Abstract:
An apparatus for imaging an animal includes a first mounting surface, a bed sized to support the animal and releasably secured to or integral with the first mounting surface. The apparatus also includes a plurality of straps, each having a first end in a fixed position relative to the bed and a second end for tightening around a limb of the animal. A method for in-vivo imaging of an animal includes providing an animal that has limbs, providing a first mounting surface, and providing a bed removably secured to or integral with the mounting surface and sized to support the animal as well as being coupled to a plurality of straps. The method also includes placing the animal on the bed between the plurality of straps and tightening at least two of the plurality of straps around at least two of the limbs such that the animal is substantially secured in place relative to the bed.

Description:
ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT 
       [0001]    This invention was made with Government support of Grant No. R24 CA 92865, awarded by the National Institute of Health, and DE-FC03-02ER63420, awarded by the Department of Energy. The Government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to in-vivo animal imaging, namely a method and apparatus for positioning and imaging small animals for in-vivo imaging. 
       BACKGROUND OF THE INVENTION 
       [0003]    In-vivo imaging of small animals to investigate biological functions, particularly those related to cancer, has become commonplace in the past few years. The advent of imaging systems such as the Concorde MicroPET, MicroSPECT, ImTek MicroCAT, MR, Xenogen IVIS optical imaging systems, and others specifically designed to image small animals, has resulted in a substantial increase in research of animal models of disease. Currently, each of these systems is designed and built independently. As such, each manufacturer has built its own stage for mounting animals or animal holders to its imaging system. Hence, there is currently no common platform for imaging animals in multiple systems without moving the animals to separate, proprietary stages. 
         [0004]    One of the main strengths of in-vivo imaging is the ability to image the same animal repeatedly over time or in different imaging devices and accurately compare the images. When an animal is used only for a single data point in a test, trends over time become more difficult to detect, as there are frequently individual differences between the animal subjects. Thus, substantially more data must be collected. Testing a single animal multiple times would thus increase the ease and efficiency of such testing. Data analysis of the resulting images is aided by reproducibly positioning the animal, such that the orientation is consistent across all the images. Current positioning systems, such as taping an animal in place or holding its head with toothbars and ear plugs, do not adequately provide such reproducible positioning of the animal&#39;s body. 
         [0005]    Positioning is also important in certain imaging systems to ensure that all of the animal is contained within a particular space, and that the animal is centered within the field of view. Some imaging devices, such as CT, are subject to considerable artifacts in the resulting images if any part of the animal extends outside the field of view. 
         [0006]    Combined or fused images are images formed by merging several images of the same subject taken at different times or using multiple imaging systems. Such fused images can be very beneficial for a researcher to review multiple biological structures, which may be visible in one imaging method but not another or for viewing changes in an animal over time. Alignment of the images when using multiple imaging systems is essential, therefore the animal must be held immobile during the entire imaging process. 
         [0007]    To compare separate images over time or to create fused images, data acquired from an image is typically measured in a device-specific coordinate system, which must be translated to a common coordinate system to compare with data from other imaging devices or sessions. This procedure is called “registration.” Accurate registration depends on knowledge of both orientation of the subject and its location within the imaging system. Since three dimensional objects, such as small animals, can be placed inside an imaging device in innumerable orientations, registration presents a difficult problem. 
         [0008]    Several types of registration are known in the art. Software registration employs software to track and correlate either landmarks on the subject or redundant data detected in the subject, such as an eye. External markers called fiducials fixed on the animal can also be used. These markers, however, may move relative to animals and create inaccuracies in the software image registration. Software registration is also limited in that only small changes in orientation can be corrected for. Software registration can be expensive, inaccurate, and time consuming, but is frequently used in small animal imaging for lack of an effective alternative. Additionally, there may be insufficient data available in one or more images for software methods to properly operate, as in the case when only a spherical tumor and nothing else is visible. 
         [0009]    Hardware registration, such as tracking the location of fiducial marker on hardware relative to the location of the animal is also used in some systems. The relative positioning of the fiducial to the animal, however, cannot typically be determined with sufficient accuracy when the orientation of the subject is changed slightly between sessions or devices, so hardware registration is typically not possible with multiple imaging sessions in small animals. 
         [0010]    Another problem with current imaging systems is that animals are exposed to pathogens. Research using small animals has increasingly utilized various types of transgenic and immuno-compromised animal models. Currently, the imaging systems used with these animals do not offer any type of pathogen barrier to shield the animals from pathogens in the open air. 
         [0011]    In-vivo imaging of live animals usually requires that the animals remain motionless during the image acquisition process. For most imaging experiments using small animals, this requires the animal to remain stationary for 10-60 minutes. Safe levels of injected anesthetics typically last only 30-50 minutes, and may not be suitable for longer experiments, or for experiments where two or more imaging systems are used to image the animal and exactly the same positioning is desired. Injected anesthetics also suffer from a variable depth of anesthesia over time, which may affect the biological processes under investigation. 
         [0012]    The use of gas anesthetics has become common. Gas provides a constant, easily controlled depth of anesthesia and offers essentially indefinite duration for longer experiments. The use of gas anesthesia is also safer for the animals since it is unlikely the animal will receive an overdose of anesthetic. Recovery times are also very short for gas compared to injected anesthetics, which reduces stress and the amount of time spent in an altered physiological condition. This is particularly important for imaging research where the same animal is frequently imaged, perhaps as often as once per day. 
         [0013]    To keep animals alive and healthy for imaging experiments where anesthesia lasts more than a few minutes, it is necessary to keep the animals warm to prevent hypothermia. Without heating, the effects of hypothermia will result in physiological stress or even death to the animals, which is likely to adversely effect uptake and metabolism of injected compounds used for examining biological functions or disease processes. Hypothermia-induced changes are typically not desirable. Therefore, animals are preferably maintained at or near normal physiological temperatures during imaging experiments. 
         [0014]    Currently, few systems offer any heating options, and there is not an integrated system available to ensure the animals are kept at normal physiological temperatures throughout the whole imaging experiment process. For microPET research, this is particularly important, since there is often a period of uptake after an imaging agent is injected and prior to image acquisition. If the animal is cold and peripheral blood supply is restricted to maintain core body temperature, there may be little or no uptake into subcutaneous tumors, thus compromising the intended investigation. One option used by some is heating of the air or gas anesthesia. However, this method delivers little heat, due to the low heat capacity of gasses, and when used for extended times can lead to dehydration of the animals. 
         [0015]    The creation of disease models in small animals is often a time consuming and expensive process. The complex nature of creating these animal disease models often requires weeks or months of preparation and analysis. Considerable investment in time and money is often spent to create and image these animals. Therefore, the loss of even a single animal can be quite substantial. There is a definite need for equipment and procedures that will aid the collection of imaging data and ensure the health of the animals. In addition, there is a need for ease of use to facilitate high throughput animal imaging to make the most efficient use of time and resources. 
       SUMMARY OF THE INVENTION 
       [0016]    To address one or more of the needs discussed above, an apparatus and method of in-vivo imaging of an animal is provided. An animal is placed on a bed that is sized to support the animal and held in place with straps coupled to the bed. The bed is removably secured to or integral with a first mounting surface. In this embodiment, the straps are tightened around at least two of the animal&#39;s limbs such that the animal is substantially secured in place relative to the bed. 
         [0017]    In a further embodiment, the first mounting surface is fixed to a second mounting surface associated with a first imaging device. The bed and animal are located within a field of view of the first imaging device and the animal is imaged to create a first image. 
         [0018]    In another embodiment, the bed is enclosed in a chamber and is environmentally isolated from the second mounting surface. Another embodiment of the method also includes separating the first and second mounting surfaces, fixing the first mounting surface to a third mounting surface associated with a second imaging device, locating the animal within a field of view of the second imaging device, and imaging the animal with the second imaging device to create a second image. Alternatively, the animal is removed from the bed after imaging, and the above steps are repeated to take a second image of the animal in the first imaging device. In a further embodiment, the first and second images from either the first imaging device or the first and second imaging devices are fused into a third image. 
         [0019]    Another embodiment of the system and method of the invention includes providing gas anesthesia through a mouthpiece. An air exhaust chamber may also be added to capture the air exiting from the chamber. The animal can also be heated through a heating element in the bed. In one embodiment of the apparatus, a cover encloses the bed to form an air sealed chamber. 
         [0020]    In one embodiment, the bed is curved. In yet another embodiment, the apparatus includes at least two posts projecting through openings in the bed and fixed to a supporting surface supporting the bed. The posts have circumferential grooves in one embodiment that receive the straps. The posts may also have a clamp at a distal end to fix the straps to the posts. The straps can then be tied or tightened around at least two of the animal&#39;s limbs. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0021]      FIG. 1  is a side perspective view of an apparatus according to one embodiment of the invention. 
           [0022]      FIG. 2  is an elevated perspective view of an unassembled chamber, bed and mounting plate shown in  FIG. 1 . 
           [0023]      FIG. 3  is a plan view of the chamber and bed of the embodiment shown in  FIGS. 1 and 2 . 
           [0024]      FIG. 4  is a side diagrammatic view of the embodiment shown in  FIGS. 1-3 . 
           [0025]      FIG. 5  is an end view of the embodiment shown in  FIG. 4 . 
           [0026]      FIG. 6  is a plan view of the embodiment shown in  FIG. 4 . 
           [0027]      FIG. 7  is a bottom view of the embodiment shown in  FIG. 4 . 
           [0028]      FIG. 8  is a side perspective view of one embodiment of a post according to the invention. 
           [0029]      FIG. 9  is a detailed perspective view of the post shown in  FIG. 8 . 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0030]    One embodiment of an apparatus for imaging an animal is shown in  FIG. 1 . An animal  10  is supported on a bed  20  that sits within a chamber  30 ,  32 . In this embodiment, both the chamber and the bed are acrylic, but it is within the scope of the invention to construct the chamber and bed out of any material suitable for housing and supporting the animal for imaging within the particular imaging device. 
         [0031]    The chamber  30 ,  32  is fixed to a first mounting surface  40  that also includes an anesthetic delivery system  42  and a heating element control  48 . The first mounting surface  40  is fixed to a second mounting surface  52  associated with a first imaging device  50 . In this embodiment, the first mounting surface  40  is a metallic plate coupled to one edge of the chamber  30 ,  32 , but it is also within the scope of the invention for the first mounting surface  40  to be any other material compatible with the imaging device  50  or a mountable surface of the bed  20  or chamber  30  itself that will provide durable, accurate positioning. 
         [0032]    In the embodiment shown in  FIGS. 1-9 , the first mounting surface  40  is attached to the second mounting surface  52  via two metal pins  54  and a capture screw  56  to firmly affix the surfaces  40 ,  52  together. 
         [0033]    The chamber  30 ,  32  is attached to the first mounting surface  40  using a single recessed screw  42  and two alignment pins (not shown). The first mounting surface  40  and chamber  30 ,  32 , however, can be fixed together using any suitable means that will hold the chamber  30 ,  32  firmly in place relative to the second mounting surface  52 . 
         [0034]    In this embodiment, only the mounting surfaces  40 ,  52  are separated between uses, with the chamber  30 ,  32  and the first mounting surface  40  left together. The first mounting surface  40  is further adapted in this embodiment to easily secure to a third mounting surface (not shown) on a second imaging device (not shown). This adaptation allows the apparatus to be easily moved from one imaging system to another with little or no alteration of the apparatus or positioning of the animal within it, increasing the ease and uniformity of positioning. 
         [0035]    The chamber  30 ,  32  in the embodiment shown positions the animal  10  in the center of the imaging field of view, maintains a pathogen barrier, supplies anesthetic gas, and keeps the animal  10  at a constant temperature. The chamber  30 ,  32  is of a split design for easy access to the internal, curved acrylic bed  20  holding the subject  10 , with the upper and lower halves  30 ,  32  held together by a machined groove  36  on the endplate  72  and a single thumbscrew  34  at the rear. However, one skilled in the art will understand that the upper and lower halves  30 ,  32  may be held together by any suitable means. 
         [0036]    Inside the chamber  30 ,  32  is a curved bed  20 , which has a resistive wire heating element  38  attached to its under side, a platinum resistance temperature sensor  39  at the rear of the upper side, and four notched posts  60  extending above the comers to serve as tie-points for securing the animal subject  10 . Also on the bed  20  is a nose cone or mouthpiece  22  for delivery of gas anesthesia. 
         [0037]    Tubes  44 ,  46  connected to the rear portion of the chamber  30 ,  32  allow for entry of gas anesthetic through the nose cone  22  at the front of the bed  20  and for the removal of gasses from inside the enclosed chamber  30 ,  32 . The chamber  30 ,  32  in this embodiment is constructed from materials allowing it to be effectively used with positron emission tomography (PET) and x-ray computed tomography (CT) imaging modalities, and is built to hold an average-size mouse. It is within the scope of the invention, however, to construct the chamber  30 ,  32  from any size and material suitable for the particular animal and imaging device being used. Factors that may determine the suitability are, for example, transparency to the imaging device, durability, rigidity, structural integrity, etc. 
         [0038]    Suture thread is used as straps  62  to tie the limbs of the animal  10  in place. However, any suitable material, such as paper tape, thread, etc., able to be tightened securely around limbs of the animal  10  can be substituted. The straps  62  are fixed to the posts  60  to maintain a fixed position of the subject  10  in the imaging system  50  field of view. These four posts  60  extend below the bed  20  and fit into holes  64  in the chamber  30 ,  32  to secure the bed  20  inside of the chamber  30 ,  32 . Alternatively, the posts  60  can be fixed directly to the bed  20  and/or lower half  30  of the chamber  30 ,  32 . 
         [0039]    One embodiment of a post  60  is shown in more detail in  FIGS. 8 and 9 . The post  60  has a lower end  66  that is insertable into openings  68 ,  64  in the lower half of the chamber  30  and the bed  20 , respectively (shown in  FIGS. 1 and 5 ). The center portion  67  of the post  60  has a larger diameter than the lower  66  and upper  69  ends. The upper end  69  includes a circumferential ridge  65  around which the straps  62  can be tightened. A clamp  63  at the upper end  69  of the post  60  can receive an end of the strap  62  to fix it in place relative to the post  60 . 
         [0040]    In a further embodiment, the posts  60  can be replaced by openings (not shown) in the corners of the bed  20  through which the straps  62  can engage. 
         [0041]    Referring again to  FIGS. 2 and 3 , the bed  20  provides gas anesthesia from underneath the animal  10  to allow the lower half of the chamber  30  to be used without the upper half  32 , if so desired. Alternatively, the bed  20  itself can make up the lower half  30  of the chamber. In a further embodiment (not shown), the upper half of the chamber  32  is flat, as may be useful, for example, in optical imaging systems. 
         [0042]    The chamber endplates  70 ,  72  contain grooves  36  that receive the two halves of the chamber  30 ,  32  to form a nearly gas tight connection. 
         [0043]    A small O-ring  24  is located within grooves near the center of one of the endplates  72  of the chamber  30 ,  32  to seal off an opening from a Luer fitting in the nose cone  22  that ensures that the gas flows to the nose of the animal  10 . The O-ring  24  also places a small amount of force against the nose cone  22 , which pushes the bed  20  against the back  70  of the chamber  30 ,  32 . The bed  20  can thereby be fixed in location relative to the chamber  30 ,  32  and the first mounting surface  40  in a reproducible manner. 
         [0044]    The temperature of the animal  20  can also be controlled in this embodiment during imaging. A thin, electrical heating element  38  is attached to the bottom of the bed  20  that holds the animal  10 . A temperature sensor  39  senses the temperature of the bed and sends this information to the electronics  48 . The electronics  48  control the heater  38  using active feedback to maintain the set temperature. To avoid overheating the animal  10 , risking death, both a minimum and maximum value of the heater  38  temperature can be set. 
         [0045]    In one embodiment, heat is applied to the underside of the bed  20  to prevent anesthetic-induced hypothermia in the animal  10 . A 19.1×101.6 mm sheet of clear polyester with an embedded, nickel-wire heating element  38 , such as Model H6701, available from Minco Products, Inc, Minneapolis, Minn., is attached by means of an acrylic pressure-sensitive adhesive. Due to the small diameter of the nickel wire (0.03 mm), minimal artifacts are introduced into any of the imaging modalities used during preliminary testing. 
         [0046]    Heater power is supplied through a miniature, on/off controller  48  such as the Minco CT325. The temperature can be monitored using a 5×12 mm, thin-ribbon, platinum resistance temperature detector  39  (Minco S665) attached to the upper, rear surface of the bed  20  over the heater  38  and connected to the controller  48 . This location places the sensor  39  outside of the imaging field. The electronics  48  for providing on-off control of the heating element  38  and to monitor the temperature, in this embodiment, are contained in a small, aluminum box mounted on the first mounting surface. 
         [0047]    In one embodiment of a method according to the invention, the animal  10  is placed on the bed  20  and its four limbs are tied with the straps  62 . The tension in the straps  62  is adjusted to substantially secure the animal  10  from movement without harming it or interfering with blood flow. The nose cone  22  is placed around the nose of the animal  10 . The top half of the chamber  32  encloses the animal  10  in its assembled state. 
         [0048]    Once assembled, the chamber  30 ,  32  can then be carried to the desired imaging system  50  and attached to the second mounting surface  52 . The first mounting surface  40  in this embodiment is aligned using two positioning pins  54  and held in place via a screw  56  designed for easy tightening by hand. Electrical power lines to the bed heater  38  and anesthesia delivery  46  and exhaust  44  tubing are then attached, and the chamber  30 ,  32  is ready for imaging. 
         [0049]    The first mounting surface  40  is fixed to the second mounting surface  52  and the second mounting surface  52  is moved to bring the animal  10  within the field of view of the imaging device  50 . The imaging device  50  can then take one or a series of images of the animal  10 . 
         [0050]    After the images are taken by the imaging device  50 , the first mounting surface  40  can be removed from the second mounting surface  52 , and the chamber  30 ,  32 , bed  20 , animal  10 , and first mounting surface  40  can be moved to a second imaging device (not shown). The first mounting surface  40  can then be fixed to a third mounting surface (not shown) associated with this second imaging device. An image taken with the second imaging device can then be co-registered through hardware registration with the first image. Creation of a third, fused image can also be accomplished through such effective co-registration. 
         [0051]    The apparatus and method described above is well suited for imaging of small animals such as mice and rats. In addition, the chamber  30 ,  32  described above can be well suited for a range of different imaging devices, including microPET, microSPECT, microCAT, small animal MRI, and optical systems. 
         [0052]    The embodiment described above also provides a reproducible method for positioning small animals used in imaging research. Through the use of tie-down posts  60  and light tension using suture thread  62 , animals can be quickly positioned on a bed  20  and enclosed in a chamber  30 ,  32 . Initial studies have shown that this embodiment of the chamber  30 ,  32  provides reproducible animal positioning for longitudinal studies with an average location difference of 790 micrometers. The anesthesia nose cone  22  on the bed  20  is designed to easily attach a Luer fitting from the tubing  46  delivering gas anesthesia. This allows the investigator as much time as needed to secure and position the animal  10  without concerns for the animal regaining consciousness. 
         [0053]    The bed  20  in this embodiment warms up quickly due to low thermal mass, and can also be preheated if desired. The bed  20  is curved to facilitate the reproducible positioning and to reduce the width of the animal  10 . By avoiding a flat platform, the animal  10  is in a more natural position and has a smaller horizontal cross section and therefore more uniform attenuation and potentially better imaging characteristics. This chamber  30 ,  32  and bed  20  thus contain the animal  10  within a certain space and hold the animal in the optimal position for the imaging device (typically the center of the imaging area). However, it is also within the scope of the invention to provide a flat bed, which may provide more effective optical imaging. 
         [0054]    The apparatus described above provides a stable platform for imaging in multiple systems without moving the animal relative to the apparatus, thus providing a fixed orientation of the animal for all the imaging modalities. 
         [0055]    Software image registration is a computationally demanding and difficult problem to solve, one that can be avoided or simplified using the chamber  30 ,  32 . With the chamber  30 ,  32 , often only a fixed offset is needed to align the images from different systems. Initial testing has shown that the movements in positioning when changing the chamber  30 ,  32  from one imaging device to another are minimal, ˜82 microns, and well below the resolution of current microPET systems. Thus, images can be registered using only hardware registration. 
         [0056]    The use of gas anesthesia and heating of the animal  10  to maintain normal physiology can reduce movement artifacts and effects of hypothermia. This can be particularly advantageous for longer experiments and especially for multiple experiments carried out over days or weeks. By maintaining a fixed temperature and depth of anesthesia, any changes in the image data can be related to the experimental intervention rather than the experimental conditions during the imaging session. 
         [0057]    Once the animal  10  is positioned on the bed  20 , it is a simple matter to place the bed  20  in the chamber  30 ,  32 , reattach the gas delivery line  46  from the nose cone  22  to the chamber  30 ,  32 , and replace the top half of the chamber  32 . The top half of the chamber  32  in this embodiment is specifically designed to fit closely, with an endplate  72  on one end that has a groove  36  to accept the lower portion of the chamber  30 . The snug fit is further enhanced by a small O ring  24  that provides a small amount of pressure to the bed  20 , as described above, to ensure the bed  20  is always in substantially the same location. The staging process of securing the animal  10  in this embodiment only takes a few minutes and typically is done just prior to the imaging session. 
         [0058]    For research with multiple animals using the same protocol and/or radioisotopes, several animals (not shown) can be prepared at once and held until ready for imaging. For experiments using multiple imaging modalities, it is easy to move the animals between systems and have the next animal prepared and waiting in a chamber  30 ,  32  to go into the next available imaging session to make maximal and efficient use of the imaging systems. 
         [0059]    Since the use of immunocompromised animals is commonplace, parts coming into contact with the animals can be sterilized. This embodiment of the chamber  30 ,  32  is designed in such a way that animals only come into contact with the bed  20 . The bed  20  can easily be sterilized using various commercially available solutions or gas. Although, in this embodiment, the animal  10  does not come into contact with the chamber walls, the entire chamber  30 ,  32  can also be sterilized if desired. 
         [0060]    By maintaining a constant flow of anesthetic gas, an even, reproducible level of anesthesia can easily be maintained. The anesthetic gas also produces a positive pressure within the chamber  30 ,  32 , therefore preventing pathogens from entering. 
         [0061]    The gas exits through the tube  44  in the back of the chamber  30 ,  32 , which serves two purposes. First, since the gas is delivered at the nose of the animal  10 , and vented at the other end of the chamber  30 ,  32 , the whole chamber  30 ,  32  is filled with the anesthetic agent, ensuring complete anesthesia, even if the animal is not well placed into the nose cone  22 . Second, the tube  44  for the exhaust has a Luer fitting for connection to the chamber, so the anesthetic agent can either be captured in an exhaust chamber (not shown) or vented. The pathogen barrier of the chamber  30 ,  32  permits in-vivo imaging of these animals using systems placed outside of barrier facilities and significantly expands the possible sites for installation and use. This reduces the expense related to barrier facilities, and increases the ease and speed of imaging studies. 
         [0062]    The heating element  38  is controlled by the electronic systems  48  and does not require adjustment or monitoring by the investigator. Since imaging experiments are typically complicated and have many details requiring attention, the ability to plug in the heater  38  and no longer worry about the temperature is advantageous. 
         [0063]    The invention has been described and illustrated by exemplary and preferred embodiments, but is not limited thereto. Persons skilled in the art will appreciate that a variety of modifications can be made without departing from the scope of the invention, which is limited only by the appended claims and equivalents thereof.