Abstract:
An optical CT scanner for small laboratory animals comprises a housing having a vertical through opening through which a test subject is passed through during a scanning session, the housing including a peripheral slot disposed transversely through the perimeter of the opening; a movable horizontal table disposed through the opening, the table being split with a horizontal slot aligned with the peripheral slot; a scanning head rotatable about the opening, the scanning head including a light beam directed toward the peripheral slot, the scanning head including a plurality of collimators directed toward the peripheral slot, the scanning head including a plurality of main photodetectors to detect the light beam after passing through the test subject and the collimators; a perimeter photodetector adapted to provide perimeter data of the test subject during a scanning session; an electrical circuit to amplify and digitize the output from the photodetectors; and a first computer programmed to reconstruct an image of the test subject from the output of the circuit.

Description:
FIELD OF THE INVENTION 
     The present invention relates generally to an optical computed tomography (CT) scanner and specifically to an optical CT scanner for scanning small laboratory animals, such as nude mice carrying cancer cells with fluorophore, such as green fluorescent protein (GFP). 
     BACKGROUND OF THE INVENTION 
     Fluorescence is often used in biology and particularly in optical microscopy for tissue identification. A fluorophore chosen for its abilities to bind to or ‘tag’ a specific type of tissue is introduced into the sample being interrogated. When the sample is illuminated by light at the excitation wavelength of the fluorophore, the tissue tagged by the fluorophore will emit light at the fluorophore&#39;s emission wavelength, thereby allowing optical detection of that tissue&#39;s presence and evaluation of the tissue&#39;s distribution within the sample. 
     A sizeable industry exists to support fluorescent optical microscopy. Several microscope manufacturers (e.g. Nikon Zeiss, Olympus) offer adapters for their instruments that allow fluorescent microscopy using the microscopes&#39; existing light sources. Biochemical suppliers such as Fluka BioChemika, Molecular Probes, Fuji Photofilm and Sigma-Aldrich supply scores of different fluorophores that are designed with specific optical and biochemical properties. Optics companies such as Omega Optical, Barr Associates, and Semrock supply optical filters for both the excitation and fluorescent emission wavelengths, allowing customization of microscopy equipment for specific fluorophores. 
     An extension of the concept of fluorescent microscopy is to image tissue-bound fluorophores in animals in vivo, or immediately post mortem, to ascertain the distribution of the tagged tissue. In this application, typically a white-light source is filtered with an excitation filter appropriate to the fluorophore, and a video camera, usually a CCD camera, views the test subject through an appropriate fluorescence emission filter. The camera produces a two-dimensional image that is a projection of the fluorophore&#39;s distribution onto a plane, much like a conventional two-dimensional x-ray. Although these two-dimensional images show the distribution of the fluorophore, accurate estimation of the quantity of tagged tissue within the sample (e.g. the volume of a tumor) is difficult without using actual three-dimensional fluorescent images of the sample. Several companies produce 2-D fluorescence imaging systems, notably Xenogen&#39;s IVIS® Imaging System, Berthold Technologies&#39; NightOWL LB98, and ART Advanced Research Technologies, Inc.&#39;s SAMI. None of these systems, however, show volumetric data that can allow quantitative estimation of tissue volumes. 
     Prior art fluorescent imaging systems, such as those described above, are used in cancer research to evaluate anticancer treatments in nude mice. These hairless mice carry a recessive gene that inhibits the development of the thymus gland. The mice are unable to generate mature T-lymphocytes and therefore are unable to mount most types of immune responses, including antibody formation and rejection of transplanted tissues. Cancerous allografts and xenografts are readily accepted and nurtured by the mice, making them excellent vehicles for the study of human cancers and their reactions to different treatments. Treatment efficacy is monitored by injecting a tumor-bearing nude mouse with a cancer-specific fluorophore and then tracking the change in tumor size with a fluorescent imaging system. 
     A technique developed in the mid-1990&#39;s creates cancer cells that are genetically altered to fluoresce, obviating an injected fluorophore. Green fluorescent protein (GFP), which is produced by certain jellyfish, emits green light when exposed to certain wavelengths of blue light. By ‘transfecting’ the appropriate DNA segment from these jellyfish into other cells, the cells are made to express GFP. In cancer research, the cancer cells are transfected with the GFP gene, and the progression of the tumor or its metastases can be monitored noninvasively by its GFP fluorescence using fluorescence imaging. 
     Recently, cells have been genetically modified to express fluorophores at other wavelengths (particularly by the Clontech division of Becton Dickinson), but GFP-expressing cells are much more widely used than any of these new cells. 
     Human cancer cells are being grown that are transfected with the GFP gene. These cancer cells are transplanted into nude mice, which do not reject the cancer, rather they nourish the cancer, allowing testing of anticancer treatments. The progression of the cancer or its metastases can be imaged with fluorescence of the GFP. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide an optical CT scanner for small laboratory animals injected with a cancer-specific fluorophore, such as green fluorescent protein (GFP), to monitor the progression of the tumor so as to evaluate the efficacy of cancer treatment being studied. 
     If is another object of the present invention to provide an optical CT scanner that can image both attenuation and fluorescence distributions in small laboratory animals injected with a fluorophore, thereby to locate and quantify the volumetric size of tumors growing in these animals for research purposes. 
     If is yet another object of the present invention to provide an optical CT scanner made to a relatively small size, for example, 400 mm cube, so as to be conveniently situated within a typically crowded laboratory setting. 
     In summary, the present invention provides an optical CT scanner for small laboratory animals, comprising a housing having a vertical through opening through which a test subject is passed through during a scanning session, the housing including a peripheral slot disposed transversely through the perimeter of the opening; a movable horizontal table disposed through the opening, the table being split with a horizontal slot aligned with the peripheral slot; a scanning head rotatable about the opening, the scanning head including a light beam directed toward the peripheral slot, the scanning head including a plurality of collimators directed toward the peripheral slot, the scanning head including a plurality of main photodetectors to detect the light beam after passing through the test subject and the collimators; a perimeter photodetector adapted to provide perimeter data of the test subject during a scanning session; an electrical circuit to amplify and digitize the output from the photodetectors; and a first computer programmed to reconstruct an image of the test subject from the output of the circuit. 
     These and other objects of the present invention will become apparent from the following detailed description. 
    
    
     
       BRIEF DESCRIPTIONS OF THE DRAWINGS 
         FIG. 1  is a front perspective view of an optical CT scanner for small laboratory animals made in accordance with the present invention. 
         FIG. 2  is a rear perspective view of  FIG. 1 . 
         FIG. 3  is the scanner of  FIG. 1  shown enclosed within a light-tight enclosure. 
         FIG. 4  is a cross-sectional view taken along line  4 — 4  of  FIG. 1 , with portions shown schematically. 
         FIG. 5  is a schematic view taken along line  5 — 5  of  FIG. 4 . 
         FIG. 6  is a partial front view of a vertical scanning head of the scanner of  FIG. 1 , with portions omitted, showing the arrangement of the collimators, photodetectors, the laser beam and the CCD cameras used for generating image data. 
         FIG. 7  is a schematic view along the scanning plane of the scanner head of  FIG. 6 . 
         FIG. 8  is an enlarged plan view of the collimator assembly, showing how a laser beam entering a test subject might exit and be picked up by the collimators. 
         FIG. 9  is a schematic view taken along the scanning plane of  FIG. 8 , showing a typical light path taken by the laser beam impinging the test subject. 
         FIG. 10  is a schematic diagram of the electronic system of the scanner of  FIG. 1 . 
         FIG. 11  is a block diagram of an algorithm used for image reconstruction. 
         FIG. 12  is an example of the excitation and emission spectra of GFP. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     An optical computed tomography scanner  2  made in accordance with the present invention is disclosed in  FIGS. 1 and 2 . The scanner  2  includes a housing  4  and horizontal front and rear tables  6  and  8 , respectively. The housing has a vertical opening  10  to allow the test subject to go from the front table  6  to the rear table  8  during scanning, as will be further described below. The housing  4  includes a well  9  having a bottom at which the opening  10  is disposed. The well  9  advantageously allows the user to observe the test subject as it progresses toward the rear table  8 . 
     The scanner  2  is placed within a light-tight enclosure  12  to prevent room light from contaminating the light transmitted through the test subject by the scanner. The enclosure  12  is provided with light-tight door  14  to provide access to the scanner  2 . 
     Video cameras  16  are provided within the enclosure  12  to provide remote monitoring of the test subject during the scanning period. Light fixtures  18  provide illumination within the enclosure  12  not detectable by the scanner, such as yellow or red light. The enclosure  12  is advantageously small, for example about 400 mm cube, so as to not occupy so much space in a crowded laboratory where space is a premium. A light-tight access port  13  for cables and other lines is provided. 
     Referring to  FIGS. 4 and 5 , the scanner  2  comprises a vertically rotating scanning head  20  comprising a rotating plate  22  mounted on a ball bearing  24 , which mounts on a stationary hub  26  supported by a vertical structure  28 . Laser diode  30  and its controller  32  are carried by the rotating plate  22 . The output of the laser diode  30  may be connected to an optic fiber  34  to a collimator  36  to project a parallel beam of light  38  across the scanning aperture  10  to illuminate a test subject  42 . Other methods of aiming the laser output to the collimator  36 , such as by mirrors, may be used. The laser beam is passed through an annular gap  11  in the housing  4  in the periphery of the opening  10 . 
     The rotating plate  22  also carries a plurality of collimators  44  (see  FIG. 8 ). Each collimator  44  is preferably connected by a respective optic fiber  46  to a respective photodetector  48 . However, each photodetector  48  may be directly coupled to the end of its respective collimator  44 , without using an optic fiber. The optic fibers  46  and the photodetectors  48  are also carried by the rotating plate  22 . The photodetectors  48  are preferably arranged in first and second arrays, where the first array  50  is above or to the left of the second array  52 . The collimators  44  can then be assigned alternately between the first and second arrays of photodetectors, so that the first array  50  may be assigned for attenuation light and the second array  52  for fluorescent light. The photodetectors  48  are carried by the rotating plate  22 . 
     There are preferably 84 collimators, spaced about 3.5° from each other, for a total 290° arc coverage. The collimators  44  and the laser beam  38  are aligned on a scanning plane through the annular gap  11 . 
     Photodetector amplifiers  54 , two CCD cameras  56  and a reference diode  58  are also carried by the rotating plate  22 . Data acquisition controller  60  is also carried by the rotating plate  22 . 
     A slip ring  62 , comprising a stationary rotor  64  and a rotating stator  66 , conveys electrical power and signals to and from the rotating electronics of the scanning head  20  to a stationary date acquisition module  68  and a stationary embedded computer  70 . 
     The rotating plate  22  is driven by a stepping motor  72  via a drive wheel  74  and an idler wheel  76 , which is spring loaded as schematically indicated at  77 . Advantageously, the speed ratio between the drive wheel  74  and the rotating plate  22  remains the same even when the idler wheel  76  wears out. The idler wheel  76  is preferably rubber surfaced for good contact with the wheels  74  and  22 , both of which are preferably made of metal. 
     Referring again to  FIG. 4 , the front table  6  is separated with a small air gap  80  from the rear table  8 . The gap  80  advantageously allows the emitted light from the test subject  42  to reach the collimators  44  and the respective photodetectors without further attenuation. The gap  80  is aligned with the scanning plane and the annular gap  11  at the opening  10 . 
     The front and rear tables  6  and  8  are functionally identical, each comprising an endless conveyor belt  82  passing over three large pulleys  84 ,  86 , and  88  and a small diameter pulley  90 . The conveyor belt  82  is a thin, preferably non-stretching materials, such as KAPTON or MYLAR. The small diameter pulley  90  is located adjacent the gap  80 , thereby advantageously allowing the gap  80  to be minimized preferably to a few millimeters. The small diameter pulley  90  can be a rotating pulley with small bearings, such as jeweled bearings, or can be highly polished stationary round pin that the conveyor belt  82  slides on, or other standard structures. The conveyor belt  82  is driven by the pulley  86 , which itself is driven from a stepping motor  92  by a toothed belt  94  via toothed-belt pulleys  96  ad  98 . The pulley  84  acts at the end of the respective table and the pulley  88  is a tensioner, moving vertically, either spring-loaded or relying on gravity or some standard means to tension the conveyor belt  82 . The top, outside surface of the conveyor belt  82  has a matte finish to better support the test subject, provide more friction against the driver pulley  86  and to minimize stray light reflection from being picked up by the photodetectors. The lower, inner side of the conveyor belt  82  is preferably smooth or shiny to minimize friction against the pulleys  84 ,  88  and  90 . 
     The stepping motors  92  that drive the conveyor belt  82 , and the stepping motor  72  that drives the scanning head  20  together provide a precise screw thread or helical pattern  99  for scanning the test subject  42 . As the scanning head  20  rotates continuously during scanning, generally indicated at  91  (see  FIG. 6 ), for example, at a rate selectable from about 5–20 sec./revolution, the tables at the same time move forward at identical rate, generally indicated at  93 , for example, at a selectable speed from about 0.05 mm–5 mm/sec., giving a minimum slice thickness at the fastest orbit time of 0.25 mm. 
     The front and rear tables  6  and  8  are mounted on respective vertical lift mechanisms  100 . The front and rear tables  6  and  8  are raised or lowered synchronously, generally indicated at  101 , remaining at the same height at all times. The tables  6  and  8  are raised or lowered to align the centerline of the test subject  42  with the center of rotation of the scanning head  20 . The lift mechanisms  100  are driven from the data acquisition module  68  under the control of the embedded computer  70 . The lift mechanisms  100  is of standard construction and may include vertical slides driven by a linear actuator comprising a lead screw powered by a stepping motor. Since the CCD cameras  56  measure the perimeter of the test subject during scanning, the height of the tables may be adjusted automatically and continuously during scanning to keep the test subject&#39;s centerline on the scanning head&#39;s centerline. 
     It should be understood that, although not shown, it is standard construction to provide the appropriates support structures to support the endless conveyor belts  82  and the associated pulleys and stepping motor with the vertical lift mechanisms  100 , such as that shown schematically at  102  for the front table  6  and  104  for the rear table  8  in  FIGS. 1 and 2 . 
     Referring to  FIGS. 6 and 7 , the CCD cameras  56  image the laser beam landing spot on the test subject through respective vertical slits  106 . The CCD cameras  56  are linear cameras, preferably 128×1 pixels, which image the lateral position of the laser beam landing spot as the distance to the surface of the test subject changes. For example, as the landing spot moves from a maximum position at  108  at the edge of the opening  10 , to a minimum position at  110  at the center of rotation of the rotating plate  22 , the spot imaged by the CCD camera  56  will move from one end of the linear camera to the opposite end, each position in between representing a specific perimeter point on the test subject. The perimeter of the test subject is thus obtained during scanning, as disclosed in U.S. Pat. No. 6,044,288. The vertical slits  106 , which are disposed perpendicular to the scanning plane (parallel to the plane of  FIG. 6 ), is advantageously used in lieu of a lens, to focus the laser spot on the CCD cameras. The vertical slits  106 , approximately 0.1 mm, will act as pinhole lens in the plane of the collimators  44 , and like a pinhole lens, will have a large depth-of-field. Being a slit perpendicular with the scanning plane, it is advantageously insensitive to aiming errors in that direction. Each slit  106  will take the landing spot and project it into a short vertical line intersecting the horizontal line of the 128×1 pixels of the respective CCD camera  56 . 
     Referring to  FIGS. 8 and 9 , the test subject  42  is illuminated by the laser beam  38  at point  112 . The laser light scatters within the test subject and is emitted from the surface of the test subject  42 , for example at points  114 ,  116 ,  118  and  120 , which are then received by the respective collimators  44 . During a scan, the assembly of collimators  44  continuously rotates while at the same time the test subject  42  moves through the opening  10  of the scanning aperture. 
     The laser diode  30  preferably has a beam diameter of about 0.3 mm–0.6 mm. at the center of rotation. 
     Referring to  FIG. 9 , the light exiting the test subject  42 , for example at point  118 , passes into a rectangular channel  122  of the collimator  44 . The centerline through the channel  122  and the laser beam  38  are preferably coplanar and preferably directed toward the center  123  of rotation of the scanning head. The collimator  44  preferably tapers in the scanning plane. At the end of the rectangular channel  122 , the light strikes a lens  124 , preferably a ball lens made of sapphire. The lens  124  focuses the light into the optical fiber  46 , which passes the light with minimal attenuation to a lens  126 , preferably a ball lens made of sapphire. The lens  126  collimates the light into an approximately parallel beam for passing through a filter  128  before striking a photodetector  130 , where the light is converted to an electrical charge and is then amplified. An opaque housing  132  contains the lens  126 , the filter  128  and the photodetector  130 . 
     The filter  128  is chosen for the fluorophore to be used in the test subject. For GFP, the filter  128  will pass light in the range 500–560 nanometers, with minimal transmission at the laser&#39;s wavelength of 440–495 nanometer. Preferably, every other collimator  44  will be filtered for fluorescence, and the rest will be assigned to measuring attenuation at the laser wavelength. The attenuation collimators are filtered to reject long wavelengths beyond 550 nanometers so that yellow and red lighting can be used to illuminate the enclosure  12  and not contaminate the scan data. The lighting in the scanning enclosure  12  advantageously allows the operator to monitor the status of the test subject via the video cameras  16  placed inside the enclosure  12 . 
     Attenuation data may be useful for providing anatomical landmarks within the test subject that otherwise would not be visible with fluorescence data. By superimposing the fluorescence image over the attenuation image, the fluorescence image may be properly located within the test subject. 
     Other assignments of the collimators  44  can be used. For example, the scanner  2  may be provided with two lasers at different wavelengths for exciting different fluorophores. In this case, one set of collimators can be assigned, with the appropriate filter, to detect fluorescence at one wavelength, another set for fluorescence at another wavelength and the rest for attenuation at one of the laser&#39;s wavelengths. 
     The assembly of collimators  44  preferably has 84 channels, with septa between channels as thin as 0.1 mm. The collimator channel  122  is preferably rectangular in cross-section. The collimator assembly is preferably fabricated by stereo lithography, a standard prototyping technology where the part is built-up additively in very thin layers with no constraint on geometry and adequate tolerances. 
     Referring to  FIG. 10 , an electrical schematic of the scanner  2  is disclosed. The laser diode  30  emits light that is attenuated and scattered by the test subject  42  and received by the photodiodes  130 , which is connected to a switch integrating operational amplifier  134 . The timing of the integration period effects a gain control, as disclosed in U.S. Pat. No. 6,150,063. Each switch integrating operational amplifier  134  is selected by a multiplexer  136  and presented to an analog-to-digital converter  138 , where the output of the amplifier is digitized. The switched amplifier circuits  134  are preferably grouped to correspond to the first and second arrays of photodetectors  50  and  52  for the embodiment of  84  photodetectors. 
     The timing and control of the multiplexer  136  and the converter  136  is provided by a field programmable gate array (FPGA)  140  located on the acquisition control module. The FPGA  140  controls the timing of the acquisition data, the CCD camera data and the reference detector data, all under the command of the embedded processor  70 . The FPGA  140  arranges (packetize) and serializes these data for transmission to the data acquisition module  68 . An example of the FPGA  140  is a Xilinx Spartan XCS40XL. 
     The reference photodiode  58  and the CCD cameras  56  connect to a multiplexer  142  where the output of each is selected and digitized by an analog-to-digital converter  144 . The digital data is presented to the FPGA  140 . The FPGA  140  serializes and de-serializes the digital data for transmission to the data acquisition module  68  over the slip ring  62 . 
     The data acquisition module  68  controls the stepping motors  72  and  92  and the stepping motor for the lifting mechanisms  100 . The data acquisition module  68  also buffers the acquisition data for the embedded processor  70 . 
     The embedded processor  70 , such as a PC/104 processor, controls the scanner operation and the collection of data. The embedded processor  70  communicates with the data acquisition module  68  via an ISA bus  146  (PC-80 bus). The embedded processor  70  uploads the acquisition data, performs some diagnostic functions and transmits the data to an operator&#39;s terminal  148  over a link  150 , preferably a 100 Mbit Ethernet link. Other types may be used, such as USB2.0 or FIREWIRE. The use of an embedded processor  70  advantageously allows multiple scanners to be controlled, from a single operator&#39;s terminal. 
     The operator&#39;s terminal  148  consists of a standard personal computer, a video monitor, keyboard, mouse and printer. The terminal  148  is used to record information on the test subject, set scan parameters and acquire image data. It is also used to reconstruct attenuation and fluorescence volume images and display multiplanar and interactive 3-D representations of the volumes. Further, the terminal  148  is used to calculate and save measurements from the volumes and output selected data and images for report writing. The terminal  148  also backups reconstructed volumes, snapshots and measurements to a storage device, such as DVD-R. Diagnostics may also be performed by the terminal  148 . 
     Fluorescence and absorption image reconstructions are accomplished with a filtered back projection algorithm as shown in  FIG. 11 . The algorithm at  151  extracts image acquisition data and CCD camera data from the helical set and create an artificial slice for reconstruction which is then used by the rest of the algorithm in a manner disclosed in U.S. Pat. No. 6,139,958. 
     The PC in the terminal  148  can also connect to the video cameras  16  in the enclosure  12 , preferably via USB 2.0 to display live images of the test subject during the scanning process. Since the test subject must remain motionless, the operator must know that the anesthetic has not worn off. 
     Referring to  FIG. 11 , an example of excitation emission spectra for GPF is disclosed. The vertical line  152  represents a 488 nm laser which will excite the GFP at close to its maximum efficiency. 
     The test subject may be a nude mouse transfected with GFP. The scanner  2 , equipped with the laser diode at about 440–495 nm, and the collimators filtered with bandpass filters in the range of 500–560 nm, will collect data over time on the growth of any tumor growing anywhere, including in the abdomen and lung, in the mouse that has been injected with GFP. The mouse is paralyzed and anaesthetized during the scanning process. A blind fold is placed over the mouse&#39;s eyes to protect them from the laser during the scanning process. The volume of the tumor can then be quantified, providing the researcher valuable information on the effectiveness of a drug under study. 
     While this invention has been described as having preferred design, it is understood that it is capable of further modification, uses and/or adaptations following in general the principle of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and as may be applied to the essential features set forth, and fall within the scope of the invention or the limits of the appended claims.