Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

Abstract

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5-2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.

Details

OriginalspracheEnglisch
Seiten (von - bis)1437-1448
Seitenumfang12
FachzeitschriftBiophysical journal
Jahrgang94
Ausgabenummer4
PublikationsstatusVeröffentlicht - 15 Feb. 2008
Peer-Review-StatusJa

Externe IDs

PubMed 17933881

Schlagworte

ASJC Scopus Sachgebiete