2.3. PDT treatment

BPD solution was used within 1 hour post-preparation for each PDT treatment. Mice were given BPD solution via tail vein injection at a dose of 0.75 mg/kg and were isolated in dark places post-injection. During PDT treatment, mice bodies were covered by tapes except for the targeted ear zone. Note that only a small portion of the ear was treated by PDT, and the rest of the ear was enclosed in the tape as a control, as shown in Fig. 3(d) and Fig. 8 (Appendix A). Mouse ear was flattened on a glass plate and the laser source was placed perpendicular to the exposed region to enable a uniform distribution of irradiation on the surface of the ear [Fig. 3(d)]. The targeted ear zone was irradiated with a continuous wave 690 nm laser diode source (Model MRL-III-690 nm-500 mW, Changchun New Industries Optoelectronics Tech) at four different combinations of fluence rate and irradiation time in four treated groups. The total radiant exposure dose (i.e., fluence rate times irradiation time) in all four groups, however, are kept the same (113 J/cm²). In our investigation, PDT treatments were performed within the time interval of 3h-4.3 h post-administration, according to the peak of PS accumulation in ear region shown in Fig. 2(b). This arrangement is to maintain PS concentration a constant during PDT treatment. As such fluence rate is the only variable between different treatment groups, which facilitates the quantitatively investigation of PDT efficacy as a function of fluence rate. Further details will be discussed in Section 2.5 and Section 3. The 690 nm wavelength of the PDT laser source was validated using a spectrometer (Model USB4000, Ocean Optics), and the fluence rate was monitored via a photodiode power sensor (Model PD300, Ophir Optronics). A liquid core optical fiber (core diameter 5 mm; NA 0.5) was applied to transmit laser beam to the ear surface. The distance between the ear surface and the output of the fiber was kept at 1 mm, leading to a light spot on the surface of the ear bigger than that of the uncovered region of the ear. Given that mice ears are considerably thin and tend to get dehydrated when exposed to a strong laser irradiation, ultrasonic coupling agents were applied on the exposed ear surface to avoid burn injuries during PDT treatment. After treatment, the covered tape was removed from the mice ears for the subsequent photoacoustic imaging.

An overview of the experimental setup. (a) The schematic diagram of the custom-built OR-PAM System. NDF, neutral density filter; FC, fiber coupler; SMF, single-mode fiber; OBJ, objective; DL, doublet lense; UST, ultrasonic transducer; DAQ, acquisition card; PC, personal computer. (b) Detailed view of the optical-acoustic beam combiner. (c) Lateral resolution measurement on a metallic blade edge. (d) A photograph of PDT treatment on a mice ear.