Fluorescence Cross-Correlation Spectroscopy (FCCS) is a powerful tool to measure interactions. In FCCS one aligns two lasers with different wavelength to the same spot and records the fluorescence of spectarlly distinct fluorophores in two detection channles. In the graphics below, the two fluorophores are depicted as blue and yellw. They will give rise to ACFs providing concentrations and diffusion coefficients for the two labels or labeled molecules. If both fluorophores reside on the same particle, e.g. due to the interaction of differently labeled moelcules, then the fluorescence signal in the two channles will be correclated. This results in a positive cross-correlation function (CCF) amplitude as shown in grey in the upper graph. If there is no interaction the CCF amplitude will be ideally zero (lower graph). The ACFs, however, still report on the concetration and diffusion coefficient of the singly labeled particles.
Note that in many cases, the CCF amplitude is non-zero even in the absence of interactions. This is due mainly to cross-talk between channels. In the case of cross-talk, some fluorescence of the shorter wavelength fluorophore (blue) will be detected oin the longer wavelength channel (yellow). This will result in a pseudo-CCF which actually more akin to an ACF as the signal from the same molecule is detected in both channels. Therefore, it is important to measure neagtive conrols in FCCS measurements to determine the lowest level of cross-correlation that can be determined. This can be avoided in pulsed interleaved excitation FCCS (PIE-FCCS) as in this case the interleaved excitation which excite either one or the other fluorophore, allows sorting photons and cross-talk can be minimized.
In a positive control, when all molecules are labeled with both fluorophores, one should obtain 100% cross-correlation and ACFs and CCFs should show the same amplitude. Again, this is very rarely achieved, as either labeling efficincy is not 100%, there are free fluorescent labelsstill in solution, or some of the labled molecules are non-fluorescent. Especially the latter is the case for many fluorescent proteins where non fluorescent moleucles can range between 20-80%. Therefore, a positive control shoudl also be determined for any FCCS measurement.
Almost 100% CCFs can be achieved in calibration measurements by splitting the emission of a single fluorophore into two channels. Then in fact, all emitting molecules emit in both channels and an almost perfcet CCF can be detected. Why only "almost perfect"? Due to different diffraction limits for the two different wavelength, the observation volumes are slightly different, leading to slightly different numbers of particles in the observation volumes and thus slightly different ACF amplitudes. Also different background signals in the two channels can influence the amplitudes differently. To see some of these effect, please go to "FCS"