Caged bioactive probes provide a unique set of tools for the experimental biologist. A wide range of bioactive molecules such as second messengers or neurotransmitters are now available with conjugated caging groups. These caging groups render the molecule inert until the cage opened by photolysis, usually achieved by the localized application of an intense source of UV light. Using this technique it is possible to precisely control in space and time the application of an experimentaly applied signal molecule.

Multiphoton excitation may be used for uncaging (Williams et al. 1994, FASEB J 8(11):804). The unique feature of multiphoton uncaging is that it can provide true 3D localization of excitation in volumes down to one cubic micron within cells or tissue. This would make this technique an apparently ideal way of releasing caged probes within cells or tissues. Although there have been some demonstrated successes in this area (Denk, 1994 PNAS 91(14), 6629), it is probably fair to say that the promise of multiphoton uncaging has not yet been fully realized. There are probably two main reasons for this situation:

• The commonly-used nitrobenzyl caging group has a low cross-section for interaction with light around 700nm (close to the shortest wavelength to which the commonly used Ti:sapphire lasers can be tuned). The one-photon action spectra of nitrobenzyl indicates that shorter wavelength two-photon excitation would be better.
• The combination of the small volume where uncaging occurs (i.e. the focal volume of the objective) with a slow uncaging reaction means that diffusion will severely limit the concentration of uncaged probe that can be attained.

We believe that, given further development, that multiphoton uncaging will become a very powerful experimental tool.