Fig. 5. In vivo NIR-IIb molecular imaging of RBD-specific cells in LNs of mice after immunization.

(A) Immunization schedule; the female BALB/c mice were immunized by subcutaneous (sc) injection at the mouse tail base with the pEr-P³-RBD (WT) nanovaccine on day 0 and day 21 treated with only PBS were used as a control group; n = 3 in each group. The mice were injected the IR840-RBD (WT) at the mouse tail base (subcutaneously) on day 35. (B) Color photograph and multiplexed NIR-II imaging were conducted on pEr-P³, IR840 in PBS buffer. Under 808-nm excitation and 1000- to 1200-nm imaging conditions, only IR840 was detectable whereas pEr-P³ was undetectable. Under 975-nm excitation and 1500- to 1700-nm imaging conditions, only pEr-P³ was detectable whereas IR840 was undetectable. (C) NIR-II luminescence images (808-nm excitation with a power density of ~70 mW cm⁻², 1000- to 1200-nm detection, and the exposure time is 10 ms) showing the IR840-RBD (WT) trafficking pathways in mice immunized by pEr-P³-RBD (WT) and (D) treated by only PBS. Images were recorded at different time points as indicated after injection. Representative images from one mouse from each group are shown here. d, days. (E) In vivo IR840-RBD (WT) signal intensity in iLNs and (F) bladder normalized by the background plotted as a function of time after subcutaneous injection of IR840-RBD (WT). Error bars represent the SD of three repeated experiments. Data are presented as mean values ± SD. (G) pEr-P³-RBD (WT) vaccine or only PBS induces RBD-specific cell response in iLNs at day 35. The percentage of RBD-specific cells in live cells was counted by flow cytometry. Error bars represent the SD of eight repeated experiments. Data are presented as mean values ± SD.