Two-Photon Two-Focus Fluorescence Correlation Spectroscopy with a Tunable Distance Between the Excitation Volumes

In the present work, a Michelson interferometer was combined with a two-photon excitation microscope to perform two-focus Fluorescence Correlation Spectroscopy. This simple and original approach allows us to tune the distance between the two excitation volumes and determine absolute diffusion constants. The technique was validated on different model systems that demonstrate the sensitivity of the approach.