# Photoacoustic Imaging in Oxygen Detection
## 3 Photoacoustic Imaging in pO\({}_{2}\) Measurement
Within this time frame, a structure of events if repeated at a defined repetition frequency, for example, for image window 1: the excitation light is switched on and off, consequently the luminescence follows with a certain delay in rise and decay, for image window 1 the camera is 'opened' by the modulation signal for a time \(\Delta_{\text{i}}\). This sequence is repeated. For image window 2 and the window 'dark', the delays after switching off the excitation light are different but the width \(\Delta_{\text{i}}\) is the same. Different from intensity-based imaging, molecular lifetime is a relatively stable parameter in certain ambient environments, and can elude effects of dye concentration, heterogeneous distribution, and light scattering in tissue. This relatively 'intrinsic' property of lifetime imaging makes it a strong candidate for imaging tissue oxygen condition, and is considered sufficiently sensitive to produce high-contrast spatial heterogeneous information of tissue oxygenation [39; 40]. In addition, quenching method is a reversible way, in which the upper state lifetime or the luminescence intensity of the oxygen-sensitive dye is reduced with high oxygen concentration, but can be recovered with low oxygen concentration. Therefore, the quenching method opens a new avenue to accomplish real-time recording of the change of pO\({}_{2}\), and hence has gained great development in recent years. The representative fluorescence lifetime microscopy (FLIM) (Figure 3c [41]) has especially contributed a lot in today's in vitro trials. As can be seen from Figure 3c, the system employs a pulsed laser whose beam is focused into a spot which is scanned across the sample, dedicated timing electronics, and a single point detector. Nevertheless, optical lifetime microscopes, similar to traditional optical methods, are incapable of yielding fine spatial resolution and deep penetration depth in tissue simultaneously due to the strong scattering of light in tissue. Recently there has been a trend to combine photoacoustic imaging with traditional optical imaging mechanisms to obtain deep tissue information. For example, photoacoustic lifetime imaging (PALI) utilizes photoacoustic technique for probing the lifetime of fluorophore's excited state (Figure 4a) [42]. In optical lifetime microscopies, a pulse wave is first applied to excite the molecules from the ground state to the upper state. Then a time gating method or TSCPC (time correlated single photon counting) scheme is used to record the fluorescence decay from the singlet state or phosphorescence decay  
Figure 3: (a) Jablonski diagram for oxygen sensing mechanisms; (**b**) Structure of timing and light signals (reproduced with permission from [38]. Copyright Elsevier, 2001); (c) A schematic time-correlated single photon counting (TCSPC)-based scanning FLIM set-up (reproduced with permission from [41]. Copyright Elsevier, 2015). from the triplet state in the time domain, or use phase modulation to get the signal in the frequency domain. By contrast, PALI detects the population of the molecules at the upper state by absorption. In a typical PALI system, as shown in Figure 4a, the measurement of an object requires two lasers. The first pulse (pump) is for excitation, which pumps the molecules to the excited triplet state where transient absorption happens. The transient absorption is then probed by a second laser to generate the photoacoustic waves from triplet-triplet absorption, of which the signal is closely related to the molecule population of the triplet state. Time delay between the pump and probe pulses are tuned to capture the time-dependent photoacoustic amplitude. The higher the pO\({}_{2}\) in the surrounding environment, the faster the excited triplet state population decreases, and hence the faster PA amplitude reduces. By changing the delay between the pump and probe laser pulses, the luminescence decay can be detected, and finally be fit into an exponential function to compute the lifetime. This principle was first proposed by [PERSON] et al.