Whole-body fluorescence imaging of mice

Whole body fluorescence imaging of mice was performed with LI-COR Biosciences Odyssey Infrared Imaging System with emission wavelength at 700 nm (for verteporfin) and 800 nm (for Cy7-BSA). The nu/nu mice were first subcutaneously inoculated with 5 × 10⁴ 4T1 cells in 50 µL PBS (1×) into the back of neck. Similar to the descriptions above, IMOD with verteporfin loaded optical fiber was implanted once tumor length was determined to be approximately 8 mm. The nu/nu mice were fed with alfalfa-free food for at least one week prior to the study in order to minimize the gastrointestinal background autofluorescence⁹⁰. For imaging studies, 100 µg/mL Cy7-BSA in 30 µL sterile PBS (1×) was injected through the refillable tubing on IMOD into the 4T1 tumor after PDT irradiation. Fluorescence images of nu/nu mice were taken at designated time points. The analysis of the results was carried out using Origin software (Northampton, MA) by fitting the normalized fluorescence intensity (I) – time (t) curve into an exponential decay model according to Eq. (¹).

where a, b, T₁ are all constants fitted by Origin software. The starting point for the curve fitting was the peak value of the normalized fluorescence intensity, usually at 4 or 12 h. The obtained T₁ value was used to calculate the decay half-life τ based on Eq. (²).

Note that the fluorescence intensity at t = 0 was normalized as 1.