# High-Resolution Phosphorescence Lifetime Imaging (PLIM) of Bones
## 3 Results and Discussion  
A photographic image of the bones samples after the heating which feature typical heat-induced color changes [16] is shown in Figure 2a. The color ranges from black (400 \({}^{\circ}\)C) to brown and grey and completely white (900 \({}^{\circ}\)C). The color changes of bones as a result of temperature are well-known and caused by changes of the chemical composition of the organic components [16]. Figure 2b shows microscopic images which were derived from the pixelwise-collected photon counts during \"laser on\" and \"laser off\" times. The laser \"on-time\" images reflect the fluorescence intensity distribution (with negligible phosphorescence contributions) and the \"laser-off times\" reflect the phosphorescence signal. Microscopic fine structures of the bone samples can be recognized in both the fluorescence and phosphorescence signals, although, the fluoresce intensity is up to two orders of magnitude higher. For example, the total photon counts  
Figure 2: (**a**) Photographs of chicken bone samples after being exposed to maximum temperatures between 400 \({}^{\circ}\)C and 900 \({}^{\circ}\)C. (**b**) Microscopic photoluminescence images of the different samples. The brightness reflects the intensity of fluorescence (laser on) and phosphorescence (laser off), respectively. Yellow and green pseudo colors were applied to increase the image clarity. were 233 \(\times\) 10\({}^{6}\) and 4 \(\times\) 10\({}^{6}\) for fluorescence and phosphorescence in the 600 \({}^{\circ}\)C images, respectively. For both, the 400 \({}^{\circ}\)C- and the 900 \({}^{\circ}\)C-heated samples, only fluorescence was detectable. The ratio of the total phosphoresce to fluorescence intensities for the different maximum exposure temperatures is shown in Figure 3.  
The figure indicates a clear dependence of the intensity ratio on the exposure temperature. The bone sample which had been subjected to 400 \({}^{\circ}\)C hardly phosphorescents. With increasing exposure temperature, the ratio significantly increases up to a maximum of 600 \({}^{\circ}\)C from where it drops to 700 \({}^{\circ}\)C and 800 \({}^{\circ}\)C until at 900 \({}^{\circ}\)C the phosphorescence vanishes again. This general trend has been observed with all measured samples, although, different regions of a sample do exhibit varying phosphorescence and fluorescence emission intensities. The temperature dependence of the data in Figure 3 is in line with the trend described by [PERSON] et al. for the phosphoresce intensity scores [3]. The phosphoresce-to-fluorescence ratio is an easy to obtain and more unbiased measure of the phosphorescence, whose absolute intensity is affected by the exact excitation and focusing conditions, without subjective observer evaluation. However, it should be noted that the described temperature dependence of the ratio does not rule out individual temperature dependencies of both the fluorescence as well as the phosphorescence. In fact, our data seems to indicate that absolute intensity values cannot be directly compared for the different samples since they depend on the region of interest and other imaging conditions. With our measurement setup, the phosphorescence decay can also be analyzed pixelwise. This phosphorescence lifetime microscopy (PLIM) is the phosphorescence analogue of the better-known fluorescent lifetime microscopy (FLIM) [9; 10; 15]: the time-dependent data are pixelwise color-coded according the to fit parameters. Usually, the day data are fitted by a mono or bi-exponential decay curve. We have used a bi-exponential fit curve for the decay time tmean = a1 \(\times\) t1 + a2 \(\times\) t2 with amplitudes a1,2 (a1 + a2 = 100%) of the short and long decay times t1,2 as fit parameters [9]. For the sample that was exposed  
Figure 3: Ratio of phosphorescence to fluorescence intensities derived from Figure 2b. The line is a guide to the eye only.