Macroscopic Optical scanning techniques image the actions of molecules and cells that are illuminated with bioluminescent or fluorescent probes in live laboratory animals. These techniques enable scientists to visualize actions of cells or molecules anywhere they occur within living small laboratory animals, and in some cases in laboratory sheep and pigs. Imaging of live laboratory animals is undertaken in a few major ways.
One approach is to use bioluminescent probes with light microscopes to image molecules in small laboratory animals, and then to superimpose the images onto MRI or CT whole body scans of the animal to identify the probes’ anatomical locations. Due to limitations of this bioluminescent imaging approach, however, the technique of choice for imaging cells and molecules in laboratory animals is tomographic optical imaging. This technology uses near infrared (NIR) fluorescent probes.
Optical tomographic imaging of live laboratory animals utilizes fluorescent probes with near infrared (NIR) light to observe biochemical activity that occurs deeper within the animals’ tissues. While animal tissue absorbs and scatters visible light, the amount of NIR light absorbed by tissues is less, enabling this imaging to penetrate further into laboratory animals’ tissues. In fact, scientists can detect biochemical cellular activity that occurs hundreds of micrometers beneath the animals’ skin. The amount of resolution provided by NIR alone, however, is insufficient for indentifying which specific cells within any location are actually emitting the light. This problem is addressed with the application of genetic and adoptive transfer techniques for creating fluorescent probes to use with NIR optical imaging. Fluorescence greatly increases the sensitivity of NIR imaging.
Optical tomography using fluorescent probes and NIR light requires an additional step compared to imaging whole animals with bioluminescence probes. In fluorescence optical tomography, light of a specific wavelength must be shined on the animal. This shined light, in turn, excites the molecule to emit light at a different wavelength from the light being shined on it. The emitted light then can be monitored by an imaging device.
Specifically, small animals are placed on a piece of glass in a dark chamber and the excitation light is switched on. Through the use of filters and a sensitive electronic camera, light emitted from the fluorescent molecule within the animal can be separated from the shined light, and quantified. Since the emitted light comes from many directions, tomographic detectors are placed in a circle around the animal to collect light coming from various directions. Computers combine the multiple individual views into three-dimensional images. Additionally, there have been increasing improvements in resolution, which now reaches millimeters
Tomographic imaging with fluorescence has two advantages over bioluminescent whole animal imaging. It allows scientists to increase the resolution to more precisely define the point that is the source of the emitted light. It also allows greater sensitivity and absolute quantification.
While PET imaging also is being used to image small animals in research, tomographic optical imaging for this purpose is now considered to be on a par with PET. Unlike PET, however, which is used in human imaging, tomographic imaging using fluorescently labeled molecules in humans is limited because the near-infrared light is not able to penetrate as far into thick tissues. Nonetheless, fluorescent optical tomography is beginning to be attempted in human breast imaging and colonoscopy, where tissues are not as dense as other human tissues. NIR probes are also being applied to endoscopy and surgical guidance in humans.