Figure 1.

Comparison of single- and two-photon excitation. The objective lens of a fluorescence microscope focuses the excitation light to a spot. The cartoon of this process (left) illustrates how the photons become increasingly concentrated as they approach the focal plane. The colored circles show that along the excitation path the flux per unit area increases while maintaining the number of photons in each z section. This is true of both single-photon (blue circles, middle) and two-photon excitation (red circles, right). The linear nature of one-photon excitation thus creates the same number of excited states in each z section (green circles, middle). In contrast, the nonlinear nature of two-photon excitation produces punctate emission in the focal plane (green and white circles, right). The photographs show the emission profiles from single- and two-photon excitation of a cuvette solution of a fluorophore.