# High-Resolution Phosphorescence Lifetime Imaging (PLIM) of Bones
## 2 Materials and Methods  
### Imaging System  
The functionality of a commercial multiphoton tomograph (MPT_flex_-CARS [10, 11, 12, 13]), JenLab, Germany) was extended by including phosphorescence lifetime imaging capability (Figure 1a). The tomograph enables, in its original configuration, in vivo and ex vivo multi-modal multiphoton microscopy including fluorescence, second harmonic generation (SHG), and coherent anti-Stokes Raman scattering microscopy (CARS) as well as fluorescence lifetime imaging (FLIM) [9, 10]. The mobile system contains a tunable 80 MHz femtosecond laser (MaiTai HP-JL, Newport Spectra Physics), that generates ultrashort pulses for two-photon excitation. The tomograph is outfitted with a TCSPC module (SPC-150, Becker& Hickl, Germany) in combination with a photo multiplier module (PMT) with a fast response time [9]. Imaging is achieved with a laser scanning setup, which is integrated inside a flexibly positionable scan-detector unit, i.e., by scanning-focused laser pulses pixelwise across a sample region through deflection by a pair of galvo-scanner mirrors [14, 15]. The laser power that reaches the sample can be regulated with an acoustic optical modulator (AOM, AA opto-electronic, France). All signals are collected in reflection geometry and can be detected in up to four spectral channels equipped with PMTs [12]. For the imaging of the bone samples, the flexible measuring head was set vertically such that the laser light was emitted upwards and an inverse microscope setup was realized. The samples were put on a cover slip and imaged from below with a high-NA objective (1.35 NA-40x Uapo; Olympus) which focused the incoming laser pulses and collected signals in reflection geometry. After the signal-laser light separation with a beamsplitter (LP 705, Semrock, Rochester, NY, USA) and the blocking of residual laser light by a short pass filter (SP680, Semrock, Rochester, NY, USA), all signals were detected in a one spectral channel within the range of 350 to 680 nm by a PMT (R9880-20, Hamamatsu, Japan). For better index of refraction matching, the samples were placed in a drop of water. All measurements were performed at room temperature. The functionality to record in addition phosphorescence was achieved by adding a delay generator card (DDG-210, Becker & Hickel, Berlin, Germany) which, in combination with the AOM, allowed to limit the laser irradiation duration for each pixel to a fraction of the pixel dwell time [9]. In contrast to fluorescence, which results from fast singlet-to-ground-state relaxation, (triplet) phosphorescence is based on the decay of a long-lived triplet state with lifetimes in the \(\upmu\)s range. The different lifetime ranges of fluorescence and phosphorescence as well as the ability to control both the pixel dwell and irradiation times are critical for the separate detection of both signals. In order to record the phosphoresce pixelwise, the duration of the laser illumination needs to be shorter than the pixel dwell time. For the measurements described here, the pixel dwell time was set to 110 \(\upmu\)s. During that time the laser was unblocked (\"laser on\") from 1-56 \(\upmu\)s and blocked (\"laser off\") afterwards (56 \(\upmu\)s-110 \(\upmu\)s), hence, the \"laser-on\" and \"laser-off\" durations were 55 \(\upmu\)s and 54 \(\upmu\)s each. During these time durations, signals from the sample were recorded in 1024-time channels with time steps of 100 ns. The \"laser-on signal\" is dominated by the fluorescence with neglectable phosphoresce contribution. During the \"laser-off time\", the fluorescence quickly fades such that only the phosphoresce signal remains which can then be exclusively (background free) detected. Figure 1b illustrates this temporal dependence of the recorded signals. For the measurements reported throughout the paper, the following laser settings were chosen: the laser wavelength was set to 800 nm which corresponded to a wavelength of 400 nm for one-photon excitation.