4.4. In Vivo NIR Fluorescence Imaging

One week after inoculation, subcutaneous A4 tumors in mice reached approximately 10 mm at the longest diameter, and tumor-bearing mice received intravenous injection with HER2-ICG (100 μg) via the tail vein. To assess the biodistribution and clearance of the conjugate, the mice were anesthetized through 2.5% isoflurane inhalation and in vivo NIR fluorescence imaging was performed. Spectral fluorescence images at the dorsal position of mice were acquired using the Pearl Trilogy Imaging system (LI-COR Biosciences, Lincoln, NE, USA) with the 800 nm channel at pre-injection, followed by various post-injection time points (0.5, 1, 2, 4, 6, and 24 h). Image analysis was conducted using the Image Studio Software. After selection of the appropriate parameters and setting a suitable background reading, the region of interest was drawn to encompass the tumor and the fluorescence SI was measured.