Near-infrared Fluorescence Imaging of Abdominal Aortic Aneurysms

The following procedure provides detailed steps needed to collect in vivo and ex vivo NIRF images from small animals:

1. Experimental Setup

1. Connect a fiber optic light source to the fluorescence imaging system using a fiber optic light guide.
2. Select the appropriate excitation filter for the experiment. The excitation filter determines the wavelength of light to be delivered to the sample and should be chosen to match the excitation spectrum of the fluorophore introduced into the sample.
3. Select the appropriate emission filter. The emission filter blocks undesired spectral components, which may be attributed to autofluorescence, and should be chosen to match the emission spectrum of the fluorophore.

2. Sample Preparation

1. In Vivo
   1. Anesthetize the animal in an induction chamber using isoflurane at a concentration of 3-4% on the flowmeter dial.
   2. Transfer the animal to a nose cone that is fixed on the imaging stage, and maintain isoflurane at a concentration of 1-2%. A heat source is not necessary because the animals are typically only imaged for a short period of time (> 5 min), and their body temperature does not substantially decrease.
   3. Secure the animal's paws to minimize motion artifacts. Remove hair from the region of interest by applying a depilatory cream.
   4. Apply depilatory cream to the smallest area necessary. After 30 s, wipe it off with a gauze pad. Wipe the area a second time with an ethyl alcohol moistened gauze pad to completely remove the depilatory cream.
   5. Apply ophthalmic ointment to the eyes to prevent drying of the corneas.
   6. Inject the activatable fluorescent molecular probe into the animal. For this specific application, MMP2 activatable probes were injected intravenously into the tail vein. At this point, the mouse can be imaged. Proceed to the "Image Acquistion" of this protocol to continue. Monitor the animal for regular breathing throughout the brief procedure.
2. Ex Vivo
   1. Following injection of the fluorescent probe, euthanize the animal in a humane way according to the 2013 AVMA Guidelines for the euthaniasia of animals. Carbon dioxide (CO₂) overdose is a standard practice for euthanizing small animals.
   2. Surgically extract the tissue or organ of interest and carefully remove excess adipose tissue with forceps.
   3. Rinse the tissue in phosphate buffered saline to remove residual blood and place the sample directly on the imaging stage.

3. Image Acquisition

1. Open the molecular imaging software and turn on both the fiber optic light source and the fluorescence imaging system.
2. Open the acquisition window and specify the type of exposure appropriate for the study. Available exposures include: Standard Exposure to capture a single image, Time Lapse Exposure to capture a series of images over a fixed time interval, and Progressive Exposure to capture a continuous sequence of exposures at different exposure times.
3. Select UV-Transillumination as the illumination source.
4. Using the previewed image as a reference, adjust the focus of the lens, the field of view, and the f-stop/aperture in the capture system chamber to optimize the sampled image quality. Adjust the exposure time and position of the sample.
5. Close the preview window and ensure that all parameters on the acquisition window match the camera and filter settings.
6. Click 'Expose' to acquire and save the image.
7. Standard molecular imaging software typically provides a variety of analytical, measurement, and image correction tools to quantify fluorescence signals for image analysis.
8. At the end of the imaging session, remove the sample/animal, turn-off the system, and clean the imaging stage to minimize damage to the system.