The invention pertains to a method of obtaining structural and functional information on proteins, based on polarization
fluorescence microscopy, which comprises subjecting a
protein tagged with a
fluorophore to two- or multi-
photon fluorescence microscopy, whereas the observed
protein is irradiated with a
laser beam with light of at least two different polarizations, which excites the
fluorescence of the
fluorophore, and wherein information on localization, intensities and polarizations of the fluorescence excited by the different polarizations of the excitation
laser beam is used to identify, localize and quantify
anisotropy of absorption and / or fluorescence, which information is then used to infer structural and functional properties of proteins. An example of a device for obtaining structural and functional information on proteins, based on polarization fluorescence
microscopy, comprises a modulator (P) for rapid modulation of the
excitation beam (1) for eliciting two- or multi-
photon fluorescence, and a
control unit (R), wherein the function of the modulator (P) and
control unit (R) is synchronized with scanning of the
microscope (M) such that information on
fluorescence intensity acquired by the
microscope (M) is attributable to a particular polarization state of the
excitation beam (1) by virtue of knowing the temporal profile of the
polarization modulation of the
excitation beam (1) effected by the modulator (P). The method and device of the invention allow determining and monitoring
structure and function of proteins, such as membrane proteins, and thereby observing physiological processes in living cells.