Multi-Photon Fluorescence Microscopy.

Abstract

Multi-photon microscopy uses a different principle in exciting fluorochromes from conventional epi-fluorescence microscopy and confocal laser scanning fluorescence microscopy. In multi-photon microscopy a femto second mode locked laser is used as an illumination light source which emits near infra-red light of high peak power (over kilo watt order) and short pulse (about 100 femto seconds at FWHM) at high repetition rate (about 100 MHz). Under illumination of very high intensity by laser scanning fluorescence microscopy, a non linear effect becomes perceivable. Though near infra-red light is not usually absorbed by commonly used fluorochromes in conventional epi-fluorescence microscopy and confocal laser scanning fluorescence microscopy, intense near infra-red light is absorbed by those commonly used fluorochromes in multi-photon microscopy. This non linear interaction of near infra-red light with the fluorochromes under laser scanning microscope introduces unique benefits into the observations of biological specimens.