Biomedical subjects

Suren Felekyan

Publications and source records attributed to Suren Felekyan.

5 recordsLinked to original sources

PubMed2006

Monitoring dynamic systems with multiparameter fluorescence imaging.

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

Algorithms↗

PubMed2006

Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis.

We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the Förster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the Förster radius are ruled out.

DNA↗

PubMed2006

Analysis of photobleaching in single-molecule multicolor excitation and Förster resonance energy transfer measurements.

Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.

Carbocyanines↗

PubMed2005

Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin.

The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association rate constants are very similar for both guests and among the highest reported for this type of systems, although much lower than the diffusion-controlled collision rate constant. A two-step model including the formation of an encounter complex allows one to identify the unimolecular inclusion reaction as the rate-limiting step. Simulations indicate that this step may be controlled by geometrical and orientational requirements. These depend on critical molecular dimensions which are only weakly affected by the different alkyl substituents of PY and PB. Diffusion coefficients of PY and PB, of their complexes, and of rhodamine 110 are given and compared to those of similar molecules.

Coloring Agents↗

PubMed2004

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Adenosine Triphosphate↗