The typical time-scales of photo-physical processes in fluorophores are in the order of nano- to microseconds.
A powerful, sensitive yet relatively simple technique is fluorescence correlation spectroscopy (FCS). This method allows for studying any dynamic process that modulates the fluorescence intensity measured within the femtoliter-sized detection volume of a confocal microscope. Mostly, FCS is applied for measuring diffusion properties but it can also be used for determining photophysical transitions, To evaluate such measurements one needs to know the excitation rate throughout the detection volume. Absolute excitation rates of fluorescent molecules can be obtained from fluorescence antibunching measurements Here, we combine fluorescence antibunching with FCS on the same experimental setup to measure intersystem crossing rates and triplet state lifetimes of Rhodamine 110 and ATTO 655 with errors less than 5%.
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