Two-Photon Fluorescence Microscopy: Basic Principles, Advantages and Risks

The application of two-photon excitation to fluorescence microscopy has become a powerful tool for studying biological function in live tissue and offers many advantages over conventional imaging techniques. Neuroscientists in particular have used this technology to image physiological functioning in microscopic and subcellular neural compartments. Neurons can be imaged deep within highly light scattering tissue with unparalleled spatial resolution and dramatically reduced photodynamic tissue damage and fluorophore photobleaching. In this chapter we describe the basics of two-photon excited fluorescence imaging. We review the biophysical principles of fluorophore excitation by the absorption of two-photons and the operation fundamentals of the mode-locked pulsed Ti:Sapphire laser that is essential for optimal two-photon excitation. The advantages of using focal volume excitation by twophotons are outlined and compared with conventional single-photon excitation for fluorescence imaging in thick, highly scattering brain tissue. Finally, a discussion of the potential caveats of using two-photon excitation to image physiological functioning is presented.