We propose the development of a new instrument which builds upon established institutional strengths and combines the advantages of fluorescence lifetime measurements with those of two-photon excitation microscopy. Lifetime measurements also provide the opportunity to study the rotational mobility of a fluorescence lifetime probe using time resolved emission anisotropy (providing the lifetime of the excited state is long enough), and can also be used to gain information about the kinetics and degree of interaction of cellular constituents on a molecular scale. Two-photon excitation microscopy provides the advantages of greatly reduced photobleaching and photodamage greater detection efficiency and deeper penetration into tissue specimens. The proposed instrument will thus provide the unique advantage of generating 2- or 3-dimensional maps of the fluorescence lifetime with high spatial and temporal resolution and minimal phototoxicity. There is a pressing need for instrumentation that will allow the real time observation of the molecular activity of cells and tissues under native conditions. The proposed instrumentation will provide new views of physically distinct environments within living cells, tissue and whole embryos. The availability of such instrumentation will provide many benefits to the large number of academic, medical and industrial scientists.
