Abstract:
A novel functional imaging system for use in the imaging of unrestrained and non-anesthetized small animals or other subjects and a method for acquiring such images and further registering them with anatomical X-ray images previously or subsequently acquired. The apparatus comprises a combination of an IR laser profilometry system and gamma, PET and/or SPECT, imaging system, all mounted on a rotating gantry, that permits simultaneous acquisition of positional and orientational information and functional images of an unrestrained subject that are registered, i.e. integrated, using image processing software to produce a functional image of the subject without the use of restraints or anesthesia. The functional image thus obtained can be registered with a previously or subsequently obtained X-ray CT image of the subject. The use of the system described herein permits functional imaging of a subject in an unrestrained/non-anesthetized condition thereby reducing the stress on the subject and eliminating any potential interference with the functional testing that such stress might induce.

Description:
The United States of America may have certain rights to this invention under Management and Operating Contract No. DE-AC05-84ER40150 from the Department of Energy. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to methods and apparatus for the in vivo detection of tagged molecules in animals, especially small animals of the type used in laboratory studies, without the use of anesthetics and their attendant risks. 
     BACKGROUND OF THE INVENTION 
     Currently, the detection and imaging of tagged molecules in small animals, requires that the animal be rendered unconscious through the inhalation or injection of anesthetics or alternatively, physically restrained. These procedures are inherently undesirable because of the likelihood that the anesthesia or restraint will introduce complicating factors to the biological phenomenon under study. This is particularly an issue in neurological and brain function studies that are being done with small animals such as rats and mice. In addition, there is a substantial risk that the animal will be killed by the procedure, which event can carry a high cost in time resources and the overall effort, especially when the animal under study has been specially bred. Also, if several studies are planned for the same animal, it is not possible to subject the animal to too many episodes of anesthesia in a given period of time. In such studies, it is often desirable to follow the functional, metabolic or molecular activity for extended periods of time, extending through phases of animal activity such as sleep, active, etc. depending upon the type of label used to monitor a particular function (for example, uptake, washout times, isotope half life, etc.). 
     There therefore exists a need for a method and apparatus to perform such studies that does not require the anesthetization or other immobilization of such animals during the performance of such studies with all of the attendant risks to the animal. 
     OBJECT OF THE INVENTION 
     It is therefore an object of the present invention to provide a method and apparatus capable of in vivo anatomic and functional imaging of radioisotope and/or optically tagged molecules in non-anesthetized, non-restrained small animals that obviates the foregoing concerns. 
     SUMMARY OF THE INVENTION 
     The present invention describes a novel functional imaging system for use in the imaging of unrestrained and non-anesthetized small animals or other subjects and a method for acquiring such images and further registering them with anatomical X-ray images previously or subsequently acquired. The apparatus of the present invention comprises a combination of an IR laser profilometry system and gamma, PET and/or SPECT, imaging system, all mounted on a rotating gantry, that permits simultaneous acquisition of positional and orientational information and functional images of an unrestrained subject that are registered, i.e. integrated, using image processing software to produce a functional image of the subject without the use of restraints or anesthesia. The use of the system described herein permits functional imaging of a subject in an unrestrained/non-anesthetized condition thereby reducing the stress on the subject and eliminating any potential interference with the functional testing that such stress might induce. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a side view of a laser profilometry tracking system and burrow in accordance with one embodiment of the present invention. 
         FIG. 2  is an end view of the apparatus depicted in  FIG. 1 . 
         FIG. 3  is a right side view of one embodiment of the imaging apparatus of the present invention. 
         FIG. 4  is a diagrammatic end view of a standard SPECT imaging apparatus in accordance with the present invention. 
         FIG. 5  is a diagrammatic end view of a PET imaging apparatus using two orthogonal gamma detectors in accordance with the present invention. 
         FIG. 6  is a front elevation showing the configuration of one embodiment of the imaging apparatus of the present invention using two detector heads. 
         FIG. 7  is a front elevation showing the configuration of an alternative embodiment of the imaging apparatus of the present invention using four detector heads. 
         FIG. 8  is a process flow diagram showing the operations used to obtain anatomical and functional images of an unrestrained or non-anesthetized subject in accordance with the method of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention makes use of recent advances in a large number of advanced and sophisticated technologies to provide an imaging system capable of anatomically and/or functionally imaging radioisotopes and/or optically tagged molecules in non-anesthetized and non-restrained small animals. Among the technologies used, are: 1) laser/IR tracking of the animal under study in a relatively open environment such as a tubular containment area or “burrow” or other suitable enclosure using laser/IR tracking devices capable of determining and recording the orientation of an animal under study; 2) imaging labeled biological markers using small gamma, PET or SPECT detectors; and 3) dynamically collecting and reconstructing images normalized to the placement of the radioisotope or optically labeled regions within the animal body as determined by the laser/IR orientation determining devices. 
     The novel method and apparatus described herein make use of the following state of the art technologies to achieve this capability: 1) compact high resolution gamma imagers for single photon emission tomography (SPECT) imaging and positron emission tomography (PET) imaging; 2) modern IR and laser detection of labels applied to small animals or other subjects to determine their orientation in a specified time interval; 3) advances in radio- and optical labels applied in functional imaging; 4) compact image digital X-ray imaging systems making use of low dosage pulsed X-ray generators to obtain X-ray computed tomography (CT) images; 5) digital camera systems sensitive in the laser emission and IR regions of the spectrum; 6) fast digital signal processing technology; 8) high speed data acquisition and processing technology; and 9) fast image processing technology. 
     Thus, according to the present invention, a state of the art tracking system comprising: 1) multiple line-shaped IR laser beams that scan across an unrestrained subject, be it a small animal or a human subject, said subject being labeled with IR laser reflective tags; and 2) IR sensitive CMOS (complimentary metal oxide semiconductor) cameras that extract a linear IR laser profile from the information generated by the acquisition of the image generated by an IR laser strobe directed at the subject and the laser light reflected from the reflective tags as the CMOS cameras sweep across the subject, is used to spatially locate and map the body of an unrestrained and non-anesthetized subject, while SPECT or PET images of the subject are acquired. The laser acquired profilometry data and functional images acquired by the SPECT or PET imaging systems are then combined using state of the art software to generate a combined and registered profile and a functional image of the subject. The image thus generated can be further combined, i.e. registered, with a previously or subsequently acquired X-ray or similar image for analytical or diagnostic purposes. 
     While the apparatus and methods described herein are related primarily to the imaging/examination of small animals, it will be readily recognized that similar apparatus and methods can be applied in the examination/imaging of large animals and humans where, under normal circumstances and using conventional imaging techniques, the particular procedure being performed conventionally requires that the subject remain relatively motionless for some extended period of time, but that anesthesia or immobilization is undesirable or adversely influences the test being performed. 
     As used herein, the term “imaging volume” is meant to refer to the volume within which the subject is allowed to range or move during the imaging operation. In the case of apparatus suitable for the examination of small animals such as mice or rats the “imaging volume” will generally comprise a “burrow” or tubular structure within which the animal may be allowed to move freely during the imaging operation. In the case of large animals or human, the “imaging volume” will comprise a larger, but well defined volume within which the large animal or human can have free, but perhaps somewhat limited, movement during the imaging operation. 
     As shown in  FIG. 8 , anatomical and functional imaging is achieved in accordance with the present invention by first optionally, but preferably, obtaining an X-ray CT scan of the subject while anesthetized or immobilized. Once the anesthesia has cleared the subject&#39;s system, high resolution PET and/or SPECT and high resolution surface images of the unrestrained subject that has been injected or ingested a suitable tracer material are simultaneously acquired. Using calculated time-varying transformation coefficients and laser profilometry data the PET and/or SPECT images are reconstructed. Finally, the PET and/or SPECT mages are registered with the baseline X-ray CT data to provide and integrated functional and anatomical image of the subject under study. While the method shown in  FIG. 8  shows obtaining the X-ray CT scan of the subject while anesthetized or immobilized before imaging using the position/location system and SPECT/PET imaging system of the present invention, it will be obvious to the skilled artisan that X-ray imaging of the subject within the imaging volume may also be performed subsequent to the functional imaging with equally satisfactory results. 
     Referring now to  FIG. 1 , that depicts the essential elements of the tracking system of the imaging apparatus of the present invention, the tracking system  10  comprises: 1) an “imaging volume”, in this case a burrow  12 , within which the subject is permitted to range during imaging; 2) a plurality (at least two) of IR-lasers  14  capable of generating linear-shaped laser beams that can be scanned over the entire volume of burrow  12  as depicted by fields of view  16  and  18 ; and 3) a plurality of IR-sensitive CMOS cameras  20  and  20 A and  21  and  21 A that together are capable of imaging the entire volume of burrow  12  as depicted by fields of view  22 . While the description herein is provided in the context of the use of CMOS IR sensitive cameras due to the inherent heightened sensitivity and accuracy of such devices, it will be apparent tot the skilled artisan that as suitable IR sensitive camera, such as an IR sensitive CCD camera could also be applied to obtain a useful albeit somewhat less accurate imaging system. 
     The foregoing arrangement of elements is seen from a different angle in the view depicted in  FIG. 2 . As shown in  FIG. 2 , the apparatus of the present invention may include, in addition to the lasers  14  and gamma detectors  28  that can rotate about burrow  12  as described hereinafter, cages  12 A and  12 B at either end of burrow  12  that provide an volume for free range of the subject animal. Cages  12 A and  12 B may be carried on supports  40  and  42 , if desired to properly orient cages  12 A and  12 B in the complete apparatus structures depicted in  FIGS. 6 and 7  described below. 
     Tracking is accomplished using a two-staged approach where, in stage  1 , extrinsic landmark points, IR-laser reflectors or markers, not shown, are applied to the animal, for example at the skull, scapula, spine, ribs, pelvis and limb joints, and then imaged to allow quick extraction of the position of the critical anatomic features. In stage  2 , a time dependent surface of the animal is acquired that is subsequently used to accurately determine the position and pose of the animal during the study operation. An IR strobe integral to lasers  14  and invisible to the animal is used in conjunction with CMOS cameras  20 ,  20 A,  21  and  21 A to image the position of these markers or reflectors. The IR markers are easily segmented from the acquired images so that the position of the various skeletal features can be determined. These extracted skeletal markers indicate roughly where the rest of the body tissue is located. This is especially true for the head, thorax pelvic area and limbs. The reflective landmark points are also radio-opaque so that they are visible in X-ray CT volume of the anesthetized animal taken prior or subsequent to any SPECT or PET imaging study. Thus, these points will be easily detectable in both the laser surface profile surface data and the X-ray CT volume so that a fast, yet roughly accurate initial registration of the two data sets can be performed. The IR marker/reflectors are strobed and imaged by CMOS cameras  20 ,  20 A,  21  and  21 A between the laser profile scans of the animal surface. Given the frequency of laser scans that is possible (˜150 msec/scan) the animal markers can be imaged approximately every 200 msec (5 times/sec.). 
     A real time representation of the outer surface of the subject is generated using a commercially available laser profilometry system consisting of two linear laser profile generators  14  and four “smart” CMOS cameras  20 ,  20 A,  21  and  21 A. The CMOS cameras are designated “smart” because the CMOS has an on-chip data processing to extract the laser profile and measure via a triangulation algorithm the height of the object from the acquired images in real time, up to about 200 profiles per second. As shown in  FIGS. 6 and 7 , the two lasers  14  are mounted on a gantry  34  180° from one another and so that the laser profiles (beams)  16  and  18  can show through the gap between the gamma detectors. Burrow  12  is transparent to IR and preferably coated with an antireflective coating to allow maximum transmission of IR the laser profile. Burrow  12  is also opaque to visible light to avoid visual stimulation of the animal. Each laser  14  and  14 A is mounted to a rotating pin (not shown) so the laser profile can be swept along the length of burrow  12  perpendicular to the orientation of the projected laser profile  16  and  18 . Two CMOS cameras  20  and  20 A are also mounted on the gantry  24  shown in  FIGS. 6 and 7  to image the laser profile  16  as it sweeps across one side of the subject. CMOS cameras  20  and  20 A are preferably mounted away from the central plane of rotation of so they can view the laser profile  16  at an angle of from about 45° to about 60° from the axis of gantry rotation. A second pair of CMOS cameras  21  and  21 A are mounted to image the profile of laser  14 A as it sweeps across the opposite side of the subject. Two cameras are needed per side to eliminate surface occlusions. The height information extracted by each camera&#39;s on-chip processor is used to build two complete surfaces of the opposite sides of the subject. Because the geometry of the system is fixed, the two surfaces can be straightforwardly aligned and merged into a single data set for later registration to the surface extracted from the X-ray CT volume. While there will be partially incomplete data at the boundary where the two surfaces meet, this will not prevent effective registration of the laser profilometry surface to the extracted X-ray CT surface. 
     A variety of laser/CMOS/gamma camera configurations are possible and two of these are depicted in  FIGS. 2 and 3 . In the configuration depicted in  FIG. 2 , which is presented looking down burrow  12  with subject  26  facing the direction of the surface of the drawing. In this configuration four gamma detectors  28  are arranged about burrow  12  in a common plane that intersects the gantry&#39;s rotational axis with the two lasers  14  and  14 A and these are all rotated about burrow  12  to obtain profilometry and gamma imaging of all aspects of subject  26  simultaneously. It is obviously important that the placement of gamma detectors  28  with respect to burrow  12  be such that there is a large enough gap D, identified in  FIG. 2 , to allow CMOS cameras  20 ,  20 A,  21  and  21 A to have an unobstructed view of the entirety of burrow  12  in order that complete profilometry data be obtained. This arrangement of lasers  14  and  14 A and gamma detectors  28  is also shown in a more complete form in  FIG. 7  that shows a gantry  34  for supporting the various elements of apparatus  36  as well as a cabinet  32  for supporting entire apparatus  36  as well as providing storage for the various electronic and computer components necessary for the operation of apparatus  36 . 
     In the embodiment of the apparatus of the present invention depicted in  FIG. 4 , only two opposed gamma detectors  28  are utilized for the acquisition of the functional image of subject  26 . Again both lasers  14  and  14 A and gamma detectors  28  are mounted on a the rotary gantry  34  as depicted in a different view in  FIG. 6  that also shows a cabinet  32  upon which gantry  34  is mounted. Cabinet  32  may also be used to store various of the electronic and computer equipment that are necessary to complete operation of apparatus  36 . 
     The PET and SPECT imaging devices  28  may be of any of a number of possible such devices. Recently introduced “mini gamma cameras” using parallel hole collimators as depicted in  FIG. 4  are one possible alternative for SPECT imaging while so-called coded aperture masks as discussed by S. R. Meikle et al, “An Investigation of Coded Aperture Imaging for Small Animal SPECT” IEEE Medical Imaging Conference, Lyons, France, 2000 and as depicted schematically in  FIG. 2  provide an alternative such SPECT camera configuration. PET imaging can be obtained using more conventional positron imaging device that are well known in the art, for example, the mini-gamma camera described in U.S. Pat. No. 6,271,525 to Majewski et al. 
     As will be apparent to the skilled artisan, a number of variations and modifications can be made to the system described above without departing from the spirit and scope of the present invention. All such modifications and changes are clearly contemplated as being within the scope of the invention as defined by the appended claims.