Introduction. ILAR 42 (3): 187.
Nuclear magnetic resonance (NMR) spectroscopy and imaging, often referred to as magnetic resonance imaging (MRI),
has been a useful clinical tool for over 20 years and is being used more frequently in animal research. Another noninvasive imaging
technique is electron paramagnetic resonance (EPR) imaging. EPR is based on principles similar to NMR, and it detects paramagnetic
species. EPR images may be used to overlay functional information onto anatomical maps. EPR applications can complement other
noninvasive imaging techniques such as positron emission tomography (PET). PET images the distribution of compounds labeled with
positron-emitting radionuclides. Although used clinically and experimentally, PET has often been limited to larger species such as
primates. Development of high-resolution dedicated animal PET scanners has been critical in utilizing PET in rodent-based research.
Ultrasonagraphy has many research applications, and evolving technology has allowed its use and application in rodent research.
Transgenic mice have exerted a tremendous impact on biomedical and laboratory animal science. Many of the imaging technologies
mentioned above have been useful in rodent applications because they are noninvasive and allow for longitudinal studies in small animals
rather than relying on euthanasia and tissue harvesting at various timepoints. The same concepts apply to larger animals as well in that
noninvasive imaging technology can replace techniques that may have required necropsy as an endpoint. In pre-clinical drug research,
these technologies may provide insight into techniques or surrogate markers important in clinical trials. The main barrier or limitation for
these noninvasive technologies will be the time required to capture images. Ultrasound lends itself best to "high-throughput"
applications, as NMR (MRI), EPR, and PET involve extended image acquisition time and have the added burdens of expense and
equipment operating skill.
Questions:
1. What do the following acronyms stand for? NMR, MRI, EPR, PET.
2. T/F Ultrasonography is not covered by the OSHA noise standards.
3. What are some common advantages of NMR, MRI, EPR, PET, and ultrasonagraphy?
4. What are some disadvantages of NMR, MRI, EPR, and PET? What advantage does ultrasonography over these techniques?
Answers:
1. NMR - nuclear magnetic resonance; MRI - magnetic resonance imaging; EPR - electron paramagnetic resonance; PET - positron
emission tomography.
2. False. If the frequency is <20 kHz, it is covered by the OSHA noise standard.
3. Noninvasive, allows for longitudinal studies in small animals.
4. Disadvantages of NMR, MRI, EPR, PET - extended image acquisition time, expense, equipment operating skill that exceeds the
level normally found in a research facility. Advantage of ultrasonography - lends itself best to "high-throughput" applications, less time
required for image acquisition compared to other techniques.

 Nuclear magnetic resonance spectroscopy and imaging in animal research. ILAR 42 (3): 189.
Nuclear magnetic resonance (NMR) spectroscopy and imaging can be used to investigate, noninvasively biological processes including protein solutions, single cells, isolated perfused organs and tissues in vivo. It is also possible to combine different NMR techniques enabling metabolic, anatomical, and physiological information to be obtained in the same experiment. This review article describes basic NMR principles, advantages, disadvantages and potential uses. Atomic nuclei are charged and spin to form a magnetic dipole similar to a single magnet. Normally atomic dipoles are randomly oriented resulting in no magnetic field. However, when placed in a strong magnetic field the dipoles align resulting in a net magnetic field moment. When a second magnetic field is applied and turned off, the dipoles quickly realign to the first magnetic field and generate a radiofrequency signal when surrounded by a detector coil. This signal forms the basis of MR imaging and spectroscopy. To be effective, however, the second applied magnetic field (excitation field) must oscillate at the same frequency at the atomic nuclei; this condition is called the 'resonance condition', hence the name 'nuclear magnetic resonance'. The radiofrequency signal generated decays (called 'relaxation') at a rate that differs for different materials. So, by placing a sample in a magnetic field and by applying a second magnetic field, information can be obtained regarding intrinsic nuclear content and the environment of those nuclei. Nuclei in different chemical environments will resonate at different frequencies, an effect called 'chemical shift'. A technique called the Fourier transform is used to identify the frequencies of the signal, generating an NMR spectrum which can be used to determine the chemical content and structure of the sample. Small freely moving molecules typically yield narrow signals and vice-versa. Thus tissues, cells, membranes, molecules and metabolites (ATP, etc.) can be identified. Imaging is achieved by applying a linear magnetic gradient across the magnetic fields. When the linear gradient is applied, different parts of the sample are in different magnetic fields and produce different signals. A mathematical technique called 'projection-reconstruction' can be used to create an image of the sample. The greatest advantage of NMR or MR techniques is their lack of invasiveness. Both biochemical and spatial information can be obtained without destroying the sample. No ionizing radiation is produced. Computer assisted tomography uses x-rays, while positron emission tomography (PET) involves radioactive tracers. A wide range of biological processes can be investigated. Glucose metabolism can be evaluated in isolated neuronal cells in culture or in the brain in vivo. NMR spectroscopy can significantly reduce the number of animals required for a specific time points because animals can be re-evaluated over many time points. The greatest disadvantage is the intrinsic insensitivity of the method. Animal tissue is 70% water. Signals from water will always be detectable at two orders of magnitude or greater than other nuclei. Thus, compounds in submilimolar and micromolar concentrations cannot practically be detected. No known intrinsic risks are associated with high magnetic fields; however, the presence of the magnetic field can affect equipment in animal areas. Most MR techniques are motion sensitive, thus anesthesia is usually essential. The most commonly used NMR sensitive nuclei are 31P-, 13C-, and 1H-NMR. The 31P- spectra are a valuable tool for tissue bioenergics over time. There is a growing interest in the use of NMR for noninvasive evaluation of tissue metabolism in transgenic mice. The 13C- spectra can be used to monitor the transfer of 13C-labeled carbons from substrates into metabolites as in glycogen metabolism. 1H-NMR spectography has been applied primarily in the brain, prostrate, perfused cells and tumors. MR imaging on animal models provides exquisite pictures of soft tissues and can be used to discern a wide variety of pathologies. MR images can be sensitized to other physical processes and lead to the generation of temperature maps, current density maps, and pH maps. The development of so called 'functional MRI has lead to a flurry of activity in neuroscience. Signal changes can be observed in the brain in local tissues that undergo activation, including visual, verbal, acoustic and emotional stimulation.
Questions:
1. How many magnets are needed, minimally, to produce an image?
2. What is the greatest advantage/disadvantage of NMR spectroscopy?
3. What are the most commonly used NMR sensitive nuclei?
Answers:
1. 3
2. Advantage: noninvasiveness; Disadvantage: insensitivity
3. 31P-, 13C-, and 1H-NMR

 Electron paramagnetic resonance for small animal imaging applications. ILAR 42 (3): 209.
Electron paramagnetic resonance (EPR) is similar to nuclear magnetic resonance (NMR). Both detect resonance absorption: EPR detects species with unpaired electrons, such as free radicals; and NMR detects nuclei with non-zero nuclear spin, such as 1-H, 31-P, and 13-C. Because of low natural abundance of of paramagnetic species (free radicals), exogenous paramagnetic species, such as nitroxide radicals, or trityl-based stable free radicals must be administered to the animal. Toxicity of these "spin probes" is minimal, and they are well-tolerated in small animals. Image data from electron paramagnetic resonance imaging (EPRI) contains both spatial distribution (i.e. anatomic location) of the spin probes, and spectral information, allowing information such as tissue oxygen status and redox status of different tissues to be extracted. This modality is potentially useful for the study of tumor hypoxia, tissue heterogenicity with respect to oxygen and redox status, and vascular deficiencies in vivo.
Questions:
1. Name two paramagnetic species that can be administered to animals.
2. Which of the following statements is not true about EPR and NMR?
a. EPR detects species with unpaired electrons, which are naturally abundant in tissue.
b. NMR does not require the use of spin probes.
c. EPR is useful for the study of tumor hypoxia.
d. NMR and EPR both detect resonance absorption.
Answers:
1. nitroxide radicals and trityl-based free radicals
2. a

 Use of positron emission tomography in animal research. ILAR 42 (3): 219.
PET allows noninvasive measurement of many different biological processes and permits longitudinal calculations of animal models. The use of non-invasive imaging techniques, like PET, can reduce the overall number of laboratory animals utilized, as well as provide valuable information in-vivo.
Basic Overview of PET: A compound labeled with a positron-emitting radionuclide is introduced in the body, usually by IV injection. When the radionuclide atoms decay, a positron (the antiparticle to the electron with the same mass but with opposite electric charge) is emitted from the nucleus, and travels a short distance (a few tenths of a mm) before undergoing an annihilation reaction with an electron in the tissue. The annihilation of the two particles results in the simultaneous emission of back to back gamma rays, each of which carry 511keV of energy. These gamma rays can then be detected by a positron emission tomography scanner. The scanner consists of a position- sensitive scintillation detector, connected to a photomultiplier tube. The later collects the flash of light which is released when the gamma rays are deposited. Data collected from a PET scanner is not directly in the form of a tomograpic image but instead, must first be reconstructed using the well-established methods of computed tomography.
PET advantages and disadvantages:
Advantages Disadvantages
Availability of positron-emitting C isotopes Short half life requires immediate imaging
Short half life requires on-site cyclotron
Quantitative measurements of radionuclides
Increased sensitivity Dual radionuclide studies not possible
PET Use in Animal Research: The fist dedicated animal PET scanners were introduced in the early 1990's, primarily for larger lab animals (brain imaging of NHP's). The recent advances made in mouse genomics have produced significant motivation to extend PET techniques to the imaging of smaller animals. In order to accomplish this, (1) the spatial resolution must be improved, (2) the scanner technology must be advanced to a small, compact, cost efficient model, and (3) the PET radionuclides must be made readily available without the need for an on-site cyclotron. During the mid-late 1990's, several different scanners were developed and studied. In order to reach the sub mm resolution needed for small animal PET scanners, substantial improvements in both spatial resolution and overall sensitivity must be met.
Applications of PET in Animal Research: One of the major roles of PET in animal research has been in the field of neuroreceptor imaging, which depends heavily on radiolabeled tracers. One of the key limitations to PET in animal research is that the synthesis of these new probes can take many months-years to develop. Thus, the development of a "universal probe" that could monitor many different molecular processes would speed up this process. Alternatively, the "reporter gene approach" could be utilized. The later is the process by which a set of cells is altered to express a gene product which leads to an enzyme or receptor that is capable of trapping a radiolabeled probe. Two examples of reporter genes for PET imaging are herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor.
Future Challenges of PET in Animal Research: (1) Necessity of immobilization of the animal during the study; (2) Necessity of direct arterial blood sampling during the study for delivery of tracer tracking; (3) General cost and tracer access.
Questions:
1. True of False: PET relies on the unique decay characteristics of positron-emitting radionuclides?
2. True or False: PET imaging requires a collimator?
3. True or False: Gamma Camera imaging requires a collimator?
4. Gamma rays emitted during PET carry an energy of?
A. 500 meV
B. 550 meV
C. 600 meV
D. 511 meV
E. 411 meV
Answers:
1. True
2. False

 Ultrasound imaging: principles and applications in rodent research. ILAR 42 (3): 233.
This article, while focused primarily on rodent applications, provides an excellent summary of traditional ultrasound concepts as well as an overview of new ultrasound technologies. One of the key advantages to ultrasound is its non-invasive nature which can be used to refine experimental techniques that would otherwise require more invasive techniques. The basic principles of ultrasound are described below. Basic Imaging Principles Ultrasound waves have frequencies greater than 20,000 cycles/sec (Hz) with diagnostic ultrasound utilizing frequencies of 2-15 MHz (106 cycles/sec). Intravascular ultrasound systems utilize frequencies of up to 30 MHz and ultrasound biomicroscopy (UBM) systems operate at up to 100 MHz. The sound waves are transmitted through soft tissues relative to the impedance of each tissue. The acoustic impedance of each tissue is the product of the transmission velocity of sound and the tissue density. For most soft tissues, the velocity of sound is around 1540 m/sec so the impedence is primarily dependent on tissue density. Tissues with varying densities create an impedance mismatch which results in reflection of sound waves - the greater the difference in densities, the more waves that are reflected to the transducer resulting in a brighter area on the image. Bone tissue transmits sounds very rapidly while air and air-filled structures transmit sound very slowly. In areas where bone or air interface with soft tissue, the degree of impedance mismatch is increased resulting in a large reflection of the sound waves. The increased reflection results in decreased sound wave penetration and can cause artifacts in the image. Some basic terminology associated with ultrasound is summarized below: Resolution - resolution refers to the ability to accurately distinguish between two closely related structures or events. The ability to resolve very small objects is critical when dealing with rodents where the left ventricle chamber of a mouse is only 2 to 3 mm in diameter. Resolution is further divided into spatial and temporal resolution. Spatial resolution - refers to the ability to resolve objects in distance - further broken down into axial and lateral resolution. Axial resolution - the ability to distinguish 2 separate but closely positioned structures that are parallel to the US beam. This is dependent on both sound wave pulse length and frequency. In order for 2 structures to be differentiated, the pulse length must be shorter than the distance between the objects. As the frequency of the sound waves increases, the pulse length dereases thus improving axial resolution. Lateral resolution - the ability to distinguish 2 separate but closely positioned structures that are perpendicular to the US beam. This is dependent on both beam width and frequency. The beam width can be minimized by focusing the sound waves produced by the transducer. As the beam width narrows, lateral resolution increases. Lateral resolution can also be increased by increasing the frequency. Because both axial and lateral resolution improve with increasing frequencies, the higher frequency transducers (5 MHz and higher) are used for rodent imaging. The trade-off is that as frequency increases, the depth of penetration decreases. This presents less of a problem with rodents than with other species but does result in potential issues when UBM is utilized Temporal - the ability to resolve 2 distinct events in time. This is dependent on the number of image frames that can be acquired per second. The more image frames that can be collected, the better the temporal resolution. Temporal resolution is particularly important when using ultrasound to obtain cardiac images for rodents because of their high heart rates. Commercially available US units can acquire frame rates of 120 to 600 Hz Equipment The basic ultrasound equipment is listed below. Central Processing unit (CPU) or computer - this component controls the majority of the US functions including input to the transducer and monitor and reception of input from the transducer. This unit also enables data analysis and allows quantitative measurements to be obtained. Most newer US models can be upgraded using software technology negating the need to purchase additional equipment. Transducer - the transducer generates transmitted sound waves and receives reflected waves. It contains piezoelectric crystals that vibrate when exposed to electrical currents thus producing sound waves. When reflected sound waves are received, the crystals generate an electrical impulse that is processed by the CPU and relayed as an image to the monitor. Transducers are generally hand-held instruments but they are also available in the form of a catheter tip and in stationary arm format. It is recommended to use the highest frequency transducer that will provide adequate depth penetration. Monitor - this unit allows the images obtained via the transducer to be displayed visually Data storage system - this unit allows for the storage of collected data. In the past videotape or paper print-outs were used; however, most systems now use digital storage systems allowing for easier archiving and retrieval. Imaging Formats B-mode - this is the most commonly used 2-D imaging mode that allows operators to obtain longitudinal and cross-sectional views of organs. This mode is mostly used to obtain information on organ structure, motion and function. B-mode imaging has been used in mice and rats to evaluate cardiac structure and function, renal function, provide guidance for biopsies and injections and is used as guidance for the other modes. M-mode - this refers to motion mode imaging which is most commonly used in cardiac imaging (echocardiography). This format is uses a single beam transmitted through the organ with the image displayed over time (y-axis represents depth, x-axis represents time). M-mode imaging requires decreased beam widths and increased acquisition of frames. This mode is generally used to evaluate heart chamber dimensions. These measurements can be used to quantitate left ventricular function and other cardiac parameters. Spectral Doppler - The doppler mode is based on the doppler effect whereby there is a shift in soundwave frequency when reflected by a moving object (e.g., blood cells). The higher the velocity of the object, the more dramatic the shift. However, the doppler effect is also dependent on the alignment of the transducer with respect to the object. As the alignment diverges from parallel, the shift is attenuated. Thus, the US beam should be oriented as close to parallel as possible in relation to the direction of the flow or movement of interest. Spectral Doppler is used to display the velocity within a region of a vessel over time which results in a velocity profile. Spectral Doppler can be further classified as pulsed or continuous wave both of which are used to evaluate cardiac and peripheral vasculature function. pulsed flow doppler - This refers to the use of a single transducer to both send and receive sound waves. Pulsed flow enables more precise determination of a velocity profile for a specific region but the maximum flow velocity that can be detected is limited. continuous flow doppler - This refers to the use of separate transducers ? one to produce sound waves and a second to receive reflected sound waves. Continuous flow can detect higher flow velocities than pulsed flow but has decreased resolution. Doppler color flow - This technique employs a color-coded display showing velocity and direction of the flow. It can be used to evaluate valve competency and regurgitation in the heart and is also used in evaluating turbulence in peripheral vessels. Ultrasound Biomicroscopy (UBM) - Also referred to as high- or very high-frequency US imaging, this technique uses ultrasound technology to visualize tissues with microscopic resolution. UBM uses single element transducers in the 30 - 100 MHz range to acquire images using 2-D, B-Mode and Doppler images. These systems have axial resolutions ranging from 19 to 60 um and lateral resolutions between 60 and 250 um. Because of the high frequencies utilized, UBM has low temporal resolution and limited depth penetration thus cardiac imaging capabilities are limited. To date, UBM has been used in ocular applications such as imaging of the cornea and anterior chamber, evaluation of the cornea for disease or trauma and imaging of anterior chamber tumors. Dermal applications include measuring of skin thickness, evaluation of wound healing and assessment of dermal blood flow. Contrast-enhanced Ultrasound imaging - This technique utilizes microbubbles that are filled with inert gases. The gas filled microbubbles cause increased sound wave reflections which results in enhanced imaging. These agents have been used to enhance 2-D nd spectral and color-flow Doppler images. The microbubbles, which can be ruptured on exposure to high energy sound waves, have also been used in the targeted delivery of substances to specific organs. Transesophageal Echocardiography (TEE) - This technique is commonly used in humans as it allows better access for cardiovascular imaging with decreased interference from the lungs and other tissues. The commercially available systems are currently too large for use in rodents; however, a system utilizing a 10 French catheter has recently been adapted for use in rabbits. A high frequency (20 - 30 MHz) intravascular transducer has been used to obtain transesophageal images in rats and mice but currently the technology is limited in these species due to the decreased temporal resolution of these systems.
Questions:
For questions 1 -3, select the word in parentheses that correctly completes the sentence.
1. Lateral resolution increases as beam (width/length) decreases.
2. Axial resolution increases as the sound wave pulse length (increases/decreases).
3. As sound wave frequency increases, resolution (increases/decreases).
4. Diagnostic ultrasound, intravascular ultrasound, and Ultrasound biomicroscopy operate at frequencies of _______, _________, and __________, respectively
a. 100 MHz, 50 MHz and 10 MHz
b. 5-15 MHz, 30 MHz and 100 MHz
c. 30 MHz, 10 MHz and 100 MHz
d. 10 MHz, 100 MHz and 1000 MHz
5. Match the following applications with the appropriate ultrasound technology
1. Measuring velocity of blood flow through the heart
2. Measuring end diastolic volume of the left venricle
3. Measuring flow velocities through ocular vessels as small as 40 um.
4. Delivering substances to target organs
5. View the gross structure of organs
a. M-mode ultrasound
b. B-mode ultrasound
c. Ultrasound Biomicroscopy
d. Contrast enhanced ultrasound
e. Doppler mode ultrasound
Answers:
1. Lateral resolution increases as beam WIDTH decreases.
2. Axial resolution increases as the sound wave pulse length DECREASES.
3. As sound wave frequency increases, resolution INCREASES.
4. b.
5. 1e, 2a, 3c, 4d, 5b

 Challenges in small animal noninvasive imaging. ILAR 42 (3): 248.
The current status and challenges of small animal noninvasive imaging are briefly reviewed. Topics covered are: noninvasive imaging role in
postmortem situations, efficiently characterizing small animal phenotypes as well as pathology, data interpretation under anesthetized conditions and five imaging technologies are discussed briefly: magnetic resonance imaging and spectroscopy, ultrasound, computer-assisted tomography, positron emission tomography, and optical imaging.
Screening, using imaging modalities, could become an integral part in the study of the "functional genomics" of the mouse over the next
decade. However, the methodology to evaluate the physiology or phenotype of the mouse and other small mammal models is still developmental at best. The idea is to evaluate numerous structures and organ systems simultaneously without necessarily targeting one system and the ability to look at large populations of animals frequently required in genetic studies, especially in the area of mutagenesis. Due to the inherent surveillance nature of most noninvasive imaging techniques, these approaches are ideal tools for discovery in evaluating the phenotype of a mouse as they are for discovery of disease in a human patient.
CONSIDERATIONS-
Scaling of target tissue- At one extreme the physiologically relevant imaging volume is nearly fixed between mouse and humans. For example, in visualizing the resistance arterioles or capillary network of the skeletal muscle, the scale of the target vascular system is the same (150-10 mm). The other extreme would be where an increase in resolution volume of several orders of magnitude is required, similar to the overall scale of the animal. For example, a measurement in the heart is the distribution of work and blood flow across the heart wall, or the so-called transmural distribution. In a human, this distribution requires a spatial resolution on the order of 2 ´ 2 ´ 7 mm, whereas in the mouse heart, the resolution must approach 0.2 ´ 0.2 ´ 1 mm. Thus, depending on the questions addressed, the experiment must take into account the required resolution for the physiological or anatomical measurement and not simply the scale of the animal alone.
Postmortem Noninvasive Imaging
Advantages
In clinical studies many times the definitive phenotype, or disease, is defined in an autopsy and noninvasive imaging of morphology can be conducted at the highest level in a cadaver because no physiological motions are present and imaging time is not as critical. Thus, a well designed system for acquiring as much information as possible from postmortem animals may provide many investigators with the morphological and biochemical information they require in a timely and cost-effective manner. Noninvasive techniques are also advantageous compared with conventional gross pathology procedures, which require sectioning the animal and organs. This advantage is realized in cost, speed of acquiring data, and the nondestructive technology permitting follow-up postmortem studies as required.
Challenges
The development of the high throughput systems to evaluate hundreds of animals for screening purposes. In addition, the automation of image interpretation and analysis must keep up with this data flow as well as just the ability to store and transfer these large data files. For example, a single whole mouse image at a 50-mm isotropic resolution would
contain approximately 5 ´ 108 numbers. This is a remarkable amount of data for a small animal and likely only obtainable on a postmortem study due to the time required and physiological motion interference at this high spatial resolution.
Live Animal Studies
The major advantage of noninvasive studies is the ability to conduct studies on living animals without significant consequence to the animal or its physiology. However, live animal imaging studies are very difficult to perform because they generally require an anesthetized animal and animal technical support to monitor the animal throughout the procedure and recovery. In addition, the physiological motion, support issues, and limited time available for the scanning generally compromise the quality of the imaging data compared with postmortem studies. Studies requiring dynamic physiologic data, Internal longitudinal controls, valuable animals, the ability to monitor manipulations and the use of imaging as a screen for inclusion in a another protocol are some examples of studies that necessitate the imaging of live animals.
Anesthesia Procedures
Active restraint of animals is possible for ultrasound and some other modalities. However, the physiological effects or reproducibility of the physical and mental stress imposed on the animal is unclear, especially in cardiovascular studies. Thus, most studies must be conducted under anesthesia. A potentially confounding issue is transgenic animals in which the phenotype might be expressed as an enhanced sensitivity to anesthesia. The anesthesia regime must be picked to minimize the impact on the function of interest. The mouse and other small mammals present a challenge to maintain a stable anesthetic plane due to problems with mechanical ventilation and the difficulty of online measures/adjustments of physiological function. Ventilation, several significant anatomical and physiological considerations must be understood about rodents before one can fully understand acid/base and respiratory balance during imaging procedures. Thoracic compression is an issue with rodents in causing ventilation/perfusion mismatching. Rodents should be positioned at a slight incline, head above tail, to allow maximal costal movement. Additionally, the plane of anesthetic is important, and apnea must be avoided as much as is possible. Investigators can modify standard electrocardiographic equipment with nonmagnetic leads for the purposes of monitoring heart rate to assess depth of anesthetic. Also of importance is that while large animals tend to adjust minute volume by increasing tidal volume and decreasing respiratory rate per minute, rodents tend to compensate in rate.
The simplest and most consistent anesthetic regime used in MRI and PET procedures usually involves inhalation anesthetics and spontaneously breathing animal models. These simple nonventilated animal protocols have been very successful especially in studies that focus mostly on structure and not physiological function.
IMAGING MODALITIES
Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) MRI and MRS are based on the detection of the oscillating magnetic field induced from a special set of nuclides that posse a net spin in the presence of a strong magnetic field MRI generally refers to the determination of the distribution of one molecule, such as water or fat, within a tissue at high spatial resolution. This information includes a diverse amount of information on blood flow and oxygenation as well as macromolecular composition and motion, tissue structure, temperature, contractile activity, nerve and muscle fiber orientation, and edema. From these data, one can gather information ranging from structure to the chemical composition of some elements. Physiological information of blood flow, oxygenation, and volume is available along with the metabolism that is supported by these processes. Finally, information on the extracellular and intracellular milieu, including ion concentrations, pH, and temperature, can also be obtained. MRS generally refers to maintaining the spectral information in the magnetic signals from the nuclides, which permits the determination of the molecules or metabolites containing a given nuclide. The collection of this additional information in MRS along with the fact that metabolites are generally at low concentration results in the MRS experiment having a low SNR. These combined effects make any images collected with MRS very poor in spatial and temporal resolution. MRS maintains the spectral properties of the nuclides permitting the determination of the chemical species with in tissues. The nuclear magnetic resonance (NMR) spectral properties of a given metabolite are a function of the local magnetic interactions within the molecule providing, in most cases, a unique spectral fingerprint. Using this fingerprint, investigators can determine the concentration of a given metabolite, noninvasively.
MRI and MRS methods have been successfully applied to the mouse and rat due to the advantageous scaling factors that occur in magnetic resonance. Small animals also permit the use of small magnetic resonance receiving coils, which increase the sensitivity to the magnetic fields generated by the nuclides. In other words, the closer a coil can be physically placed to a target organ, the better the SNR of the measurement. Smaller subjects also mean that smaller magnets with higher magnetic fields can be used. It is apparent from the progress in small animal MRI and MRS studies that much of the utility of these approaches in man will translate to the evaluation of small animal physiology.
Ultrasound
Ultrasound relies on the modification of an induced acoustic wave traveling through tissue. Ultrasound studies are conducted using a probe to project sound into the animal and recording the time and magnitude of the reflected sound wave using the same probe. The analysis of this acoustic echo permits the imaging and measurement of tissue acoustic properties. Ultrasound is used primarily for monitoring tissue structure and motion. The amount of tissue characterization that can be accomplished with ultrasound is limited. Ultrasound is not very useful in the developed brain with an intact skull. Another limitation of ultrasound is the fact that the acquisition of data is very user dependent in finding the appropriate acoustic "windows" for access of internal organs and requires a skilled user. Despite these limitations, ultrasound has been the mainstay in the evaluation of cardiac wall function, blood flow, and valve performance as well as an important tool in monitoring fetus development. The advantages of ultrasound include its ease of use, portability, and relatively low cost compared with MRI, CT, and PET. Due to its portability and the rapid frame rate that is relatively insensitive to motion, it is conceivable that nonanesthetized imaging studies could be conducted on restrained animals if the stress on the animal were not too great or influenced the results significantly. Recently, the development of bubble-based contrast agents has improved the amount and quality of information in ultrasound imaging. Other important contributions have been harmonic-based ultrasound echo permitting higher tissue and agent-generated contrast and real-time three-dimensional ultrasound performed on small animals which may also remove some of the operator limitations as well as provide rapid whole animal studies. While ultrasound does scale appropriately with small animals the SNR of uultrasound is roughly constant, because the noise is a coherent "speckle" from internal reflections. Compared with MRI, challenges in ultrasound imaging are tissue contrast and SNR. Specific contrast agents may improve the utility of ultrasound beyond the structure/function studies now under way in animals. Ultrasound remains one of the least expensive and easiest to use of the imaging tools available for small animal evaluations concerning heart function and soft tissue structure outside the brain. Its ability to monitor fetal development under nonanesthetized conditions is also a valuable asset to the evaluation of development in transgenic animals.
CT
CT is basically a three-dimensional x-ray technique that is sensitive to the x-ray absorption of the tissue. Contrast can be generated by the differences in tissue absorption, with bone providing the most striking intrinsic contrast, or by using contrast agents to enhance the vasculature or specific tissues and conditions. The inherent SNR of CT is very high. Smaller animals provide some advantage in CT by permitting the use of low energy irradiation so that the size of the device can be greatly reduced, decreasing price, ease of shielding, and siting within an animal facility. Using current technology, full three-dimensional mouse images with 100 ´ 100 ´ 100 mm resolution can be obtained in a few minutes The high speed and high resolution of CT will clearly make it a valuable tool in the screening of large mouse populations. The major limitation of CT for use in small animals is the lack of information on tissue characterization and physiological function. Of all of the methods, CT will likely provide the greatest challenge in terms of data processing and data interpretation
PET
PET relies on detection of radioactive probes emitted in the body. Imaging of this emission is performed using a combination of detector geometry along with the timing of the emissions detection. PET is one of the most sensitive imaging techniques and is capable of detecting small amounts of radiolabeled material. The use of PET tracers and tracer chemistry is rivaled only by MRI/MRS in information content in an imaging modality. In addition to flow and metabolism markers similar in both PET and MRI, the sensitivity of PET has resulted in a unique ability to monitor receptor ligand interactions in humans and animals with remarkable success. One of the major drawbacks of PET is the requirement for a local cyclotron to generate the probes and synthesis unit to produce the biologically useful probes but most major medical centers already have such facilities where the tiny quantities required for small animal imaging can be easily obtained. Because radioisotopes must be used in these studies, vascular access or direct injection of the tracers into the organ of interest is required. The major advantage of PET is high sensitivity without the penetration limitations of optical techniques. The successful application of this approach will depend on the development of appropriate probes that will determine the specificity and sensitivity of the measurements.
Optical Imaging
Optical imaging is an extremely sensitive measurement that can detect a single molecule using fluorescence techniques. Optical imaging is usually performed in two modes: simple transmission absorption imaging and fluorescence imaging. In simple transmission absorption imaging, either transmitted or reflected light is used with tissue or optical probes, providing differential absorption to generate useful tissue contrast.
The major limitation of light is the high absorption and scattering that occur in biological tissues and limit the penetration of the light through the body. However, in small animals the required path-length of light is much shorter, which makes the use optics much more feasible. Using multiphoton fluorescence with specific protein fluorescence probes, the morphology and plasticity of neurons have been directly observed in rats. Whole adult animal screening has recently been shown using whole body IR fluorescence. The optical detection of molecular events is the most sensitive molecular imaging tool available in vivo. Due to the small size of the mouse and the ability to create optical markers for monitoring a wide variety of gene functions or even simple physiological and anatomical questions, it is clear that this approach will play a growing role in the noninvasive evaluation of the mouse phenotype.
Summary
Most of the standard imaging modalities used scale favorably to the size of a mouse or rat. These improvements in performance result in the maintenance of the physiologically relevant information in these images even though the size of the subjects has been reduced by several orders of magnitude. Due to its high sensitivity and specificity coupled with the ability to create genetically coded probes, optical imaging is likely to play a growing role in small animal imaging.
Major challenges in this approach are the maintenance and monitoring of an appropriate physiological state while conducting these studies. All of the modalities must be further modified to optimize their performance in the study of small animals; however, as noted in this review good progress is being made. Potentially, the largest technical challenge involves handling and processing the enormous amount of data provided by the approaches. One of the advantages of studying small mammals is that large numbers can be evaluated for genetic or mutant screening.
Imaging these large numbers will result in the mandatory development of computational systems capable of handling these large data sets. In addition, analysis of these data must be automated in some form to reduce the need for the investigator to screen each of these images. These automated image interpretation systems are also under development in the clinical radiology community where useful approaches may already be undergoing evaluation.
With the growing interest in the function of genes in development and function as well as the study of intact biological systems in mammals, it is clear that these screening imaging tools will play a critical role.
Questions:
1. Match the imaging modality with its description
1.MRI and MRS
2. Ultrasound
3. CT
4. PET.
5. simple transmission optical imaging
A relies on detection of radioactive probes emitted in the body
B. relies on the modification of an induced acoustic wave traveling through tissue.
C. detection of the oscillating magnetic field induced from a special set of nuclides that posse a net spin in the presence of a strong magnetic field
D. either transmitted or reflected light is used with tissue or optical probes, providing differential absorption to generate useful tissue contrast.
E. three-dimensional x-ray technique that is sensitive to the x-ray absorption of the tissue.
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