Recent advancements in photomultiplier tubes and detectors, along with improved filter contrast and transmission, have allowed what was once impossible: detection and analysis of spontaneous movements on the nanometer level using fluorescence correlation spectroscopy (FCS). Here, the basic techniques of FCS are discussed along with several novel applications, such as nanoparticle dispersion studies. Insights into solving potential problems, such as photobleaching, also are provided.

Fluorescence correlation spectroscopy (FCS) is an extremely powerful, versatile technique that is useful both in vivo and in vitro for biochemistry and biophysics. Using FCS, one can determine and obtain a great deal of quantitative information, such as diffusion coefficients, hydrodynamic radii, kinetic chemical reaction rates, photophysical processes, and a wide variety of internal and external dynamic characteristics of biological molecules. These measurements can be done quickly and easily by modifying any standard fluorescence or confocal microscope. Here, we cover the important details of FCS in a few specific examples as well as focus on the material side and see how improved filters and detectors have resurrected this once popular technique.

How Does the Technique Work?

The technique of fluorescence correlation spectroscopy, in conjunction with confocal or two-photon excitation microscopy, begins, as most fluorescent microscopy systems do, with focusing light on a sample. The sample's emission is measured over time and the fluorescence intensity fluctuations are analyzed using what is called temporal autocorrelation (1). Temporal autocorrelation stems from spatial dependency and analysis of demographics or geography and is a very useful method of measurement and analysis when working with small fluctuations in a known sample size. Essentially, it refers to the association between time-shifted values given a voxel time series, and those particular signal values at present time are independent of past and future signals (2). Temporal autocorrelation is important to consider because cellular and subcellular activity is completely random and minute. Analysis of the signal during a short time domain should assist in improving the understanding of intracellular activity. The intensity fluctuations vary depending on the sample under inspection. This is crucial because the measured fluorescence is directly related to the intensity of the entity's overall fluctuations. In this technique, it is very important to ensure that the number of molecules remains constant during inspection; no samples should enter or leave the observation volume. Additionally, when the threshold of units is exceeded, fluctuations in the observation volume become undetectable. This threshold is not a specific value but rather an observational determinant. If the inspection region is saturated with noise and light, or if there is no noticeable flux, then the threshold more than likely has been crossed. A consistent concentration ratio and observation volume of a variety of specimens allows researchers to conduct experiments in a wide variety of fields, such as materials science and biology. This is not the easiest of tasks but as the saying goes, "practice makes perfect."