Fig. 7: In vivo NIR-IIb fluorescence ratiometric imaging and immunotherapy efficacy monitoring.

a Illustration of long-term imaging and immunotherapy schedule (n = 6 mice for non-treatment and treatment group, respectively). b NIR-IIb fluorescence ratiometric imaging and (c) NIR-IIb fluorescence quantification/ratiometric analysis of tumor-bearing mice without/with immunotherapy after intravenous injection of Bi-HCy-ErNPs. d Illustration of long-term imaging and immunotherapy schedule. e NIR-IIb fluorescence ratiometric long-term imaging and (f) NIR-IIb fluorescence quantification/ratiometric analysis of tumor-bearing mice without/with immunotherapy after intravenous injection of Bi-HCy-ErNPs (n = 6 mice). g Tumor growth curves, excised tumor weight, and body weight change of tumor-bearing mice without/with immunotherapy (n = 6 mice). Absolute tumor volume measured for each group is included in Source Data file. h Correlation between NIR-IIb fluorescence ratiometric signal and tumor volumes of tumor-bearing mice with immunotherapy via a simple linear regression model. Centre line in black shows the best-fit linear regression, and error band in gray shows 95% confidence intervals of linear regression line. i Flow cytometric assay of TANs at the end of imaging. j Flow cytometric assay of mDCs (CD11c⁺CD80⁺CD86⁺) in tumor-draining lymph nodes (TDLNs), Tems (CD3⁺CD8⁺CD62L^lowCD44^hi) in spleen, tumor-infiltrating CD8⁺ Ts in CD3⁺ Ts (CD3⁺CD8⁺), M1-TAMs (CD11b⁺F4/80⁺CD80^hi), M2-TAMs (CD11b⁺F4/80⁺CD206^hi), and tumor-infiltrating Tregs (CD4⁺Foxp³⁺) (n = 6 mice). Dashed line indicated the basic outline of a mouse model. Data are presented as mean ± standard deviation (c−j). Data are compared through one-way ANOVA with Tukey’s post-hoc test for multiple comparisons (c and f) and two-tailed unpaired Student’s t-test (g and j). P-value (0/1/4 d vs. 5/8/9/12/13 d) is <0.0001 in F_808ex detection (c). P-value (0/1/4/5 d vs. 8/9/12/13 d) is <0.0001 in ratiometric signal detection (c). P-values (0 d vs. 7/14/21 d) are <0.0001 in F_808ex and ratiometric signal detection (f). P-values (non-treatment vs. treatment) in tumor volume and tumor weight measurement are <0.0001 (g). P-values (non-treatment vs. treatment) in are <0.0001 in mDCs, Tems, CD8⁺ Ts, M1-TAMs, M2-TAMs, and Tregs detection (j).