Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry.

With the development of sensitive and specific fluorescent indicators, modern laser scanning microscopies enable visualization and measurement of submicron, dynamic processes inside living cells and tissues. Here we describe the working principles of new, nonlinear laser microscopies based on two-photon molecular excitation. In these techniques, a pulsed laser produces peak photon densities high enough that when focused into an appropriate medium, excitation by photon energy combinations can occur. For example, two red photons interacting simultaneously with a fluorescent molecule can excite within it a UV electronic transition, one corresponding to twice the energy of each single photon. Because the amount of two-photon excitation depends on the square of the local illumination intensity, this process exhibits a unique localization to the diffraction-limited spot of the beam focus. Elsewhere along the beam, excitation of background and photodamage is virtually nonexistent. Focal point localization of two-photon excitation lends to all visualization, measurement, and photopharmacology studies an intrinsic, three-dimensional resolution. We describe some preliminary biological applications, specifically, imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules.