Principles of Two-Photon Excitation Microscopy Photobleaching and phototoxicity, together referred to as photodamage, limit the application of fluorescence microscopy to living systems. Each excitation event carries the risk of photodamage. Optimizing fluorescence microscopy often means to minimize photodamage by maximizing the probability of detecting a signal photon per excitation event. Compared to other techniques, 2PE microscopy dramatically improves the detection of signal photons per excitation event, especially when imaging deep in highly scattering environments. In 2PE of fluorescence, two low-energy photons (typically from the same laser) cooperate to cause a higher-energy electronic transition in a fluorescent molecule. 2PE is a nonlinear process in that the absorption rate depends on the second power of the light intensity. In a focused laser, the intensity is highest in the vicinity of the focus and drops off quadratically with distance above and below. As a result, fluorophores are excited almost exclusively in a tiny diffraction-limited focal volume (Figure 1B). If the beam is focused by a high numerical aperture (NA) objective, the vast majority of fluorescence excitation occurs in a focal volume that can be as small as ∼0.1 μm3 (Zipfel et al., 2003).