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Biophys J. 1999 Mar;76(3):1619-31.

Resolution of fluorescence correlation measurements.

Author information

1
Department of Chemistry, LCPPM, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland.

Abstract

The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number 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 comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.

PMID:
10049342
PMCID:
PMC1300138
DOI:
10.1016/S0006-3495(99)77321-2
[Indexed for MEDLINE]
Free PMC Article

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