Since its inception more than 15 years ago, two-photon laser scanning microscopy (2PLSM) has found widespread use in biological and medical research. Two-photon microscopy is based on simultaneous absorption of two photons by fluorophores and subsequent fluorescence emission, a process which under normal illumination conditions is highly improbable. Theoretically described around 1930 by Maria Göppert-Mayer , the first experimental demonstration of two-photon excitation had to await the invention of the laser, which produced sufficiently high light intensities to observe two-photon absorption events . Only after the development of ultrafast lasers providing subpicosecond light pulses with high peak power intensities, however, two-photon-excited fluorescence became practical in a laser-scanning microscope . Since then 2PLSM has developed into the method of choice for high-resolution imaging in living animals (reviewed in [4,5]). One of the main reasons is the low sensitivity of 2PLSM to light scattering, which enables imaging relatively deep inside biological tissue and direct observation of the dynamic behavior of cells in their native environment. In this chapter, we introduce the physical principles governing 2PLSM and briefly describe the key instrument components. We give an overview of fluorescence labeling techniques and how they are combined with 2PLSM for functional imaging and photomanipulation in living tissue. Finally, we discuss limitations and provide some future perspectives.