High-Resolution Phosphorescence Lifetime Imaging (PLIM) of Bones

Hans Georg Breunig,Karsten König

doi:10.3390/app12031066

Abstract

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 photoluminescence 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 measurements 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 phosphorescence 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.

Highlights

IntroductionThe photoluminescence emission, in particular, of heated bones has been found to be affected by the exposure temperature [1,2,3]
Luminescence examinations of bone samples can provide valuable forensic information and archeological insight into the environmental conditions a find was exposed to.Recently, the photoluminescence emission, in particular, of heated bones has been found to be affected by the exposure temperature [1,2,3]
The study of the phosphorescence could aid in the search of human skeletal remains or indicate the maximum temperature a bone has been exposed to, for example during a previous fire as has been proposed by Krap et al [1,3]

Summary

Introduction

The photoluminescence emission, in particular, of heated bones has been found to be affected by the exposure temperature [1,2,3]. Photoluminescence consists of both quickly decaying fluorescence with lifetimes in the picosecond and nanosecond range and longer prevailing phosphoresce with lifetimes in the microsecond to minute range. The authors found the visibly observable phosphorescence intensity to depend strongly on the maximum exposure temperature, with the highest emissions observed in the range from 450 ◦ C to 800 ◦ C, and to a much lesser degree to depend on exposure duration. Photoluminescence of bones has been observed before [4] and originally assumed to stem mainly from organic substances like collagen, which is known to fluoresce but does not phosphoresce, and to a lower degree to bone minerals (e.g., apatite)

Methods

Discussion

Conclusion