Track topics on Twitter Track topics that are important to you
Inspired by recent publications doubtful of the FCS technique, we scrutinize how irreversible ("static") and reversible ("dynamic") quenching can influence the interpretation of such data. Textbook presentations often emphasize only how to analyze data in extremes, the absence of quenching or the presence of substantial quenching. Here, we consider intermediate cases where the assessment of photophysics (static quenching, blinking-like triplet-state relaxation) influence on autocorrelation curves can be delicate if dye-labeled objects diffuse on comparably rapid time scales. We used the amino acid, tryptophan, as the quencher. As our example of small-molecule dye that diffuses rapidly, we mix the quencher with the fluorescence dye, Alexa 488. The translational diffusion coefficient, inferred from fit to the standard one-component Fickian diffusion model, speeds up without the loss of quality of fit, but quenching is reflected in the fact that the data become exceptionally noisy. This reflects the bidisperse population of quenched and unquenched dyes when the time scales overlap between the processes of translational diffusion, quenching, and blinking. As our example of the large-molecule dye-labeled object that diffuses relatively slowly, we mixed the quencher with dye-labeled BSA, bovine serum albumin. Diffusion, static quenching, and blinking time scales are now separated. In spite of quenching contribution to the autocorrelation function when the delay time is relatively short, the inferred translational diffusion coefficient now depends weakly on the presence of a quencher. We conclude that when the diffusing molecule is substantially slower to diffuse than the time scale of photophysical processes of the fluorescent dye to which it is attached, the influence of quenching is self-evident and the FCS autocorrelation curves give an appropriate diffusion coefficient if correct fitting functions are chosen in the analysis.
This article was published in the following journal.
Name: The journal of physical chemistry. A
In biological fluids, nanoparticles (NPs) are in contact with proteins and other biomolecules. Proteins adsorb to NPs and form a coating called a protein corona (PC). The PC is known to greatly affect...
Fluorescence encoded infrared (FEIR) spectroscopy is an ultrafast technique that uses a visible pulse to up-convert information about IR-driven vibrations into a fluorescent electronic population. Her...
This study attempted to develop a probe-based fluorescence technology as a rapid method to discriminate the oxidation degree of oil. The fluorescence probe was made by dissolving the selected probe in...
Among the innumerable possibilities of use of technologies that employ fluorescence spectroscopy in the field of health, their applicability is focused on the diagnosis and control of infectious proce...
Elimination of fluorescence reabsorption effects is necessary to obtain reliable kinetic data in fluorescence spectroscopy. This effect must also be considered in transient absorption spectroscopy. We...
Intraoperative surgical fluorescence microscopy is a useful technique for the surgical resection of glioma. However the accuracy of this method is limited by its too low sensitivity. Fluo...
RATIONALE: Diagnostic procedures, such as laser spectroscopy, may help find and diagnose breast cancer. PURPOSE: This phase I trial is studying laser spectroscopy to see how well it works...
The goal of this clinical research study is to evaluate fluorescence imaging, widefield fluorescence imaging, point spectroscopy imaging methods, and or/ oral brush cytology that may help ...
The overall objective of this study is to identify potential improvements for a noninvasive method of diagnosing dysplasia and neoplasia in the cervix using digital colposcopy for colposco...
RATIONALE: New diagnostic procedures such as fluorescence and reflectance spectroscopy (shining light on tissue and measuring patterns of light reflected) may improve the ability to noninv...
A type of FLUORESCENCE SPECTROSCOPY using two FLUORESCENT DYES with overlapping emission and absorption spectra, which is used to indicate proximity of labeled molecules. This technique is useful for studying interactions of molecules and PROTEIN FOLDING.
Spectroscopy technique which measures changes in organic compounds by tracking the spectral energy of absorption of HYDROGEN atoms.
Measurement of the intensity and quality of fluorescence.
An analytical method for detecting and measuring FLUORESCENCE in compounds or targets such as cells, proteins, or nucleotides, or targets previously labeled with FLUORESCENCE AGENTS.
The simultaneous identification of all chromosomes from a cell by fluorescence in situ hybridization (IN SITU HYBRIDIZATION, FLUORESCENCE) with chromosome-specific florescent probes that are discerned by their different emission spectra.