in vivo imaging reveals ongoing molecular mechanisms in living animals.
Given the choice, a biologist would rather see something as it exists in nature. “Cells in a test tube often behave differently than they do in a living organism,” says Alex Kleinman, vice president marketing and business development at Bioscan. “With in vivo imaging,” Kleinman says, “you can study the natural progression of disease and treatment in an animal, which improves efficiency and reduces errors.”
In some cases, researchers might wonder when imaging could benefit a particular project. As a rule of thumb, says Vivek Shinde Patil, manager of technical applications at Caliper Life Sciences, “if you can label it, you can image it. Labeling functional events in biological context is critical in truly characterizing disease progression and treatment.”
Lots of new tools make labeling and imaging easier and expand the capabilities of in vivo approaches.
Collecting images from living animals usually requires some way to label things. Promega makes a range of imaging vectors, including ones based on luciferase biosensors and HaloTag technology. “The luciferase biosensors, known as GloSensor technology, use genetically modified forms of luciferase that are either converted to an active form upon binding second messenger molecules—for example, cAMP, cGMP—or by cleavage of a peptide sequence by the cognate protease inside live cells,” says Jeff Kelly, Promega’s strategic marketing manager, cell analysis. “Examples include a GloSensor cAMP Assay and a GloSensor-DEVDG construct for caspase-3.”
HaloTag technology creates fusion proteins. As Kelly explains: “The HaloTag protein domain forms a covalent bond with synthetic ligands, which can be labeled with fluorescent dyes for imaging.” He adds, “Vectors are available for constructing HaloTag fusions with any protein of interest, as well as libraries of open reading frame clones expressing more than 7,000 human proteins as HaloTag fusions.”
In discussing the benefits of such imaging vectors, Kelly says, “The GloSensor-DEVDG—when used in a stable cell line engineered from a cancer cell line and implanted into a mouse xenograft model—provides an unprecedented fold increase of signal upon apoptosis induction, due to the extremely low background signal and the brightness of the active biosensor.”
Reaching in with Infrared
How deep a researcher can image inside an animal depends in part on the wavelength of light being used. “You want to be able to detect tumors or other targets deep inside the animal,” says Jeff Harford, senior marketing product manager at LI-COR Biosciences. Doing that requires the right imaging system and dye.
For example, LI-CORs’ FieldBrite Xi comes with lasers for excitation at 685 and 785 nanometers. “A lot of fluorescence-based imagers use white light,” says Harford, “and that won’t penetrate tissue deeply. Lasers get light deep into tissue. The FieldBrite Xi provides uniform illumination, with a coefficient of variation that is less than 5 percent.”
To maximize the potential of this approach, LI-COR makes IRDye 680RD and 800CW. “These are well suited for animal physiology,” Harford adds.
In the Fall of 2011, Bioscan will launch its BioFLECT, which stands for fluorescence emission computed tomography. “Most optical imaging systems utilize reflectance or transillumination,” says Kleinman, “and provide good information from only one side of the animal. The BioFLECT uses a rotating laser and detectors to gather information from 360 degrees.”
In addition, tomographic data collected with the BioFLECT can be acquired and integrated with data from the system’s inline x-ray CT. “This provides a truly integrated image of quantifiable functional activity together with anatomical context,” says Kleinman. “It provides consistent resolution of approximately 1 millimeter throughout a mouse or small rat.”
To track an animal over time with x-ray CT imaging, radiation exposure to the animal must be low. The additive effects of radiation can impact an animal’s physiology and introduce artifacts. Caliper Life Sciences recently launched the Quantum FX preclinical microCT system, which offers ultralow-dose, fast, high-resolution, whole-animal scanning for trabecular and cortical bone analysis. “With a rapid scan of 18 seconds, it only exposes animals to about 11 milligrays per image,” says Patil. “Longitudinal imaging protocols of scanning an animal once a week for two months will still result in less than 5 percent of the so-called lethal ablation dose.” Researchers can also co-register their anatomical images with three-dimensional bioluminescent and fluorescent tomographic reconstructions from Caliper’s IVIS Spectrum. The IVIS Spectrum enables quantitation of functional targeting down to, for example, the picomole of fluorescent probe.
With the speed of the Quantum FX, researchers can use the contrast agents that are given to humans in clinical settings. This is more cost-effective and simplifies translation to clinical research.
Bigger Can Be Better
As researchers expand the use of in vivo imaging, they want to examine larger animals. To help with that, PerkinElmer developed its recent Multispecies Imaging Module, which is available for PerkinElmer Fluorescence Molecular Tomography (FMT). With this module, says David Daniels, global product leader, pre-clinical imaging at PerkinElmer, “a researcher can scan mice and then shift to larger animals, like rats or guinea pigs.” He adds, “FMT can image in 3D in all depths within an animal.” An important feature of the solution is the ability to export FMT data in the DICOM format, which allows coregistering functional data with anatomical data from other imaging modes like CT or MR.
By being able to use different animals, Daniels says, this imaging system can be applied to different diseases. For example, he points out that rats work best when studying arthritis.
Beyond the ability to work with larger animals, the PerkinElmer imaging systems grab more data with the company’s TrueQuant analysis software. “The algorithm in our TrueQuant software captures every signal that is emitted from an imaging agent,” Daniels says. Using near-infrared dyes also allows for more penetration.
Fishing for Toxicity
At GE Healthcare, multiwell plates filled with 4-millimeter long zebrafish could unveil new aspects of developmental biology and be used in the pharmaceutical industry.
With the IN Cell Analyzer 2000 set on its 2× objective, one well of a 96-well plate fills the field of view. The brightfield mode—this automated imaging system also works with fluorescence—reveals structural details inside the zebrafish without using any labels.
“We’ve built an image analysis algorithm providing 14 organ-based measurements on five-day post-fertilization zebrafish,” says Robert Graves, senior application scientist at GE Healthcare. The morphology of untreated and compound-treated zebrafish can easily be compared using this algorithm. “This will generate a score of any deformity or abnormality in the experimental fish,” Graves says. So this makes an ideal platform for toxicity screening for pharmaceuticals being developed, because it can examine many animals under many conditions. Incorporating fluorescent imaging allows for up to four additional molecular markers to be studied.
With in vivo imaging, researchers can explore new aspects of biology, including how systems work and fail. These imaging systems supply a real look at life and how to maintain it.