Biomedical subjects

C Eggeling

Publications and source records attributed to C Eggeling.

4 recordsLinked to original sources

PubMed2001

Data registration and selective single-molecule analysis using multi-parameter fluorescence detection.

A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.

Fluorescent Dyes↗

PubMed1998

Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence.

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

Amino Acid Substitution↗

PubMed1998

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy.

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximately 10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.

Kinetics↗

PubMedDate not supplied

The psychosocial implications of live-related kidney donation.

The aim of the study was to explore the donor's experience of the live related transplantation process whilst seeking to identify the external and internal resources and ethical considerations which were reflected in the decision-making process. The outcome would indicate that altruism and a sense of moral obligation were the prime motivational influences with the spontaneity of decision-making casting doubt on the ability to give informed consent. These findings are also discussed in relation to implications for professional practice and would suggest that potential donors benefit from having access to a counsellor during the donor-search and throughout the transplant work up programme.

Adult↗