Source: Arvin H. Soepriatna1, Kelsey A. Bullens2, and Craig J. Goergen1
1 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
2 Department of Biochemistry, Purdue University, West Lafayette, Indiana
Near-infrared fluorescence (NIRF) imaging is an exciting optical technique that utilizes fluorescent probes to visualize complex biomolecular assemblies in tissues. NIRF imaging has many advantages over conventional imaging methods for noninvasive imaging of diseases. Unlike single photon emission computed tomography (SPECT) and positron emission tomography (PET), NIRF imaging is rapid, high-throughput, and does not involve ionizing radiation. Furthermore, recent developments in engineering target-specific and activatable fluorescent probes provide NIRF with high specificity and sensitivity, making it an attractive modality in studying cancer and cardiovascular disease. The presented procedure is designed to demonstrate the principles behind NIRF imaging and how to conduct in vivo and ex vivo experiments in small animals to study a variety of diseases. The specific example shown here employs an activatable fluorescent probe for matrix metalloproteinase-2 (MMP2) to study its uptake in two different rodent models of abdominal aortic aneurysms (AAAs).
As the name suggests, NIRF imaging utilizes light within the first near-infrared window, ranging from 650 nm to 900 nm, to deliver photons into tissue. The energy, E, of a photon is characterized by Equation 1, where h is the Planck's constant, c is the speed of light in a vacuum, and λ is the wavelength of light.
= (Equation 1)
Target-specific fluorescent molecules called fluorophores are typically introduced into the animal through genetic engineering or via tail vein 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'. Due to the instability of the S1' state, the molecules relax to the lowest vibrational energy level within that state and release energy in the form of heat. The fluorophores, now in the relaxed, excited state S1, then return to the ground state S0, emitting light at a specific wavelength. The emitted light, which has a longer wavelength due to the dissipation of energy in the form of heat, is then captured and recorded using a fluorescence imaging system. The fundamental shift between the absorption and emission spectra is called the Stokes shift and is important as it makes it possible to differentiate between the excitation and emission light.
The following procedure provides detailed steps needed to collect in vivo and ex vivo NIRF images from small animals:
1. Experimental Setup
- Connect a fiber optic light source to the fluorescence imaging system using a fiber optic light guide.
- 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.
- 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
- In Vivo
- Anesthetize the animal in an induction chamber using isoflurane at a concentration of 3-4% on the flowmeter dial.
- 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.
- Secure the animal's paws to minimize motion artifacts. Remove hair from the region of interest by applying a depilatory cream.
- 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.
- Apply ophthalmic ointment to the eyes to prevent drying of the corneas.
- 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.
- Ex Vivo
- 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.
- Surgically extract the tissue or organ of interest and carefully remove excess adipose tissue with forceps.
- Rinse the tissue in phosphate buffered saline to remove residual blood and place the sample directly on the imaging stage.
3. Image Acquisition
- Open the molecular imaging software and turn on both the fiber optic light source and the fluorescence imaging system.
- 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.
- Select UV-Transillumination as the illumination source.
- 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.
- Close the preview window and ensure that all parameters on the acquisition window match the camera and filter settings.
- Click 'Expose' to acquire and save the image.
- Standard molecular imaging software typically provides a variety of analytical, measurement, and image correction tools to quantify fluorescence signals for image analysis.
- 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!
Representative in vivo and ex vivo NIRF images taken from rodents with abdominal aortic aneurysms (AAAs) are shown in Figures 1-2. An activatable fluorescent probe was injected systemically via the tail vein to visualize matrix metalloproteinase-2 (MMP2) activity. MMP2 is an elastolytic enzyme involved in the degradation of the extracellular matrix that plays a major role in the initiation and progression of AAA. All images were acquired using a 625 nm excitation filter, a 700 nm emission filter, and 60 seconds exposure time.
Figure 1: A representative in vivo NIRF image of an apolipoprotein E-deficient mouse that developed an AAA following infusion of angiotensin-II. Most of the small spots showing high signal are from skin autofluorescence (yellow arrows). The vasculature can be visualized as tubular structures with high fluorescence signals (red arrow). Scalebar: 1 cm.
Figure 2 shows an increase in MMP2 activity in the aneurysmal region of the abdominal aorta, as seen by the observed increase in signal intensity relative to healthy regions of the abdominal aorta. This result is consistent with results in the literature that show elevated MMP2 levels within AAAs. Excess fluorescent probes were filtered and accumulated in the kidneys, leading to bright fluorescent signals.
Figure 2: NIRF images of AAAs from two different animal models: (A) a suprarenal AAA in angiotensin II-infused apolipoprotein-E deficient mouse and (B) an infrarenal AAA in rat infused with porcine pancreatic elastase. Yellow arrows point to the AAAs. Scalebars: 3 mm.
Applications and Summary
NIRF imaging relies on fluorescent probes to quantify and visualize biomolecular assemblies in tissues. Absorbed photon energy from near-infrared light excites fluorescent molecules to a higher energy state, and the emitted light with a longer characteristic wavelength is captured by a fluorescence imaging system. Here, the application of NIRF imaging to study MMP2 activity in abdominal aortic aneurysms was demonstrated in vivo and ex vivo. Unlike SPECT or PET, which are considered to be the gold standards in studying metabolic processes in the body noninvasively, NIRF imaging is a rapid and high-throughput imaging technique that does not involve ionizing radiation. One of the limitations of this modality is its relatively small penetration depth. Although this limitation makes clinical imaging of deep tissues challenging, NIRF imaging plays an important role studying tumors and cardiovascular diseases in small animals.
Given the appropriate fluorescent probe, many molecular structures can be visualized using the presented NIRF imaging procedures to study both disease initiation and progression in small animal models. Specific ex vivo and in vivo applications include 1) evaluation of MMP activity in rodent vasculature, 2) early tumor detection in different types of cancers, and 3) assessment of nanoparticle pharmacokinetics and biodistribution for therapeutic applications. In addition to increased MMP2 activity within AAAs, other MMP fluorescent probes have been utilized to study atherosclerosis progression and to characterize cardiac extracellular matrix composition following a myocardial infarction. Furthermore, the fluorophore indocyanine green has been used to study tissue perfusion in murine models of hindlimb ischemia. To elaborate more on the application of NIRF imaging on early cancer detection, tumor-targeting NIRF dyes can be used to assess tumor margins and assist in resection procedures. The integration of near-infrared fluorophores into nanoparticles developed for drug delivery allows scientists to develop more effective nanoparticle-based therapeutics for a variety of diseases. Lastly, the ability to spatially localize the fluorescent signal in whole animals or intact tissue is a clear advantage over other conventional enzymatic assays (gel zymography) and protein analysis (western blot) that requires animals to be sacrificed and tissues to be homogenized.