Jörg Enderlein
fluorescenCE correlation spectroscopy: BASICS AND APPLICATIONS
Several decades ago, Magde, Elson and Webb invented an ingenuous method for measuring diffusion coefficients of fluorescent molecules: Fluorescence Correlation Spectroscopy (FCS) [1]. In FCS, the fluctuations of a fluorescence signal which is generated in a femtoliter-sized volume of a sample solution is analyzed by a correlation analysis. This analysis can then be used for extracting information about molecular diffusion, intramolecular conformational dynamics, intermolecular interactions, or photophysical processes. The presentation will give a comprehensive introduction into and overview on the method of FCS and its various applications.
In particular, I will discuss in detail a recently developed modification of conventional FCS, which is called dual-focus FCS (2fFCS) [2], that allows for very precise absolute sizing of molecules at pico- to nanomolar concentrations [3]. The basic set-up of a 2fFCS system is shown in the following figure.
Figure: Schematic of a 2fFCS setup. Excitation is done by two interleaved pulsed lasers of the same wavelength. The polarization of each laser is linear but orthogonal to each other. Light is then combined by a polarizing beam splitter and coupled into a polarization maintaining optical single-mode fiber. After exiting the fiber, the laser light is collimated by an appropriate lens and reflected by a dichroic beam splitter through a differential interference contrast prism. The DIC-prism separates the laser light into two beams according to the polarization of the incoming laser pulses. The microscope objective focuses the two beams into two laterally shifted foci of close-to-diffraction limited size. Fluorescence is collected by the same objective. A tube lens focuses the detected fluorescence from both excitation foci through a confocal pinhole for background supression. Subsequently, the fluorescence light is split by a 50/50 beam splitter and detected by two single photon avalanche diodes.
In the presentation, the method is described in detail, and manifold applications of 2fFCS will be given. Among them are: Precise sizing proteins and peptides, monitoring conformational changes of proteins, monitoring protein-protein interactions, measuring diffusion of transmembrane proteins in lipid bilayers [4], or measuring flow profiles in microchips [5]. Besides the use of 2fFCS for sizing biomolecules at pico- to nanomolar concentrations, FCS is also a powerful tool for looking at the intrinsic conformational flexibility of macromolecules [6], for elucidating the stoichiometry of macromolecular complexes [7], or watching molecular rotation [8].
The last part of the presentation will be used to discuss one particular variant of FCS: fluorescence-lifetime correlation spectroscopy or FLCS, which allows for correlating fluorescence signals in a lifetime-specific manner [9]. FLCS is extremely useful for monitoring binding interactions close to interfaces, or to monitor macromolecular conformational changes that induce fluorescence lifetime changes and can thus be another valuable tool in bio-analysis and diagnostics.
References
1. Magde, D., E. Elson, and W.W. Webb, Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett., 1972. 29: p. 705-708.
2. Dertinger, T., et al., Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements. ChemPhysChem, 2007. 8: p. 433-443.
3. Dertinger, T., et al., The optics and performance of dual-focus fluorescence correlation spectroscopy. Opt. Express, 2008. 16: p. 14535-14368.
4. Kriegsmann, J., et al., Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual-Focus FCS. ChemBioChem, 2009. 10(11): p. 1823-1829.
5. Arbour, T. and J. Enderlein, Application of dual-focus fluorescence correlation spectroscopy to microfluidic flow-velocity measurement. Lab on a Chip, 2010. 10: p. 1286-1292.
6. Neuweiler, H., et al., Measurement of Submicrosecond Intramolecular Contact Formation in Peptides at the Single-Molecule Level. J. Am. Chem. Soc., 2003. 125: p. 5324-5330.
7. Sýkora, J., et al., Exploring fluorescence antibunching in solution for determining the stoichiometry of molecular complexes. Anal. Chem., 2007. 79: p. 4040-4049.
8. Loman, A., et al., Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy. Photochem. Photobiol. Sci., 2010. 9: p. 627-636.
9. Gregor, I. and J. Enderlein, Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy. Photochem. Photobiol. Sci., 2007. 6: p. 13-18.
