# High-Resolution Phosphorescence Lifetime Imaging (PLIM) of Bones
## 1 Introduction  
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]. 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. In particular, 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 [PERSON] et al. [1, 3]. In Ref. [3], the authors have quantified the phosphorescence intensity by visual inspection and observer assessment through a scoring index scheme. The authors found the visibility observable phosphorescence intensity to depend strongly on the maximum exposure temperature, with the highest emissions observed in the range from 450 \({}^{\circ}\)C to 800 \({}^{\circ}\)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). Recent studies have suggested, however, that inorganic material of the bone generates the temperature-dependent phosphorescence, in particular, the main inorganic component hydroxyapatite [1, 5]. The detailed microscopic origins and the components contributing tophosphorescence in particular in the range of 700 \({}^{\circ}\)C to 800 \({}^{\circ}\)C are not fully understood and for lower exposure temperatures even contradicting findings on the role of organic and inorganic components have been reported [3]. So far mainly spectroscopy methods, which have been based on continuous excitation with UV or blue light, have been used to investigate the bone photoluminescence. [PERSON] et al. suggested to distinguish between \"short-decay\" and \"long-decay\" phosphorescence with the short-decay phosphoresce being the part which is \"visibly not observable\" but has a longer decay time than fluorescence [3]. In life sciences, phosphorescence lifetime microscopy (PLIM) has been used so far mainly for studies in cell biology to investigate the oxygen consumption of single cells during the cell metabolism by recording phosphorescence from exogenous markers (metal complexes) [6, 7]. The marker phosphorescence emission can be affected by the system under study, e.g., a cell, and thereby indirectly indicates the sample condition. The technique can be combined with fluorescence lifetime imaging (FLIM) [8, 9, 10]. The novelty of this study lies in the measurement of autophosphorescence, an intrinsic sample property, in contrast to measuring phosphorescence from an externally added marker. Autophosphorescence is usually very weak or absent in biological samples but can be considered as a \"direct signal\" from the specimen. To the best of our knowledge the autophosphorescence of biological specimens has not been studied (i) with high-resolution microscopy/tomography, (ii) with two-photon excitation, and (iii) with precise time resolution. In this paper, we have used high-resolution femtosecond laser scanning imaging in combination with time-correlated single-photon counting (TCSPC, [9]) to investigate the spatial and temporal distributions of the two-photon excited autophosphorescence and autofluorescence emission from bones without any additional labeling with phosphorescent markers. The measurements provided the microscopic distribution of autophosphorescent substances in heated bone samples within a temporal measurement range of tens of microseconds as well as the simultaneous measurement of fast decaying fluorescence in the picosecond/nanosecond range.