An in vivo imaging modalities overview
In vivo imaging in laboratory animals is a relatively new methodology that is gaining fast acceptance in biomedical research. Traditionally, most data obtained from animal studies is derived from in vitro analyses of tissue samples collected from cohorts of treatment groups. The two principal motivations advocating implementation of in vivo imaging are: 1) the obvious advantage of gaining data non-invasively and longitudinally from the same live animal; and 2) the 3 R’s principle of animal research regulatory effort (Reduction, Replacement and Refinement). Consequently, one gains more data points from fewer animals.
Imaging modalities frequently utilized in in vivo research are mostly adaptations from familiar, clinical imaging instrumentation such as X-ray, ultrasound, CT, SPECT, PET and MRI. However, newer technologies such as Optical Imaging and Intravital Microscopy originated with lab animal applications in mind, not ruling out longterm translational applications.
The diverse choice in imaging platforms available may foster the question of which modality is the best fit for your research model. Three parameters dictate your choice: a) disease model, b) probe and, c) imaging modality. “We let the biology choose the assay, not the other way around,” comments Kevin Foley, Ph.D., director of in vivo pharmacology – Synta Pharmaceuticals (Genetic Engineering News Aug 1 2009 - Vol. 29, No. 14). This statement is key to the success of effective animal disease modeling. Drug development today requires animal models that specifically address biological events targeted by novel therapeutics for human disease. We need imaging to assess this drug efficacy non-invasively in vivo. These novel approaches may well deviate from conventional models and methods, but will provide better insight and highly reproducible data necessary to translate animal model observations to treatment of human disease. Molecular imaging in particular can provide faster insight using pharmacodynamic endpoints (e.g biomarkers specific for molecular drug or disease effects) as opposed to traditional lengthy efficacy studies with death as an endpoint. The ultimate goal is to refine, streamline and expedite the drug discovery process.
Different imaging techniques assess different parameters (anatomical, physiological, pharmacological and molecular). In certain cases, multi-modality approaches may be necessary. With this white paper we hope to provide a basic guideline outlining realistic expectations, advantages and disadvantages of the most commonly used imaging technologies. Each of the technologies described subsequently allow for non-invasive, non-traumatic imaging over a time. Anesthesia is recommended during most procedures. Acquisition times vary, as well as sensitivity, specificity, resolution, ease of use and cost. These methods allow for longitudinal assessment of disease and therapy within the same animal over time. Consequently, a significant reduction in animal numbers, an increase in biostatistical power, a decrease in in vitro processing, etc. are significantly advantageous.
Fig 1. In vivo imaging modalities: a sensitivity, resolution and cost comparison.
Fig 2. The Electromagnetic Spectrum and Biomedical Imaging.
Lab Animal in vivo Imaging Modalities (Part 1):
Ultrasound:
Ultrasound imaging utilizes the interaction of sound waves with living tissue to produce an anatomical image or, in Doppler-based modes, determine blood flow. Micro ultrasound allows visualizing anatomical structures with high spatial resolution and quantifying hemodynamic function in vivo with high temporal resolution, longitudinally in a non-invasive manner. For cardiovascular assessments dynamic, real-time images that can be analyzed to obtain quantitative structural and functional information create a significant advantage. 3D volumetric reconstructions can be generated of multiple 2D images. High resolution imaging on the other hand, is fundamental in developmental biology. E.g. mouse embryos can be imaged in real time from 5.5 days onward. With the development of high frequency imaging systems (25-80MHz), resolution of 30 micrometer at 15 mm depth are now reality. Contrast can be enhanced with the use of microbubble contrast agents which simultaneously can be used for the delivery of drugs or gene therapy. Ultrasound guided procedures such as intracardiac injections can facilitate and improve accuracy. Imaging obstacles are bone and air which can cause artifacts; tissue depth is also limiting. There is a requirement for higher technical skill and throughput is lower. Ultrasound is widely used and directly translational to the Clinic.Optical:
Non-invasive, whole body in vivo Optical Imaging allows you to monitor and assess disease, molecule biodistribution and molecular events in small animals through labeling with light emitting reporters. Bioluminescent, chemiluminescent and fluorescent reporters can be used to tag. Light emission from within the animal is detected by means of a cooled charged-coupled device camera. This technology is longitudinal, quantitative, non-invasive and allows for real time 2D imaging and 3D reconstruction. Tumor cells, stem cells, immunological cells or infectious disease can be labeled with a constitutively expressed luminescent or fluorescent protein. Transgene expression can be monitored by reporter constructs consisting of the promoter of the gene of interest driving expression of the light emitting protein. Molecular pathways can be assessed by means of inducible reporters, fusion protein or split reporter protein reporters. Enzymatic activity can be detected by means of chemiluminescent or enzyme sensitive substrates (eg caged luciferins, luminol, lugal, etc.) or fluorescent activatable probes. In vivo immunodetection with fluorescently labeled antibodies or biomarkers can target and identify e.g. tumors. Biodistribution of molecules or biologicals can be monitored by means of labeling the moiety with a fluorescent dye or quantum dots. This technology is non-invasive, longitudinal, high throughput, user friendly, radioactivity free and relatively economical. Optical imaging has excellent sensitivity (pmol concentrations, few cells) and luciferase imaging has high specificity. Anatomical Resolution is relatively poor (1mm) due to scattering of light in tissue. Light absorption by tissue limits detection to a couple of centimeters in tissue depth. Optical Imaging is currently minimally used in the Clinic, but translational applications are under development.Select references: Lyons et al., 2005; Luker and Luker, 2008; Pomper, 2005; van der Meel et al., 2010, Hickson, 2009; Leblond et al., 2010.
MRI:
Magnetic resonance imaging (MRI) is an anatomical imaging modality independent of ionizing radiation. Due to its non-invasiveness, MRI is well suited to longitudinal studies. MRI offers excellent anatomic organ and pathology (e.g. tumor) definition and accurate 3D, volumetric measurement with 10-100 micrometer spatial resolution. Stronger magnetic fields allow for better signal-to noise ratios and improve the spatial resolution of MRI images. For mouse MRI imaging, it is best to minimally have 4.7 T and preferably at least 7.0 Tesla.MRI imaging is based upon the proton movement of the hydrogen atom (H+), abundant as H2O in tissue. Protons are positively charged particles that spin in random directions in the absence of an external magnetic field. When caught in a strong magnetic field however, the protons align like compass needles and result in a net magnetization vector (NMV). When hit with precisely tuned radio frequency (RF) waves, the NMV flips from the longitudinal to the transverse plane. When the RF is turned off, the transverse component (T2) decays and the longitudinal component (T1) recovers. A brief radio signal is emitted and its intensity reflects the number of protons in a particular "slice" of matter. Contrast arises because different tissues have different decay and recovery times (T2 and T1 values).
In addition to inherent tissue contrast, exogenous contrast agents (CA) can be injected intravenously. Upon accumulation in tissues, CAs cause significant alterations in the local T1 and/or T2. MRI CAs can cause either positive (increased signal intensity) or negative (decreased signal intensity) contrast enhancement in MRI images of tissues or organs. Examples are chelated gadolinium (Gd3+), manganese (Mn2+), and iron (Fe3+). T1-weighted CAs such as Gd-DTPA or Mn2+ chelates shorten the T1 of tissue H2O, resulting in an increase in signal intensity in the tissues where the contrast agent has accumulated. Ultrasmall Superparamagnetic Iron Oxide (USPIO) are contrast agents consisting of suspended colloids of iron oxide nanoparticles. When injected during imaging they reduce the T2 signals of absorbing tissues.
There are three types of mouse MRI imaging: 1) anatomical imaging, 2) functional imaging and 3) molecular imaging.
Anatomical imaging is straightforward. Longitudinal changes of T1 and T2 values of tissues can reveal disease state such as tumor growth; or embryonic development.
Functional or dynamic imaging is mostly based upon blood flow, blood volume and blood oxygenation. Changes induced by these parameters alter the MRI image and allow for neurological, renal, cardiovascular or gastrointestinal pathophysiological assessments. An example of functional imaging is Dynamic Contrast-Enhanced MRI. DCE-MRI provides insight in tissue perfusion and vascular permeability. DCE-MRI has high-temporal resolution and can be used to detect the first-pass of a contrast agent. Clinical studies have shown that DCE-MRI can measure and predict tumor response to therapy (Taylor et al., 1999).
Molecular MRI imaging is a relatively new methodology. Different approaches have been described. As opposed to the before mentioned passive accumulation of contrast agents (CA) due to blood pooling, CAs can generate contrast “actively” due to target-specific binding of receptors by antibodies, peptides, etc. Recently, a dual modality magnetofluorescent nanoparticle with annexin for apoptosis imaging was published (Schellenberger et al., 2004). Responsive CAs such as enzyme sensitive reporters that alter relaxivity upon molecular processes have been developed. Cells can be loaded in vitro with e.g. iron oxide nanoparticles for in vivo adoptive transfer. And most recently, cells can be labeled with MRI imaging reporter enzymes. An example of such an enzyme is ferritin, a cellular iron storage protein that can be constitutively or inducibly expressed. Upon expression, cells massively take up iron which is sequestered in the iron storage protein ferritin. The accumulation of iron led to a pronounced increase in T2 relaxation rate. (Hoehn et al., 2008). Biosensors that change relaxivity under e.g. glucose concentration changes, presence of proteases etc. are in development. These magnetic relaxation switches consist of iron oxide nanoparticles that undergo reversible assembly and disassembly in the presence of a specific stimulus and change transverse magnetic relaxivity (Sosnovik and Weissleder, 2007).
MRI offers non-invasive, radiation free, high anatomical resolution and functional imaging. This is offset by low micromolar sensitivity, lower specificity, long acquisition and image process times, high technical expertise and relatively low throughput. Multispectral (multiple reporters) MRI is under development.
To Be Continued in the next Newsletter (Part 2: CT, SPECT, PET and Multi-modality)
