. Simultaneous calculation of two multiple-tau autocorrelation (ACFs) and two cross-correlation functions (CCFs) is implemented in LabVIEW on a National Instruments field programmable gate array (FPGA) card with a minimal sampling time of 400 ns. Raw data are stored with a time resolution of 50 ns. The design can be easily adapted to other FPGA cards and extended to more inputs.

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells.

Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

New trends in studying structure and function of biological membranes.

Thirty years ago Singer and Nicolson constructed the "fluid mosaic model" of the membrane, which described the structural and functional characteristics of the plasma membrane of non-polarized cells like circulating blood lymphocytes as a fluid lipid phase accommodating proteins with a relatively free mobility. It is a rare phenomenon in biology that such a model could survive 30 years and even today it has a high degree of validity. However, in the light of new data it demands some modifications. In this minireview we present a new concept, which revives the SN model, by shifting the emphasis from fluidity to mosaicism, i.e. to lipid microdomains and rafts. A concise summary of data and key methods is given, proving the existence of non-random co-distribution patterns of different receptor kinds in the microdomain system of the plasma membrane. Furthermore we present evidence that proteins are not only accommodated by the lipid phase, but they are integral structural elements of it. Novel suggestions to the SN model help to develop a modernized version of the old paradigm in the light of new data.

A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma.

BACKGROUND: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca(2+). FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. METHODS: In the assay, FXIII was activated by thrombin and Ca(2+). Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH. RESULTS: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108-224 U/L (69-143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII. CONCLUSIONS: The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.

Cholesterol-dependent clustering of IL-2Ralpha and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts.

Immunogold staining and electron microscopy show that IL-2 receptor alpha-subunits exhibit nonrandom surface distribution on human T lymphoma cells. Analysis of interparticle distances reveals that this clustering on the scale of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R beta-subunit. Clustering of IL-2Ralpha is confirmed by confocal microscopy, yielding the same average cluster size, approximately 600-800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-rich lipid rafts with filipin or depletion of membrane cholesterol with methyl-beta-cyclodextrin results in the blurring of cluster boundaries and an apparent dispersion of clusters for all four proteins. Interestingly, the transferrin receptor, which is thought to be located outside lipid rafts, exhibits clusters that are only 300 nm in size and are less affected by modifying the membrane cholesterol content. Furthermore, transferrin receptor clusters hardly colocalize with IL-2Ralpha, HLA, and CD48 molecules (crosscorrelation coefficient is 0.05), whereas IL-2Ralpha colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46). This coclustering is confirmed by electron microscopy. The submicron clusters of IL-2Ralpha chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.

Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy.

Fluorescence correlation microscopy (FCM) was applied to characterize fusion proteins of the green fluorescent protein (GFP) on the cellular as well as molecular level within seconds in an integrated instrument. FCM combines the inherent sensitivity and high spatial resolution of fluorescence correlation spectroscopy with fluorescence imaging and micropositioning, thereby providing a spectrum of molecular information in the cellular context. Signatures of characteristic parameters derived from the autocorrelation functions served to distinguish a GFP fusion protein of the epidermal growth factor receptor from GFP fluorescence in the endoplasmic reticulum and cytoplasm. Diffusion constants measured for free transiently expressed GFP reproduced values reported previously with other techniques. The accessible concentration range extends from millions to only a few thousand molecules per cell, with single molecule detectability in the femtoliter detection volume. The detailed molecular characterization offered by FCM is fully compatible with automation in sample identification and detection, offering new possibilities for highly integrated high-throughput screening.

The helix-coil transition of DNA duplexes and hairpins observed by multiple fluorescence parameters.

The thermal denaturation of 8-20-bp DNA duplexes labeled with fluorescein and tetramethylrhodamine at opposing 5'-ends was investigated by monitoring the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, the fluorescence resonance energy transfer between fluorescein and rhodamine, and, for the 20-bp duplex, the UV absorption. Melting experiments with the single strands of the duplexes revealed that the single strands can form hairpins stabilized by only a few base pairs. The thermal denaturation curves of the duplexes were fitted well to an extended all-or-none model assuming that only the fully base-paired duplex, the maximally base-paired hairpin, and the random coil conformations are present simultaneously. The extent-of-melting versus temperature curves derived from the different spectroscopic parameters are nearly identical, provided that the analysis of the baselines is carried out correctly; the DeltaH and DeltaS of the dissociation compare well with predictions based on nearest neighbor interaction values available in the literature. Our results imply that for all the oligonucleotides other than the 34-bp oligomer, no partially melted intermediates other than hairpins are present in the reaction mixture in amounts that can be detected by our methods. The melting of the hairpins was also studied directly using single-stranded oligonucleotides. The melting of a 34-bp duplex can be accounted for by a statistical zipper model.

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy.

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Intensity-based energy transfer measurements in digital imaging microscopy.

Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by -pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).

Comparison of different keratometers with the EyeSys videokeratoscope.

We compared the accuracy and the reproducibility of the measurements using a Haag-Streit, a Shin-Nippon, and a Carl Zeiss keratometer and an EyeSys videokeratoscope. Two investigators performed 20 measurements on 3 calibrated steel balls and 5 measurements on 22 normal corneas with all instruments. Calculating the accuracy of the measurements the limits of the 95% confidence intervals of the bias were determined. The accuracy was better than +/-0.1D for the keratometers, whereas it was better than +/-0.25D for the EyeSys videokeratoscope. The coefficient of reproducibility (1.96 x standard deviation) on test balls and corneas was smaller than 0.25D in the case of the Shin-Nippon and Carl Zeiss keratometers and the EyeSys videokeratoscope whereas it was between 0.25-0.5D in the case of the Haag-Streit keratometer. The Carl Zeiss keratometer was the best with respect to the accuracy and reproducibility of the measurements. Paired t-test was used to find possible significant differences between the results of the two investigators, but only clinically insignificant differences were found.

Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.