Latest applications for 2-focus fluorescence correlation spectroscopy

T. Dertinger; I. von der Hocht; A. Loman; R. Erdmann; J. Enderlein

doi:10.1117/12.762365

21 February 2008 Latest applications for 2-focus fluorescence correlation spectroscopy

T. Dertinger, I. von der Hocht, A. Loman, R. Erdmann, J. Enderlein

Proceedings Volume 6862, Single Molecule Spectroscopy and Imaging; 686202 (2008) https://doi.org/10.1117/12.762365
Event: SPIE BiOS, 2008, San Jose, California, United States

Abstract

Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics. However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable estimation of the diffusion coefficient is no longer possible. Here, we report on the development, implementation, and application of a new and robust modification of FCS that we termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and corresponding detection regions are generated in such a way that the corresponding molecule detection functions (MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when applied to 2fFCS data analysis. Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent. Moreover, it turns out that the new technique is robust against refractive index mismatch, coverslide thickness deviations, and optical saturation effects, which so often trouble conventional FCS measurements. Additionally, we will show data that indicates that with 2fFCS it is even possible to monitor conformational changes of a calcium bindig protein affecting the hydrodynamic radius by as little as two Angstrom.

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T. Dertinger, I. von der Hocht, A. Loman, R. Erdmann, and J. Enderlein "Latest applications for 2-focus fluorescence correlation spectroscopy", Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 686202 (21 February 2008); https://doi.org/10.1117/12.762365

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KEYWORDS

Fluorescence correlation spectroscopy

Diffusion

Refractive index

Calcium

Molecules

Luminescence

Glasses

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