6.9: 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.

Near-infrared fluorescence imaging is an optical technique that utilizes fluorescent probes to visualize complex biomolecular assemblies in tissues. This noninvasive imaging technique, which is also known as NIRF, is rapid and does not require ionizing radiation.

In NIRF, fluorescent probes can be conjugated with small molecules for higher specificity to study cancer and cardiovascular disease progression. They are excited by near-infrared light which penetrates deep into tissue and may be used to delineate healthy tissue from diseased tissue that alter the concentration of these target molecules.

This video will illustrate the principles behind near-infrared fluorescence imaging as well as how to perform in vivo and ex vivo experiments in small animals to study a variety of diseases.

As the name suggests, near-infrared fluorescence imaging utilizes light within the first near-infrared window which ranges from 650 nanometers to 900 nanometers to deliver photons into tissue. Target-specific fluorescent molecules called fluorophores are typically introduced into an animal through either genetic engineering or injection prior to imaging.

These fluorophores absorb photon energy which raises the energy of the molecules from the ground state S0 to the unstable excited state S1 prime. Because this state is unstable, the molecules will relax to the lowest vibrational energy level within the excited state releasing their energy in the form of heat. The fluorophores, now in the relaxed excited state S1, then return to the ground state, emitting light of a specific wavelength.

This light has a longer wavelength than the light originally introduced into the fluorophore due to the energy that dissipates in the form of heat as the molecule relaxes to the lowest vibrational energy level. The emitted light is then captured and recorded using a fluorescence imaging system.

A graph of the absorption and emission spectra for the fluorophore shows the range of wavelengths the fluorophore can absorb and emit respectively. This fundamental shift, which is the difference in nanometers between the peak absorption and the peak emission wavelengths, is called the Stokes shift. Each fluorophore has a distinct Stokes shift which allows the emission light to be distinguished from the exciting light and makes imaging techniques such as NIRF possible.

Having reviewed the main principles of near-infrared fluorescence imaging, let's now walk through the step-by-step procedure to prepare and image an animal.

First, use a fiber-optic light guide to connect a fiber-optic light source to the fluorescence imaging system. Select the excitation filter that matches the excitation spectrum of the fluorescence to be introduced into the sample to ensure that the correct wavelength of light is delivered.

Next, select the appropriate emission filter to match the emission spectrum of the fluorophore which will block undesired spectral components that may be attributed to autofluorescence.

To begin preparing for in vivo imaging, use isoflurane to anesthetize the animal in a knockdown chamber. Transfer the animal to a nose cone that is fixed on the imaging stage. Secure the animal's paws to minimize motion artifacts. Apply a depilatory cream to remove the hair from the area of interest. Then, apply ophthalmic ointment to the animal's eyes to prevent the corneas from drying.

After this, inject the activatable fluorescent molecular probe into the animal. To begin image acquisition, open the molecular imaging software. Turn on both the fiber-optic light source and the fluorescence imaging system.

Next, 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.

Then, select UV Transillumination as the illumination source. Using the preview image as a reference, adjust the focus, field of view, and F-stop in the capture system chamber to optimize the sampled image quality. Adjust the exposure time and position of the sample as needed. After this, close the preview window. Ensure that all of the parameters on the acquisition window match the camera and filter settings. Click "Expose" to acquire and save the image.

To prepare for ex vivo imaging, euthanize the animal in a humane fashion following the injection of the fluorescent probe. Using forceps, carefully remove any excess periaortic fat. Next, surgically extract the tissue or organ of interest. Rinse the tissue in phosphate buffered saline to remove residual blood. Then, place the sample directly on the imaging stage.

Image the ex vivo tissue following the same protocol as described for in vivo imaging. When complete, remove the sample from the stage. Turn off the system and clean the imaging stage.

Now that we have completed the protocol for obtaining near-infrared field images, let's review the results of those scans.

In these representative images, an activatable fluorescent probe is injected systemically via the tail vein to visualize the Matrix Metalloproteinase or MMP2. Here we see an in vivo NIRF image of an apolipoprotein-E deficient mouse that developed an abdominal aortic aneurysm following infusion of angiotensin II. While most of the small high signal spots are from skin autofluorescence, the vasculature presents visually as tubular structures with high fluorescent signals.

The second representative image compares NIRF images of abdominal aortic aneurysms from two different animal models. One, a suprarenal abdominal aortic aneurysm in an angiotensin II-infused apolipoprotein-E deficient mouse. And two, an infrarenal abdominal aortic aneurysm in a rat infused with porcine pancreatic elastase.

In each, we see an increase in MMP2 activity in the aneurysmal region of the abdominal aorta. Excess fluorescent probes are filtered and accumulated in the kidneys, explaining the bright fluorescent signals observed there.

Let us now look at some other applications of near-infrared field imaging. First, NIRF imaging can be used to study cardiovascular disease in murine models.

In this study, knockout mice are injected with two different near-infrared fluorescent probes. The aortas are harvested 24 hours later and assessed via NIRF imaging. The results show significant NIRF response, indicating the presence of extensive calcification that co-locates with macrophage accumulation.

NIRF imaging can also be used to locate and evaluate tumors in vivo. In this study, tissue simulating breast phantoms containing fluorescent tumor simulating inclusions are created. The applications of NIRF imaging during breast conservation surgery are then simulated.

The results show that tumor-like inclusions are detectable by NIRF up to a depth of approximately two centimeters. Inclusions deeper than this are detectable after incisions are made into the overlaying phantom tissue. After the inclusions are removed, the surgeon evaluates the NIRF images. Any remaining fluorescence, which indicates the presence of tumors, indicates incomplete removal and is then excised.

You've just watched JoVE's introduction to near-infrared imaging. You should now understand the principles of fluorophore excitation and emission, how to prepare an animal for in vivo and ex vivo NIRF imaging, and some biomedical applications. Thanks for watching!