Bioluminescence Imaging System Image Acquisition and Analysis

Bioluminescence images were captured by the In Vivo Imaging System (IVIS) spectrum (Perkin Elmer, Santa Clara, CA, USA) and analyzed by IVIS imaging software (Perkin Elmer). Imaging examination was performed 1 day after conidia infection of A. fumigatus. Seven minutes before imaging, the luminescent reagent (MPO XenoLight RediJect inflammation Probe, Perkin Elmer) was dropped onto the corneal surface of mice. Animals were placed on the warm phase of IVIS, and the infected eyes were placed in the lateral decubitus position. The ocular surface directly facing the camera sensor. The standard optimization scheme was used to determine the imaging parameters in vivo. Mice were imaged with “C” field of vision and using cultured medium for 1 minute. The total bioluminescence of infected eye was determined as the sum of the background-subtracted ocular region of interest (ROI) flux from 2 consecutive 5-minute imaging windows. Normal eye total bioluminescence was determined as 2 times the background-subtracted ocular ROI flux from 5-minute imaging window. Shapiro Wilk analysis was used to test the normality of different paired data, then paired t-test in prism-5 software was used to compare the mean and the peak of bioluminescence.^23,24