## 3 Results
### Characterization of Au Nanopeanuts
or TBO-PEGylated nanopeanuts, apparent two-photon fluorescence (TPF, yellow color) contrasts can be observed (Figure 7b,c) within cells, indicating that the cells took up these nanopeanuts. As mentioned above, the TPF spectrum (Figure 5, black curve) of TBO-PEGylated nanopeanuts shows an extra contribution (\(\sim\)670 nm) from TBO, which could be the reason that TBO-PEGylated nanopeanuts (Figure 7c) show stronger TPF contrast than PEGylated nanopeanuts alone (Figure 7b). We surveyed a power level below the regime of photothermal effects while maintaining large enough photodynamic effects. As shown in Figure 8a, after 5 min of illumination, no apparent photodamage was done to the PEGylated nanopeanut-treated cells. No effect on cell morphology was seen even when the irradiation time was prolonged up to 15 min. In contrast, cells with the TBO-PEGylated nanopeanuts effectively shrank after 3 min of illumination (Figure 8b). These effects of the NIR laser on cells agree well with the results of singlet oxygen tests and MTT assays. They indicate that under 1230 nm femtosecond laser illumination, TBO-PEGylated nanopeanuts can produce singlet oxygen and cause cell death quickly. We also examined the in vivo distribution of interstitially administered TBO-PEGylated nanopeanuts in the mouse ear pinna using the same imaging system. An ICR mouse was anesthetized, and the nanopeanuts were subcutaneously injected into the ear pinna. Then, the ear was mounted on the stage of the microscope for further observation. In a typical image, the TPF (Figure 9a) and THG (Figure 9b) contrasts revealed the distribution of nanopeanuts (indicated by white arrows and the area enclosed by white dashed contour). Strong THG contrast in capillaries (outlined by the yellow dashed line in Figure 9b) originates from the flowing red blood cells. These nanopeanuts were drained by a lymphatic vessel (outlined by the yellow dashed line in Figure 9c) near the capillary. These data showed that the pharmacokinetics and efficacy of TBO-PEGylated nanopeanuts can be tracked in vivo in microenvironments, which is a potential theranostic agent for photodynamic therapy in deep tissues. Figure 6: Cell viability studies of A549 cells from PEGylated and TBO-PEGylated nanopeanuts (20 \(\upmu\)g/mL) in the MTT assay. A549 cells were incubated with nanopeanuts for 8 h at 37 \({}^{\circ}\)C. After removal of uninternalized nanopeanuts by PBS wash, A549 cells were subjected to 3-min exposure to a 1230-nm femtosecond Cr: forsterite laser, followed by an additional 16 h incubation. The photodynamic efficacy was evaluated using two different beam size conditions: Focused and collimated beam. The collimated beam has 104 mW/cm\({}^{2}\) in power density with a beam diameter of 3-4 mm. The focused beam has \(5.7\times 10^{5}\) W/cm\({}^{2}\) with a beam diameter of 1.5 \(\upmu\)m, estimated based on Radius = 0.61 \(\times\) (\(\uplambda\)/NA). The cells -alone group were also subjected to laser irradiation. The dark group represents A549 cells treated with no laser irradiation. Figure 8: Dynamic THG (magenta color) and TPF (yellow color) images of A549 lung cancer cells treated with **(a)** PEGylated nanopeanuts and **(b)** TBO-PEGylated nanopeanuts. After 5 min of illumination with a femtosecond Cr: forsterite laser at a power density of 6.5 \(\times\) 10\({}^{6}\) W/cm\({}^{2}\), cells treated with PEGylated nanopeanuts were not affected, while cells treated with TBO-PEGylated nanopeanuts apparently shrank after 3 min of exposure (yellow dashed contour indicates the original size of cells). Fields of view: **(a)** 90 \(\upmu\)m \(\times\) 50 \(\upmu\)m; **(b)** 40 \(\upmu\)m \(\times\) 40 \(\upmu\)m.  
Figure 7: **(a)** Microscopy image of A549 lung cancer cells alone. Cells treated with **(b)** PEGylated nanopeanuts and **(c)** TBO-PEGylated nanopeanuts show strong TPF contrast. The magenta color and yellow color represent THG and TPF, respectively.