Figure 1. The petrie dish shows glowing bacteria on agar. (Xenogen)
Advances in genetic splicing of bioluminescent enzymes into living cells, combined with highly sensitive cameras, are giving researchers a new tool to explore in test animals a variety of conditions, including cancer, the interaction and screening of potential drug treatments, and the toxicological effects of new compounds and potential treatments. Today, as part of an experiment to judge the effectiveness of a new drug on tumors, for instance, biologists and chemists must resort to expensive 3-D imaging systems or sacrifice dozens of animals. Most 3-D imaging modalities are slow to acquire images and only show macrostructures within the living tissue instead of what is happening at the cellular level where drugs succeed or fail.
A better system would be faster, use self-multiplying tracers (or probes or reporters), and, of course, be cheaper, so that more laboratories could afford it. Researchers at Xenogen (Alameda, CA) have developed an in vivo imaging system (IVIS) that uses the light-emitting enzyme luciferase found in fireflies and soil bacterium to noninvasively track tumors and drug interactions in laboratory mice and rats.
Bioluminescence has been limited in research to in vitro assays because most of the emission is in the blue and green portions of the spectrum, where tissue absorbs the most light. "One thing that was not widely recognized is that luciferase peaks around 560 nm, which is not good for penetrating tissue," explains Brad Rice, Xenogen's director of physics. "The [luciferase] spectrum is quite wide, extending above 600 nm. This red component of the emission penetrates tissue very well."
For oncology applications, the IVIS imaging process starts by splicing the luc gene into tumor cells. The engineered cells are injected into immune-compromised animals ('nude' or SCID mice), and tumors begin to grow. Prior to imaging, a substrate called luciferin is injected into the animal, which, when combined with the luciferase protein, oxygen, and adenosine triphosphate, generates light. The animal can then be imaged and the light emission quantified. Because light emission is proportional to the number of tumor cells, IVIS provides quantitative measurement of tumor growth and, therefore, allows researchers to assess the effectiveness of drug treatments.
According to Rice, the advent of cooled CCD cameras made it possible to capture bioluminescence through tissue. IVIS uses a specially sealed chamber to hold the animal stage and a cryogenically cooled 1-in. CCD megapixel camera with an f/1 lens to capture the scattered photons. Software then overlays a false color luminescence image over a black and white photographic image of the anesthetized test animals.
Figure 2. The metastatic mouse was created by injecting 5 x 106 breast-cancer cells (MDA-MB-231-luc) into a tail vein. This image shows metastatic lesions from both the ventral and dorsal view at week 14. Signals were identified in the lungs, adrenals, spine, and lymph nodes.
The team also tested intensified cameras, but these cameras tend to use bi-alkali photocathode tubes with low quantum efficiencies in the red spectrum, or consist of smaller arrays, as with multi-alkali and gallium arsenide photocathode tubes.
Using the IVIS software, the operator can bin pixels into blocks to boost the signal-to-noise ratio and estimate the location and number of affected cells based on the emission spectra from the luciferase and diffusion models that take into account what types of cells (muscle, skin, etc.) lay between the luciferase-producing cells and the camera.
Alnawaz Rehemtulla, an associate professor of radiation oncology at the University of Michigan (Ann Arbor, MI), says that IVIS' ability to accept multiple animals and give a quick positive/negative read on the animals' condition has proven very useful in their experiments. "It has the advantage that it's rapid and can image a lot of animals at once, but the spatial resolution of the data is limited," Rehemtulla says. The diffuse light coming from the luciferase cells means that while IVIS can detect a brain tumor and provide a size estimate, a few thousand cells can create a pattern a couple of centimeters across (or the size of the mouse's brain), while a signal indicating a tumor in the midsection could arise from the ribs, spleen, or other organs. MRI/CT scans are typically done to reveal 3-D data on larger tumors, Rehemtulla adds.
Xenogen's Rice says that next generations of IVIS will feature improved spatial resolution.