The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy
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- 作者：Dominik Drabik ; Magda Przybyło ; Aleksander Sikorski…
- 关键词：Fluorescence correlation spectroscopy ; Dynamic light scattering ; Photo ; bleaching ; Blinking ; Fluorescent probes
- 刊名：Journal of Fluorescence
- 出版年：2016
- 出版时间：March 2016
- 年：2016
- 卷：26
- 期：2
- 页码：661-669
- 全文大小：1,180 KB
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Biomedicine
Biomedicine
Biophysics and Biomedical Physics
Biotechnology
Biochemistry
Analytical Chemistry - 出版者：Springer Netherlands
- ISSN：1573-4994
文摘
Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.