Biomedical subjects

Dror Fixler

Publications and source records attributed to Dror Fixler.

7 recordsLinked to original sources

PubMed2006

Concomitant real-time monitoring of intracellular reactive oxygen species and mitochondrial membrane potential in individual living promonocytic cells.

Reactive oxygen species (ROS) have recently been shown to be involved in multiple physiological responses through modulation of signaling pathways. Inappropriate production of these radicals, and their metabolites, leads to the development of various pathologies. Free radicals can induce both positive and negative effects in cells, and their metabolic pathways are very complex. Hence, it is crucial to be able to simultaneously and directly determine their production dynamics and concentrations in individual living cells, in physiological or pathological states, and in response to drugs. The aim of the present study was to monitor in real time the rates of ROS generation in promonocytic cells upon stimulation with hydrogen peroxide and oxidized lipid. Quantitative detection of intracellular ROS concentration in intact living U937 cells was performed by fluorescence intensity (FI) and polarization (FP) measurements utilizing the Optical LiveCell Array technology. The "dihydro" derivative probes of fluorescein (DCF-DA) and rhodamine (DHR123) were used to assess the intracellular levels of ROS. Each probe molecule exhibited a characteristic FI and FP in its non-fluorescent or oxidized form. Analysis of the temporal relationship between the kinetics of ROS generation and the onset of changes in mitochondrial membrane potential shows high variability within a cell population with regard to both processes. The data demonstrated that temporal measurement of ROS generation, in identifiable individual cells, reveals kinetic behavior that otherwise would be undetected.

Dicarbethoxydihydrocollidine↗

PubMed2006

Pattern projection for subpixel resolved imaging in microscopy.

In this paper, we present a new approach providing super resolved images exceeding the geometrical limitation given by the detector pixel size of the imaging camera. The concept involves the projection of periodic patterns on top of the sample, which are then investigated under a microscope. Combining spatial scanning together with proper digital post-processing algorithm yields the improved geometrical resolution enhancement. This new method is especially interesting for microscopic imaging when the resolution of the detector is lower than the resolution due to diffraction.

Journal Article↗

PubMed2006

Speckle random coding for 2D super resolving fluorescent microscopic imaging.

In this manuscript we present a novel super resolving approach based upon projection of a random speckle pattern onto samples observed through a microscope. The projection of the speckle pattern is created by coherent illumination of the inspected pattern through a diffuser. Due to local interference of the coherent wave front with itself, a random speckle pattern is superimposed on the sample. This speckle pattern can be scanned over the object. A super-resolved image can be extracted from a temporal sequence of images by appropriate digital processing of the image stream. The resulting resolution is significantly higher than the diffraction limitation of the microscope objective. The new super-resolution method is demonstrated by application to fluorescence of biological samples.

Journal Article↗

PubMed2006

Influence of fluorescence anisotropy on fluorescence intensity and lifetime measurement: theory, simulations and experiments.

The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements--including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy--it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2) the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.

Algorithms↗

PubMed2003

Ratiometric fluorescence polarization as a cytometric functional parameter: theory and practice.

The use of ratiometric fluorescence polarization (RFP) as a functional parameter in monitoring cellular activation is suggested, based on the physical phenomenon of fluorescence polarization dependency on emission wavelengths in multiple (at least binary) solutions. The theoretical basis of this dependency is thoroughly discussed and examined via simulation. For simulation, aimed to imitate a fluorophore-stained cell, real values of the fluorescence spectrum and polarization of different single fluorophore solutions were used. The simulation as well as the experimentally obtained values of RFP indicated the high sensitivity of this measure. Finally, the RFP parameter was utilized as a cytometric measure in three exemplary cellular bioassays. In the first, the apoptotic effect of oxLDL in a human Jurkat FDA-stained T cell line was monitored by RFP. In the second, the interaction between cell surface membrane receptors of human T lymphocyte cells was monitored by RFP measurements as a complementary means to the fluorescence resonance energy transfer (FRET) technique. In the third bioassay, cellular thiol level of FDA- and CMFDA-labelled Jurkat T cells was monitored via RFP.

Computer Simulation↗

PubMed2003

Fluorescence polarization: a novel indicator of cardiomyocyte contraction.

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.

Animals↗

PubMedDate not supplied

Tracing apoptosis and stimulation in individual cells by fluorescence intensity and anisotropy decay.

Presented is the use of fluorescence lifetime (FLT), anisotropy decay, and associated parameters as differential indicators of cellular activity. A specially designed combination of a frequency mode based time resolved microscope and a picoliter well-per-cell array have been used to perform temporal measurements in individual cells under various biological conditions. Two biological models have been examined: mitogenic activation of peripheral blood mononuclear cells (PBMC) and induction of programmed cell death (apoptosis) in Jurkat T cells (JTC). The FLT of fluorescein stained PBMC was found to increase from 4+/-0.02 to 4.5+/-0.025 ns due to mitogenic activation, whereas during apoptosis in fluorescein stained JTC, the FLT remained constant. Notably, the rotational correlation times changed in both models: decreased in PBMC from 2.5+/-0.08 to 2+/-0.1 ns, and increased in JTC from 2.1+/-0.07 to 3.3+/-0.09 ns. FLT and rotational correlation time were used to calculate the steady state fluorescence anisotropy (FA) which was compared to directly measured FA values. The present study suggests that in addition to bioindication, the said parameters can provide valuable information about cellular mechanisms that may involve complex molecular diffusion dynamics, as well as information about structural changes that a cellular fluorophore undergoes in the course of cell activation.

Anisotropy↗