Fluorescence correlation microscopy by two-photon excitation: investigation of biological probes at the single molecule level

Summary form only. Fluorescence correlation microscopy (FCM) is based on intensity fluorescence fluctuations occurring in very small sample volumes containing few molecules. From the analysis of the fluorescence signal autocorrelation, parameters such as molecular concentration, diffusion constant and photophysical properties can be investigated. A method to define a very small volume is based on two-photon excitation (TPE) which inherently confines the excitation volume to the focus region. We have developed an experimental method for the study of diffusion of molecules in solution and/or in cells. This method is based on FCM associated with two-photon transitions excited by a femtosecond Ti:sapphire laser.