For the first time, the time-resolved two-photon excited autophosphorescence of non labeled biological specimens was investigated by phosphoresce lifetime imaging with microscopic
spatial resolution. A modified multiphoton tomograph was employed to record both photolumi nescence contributions, autofluorescence and autophosphorescence, simultaneously, induced by
two-photon excitation using an 80 MHz near infrared femtosecond-pulse-laser scanning beam, an
acousto-optic modulator, and a time-correlated single-photon counting module for lifetime mea surements from the picosecond to the microsecond range. In particular, the two-photon-excited
luminescence of thermally altered bones was imaged. A strong dependence of the phosphores cence intensity on exposure temperature, with a maximum emission for an exposure temperature
of approximately 600 ◦C was observed. Furthermore, the phosphorescence lifetime data indicated
a bi-exponential signal decay with both a faster few µs decay time in the range of 3–10 µs and a
slower one in the range of 30–60 µs. The recording of fluorescence and phosphorescence allowed
deriving the relative signal proportion as an unbiased measure of the temperature dependence. The
measurements on thermally altered bones are of particular interest for application to forensic and
archeological investigations
