Fluorescence anisotropy in the frequency domain by an optical microscope

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.