Biomedical subjects
Publications and source records attributed to Ingo Gregor.
We present a thorough introduction into the recently developed fluorescence lifetime correlation spectroscopy (FLCS). The theoretical basis of FLCS is explained, and the method is applied to the study of a dynamic transition between two fluorescence lifetime states in a dye-protein complex.
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.
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.
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.
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.
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.