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D L Farkas

Publications and source records attributed to D L Farkas.

29 records · Page 2Linked to original sources
PubMed1993

Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation.

The use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope. Whether by direct imaging or by confocal scanning, diffraction effects and noise generally limit axial resolution to about 0.5 microns. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 microns by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibres and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.

3T3 Cells↗
PubMed1993

Remapping disparate images for coincidence.

With the development of complex multimode computerized microscope systems, it is possible and necessary to obtain images of the same area of the microscopical preparation by several methods of microscopy, such as differential interference contrast, reflection interference microscopy, several wavelengths of fluorescence microscopy, laser scanning and confocal modes. Thus, varied information may be obtained about a single field, in the form of a set of images, taken at different ports of the microscope, using different digitizing cameras, each appropriate to certain tasks. For comparative purposes, the images should be superimposable, pixel by pixel, but in general they are not--they differ in image shape and size, magnification, distortion, centration and orientation. This paper shows how the problem may be approached, using an extension of the remapping procedures described in a previous paper, in which images of a separate grid reference slide are used to detect, quantify and correct the image errors. Affine remapping, without the use of grid images, is also described.

Cells, Cultured↗
PubMed1989

Dual-wavelength ratiometric fluorescence measurements of membrane potential.

This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.

Electric Conductivity↗
PubMed1989

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.

Cell Membrane↗
PubMed1989

Simultaneous imaging of cell and mitochondrial membrane potentials.

The distribution of charged membrane-permeable molecular probes between intracellular organelles, the cytoplasm, and the outside medium is governed by the relative membrane electrical potentials of these regions through coupled equilibria described by the Nernst equation. A series of highly fluorescent cationic dyes of low membrane binding and toxicity (Ehrenberg, B., V. Montana, M.-D. Wei, J. P. Wuskell, and L. M. Loew, 1988. Biophys. J. 53:785-794) allows the monitoring of these equilibria through digital imaging video microscopy. We employ this combination of technologies to assess, simultaneously, the membrane potentials of cells and of their organelles in situ. We describe the methodology and optimal conditions for such measurements, and apply the technique to concomitantly follow, with good time resolution, the mitochondrial and plasma membrane potentials in several cultured cell lines. The time course of variations induced by chemical agents (ionophores, uncouplers, electron transport, and energy transfer inhibitors) in either or both these potentials is easily quantitated, and in accordance with mechanistic expectations. The methodology should therefore be applicable to the study of more subtle and specific, biologically induced potential changes in cells.

Adenosine Triphosphate↗
PubMed1987

Membrane potential induced by external electric field pulses can be followed with a potentiometric dye.

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.

Cell Membrane↗
PubMed1984

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. I. Initial kinetics and the charging capacitance of the membrane.

Preilluminated chloroplast membranes, and particularly hypotonically swollen vesicles (blebs), give rise to a strong characteristic luminescence (electrophotoluminescence, EPL; Ellenson and Sauer, 1976, Photochem. Photobiol., 23:113-123; Arnold and Azzi, 1971, Photochem. Photobiol., 14:233-240) during the application of a strong external electric field. A detailed kinetic study of EPL was carried out and the initial kinetics from the field onset are reported here. The fast rise time (less than 0.2 mus) of the applied external electric field together with a high instrumental time resolution allowed the observation of a characteristic delay (lag time) between the field onset and the appearance of the induced emission. The lag time decreased with increase in the applied field strength and/or the conductivity of the suspension and is interpreted to be a consequence of (a) the necessity to reach a threshold electrical potential difference in the bleb membrane, below which no emission can be triggered, and (b) the finite time required to attain such a transmembranal field during the charging process of the membrane. A quantitative analysis, connecting the lag time, the controllable experimental parameters, and the membrane electrical characteristics is presented. Its verification was carried out in both size-selected and heterogeneous bleb populations. In the latter, experiments were consistent with the assumption that the lag time reflects the charging of the largest blebs. The results indicate (a) the possibility of directly measuring the specific membrane capacitance, yielding an estimate of Cm = 1.2 +/- 0.3 microF/cm2 (the precision being particle size-homogeneity dependent); (b) A minimal transmembranal potential difference (of approximately 240 mV) is necessary to induce electrophotoluminescence; and (c) the lag duration depends on the time elapsed between the preillumination and the external field application. Correlated with the study of ionophore effects on the lag time, this suggests additivity of the light- and field-induced transmembrane potentials in attaining the threshold for emission.

Chloroplasts↗
PubMed1979

Cold storage of isolated class C chloroplasts: optimal conditions for stabilization of photosynthetic activities.

Preservation of photosynthetic activities (photophosphorylation, electron transport, fluorescence induction, 0.3-second delayed light emission) of isolated broken (class C) chloroplasts by low temperature storage was investigated under a wide range of conditions in order to optimize long time activity retention.The more labile functions (photophosphorylation and electron transport) required very low temperatures (below -79 C) and relatively high (above 20%, v/v) concentrations of cryoprotectives for satisfactory stabilization. Fluorescence induction and delayed light emission were less sensitive, especially during the 1st month of storage.Taking into account the effect of cryoprotectives on absolute activities prior to freezing, optimum activity retention was observed with a medium containing ethylene glycol (30%, v/v) and a storage temperature of -100 C or below. In this case, given fast thawing and high chloroplast concentration, practically 100% preservation of all of the photosynthetic activities investigated was obtained for at least 10 months, even with very simple freezing and storage procedures.The same optimal medium at somewhat higher temperatures (-79 C and to a lesser extent at -41 C) caused a dramatic uncoupling effect: photophosphorylation was inhibited in a few hours, while electron transport increased 3- to 5-fold. The enhanced electron transport was stable for almost a month and then declined sharply. This uncoupling effect was specific only to ethylene glycol.

Journal Article↗
PubMedDate not supplied

Non-invasive image acquisition and advanced processing in optical bioimaging.

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Fluorescence↗
PubMedDate not supplied

Tumor detection and visualization using cyanine fluorochrome-labeled antibodies.

Tumor localization using fluorescence has been made practical by current improvements in tumor targeting molecules, especially monoclonal antibodies and their derivatives, by the development of convenient near-infrared emitting fluorochromes and by the availability of digital cameras having high sensitivity in this spectral region. Recent studies in animals have demonstrated that fluorochrome labeling of monoclonal antibodies confers adequate sensitivity and improved resolution. Distribution and catabolism of fluorochrome-labeled and radiolabeled antibodies are similar. Simultaneous localization of multiple reagents is made possible by labeling with several different near-infrared emitting fluorochromes; thus background subtraction and differential labeling of multiple tumor-associated components can be performed. Difficulties in using the fluorochrome labels are mainly related to light scattering and absorption in tissues, but detection of small tumors at depths of several millimeters is feasible. The major medical use of this new technology is likely to be endoscopic location of tumors. Scientific uses include studies of tumor metastasis, uptake and distribution of drugs and tumor-targeting molecules by tumors, and migration patterns of near-infrared labeled cells in vivo.

Animals↗