Advanced Fluorescence Correlation Techniques to Study Membrane Dynamics
Fluorescence Correlation Spectroscopy (FCS) is a powerful tool to measure important physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in solution and in membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we develop several novel implementations of FCS which overcome these limitations and facilitate accurate and quantitative determination of dynamic parameters in membranes. Two-focus FCS with camera-detection allows for accurate and calibration-free determination of diffusion coefficients. Confocal FCS using a laser scanning microscope provides an unprecedented positioning accuracy which enabled us to study, for the first time with FCS, dynamics in bacterial membranes. Scanning FCS with a scan path perpendicular to the membrane plane allows to correct for instabilities permitting long measurement times necessary to study slow diffusion. It can easily be extended to measure calibration-free diffusion coefficients with two-focus scanning FCS and to quantify binding with dual color scanning FCS. Spectral crosstalk can be avoided effectively by using alternating excitation. Using this method we were able to perform measurements in systems previously not accessible with FCS, such as yeast cell membranes or membranes of living zebrafish embryos. Line-scan FCS with a scan path in the membrane plane uses the parallel acquisition along the line to increase the statistical accuracy and decrease the measurement times. Knowledge of the scan speed serves as an internal calibration, enabling accurate diffusion and concentration measurements within seconds, hardly affected by photobleaching. Both realizations of scanning FCS can be easily implemented with commercial laser scanning microscopes. Often, a fluorescence background around the membrane cannot be avoided. The high surface selectivity needed in this case can be achieved efficiently by using a novel objective for FCS, the supercritical angle objective, which produces a very flat and laterally confined detection volume. Another technique with similar surface selectivity is FCS with total internal reflection excitation (TIRFCS). Due to the lack of a correct model, the accurate analysis of TIR-FCS data was previously not possible. In this work we develop such a model, enabling quantitative measurements of membrane dynamics with TIR-FCS. The novel FCS techniques developed here will have a high impact on the use of FCS to address key questions in biological systems, previously inaccessible by other methods.
FCS, membranes, diffusion,TIRF, Scanning-FCS, ICS, Membranen, Diffusion, TIRF
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