Lab Animal in vivo Imaging Modalities (Part 2):
(Part 1: Ultrasound, Optical, MRI)
CT:
X-ray computed tomography (CT) is an anatomical imaging modality which provides high spatial resolution, three-dimensional anatomical images. This technology provides anatomic localization, density and volume measurement and anatomic reference for coregistration with PET, SPECT and optical imaging. Image data is based upon differential x-ray absorption by different tissues. Most systems utilize a cone-beam x-ray source and a solid-state, flat panel detector rotating in a gantry around the subject, enabling the acquisition of a whole mouse in a single scan. For imaging systems dedicated to in vivo studies, the optimal resolution is 50–100 micrometer. Specimen instruments have resolutions down to less than 10 micrometer and can visualize e.g. trabecular bone structure.Tissue contrast depends mainly on differences in tissue density and on the presence of contrast agents. However, soft tissue contrast is poor even with using CAs. Living animal tissues best suited to image with CT and quantify pathogenesis are lung and bone. Common applications are detailed studies of bone and joint structure, bone metastasis scans, measurement of tumor size and location, and visualization of airway structure in the lungs. Respiratory gating does enhance thoracic imaging.
The major drawback of the technology is high radiation dose which can be lethal, mutagenic or therapeutic. Newer micro-CT instruments for in vivo imaging of small animals strive for a reduced dose, shorter exposure and therefore the possibility of longitudinal studies. Since iodinated contrast agents have a short half-life in circulation (10min) faster scan times permit the use of clinical contrast agents and the possibility of performing perfusion studies. Gold nanoparticles offer a new alternative to CT functional imaging. Gold is a better contrast agent than iodine and has a longer half life and can potentially be targeting.
SPECT:
Single photon emission computed tomography (SPECT) is a nuclear medicine tomographic imaging modality commonly used in the Clinic. SPECT uses gamma-emitting radioisotopes (99mTc, 111I, 123I, 131I, 125I) where a single γ-ray is emitted per nuclear disintegration. The basic technique requires injection of a gamma-emitting radioisotope that has been attached to a special ligand, which is of interest for its chemical binding properties to certain types of tissues. The specific binding of the radiopharmaceutical allows the detection of disease upon imaging with a gamma-camera. The use of targeted radiolabeled ligands to determine the tissue distributions of receptors is termed autoradiography. For in vivo 2D planar imaging, the method is called scintigraphy. With SPECT imaging is performed by using a gamma camera to acquire multiple 2D image projections from multiple angles. Tomographic 3D reconstruction algorithms then allows for visualization of the 3D biodistribution of the radionuclides. Small animal SPECT can provide resolutions up to 200 micrometer. Sensitivity, resolution, and field of view are dependent upon the pinhole collimator of the instrument. The collimator filters projections of certain directions in order to determine the direction of flight and the localization of the radioisotope. Multiple-pinhole and multiple-solid state detector systems are now available increasing the detection sensitivity with lower radiation dose and a scan time of just a couple of minutes. As opposed to PET, SPECT uses radioisotopes with longer half lives, for which synthesis does not require a cyclotron, thus offer greater accessibility. A large number of gamma-emitting radiopharmaceuticals, small molecules, peptides and antibodies, are routinely used in the Clinic. SPECT imaging can be used to monitor physiological functions, track metabolic processes and quantify receptor density.Dual-tracer imaging using SPECT, where multiple energy windows are used for simultaneous imaging of radiotracers using radionuclides emitting γ-rays at different energies, is one of the unique advantages inherent to SPECT technology. Per example 99mTc (140 keV) and 123I (159 keV) can be discriminated (Stout and Zaidi, 2008).
Imaging reporter genes specifically expressed in transduced cells have also been developed for PET and SPECT. An example of a commonly used reporter gene is thymidine kinase from herpes simplex virus (HSV-TK), which specifically phosphorylates and traps trace amounts of radiolabeled nucleoside analogues e.g. FIAU. This reporter can be expressed constitutively to track cell distribution or tumor growth or with inducible expression to e.g. evaluate molecular pathway activation.
PET:
Positron emission tomography is a clinical nuclear medicine imaging technique which produces a three-dimensional image or picture of functional processes in the body. PET uses positron-emitting radioisotopes (e.g.15O, 13N, 11C, 18F, 124I). The imaging system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide. When the positron emitting radioisotope undergoes positron emission decay, it emits a positron from its nucleus which then annihilates with an electron in close proximity, producing two 511 keV gamma photons that emit in opposite directions. The photons are detected when they reach a scintillator, creating a burst of light which is detected by photomultiplier tubes or silicon avalanche photodiodes. The coincidence detection of both γ-rays in PET within nanoseconds of each other defines the direction of flight and localization of the radioisotope.A commonly used positron emitting isotope is 18F. Fluorine-18 is produced in a cyclotron and has a short half-life of 110min. Since Positron-emitting isotopes are small, they can directly substitute atoms within the molecule of interest (e.g. 18F for a hydrogen atom) and readily label naturally occurring molecules or drugs for imaging molecular events and PK/PD studies. This is a significant advantage towards SPECT and fluorescence imaging in which the bulk of the tag potentially may modify the biodistribution and/or activity of the molecule. A commonly used PET imaging probe is 18F-labeled glucose, which accumulates in tumor based upon higher tumor metabolic rate and related higher glucose uptake compared to normal tissues. As described for SPECT, molecular reporter genes such as the thymidine kinase, dopamine D2 receptor, somatostatin-2 receptor and NIS (sodium-iodine symporter) can also be imaged with radiolabeled substrates. Of course, various radiolabeled targeting moieties and ligands can also be imaged.
As opposed to SPECT and optical imaging, it is not possible to accomplish multispectral imaging with PET, since all positron emitting isotopes emit two γ-rays of the same energy.
When comparing PET versus SPECT a couple of parameters can offer guidance: PET is best suited for small molecules and molecules with fast kinetics, PET has the highest sensitivity (pmoles vs nanomoles), PET tracers have lower specific acitivty, have shorter half-lives, are more difficult to synthesize and are more expensive and therefore less accessible. PET radiation dose is 10 fold higher than SPECT. SPECT suffers higher attenuation in larger species.
Most recently, a couple of laboratories have described the phenomenon of Cerenkov light emission by positron emitting radioisotopes (Robertson et al., 2009; Liu et al., 2010) and the possibility to detect these photons with optical imaging instrumentation.
Multi-modality imaging:
No single imaging modality can address all biological questions. Multimodality imaging resolves this issue. A perfect example is the combination of molecular imaging modalities (Optical, SPECT, PET) with anatomical imaging modalities (CT, MRI). This co-registration method allows visualization of molecular information within an adequate anatomic framework. Imaging Systems can be integrated or side by side. Feducial markers and multimodality imaging beds can enhance co-registration. Trimodality fusion reporter gene constructs Fluc-mRFP-tTK encompass bioluminescence (BLI), fluorescence and SPECT/PET imaging (Cao et al., 2006). BLI has the highest sensitivity in mice and rats and BLI scanning should be used initially to monitor the animals while the metastases are below the detection thresholds of PET and CT. When BLI shows lesions that have reached the detection thresholds of PET or CT, combined PET/CT scans can then be used for detection. This methodology facilitates translation to the Clinic (Deroose et al., 2007).