To begin, lace the anesthetized mouse supine in the imaging system with the snout in a nose cone. Position the mouse such that the lower legs are extended, and the knees are pointed slightly in the air. Open the compatible software on the imaging system computer.
From the acquisition control panel, click on initialize to warm up the system, and wait until the temperature light turns green. Then click on Imaging Wizard. After clicking on filter pair, ensure that the setting is configured for epi-illumination.
Then press next. From the pull down list, find the probe of interest to select the correct excitation and emission settings, and click next. Choose mouse for imaging subject.
In exposure parameters, ensure the auto settings is checked and the fluorescence and photograph options are selected. In the checklist of field of view, select D to 22.6 centimeters, and press next. After confirming all settings are correct, press the acquire sequence button, and confirm that the image got adequate exposure.
To analyze the image, place a region of interest circle of consistent size over each knee joint on the black and white image. Calculate the total radient efficiency and average radient efficiency for each knee joint. If the radient efficiency is also calculated on the contralateral legs, normalize the data by dividing the radient efficiency measurement for the injured leg by that of the contralateral leg.
Fluorescence reflectance imaging showed significantly greater protease activity in the injured joints of mice compared to the uninjured mice. Injured joints showed 43%greater average radient efficiency compared to contralateral joints, and joints from uninjured mice. A 30 to 80%greater radiant efficiency was observed in injured joints, compared to contralateral joints at two weeks post-injury.
In contrast, surgically operated joints exhibited 300%greater radiant efficiency at week four compared to contralateral joints, suggesting a notable confounding effect of surgery.
