Home | Contacts | FAQ’s | Maps | Cornell Biophysics | Jobs | Funding | Links | Search DRBIO

 
 

Infrastructure for Bio-Optical Investigations
Dynamic Spectroscopies

EXPERIMENTAL PRINCIPLES OF FLUORESCENCE CORRELATION SPECTROSCOPY
 
The concept of fluorescence correlation spectroscopy (FCS) was introduced in the early 1970s [Magde, D., Elson, E., and Webb, W.W. (1972) Phys. Rev. Lett. 29, 705]. Since then, the technique has been developed into a powerful tool in analytical chemistry and biological research. The elegance of FCS lies in its ability to extract a wealth of molecular and environmental information from a weak fluorescence signal that is comparable with the background noise, using correlation analysis of the fluorescence fluctuations of very small samples of molecules at nanomolar concentrations. Fluorescence fluctuations due to concentration fluctuations via molecular diffusion, chemical reactions, and physical processes of a few fluorescent molecules in a an optically restricted sub-micron observation volume (~1 fL=10-15 L) can be studied with temporal resolution typically from 1 ms to >10 s. Recent technological advances have enhanced the number of biological and chemical applications of FCS, such as binding interactions between biomolecules, sparse molecule detection, intramolecular protein dynamics, and diffusion in the membranes of living cells. For a recent review, see Hess, Huang, Heikal, and Webb, 2002, Biochemistry 41: 697.
 
 

(Left) Experimental setup for FCS. (a) A laser beam is first expanded by a telescope (L1 and L2), then focused by a high-NA objective lens (OBJ) on a fluorescent sample (S). The epi-fluorescence is collected by the same objective, reflected by a dichroic mirror (DM), focused by a tube lens (TL), filtered (F), and passed through a confocal aperture (P) onto the detector (DET). (b) Magnified focal volume (green) within which the sample particles (black circles) are illuminated. The focal volume is the distribution of laser illumination at the focus of the objective. On the other hand, the observation volume, contained within the focal volume, is the region in space where fluorescent molecules are both excited and detected.

(Right) (c) A typical fluorescence signal, as a function of time, measured for rhodamine green (RG) with excitation wavelength lx=488 nm. (d) Portion of same signal in (c), binned, with expanded time axis and average fluorescence Fbar. The signal F(t) at time t is correlated with itself at a later time (t+t) to produce the autocorrelation G(t). (e) Measured G(t) describing the fluorescence fluctuation of RG molecules due to diffusion only as observed by FCS. Assuming a Gaussian observation volume, G(t) can be least-squares fitted using various analytic functions to extract information about molecular concentration, brightness, diffusion, and chemical kinetics, for one or more diffusing fluorescent species.