Abstract

Two-photon excitation provides attractive advantages over confocal microscopy for three-dimensional fluorescence imaging and photochemistry, such as release of caged compounds. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. The most important property of two-photon excitation is that the excitation is limited to the focal volume because of the intensity-squared dependence of the two-photon absorption. This inherent localization provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter. Confinement of excitation to the focal volume also minimizes photobleaching and photodamage, and allows for three-dimensionally resolved point photochemistry. Cellular biophysics applications include three-dimensionally resolved, dynamic quantification of intracellular calcium activity by Indo-1, redox imaging of cell autofluorescence arising from NADH, and imaging of nuclear division by Hoechst 33342. The pulsed nature of mode- locked excitation has also been utilized to efficiently realize fluorescence decay time imaging. Finally, point photorelease has been used to marked cells in developing embryos, map receptor distributions in neural cells, and trigger local calcium release in muscle cells.

© 1995 Optical Society of America