Featured Article
Monday April 20, 2009
by Catherine Shaffer
In vivo imaging is an optical imaging technique which looks deep into the tissues of living test subjects. Applications for in vivo imaging include tracking grafted stem cells, studying biological signaling networks and pathways, gene expression, angiogenesis, and apoptosis, to name just a few. In order to take a picture of a tissue a centimeter or more into the animal, and accurately visualize the processes inside, it is necessary to use near-infrared light. The tools and technologies associated with working in the near-infrared window vary somewhat from standard fluorescence imaging tools. A number of CCD camera systems optimized for near-infrared imaging are now available.
Imaging Systems for the Near-Infrared Neighborhood
Anin vivo imager generally consists of a box for the animal, a near-infrared-capable CCD imaging system, and a computer interface. The animal box is designed to keep the animal comfortable and, typically, anaesthetized. Ports are included for connecting supplies of anaesthetic gas, oxygen, and heat. A number of systems include additional features for the animal containment box, but the heart of the system is the optical imaging technology. Normally, optical imaging systems can only look at the surface of an object. However, in the near-infrared window of the spectrum, living tissues become "transparent" and the light source will penetrate the tissue and pick up fluorescence from near-infrared dye molecules.
The IVIS system, by Caliper Life Sciences, is an example of an imaging system purpose-built for in vivo imaging. IVIS uses a large CCD camera, cooled to -90°C. This gives the camera a very high sensitivity for deep image sources. The IVIS system is also capable of three-dimensional imaging using fluorescent or bioluminescent reporters. In order to be able to handle these two types of signal, the system needs to have some flexibility in how background is managed. Stephen Oldfield, PhD, senior director of imaging marketing for Caliper Life Sciences explains, "For fluorescence, you're dealing with illumination and light collection and you have to reject background autofluorescence. You do that with optical filtering or spectral unmixing using high resolution filters with a sharp cutoff. For bioluminescence, you're dealing with absolute sensitivity. You're trying to detect photons when there's almost no background. You do that by having the most sensitive camera available."
A new containment box allows the IVIS KInetic to image a non-anaesthetized animal. Some disease progression studies have found differences between active and inactive animals. Caliper continues to marry biology and hardware innovation in its development program for in vivo imaging, and anticipates expanding its line of reagents to include novel biologicals such as dual-labeled fluorescent and bioluminescent tumor cells. As with the IVIS KInetic, new developments in biology inform the development of new instruments.
Sensitivity Meets Convenience
LI-COR’s Pearl Imager is another specialized system for in vivo imaging. The Pearl Imager is designed for near-infrared fluorescent imaging only (not bioluminescent). Its advantages include sensitivity, ease of use, and affordability. Says Jeff Harford, senior product marketing manager for LI-COR, "The sensitivity is useful in situations where early target detection is important or when imaging deep targets such as orthotopic tumors. People really like the Pearl because, not only is it exquisitely sensitive, but you can train somebody to use it in two or three minutes. It doesn't require a lot of manipulation of cameras and optics and so on...our expertise in near-infrared optical design allowed us to make our own optical system...our cost is much lower, and we pass on our savings to the researcher."
Accessories available for the Pearl Imager include a suite of IRDye imaging agents and dyes, particularly targeting agents like labeled 2-deoxyglucose, EGF and RGD. They have also recently released the Clean Box. Says Harford, "As you're working with these animals, especially immunocompromised animals, you want to be able to transport them safely to and from the imager, without exposing them to outside elements because of their lack of immune system." The Clean Box keeps the animal isolated during transport to and from the imager, and throughout the imaging process.
Coregistration with Multiple Imaging Modalities
Due to background and other factors, it is often difficult to know the exact location of a fluorescent signal in the animal. The conventional method of solving this problem is to perform a different type of imaging on the animal and compare it with the near-infrared optical imaging in order to pinpoint the precise location of a tumor or other target structure or event. New advances in technology are making it easier to do these companion imaging studies. The Kodak In Vivo Multispectral Imaging System by Carestream combines multiple imaging modalities into one system, allowing nearly simultaneous optical imaging, X-ray, luminescent, fluorescent, or radioisotopic imaging. Says Bill McLaughlin, director of research and advanced applications for Carestream Molecular Imaging, "That system is the only system in the market that will allow researchers to do our modalities all in one system...What's really neat about it is that all of those modalities can be done without moving the animal. You don't change the zoom, you don't change the focal frame. It can be very precisely coregistered."
Dyes and Dots
Every imaging system uses reagents and consumables, and in vivo applications are no exception. Typically, infrared dyes are targeted to tissues by coupling them to antibodies. The optimal dye-molecule ratio for tagging antibodies is between 1.5 and 3 molecules. Kits such as Invitrogen's SAVI (small animal in vivo imaging) expedite the process of labeling using a patent-pending protocol that puts the right number of dye molecules on each antibody.
Creative uses of infrared probes and dyes can also have interesting results. In February, a research team from the National Institutes of Health (NIH) reported the use of a self-quenching probe that activates itself inside the cell for in vivo clinical studies. Currently, in vivo imaging has limited clinical applications, because indocyanin green is the only near-infrared dye approved for that purpose. The NIH group overloaded the targeting antibody, in this case, trastuzumab (herceptin), with seven dye molecules. Constructed in this fashion, the dye-antibody complex self-quenches, and is invisible under the imager. But when bound by an affected cell and internalized, the antibody is degraded, and the fluorescent dyes become activated and begin to glow. This lights up the tumor and shows the therapy is acting as intended, while avoiding two major causes of high fluorescent backgrounds: unbound reagent in the circulation, and reagent that non-specifically pools in tumors due to their leaky vasculature.1
Quantum Dots® are also being used for in vivo imaging. Organic dyes tend to be photolabile in the near-infrared window. Quantum Dots are photostable. Another advantage of Quantum Dots is that one excitation source can give you multiple colors, meaning you can multiplex in real time. It is also possible to do two photon excitation. In two photon excitation, the laser uses 1000 nm light, but by pulsing it, you deliver a concentrated excitation source that acts like half of its wavelength. The longer wavelength 1000 nm pulsed light can penetrate further into tissue, but excites with the combined energy of two photons instead of one. Quantum Dots also offer continuity between in vivo and in vitro studies. Says Mike Ignatius, PhD, product manager for in vivo imaging products at Molecular Probes, "Quantum Dots give you the ability to go from the macro view of a whole animal to the micro, which would be a fluorescent microscope, to the nano, which is an electron microscope...and that's pretty cool. One can image a target in a live animal at the macro level, at the micro level in a fluorescent microscope and eventually get down to the nanoworld in an electron microscope, using the same reagent."
Although in vivo imaging is predominantly a basic research tool, it has potential also for clinical applications and uses in live human patients in the future. As innovative tools continue to move the capabilities of in vivo imaging forward, we are sure to see what’s going on inside our tissues and cells with greater sensitivity. The knowledge and understanding that follows can only be a gain for the basic researcher, the clinician and the future patient.
References:
1Ogawa, M et al., “In vivo Molecular Imaging of Cancer with a Quenching Near-Infrared Fluorescent Probe Using Conjugates of Monoclonal Antibodies and Indocyanine Green,” Cancer Research 69, 1268, February 15, 2009. Published Online First January 27, 2009.