Two-photon excited fluorescence microscopy using spectroscopic detection techniques

Abstract

Fluorescence microscopy in combination with spectroscopic detection is a powerful tool for simultaneously studying several independent fluorophores in a sample. In medicine, such a tool is interesting for studies of the localisation of various photosensitisers in the development of photodynamic therapy. In these studies one needs to be able to measure the weak signal from a sensitiser without any interference from the tissue autofluorescence. Also, it is interesting to measure the fluorescence emission characteristics as they shift with the local environment of the sensitiser. Two-photon excited scanning fluorescence microscopy is interesting for such studies, due to its simplicity and straight forward compatibility with temporally or spectrally resolved detection techniques. An advantage of two-photon excited fluorescence microscopy, compared with conventional fluorescence microscopy is that the excitation is made only in the small volume defined by the focus of the laser beam. This directly gives a high three-dimensional spatial resolution.