Amplitude of Relaxations in Fluorescence Correlation Spectroscopy for Fluorophores That Diffuse Together

The amplitude of chemical relaxations in fluorescence correlation spectroscopy (FCS) is an important parameter that directly relates to not only the equilibrium constant of the relaxations but also the number of individual fluorophores that diffuse together. In this Letter we answer the question how exactly the amplitude of the relaxations in FCS changes with respect to the number of identical fluorophores on one cargo. We anchored tetramethylrhodamine molecules onto each arm of a DNA Holliday junction molecule so that the codiffusing dyes were capable of performing independent fluorescent fluctuations. We found that the amplitudes of the relaxations were inversely proportional to the number of the dyes on each cargo molecule, well agreeing with the theoretical prediction derived in this Letter. The result provides a guideline for the FCS data analysis and points out a simple way to determine the number of molecules that a cargo carries.