Bioluminescence and Fluorescence
The two types of visible light imaging are bioluminescence and fluorescence. Xenogen (Alameda, CA) is one of the industry leaders in the usage of bioluminescent markers, while AntiCancer (San Diego) has pioneered the use of fluorescent markers like Green Fluorescence Protein (GFP). Cambridge Research & Instrumentation (CRI; Woburn, MA) develops and manufactures instrumentation systems for small animal fluorescence imaging.
"When I think about the activities that a pharmaceutical company is involved inpicking a disease mechanism to target, testing a drug against a particular target, and if you use a drug, what are the side effects and safety issues that arisewe can do all of that," says Pamela R. Contag, Ph.D., president and co-founder of Xenogen.
In vivo imaging is playing an increasingly important role in bridging early preclinical drug discovery, which is often centered around in vitro and/or cellular assays, to later stages of preclinical and clinical development, which aim to elucidate and quantify a drug's effects in living organisms.
Xenogen uses the light-emitting reporter luciferase to tag genes and proteins within living animals. They create transcriptional fusions, with a promoter of interest driving luciferase expression, or translational fusions to create protein/luciferase chimeras. The tools of molecular biology and the power of mouse genetics can be harnessed to allow tremendous flexibility in the kinds of experiments that can be performed.
In a typical experiment, "you essentially set up a model, a mouse line, that represents something that occurs in a disease state. Once you have the model established, you can run a series of compounds against the model," Dr. Contag says. One example is the development of models for testing cancer compounds.
"Using our imaging system, you can watch the tumor cells emit light, grow, and calculate the rate of growth. Then you can administer drug and watch the light go away. Since it's nondestructive, you can watch the animal for a long period of time. If the tumor cells form resistance to the drug, you can see the light come back," she adds.
Fluorescent markers and imaging systems can also be employed for a variety of purposes, including tumor models. Both bioluminescence and fluorescence have explicit advantages over the measurement of tumor growth in animals using palpation, calipers, and/or histology.
"Each mouse has only one timepoint, and the caliper isn't reliable. You have to sacrifice lots and lots of animals," says Richard Levenson, M.D., director of R&D, biomedical systems.
"With imaging, you can get a full curve from one injection. You don't have to sacrifice animals, and the data is better quality, since each animal serves as its own control. It's not quicker or cheaper, but it's competitive in its cost effectiveness, and it's more ethical," explains Dr. Maclean.
According to Dr. Lassota, visible light is particularly useful for reporter assays in preclinical development. "I think we're making steady progress in reporter assays that can be used in vivo, and that at some point, these can replace efficacy assays.
"With reporter assays, you can get a mechanistic readout pretty quickly in mice. You don't have to wait for the tumor to shrink. You can effectively screen compounds with reporter assays, but they take a lot of work upfront."
CRI's fluorescence imaging system uses multispectral technology to reduce autofluorescence and improve sensitivity, allowing the detection of smaller and fainter targets.
"Our system enables the imaging of low level fluorescence in vivo. Before, it was completely obscured by background autofluorescence except for very bright signals," says Cliff Hoyt, vp and CTO of CRI.
"Fluorescence-based imaging also allows multiplexing," adds Dr. Levenson. "You can look at two, three, four things at once by taking advantage of different wavelengths. You unmix each from autofluorescence but also each from each other."
One of the problems with visible light imaging is that deep imaging is more difficult due to the scattering and absorbance of light. "The best results are for things that are more superficial," says Dr. Levenson, who adds that, by moving into the near-infrared range (750900 nm), signals can be detected from all the way through a mouse.
One of the hurdles can be finding appropriate molecular probes. "The imaging side is easier than the biology and the strategy. There can be problems with specificity and in where they gofor example, they can all end up in the liver and bladder," says Dr. Levenson.