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Jörg Enderlein

Publications and source records attributed to Jörg Enderlein.

18 recordsLinked to original sources
PubMed2006

Fluorescence lifetime correlation spectroscopy.

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

Journal Article↗
PubMed2006

Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements.

A recent publication reported on measurements of Exonuclease I activity using a real-time fluorescence method that measures the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. The observed fluorescence-intensity curves were interpreted as a sign of strong heterogeneity of the activity of Exonuclease I. Here, I propose a different model, which assumes that Exonuclease I activity is nucleotide-dependent, and that a fluorescent label bound to a nucleotide significantly slows its cleavage rate. The presented model fits the observed data equally well, but can be used to make specific predictions upon observable sequence dependence of measured fluorescence-intensity curves.

DNA, Single-Stranded↗
PubMed2006

Surface sticking and lateral diffusion of lipids in supported bilayers.

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes.

Diffusion↗
PubMed2005

Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration.

Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.

Diffusion↗
PubMed2005

Focusing astigmatic Gaussian beams through optical systems with a high numerical aperture.

We theoretically derive the electric field distribution of an astigmatic Gaussian laser beam after it is focused through a high-aperture objective. We show that astigmatism values that are hard to detect in the collimated laser beam can have a large effect after diffraction-limited focusing. Such astigmatic beams may be frequently encountered in fluorescence correlation measurements and in laser-scanning confocal microscopy. We present experimental measurements of the excitation intensity distribution measured by 3D scanning of single fluorescent molecules immobilized on a glass surface.

Journal Article↗
PubMed2005

Cell-transistor coupling: investigation of potassium currents recorded with p- and n-channel FETs.

Microelectronic-based biosensors that allow noninvasive measurement of cell activity are in the focus of current developments, however, the mechanisms underlying the cell-transistor coupling are not completely understood. In particular, characteristic properties of the extracellular voltage response such as the waveform and amplitude are not satisfactorily described by electrical circuit models. Here we examine the electrical coupling between a nonmetallized field-effect transistor (FET) and a cell line expressing a voltage-gated EAG K+ channel. The activation kinetics of this channel depends on the voltage pulse protocol and extracellular divalent cations. This feature allows testing, whether the extracellular voltage signal recorded with the FET faithfully tracks the current simultaneously recorded with the patch-clamp technique. We find that the FET signals contain different kinetic components that cannot be entirely explained by equivalent electrical-circuit models. Rather, we suggest that changes in ion concentration in the small cleft between cell and FET may change the surface potential of the FET. This study provides evidence that the electrochemical processes at the cell-transistor interface are complex and that at least two different mechanisms contribute to the shape and amplitude of transistor signals.

Action Potentials↗
PubMed2005

Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation.

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.

Journal Article↗
PubMed2005

Statistical analysis of diffusion coefficient determination by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.

Journal Article↗
PubMed2004

Fluorescence lifetimes and emission patterns probe the 3D orientation of the emitting chromophore in a multichromophoric system.

In this Communication, we report on the fluorescence behavior of an individual first generation multichromophoric dendrimer. The fact that each of the chromophores in time acts as a fluorescent trap is demonstrated by directly probing the dipole orientation of the emitting chromophore by means of defocused wide-field imaging and comparing experimental and calculated emission patterns. It is shown that in such cases the electromagnetic boundary condition effect results in discrete changes in the fluorescence lifetime as a function of time for individual dendrimers.

Journal Article↗
PubMed2004

Unified operator approach for deriving Hermite-Gaussian and Laguerre-Gaussian laser modes.

A unified operator approach is described for deriving Hermite-Gaussian and Laguerre-Gaussian laser beams by using as a starting point a plane-wave-spectrum representation of the electromagnetic field. We show that by using the plane-wave representation of the fundamental Gaussian mode as a seed function, all higher-order beam modes can be derived by acting with differential operators on this fundamental solution. The approach presented can be easily generalized to nonparaxial situations and to include vector effects of the electromagnetic field.

Journal Article↗
PubMed2004

Art and artefacts of fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an important technique for studying low concentrations of analyte molecules in solution. The core molecular characteristic that can be addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for i.e. studying molecular binding and reactions, or conformational changes of macromolecules. The present paper discusses several possible optical and photophysical effects that can influence the outcome of a FCS measurement and thus can bias the value of the derived diffusion coefficient.

Artifacts↗
PubMed2003

Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology.

This review presents an overview of the fluorescence detection and spectroscopy of single molecules (SMS) in liquids and on surfaces under ambient conditions. The various techniques of SMS, such as confocal epifluorescence detection and wide-field imaging are presented and discussed, together with the different methods of data analysis such as fluorescence correlation spectroscopy and burst-by-burst analysis. Selected applications of the various techniques in physics, chemistry, and biology are described.

Biophysical Phenomena↗
PubMed2003

Influence of interface-dipole interactions on the efficiency of fluorescence light collection near surfaces.

Recently Török and colleagues published in a series of papers a general wave-optical approach to calculating the light-collection efficiency function (CEF) for confocal imaging of dipole emitters [J. Mod. Opt. 45, 1681 (1998); J. Microec. 194, 127 (1999); Opt. Lett. 25, 1463 (2000)]. In their theory they did not address the possibility that the lifetimes of fluorescing molecules can change significantly near interfaces, which has a direct effect on the CEF. The research of Török and colleagues is extended here to include this effect, which may become important for imaging near surfaces.

Journal Article↗
PubMed2003

Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level.

We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

Fluorescent Dyes↗
PubMed2003

Time-resolved luminescence imaging of hydrogen peroxide using sensor membranes in a microwell format.

We demonstrate an optical imaging scheme for hydrogen peroxide in a microwell-based format using the europium(III) tetracycline complex as the fluorescent probe, which is incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix. The resulting sensor membranes are integrated into a 96-microwell plate. Hydrogen peroxide can be visualized by means of time-resolved luminescence lifetime imaging. The imaging system consists of a fast, gated charge-coupled device (CCD) camera and a pulsed array of 96 light emitting diodes (LEDs). Fluorescence lifetime images are acquired in different modes (rapid lifetime determination, RLD, and phase delay rationing, PDR) and compared with conventional intensity-based methods with respect to sensitivity and the dynamic range of the sensor. The lowest limits of detection can be achieved by the RLD method. The response time of the sensor is comparatively high, typically in the range of 10 to 20 minutes, but the response is reversible. The largest signal changes are observed at pH values between 6.5 and 7.5.

Adsorption↗
PubMed2003

Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA.

Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC DNA and 1.8 ns in poly-AT DNA. The distribution of excited state lifetimes of single molecules of TOTO intercalated into oligonucleotides having varying proportions of poly-GC sequences relative to poly-AT sequences were analyzed as a function of the fraction of poly-GC. By using excited state lifetime distributions from the purely GC and purely AT oligonucleotides as a basis set, it was possible to estimate the GC content of oligonucleotides with intermediate GC composition to within a few percent error. This serves as a model for the analysis of equilibrium binding distributions in DNA using single-molecule methods.

Base Sequence↗