## 3 Results
### Characterization of Au Nanopeanuts
The resonantly enhanced surface plasmon field of the metal nanostructure is known to be able to interact with the transitional states of the nanomaterials, thereby resulting in enhanced luminescence intensity and the efficiency of excitation [28; 29; 30]. In the SPR-enhanced nonlinear optical processes, not only the local excitation electric field, but also the nonlinear polarization is enhanced. For nonlinear optical processes such as two-photon fluorescence (TPF), harmonic generation, and Raman scattering [31], the enhancement of  
Figure 4: Darkfield scattering and fluorescence images of A549 cells exposed to either nil or 633-nm diode laser irradiation. A549 cells were incubated with TBO-PEGylated nanopeanuts (10 \(\upmu\)g/mL) for 4 h at 37 \({}^{\circ}\)C. After removing uninternalized colloids by PBS wash, A549 cells were subjected to 3 min of exposure to a 633-nm diode laser (200 mW/cm\({}^{2}\)), followed by an additional 16 h incubation. The images were acquired using a 10\(\times\) objective lens (CytoViva microscope). overall signal yields could be more than 100 [9; 31; 32; 33; 34]. In our gold nanopeanuts, the local field enhancement factor of surface plasmon resonance at 1230 nm is about 9 [14], which benefits the yield of two-photon luminescence and the singlet oxygen generation. Upon exciting 1230-nm femtosecond laser pulses for 3 min, the TBO-PEGylated nanopeanuts showed significant singlet oxygen generation, while no singlet oxygen was detected using PEGylated nanopeanuts (Figure S7). Then, we conducted NIR PDT in vitro in A549 malignant cells using the MTT assay (Figure 6). The photodynamic efficacy was evaluated using two different beam size conditions: A focused beam (5.7 \(\times\) 10\({}^{5}\) W/cm\({}^{2}\); beam diameter: 1.5 \(\upmu\)m) and a collimated beam (104 mW/cm\({}^{2}\); beam diameter: 3-4 mm). Without laser exposure, cell viability was not affected by either PEGylated or TBO-PEGylated nanopeanut incubation. Additionally, no decrease in cell viability was observed for cells alone or for PEGylated nanopeanut-treated cells irradiated by NIR femtosecond laser. There are no photothermal damages on cells. For cells treated with TBO-PEGylated nanopeanuts, apparent cell death was observed from focused and collimated beam irradiation, giving 63% and 45% (<IC\({}_{50}\)) viability, respectively. Higher cell death in the collimated beam illumination can be attributed to a larger exposure area and more production of singlet oxygen. These results verify the PDT capability of TBO-PEGylated nanopeanuts in the second biological window. Subsequently, we employed a multiphoton nonlinear optical microscope to observe microscopic evidence of PDT action. The cells were cultured and attached well to the bottom of glass Petri dishes. On a laser-scanned inverted microscope, these dishes were mounted within a micro incubator to keep cells in their normal physiological conditions. The femtosecond Cr: forsterite laser was used as the excitation source of the nonlinear microscope. Its minimal invasiveness for nonlinear microscopy imaging has been demonstrated in zebrafish embryos [31], mouse embryos, and human skin [32; 33]. Without any treatment of nanopeanuts, the morphology of attached cells can be observed through a third harmonic generation (THG) modality without labeling (Figure 7a). Due to the 1230 nm excitation wavelength of the femtosecond Cr: forsterite laser, most endogenous fluorophores would not be resonantly excited and show no fluorescence background (Figure 7a), even with the highest photomultiplier gain. Long-term observation for 30 min did not change the attachments and morphologies of cells. For cells treated with PEGylated  
Figure 5: Multiphoton emission spectra of nanopeanuts alone (no surface modification, red curve) and TBO-PEGylated nanopeanuts (black curve). The emission peaks at approximately 410 nm and 615 nm are third-harmonic generation (THG) and second-harmonic generation (SHG) of nanopeanuts. The arising band at approximately 670 nm is the fluorescence from TBO.