Two-Photon Excited Fluorescence Microscopy

Several laser-scanning microscopy techniques have been developed in the last twenty years based on non-linear optical phenomena.1 This has led to a variety of powerful imaging tools, such as two-photon excited fluorescence microscopy (TPM),2 second harmonic generation (SHG) microscopy,3 and coherent anti-Stokes Raman scattering (CARS) microscopy.4 The advantages these novel techniques present versus conventional microscopy methods have enabled great developments in biological imaging, especially in thick tissue and live animal studies.

The common denominator of these techniques, which can be grouped in the family of multiphoton microscopy (MPM), is the non-linear dependence of the signal with the intensity of the excitation light. Some of the consequences of this phenomenon are the localization of signal within a femtoliter volume, the reduction of photodamage in out-of-focus planes, and the ability to excite areas hundreds of microns deep within samples.