Resolution of fluorescence correlation measurements

The resoln. limit of fluorescence correlation spectroscopy for two-component solns. is investigated theor. and exptl. The autocorrelation function for two different particles in soln. 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 mol. species in soln. depends strongly on the no. of photons detected from each particle, their difference in size, and the concn. of each component in soln. 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. Expts. were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The exptl. results support the simulations. In addn., 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 concn. threshold for the less abundant component below which it is not possible to det. with statistical means alone that two particles are in soln. [on SciFinder (R)]

Published in:
Biophysical Journal, 76, 3, 1619-1631

 Record created 2006-02-27, last modified 2018-03-17

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