Resolution of fluorescence correlation measurements

The resolution limit of fluorescence correlation spectroscopy for two-compo nent solutions is investigated theoretically and experimentally. The autoco rrelation function for two different particles in solution were computed, s tatistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish betwee n two different molecular species in solution depends strongly on the numbe r of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components , their diffusion times must differ by at least a factor of 1.6 for compara ble quantum yields and a high fluorescence signal. Experiments were conduct ed with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experi mental results support the simulations. In addition, they show that even wi th a high fluorescence signal but significantly different quantum yields, t he diffusion times must differ by a factor much bigger than 1.6 to distingu ish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant compo nent below which it is not possible to determine with statistical means alo ne that two particles are in solution.