## 4 Discussion  
We have demonstrated a new NIR-active photodynamic platform by plasmon resonant-enhanced two-photon luminescence and singlet oxygen generation from conjugated PS. The NIR surface plasmon bands that appeared beyond 1000 nm allowed us to excite the unique nanopeanut structure in the second biological transparency window. Two-photon excited electrons in TBO may either radiate fluorescence or be coupled to the non-radiative triplet states through intersystem crossing (ISC). The dark state triplet electrons could further generate single oxygens in solutions. In addition, the enhanced fluorescence from TBO-conjugated nanopeanuts, giving stronger contrast than nanopeanuts alone, makes TBO-conjugated nanopeanuts more promising theranostic agents in deep tissues. In-evitably, direct conjugation of TBO to the gold surface will bring severe quenching even though it could still emit substantial fluorescence for two-photon imaging. The reason TBO fluorescence was not completely quenched by gold nanopeanuts could be due to the residual CTAB surfactant on the surface of gold [35]. Many other works that have also employed TBO conjugation have similar issues [36; 37]. Thin-layer polyelectrolyte deposition could mitigate this problem [37]. Following this surface modification strategy, we could improve the two-photon fluorescence yields of TBO-PEGylated nanopeanuts in the future. **Supplementary Materials:** The following are available online at [[https://www.mdpi.com/article/10.3390/app112210875/s1](https://www.mdpi.com/article/10.3390/app112210875/s1)]([https://www.mdpi.com/article/10.3390/app112210875/s1](https://www.mdpi.com/article/10.3390/app112210875/s1)). Figure S1: HRTEM image and EDX analysis of Au nanopeanuts. Spectra 1-5 represent different sites analyzed in the Ag/Au nanoshell. Au nanopeanuts were prepared by adding 700 \(\mu\)L of HAuCl\({}_{4}\) into Au NR@Ag colloidal solution containing CTAB and AA., Figure S2: (a) TEM images and (b) UV-Vis-NIR spectra of Au nanopeanuts obtained from the addition of different volumes (140, 280, 420, and 700 \(\mu\)L) of HAuCl\({}_{4}\) into Au NR@Ag colloidal solutions containing CTAB and AA. Insets in each figure present the corresponding colloidal color and the amplified TEM of the single nanopeanut. Figure S3: HRTEM images and EDX analysis of Au nanopeanuts obtained by adding different volumes (140, 280, and 420 \(\mu\)L) of HAuCl\({}_{4}\) into Au NR@Ag colloidal solutions containing CTAB and AA. Spectra 1-5 shown for each nanopeanut represent different spot sites  
Figure 9: In vivo **(a)** TPF (yellow color), **(b)** THG (magenta), **(c)** second harmonic generation (green color), and **(d,e)** combined microscopy images of TBO-PEGylated nanopeanuts in the ears of ICR mice. SG: Sebaceous gland. White arrows and the area enclosed by the white dashed contour in **(b)** indicate the positions of nanopeanuts. Yellow dashed lines in **(b)** and **(c)** outline the capillary and lymphatic vessels, respectively. analyzed in the Ag/Au shell. Figure S4: UV-Vis spectra taken as a function of TBO concentration. The inset shows the standard linear calibration curve of TBO. Figure S5: Fluorescence intensity of Singlet Oxygen Sensor Green mixed with TBO-PEGylated nanopeanuts (20 ug/mL) exposed to a 633-nm (200 mW/cm\({}^{2}\)) diode laser for different irradiation times. Figure S6: Fluorescence intensity of TBO-PEGylated nanopeanuts and free TBO measured from a conventional HORIBA Fluoromax-4 spectrometer by the selection of excitation wavelength 580 nm of xenon arc-lamp. The TBO concentration was fixed at 0.23 uM for both TBO-PEGylated nanopeanuts and free TBO molecules. Figure S7: Fluorescence intensity of Singlet Oxygen Sensor Green mixed with nanopeanuts (20 ug/mL) exposed to a 1230-nm femtosecond Cr: forsterite laser (104 mW/cm\({}^{2}\)) for 3 min. Data processing and multiphoton imaging, L.-Z.G. ; animal handling and multiphoton imaging, C.-H.W. ; synthesis of Au nanopeanuts and material characterization, M.-F.T., F.-Y.C. and V.S.