Patent Publication Number: US-7595838-B2

Title: Multi-view imaging apparatus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under U.S.C. §120 from co-pending U.S. patent application Ser. No. 09/905,668, filed Jul. 13, 2001 and entitled, “MULTI-VIEW IMAGING APPARATUS”, which is incorporated herein for all purposes. 
     FIELD OF THE INVENTION 
     The present invention relates generally to imaging systems and their methods of use. More specifically, the present invention relates to imaging systems and methods used in capturing images from multiple views. 
     BACKGROUND OF THE INVENTION 
     One specialized type of imaging involves the capture of low intensity light (on the order of individual photons) from a light emitting sample, and the construction of images based on the photon emission data. This source of light in the sample visually indicates the origin of the activity of interest. For example, specialized in-vivo imaging applications may include analysis of one or more representations of photon emissions from internal portions of a specimen superimposed on a photographic representation of the specimen. The luminescence representation indicates portions of the specimen where an activity of interest may be taking place. The photographic representation provides the user with a pictorial reference of the specimen. 
     In-vivo imaging is performed by capturing an image of the sample using a camera. Intensified or cooled charge-coupled device (CCD) cameras are often used to detect the localization of low intensity light-producing cells in the sample. These cameras are considerably complex, require specialized cooling, and are fixed to a single location on the top of a specimen chamber. A user places a sample at a predetermined position on the bottom of the specimen chamber within the field of view for the overhead camera. This static relationship between camera and sample limits image capture to overhead images only. 
     Often, it is desirable to capture different views of the sample. For example, the detection of internal light-producing cells from the underside of a mammalian sample may be affected by covering tissue which the light must penetrate before being captured by the overhead camera. By gathering data from different angles, a user can obtain more information about the location and intensity of a light source in the animal than possible using only a single view. However, it may be impractical to reposition the sample to capture a different view when using an overhead camera. 
     In view of the foregoing, an improved imaging system that enables the capture of images from different views without repositioning the posture of the sample would be highly desirable. 
     SUMMARY OF THE INVENTION 
     The present invention relates to systems and methods for capturing an image of a sample. The sample is placed on a moveable stage in an imaging box. The moveable stage allows an image of the sample, or portions thereof, to be captured by a camera from different views, angles, and positions within the imaging box without repositioning the posture of the sample. As the sample is variably located within the imaging box, a light transmission device assists image capture by transmitting light emitted or reflected from the sample to a common datum associated with a camera. 
     In one aspect, the present invention includes a processor which is electrically coupled to the camera. The processor may also provide control of the moveable stage. In one embodiment, a transparent stage is used to allow image capture from angles beneath the stage. 
     In another configuration, the imaging system comprises an imaging box having a set of walls enclosing an interior cavity. The imaging system also includes a camera mount configured to position the camera relative to a fixed datum on one of the walls for viewing by the camera and a light transmission device. The imaging system additionally comprises a moveable stage apparatus including a transport mechanism and a stage configured to support the sample within the interior cavity. The stage is coupled to the transport mechanism for movement of the sample to one of a plurality of positions in the interior cavity. The transport mechanism and the light transmission device cooperate to direct light reflected or emitted from the sample to the fixed datum to capture the image by the camera. 
     In another aspect, the imaging apparatus comprises an imaging box including an interior cavity for receiving the sample and a stage for supporting the sample. The imaging apparatus further comprises a first linear actuator attached to the imaging box and capable of positioning the moveable stage in a first direction. The imaging apparatus additionally comprises a second linear actuator attached to the first linear actuator, attached to the stage, and capable of positioning the moveable stage in a second direction. The first linear actuator and the second linear actuator cooperate to position the stage at one of a plurality of positions in the interior cavity. 
     In yet another aspect, the imaging apparatus includes a positioning arm rotably coupled to the stage and rotably coupled to the imaging box such that the stage remains substantially horizontal for any rotational position of the positioning arm relative the imaging box. The imaging apparatus additionally includes a mirror attached to positioning arm. The mirror is configured to reflect light emitted from the sample at least partially along a fixed datum. 
     In another aspect, the invention relates to a method for imaging a sample. The sample is supported by a stage moveable within an imaging box that is coupled to a camera configured to capture an image of the sample. The method includes moving the stage to a first position in the imaging box. The method also includes capturing a first image of the sample from the first position using the camera. The method further includes moving the stage to a second position in the imaging box. The second position has a different angle relative to a fixed datum associated with the camera than the first position. The method additionally includes capturing a second image of the sample from the second position using the camera. 
     In still another aspect, the invention relates to a stage apparatus for use with an imaging system for capturing an image of a sample with a camera. The imaging system includes an imaging box having a set of walls defining an interior cavity, and a camera mounted relative to a fixed datum on one of the walls. The stage apparatus comprises a light transmission device, and a transport mechanism. The stage apparatus further includes a stage configured to support the sample within the interior cavity where the stage is coupled to the transport mechanism for movement of the sample to one of a plurality of positions in the interior cavity. The transport mechanism and the light transmission device cooperate to direct light reflected or emitted from the sample on the stage to the fixed datum to capture the image by the camera. 
     These and other features of the present invention will be described in more detail below in the detailed description of the invention and in conjunction with the following figures. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which: 
         FIG. 1A  is a perspective view of an imaging system including an imaging box adapted to capture images in accordance with one embodiment of the invention. 
         FIG. 1B  illustrates the structural components of the imaging box of  FIG. 1A  in accordance with one embodiment of the present invention. 
         FIG. 2A  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage directly below a fixed datum in accordance with one embodiment of the present invention. 
         FIG. 2B  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage below and off-center from the fixed datum in accordance with one embodiment of the present invention. 
         FIG. 2C  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage above and off center from the fixed datum in accordance with one embodiment of the present invention. 
         FIG. 2D  illustrates an internal side view of a side wall and housing included for the box of  FIG. 1A  in accordance with one embodiment of the present invention. 
         FIG. 2E  illustrates an internal top perspective view of a side wall and housing for the box of  FIG. 1A  in accordance with one embodiment of the present invention. 
         FIG. 2F  illustrates a simplified view of light transmission within box using the light transmission device included in box of  FIG. 1A . 
         FIGS. 3A and 3B  illustrate a top and side view, respectively, of the stage included in the imaging box of  FIG. 1A  in accordance with one embodiment of the present invention. 
         FIG. 3C  illustrates a top perspective view of drawer and electronic components housed therein in accordance with one embodiment of the present invention. 
         FIG. 4A  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage directly below a fixed datum in accordance with another embodiment of the present invention. 
         FIG. 4B  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage below and off-center from the fixed datum in accordance with another embodiment of the present invention. 
         FIG. 4C  illustrates a top perspective view of the components in the box of  FIG. 1A  with the exterior walls removed showing the moveable stage above and off center from the fixed datum in accordance with another embodiment of the present invention. 
         FIG. 4D  illustrates a gearing mechanism used to maintain the horizontal position of the moveable stage of  FIG. 4A  in accordance with another embodiment of the present invention. 
         FIG. 5  is a process flow illustrating a method of capturing photographic and luminescence images using the imaging apparatus of  FIG. 1A  in accordance with one embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description of the present invention, numerous specific embodiments are set forth in order to provide a thorough understanding of the invention. However, as will be apparent to those skilled in the art, the present invention may be practiced without these specific details or by using alternate elements or processes. In other instances well known processes, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present invention. 
     I. Imaging System 
     In one aspect, the present invention relates generally to improved imaging systems.  FIG. 1A  illustrates an imaging system  10  adapted to capture photographic and luminescence images in accordance with one embodiment of the present invention. The system  10  provides user automated control of image capture in an imaging box  12 . The imaging system  10  is also useful for capturing and constructing structured light images. 
     The imaging system  10  comprises an imaging box  12  adapted to receive a light-emitting sample in which low intensity light, e.g., luciferase-based luminescence, is to be detected. The imaging box  12  includes a housing  16  on a side vertical wall of the box having a camera mount  109  ( FIGS. 2A-2C ) adapted to receive a camera. The imaging box  12  is configured to be “light-tight”, i.e., essentially all external light is prevented from entering the box  12  from the ambient room. 
     A high sensitivity camera, e.g., an intensified or a charge-coupled device (CCD) camera  20 , is attached to the imaging box  12  preferably through the camera mount  109  affixed to the housing  16 . The CCD camera  20  is capable of capturing luminescent and photographic (i.e., reflection based images) images of the sample within the imaging box  12 . The CCD camera  20  may optionally be cooled by a suitable source such as a refrigeration device  22  that cycles a cryogenic fluid through the CCD camera via conduits  24 . A suitable refrigeration device is the “CRYOTIGER®” compressor, which can be obtained from IGC-APD Cryogenics Inc., Allentown, Pa. Other refrigerants, such as liquid nitrogen or solid state devices, may be used to cool the CCD camera  20 . 
     An image processing unit  26  optionally interfaces between camera  20  and a computer  28  through cables  30  and  32 , respectively. The computer  28 , which may be of any suitable type, typically comprises a main unit  36  that contains hardware including a processor, memory components such as random-access memory (RAM) and read-only memory (ROM), and disk drive components (e.g., hard drive, CD, floppy drive, etc.). The computer  28  also includes a display  38  and input devices such as a keyboard  40  and mouse  42 . The computer  28  is in communication with various components in the imaging box  12  via cable  34 . 
     To provide communication and control for these components, the computer  28  includes suitable processing hardware and software configured to provide output for controlling any of the devices in the imaging box  12 . The processing hardware and software may include an I/O card, control logic for controlling any of the components of the imaging system  10 , and a suitable graphical user interface for the imaging system  10 . The computer  28  also includes suitable processing hardware and software for the camera  20  such as additional imaging hardware, software, and image processing logic for processing information obtained by the camera  20 . Components controlled by the computer  28  may include the camera  20 , the motors responsible for camera  20  focus, one or more motors responsible for position control of a stage supporting the sample, the camera lens, f-stop, etc. The logic in computer  28  may take the form of software, hardware or a combination thereof. The computer  28  also communicates with a display  38  for presenting imaging information to the user. By way of example, the display  38  may be a monitor, which presents an image measurement graphical user interface (GUI) that allows the user to view imaging results and also acts an interface to control the imaging system  10 . 
     The processing hardware and software may also include a suitable processor configured to provide control signals to a motor coupled to a moveable stage included in box  12 . The processor may also be configured to prevent the stage from contacting the light transmission device during movement of the stage. In addition to control functions, the processor may also be applied to perform various image processing functions described herein. For example, the processor may be configured to produce a structured light representations using 2-D structured light images taken from one or more positions of the stage in the interior cavity. 
     The imaging system  10  is suitable for capturing images from a variety of views and positions of the sample relative to the camera  20 . These images may be used in in-vivo imaging applications that include analysis of one or more representations of emissions from internal portions of a specimen superimposed on a photographic representation of the specimen. In one embodiment, the imaging system  10  is used for 2-D and structured light imaging of a low intensity light source, such as luminescence from luciferase-expressing cells, fluorescence from fluorescing molecules, and the like. The low intensity light source may be emitted from any of a variety of light-emitting objects or samples which may include, for example, tissue culture plates, multi-well plates (including 96, 384 and 864 well plates), and animals or plants containing light-emitting molecules, such as various mammalian subjects including mice containing luciferase expressing cells. 
     In one application, the sample is a biological specimen containing light producing cells. The resulting luminescence image may therefore be captured without using any light sources other than the sample itself. Luminescence from the sample is recorded as a function of position to produce the luminescence image. One approach to generating such composite photographic/luminescence images is described in U.S. Pat. No. 5,650,135 issued to Contag et al. on Jul. 22, 1997. The entire disclosure of that patent is incorporated herein by reference for all purposes. 
     In one particular embodiment, a 2-D luminescence image represents a collection of emitted photons received by each detector pixel of the CCD camera  20  over a defined length of time. In other words, the luminescence image may display magnitude values representing the photon counts at the individual detector pixels. Regions of the sample emitting radiation (e.g., photons) will appear in the luminescence image. The luminescence images may indicate the presence of a biocompatible entity, for example. The entity can be a molecule, macromolecule, cell, microorganism, a particle or the like. Thus, an in-vivo analysis may include detecting localization of a biocompatible entity in a mammalian subject. Alternatively, the information in the live mode may be used to track the localization of the entity over time. For more examples of analysis applications for a digital overlay image suitable for use with the present invention, the reader is referred to in U.S. Pat. No. 5,650,135, which was previously incorporated by reference. 
     II. Imaging Box 
     In one aspect, the present invention relates to an imaging apparatus suitable for various imaging operations.  FIG. 1B  illustrates the external components of imaging box  12  of  FIG. 1A  in accordance with one embodiment of the present invention.  FIGS. 2A-E  and  4 A-D illustrate internal components of box  12  in accordance with various embodiments of the present invention. Each of the imaging apparatus described are capable of capturing an image of a sample in box  12  using a camera coupled thereto. 
     As shown in  FIG. 1B , the imaging box  12  is illustrated with a door  18  in an open position, showing an interior cavity  44  for receiving the sample. The interior cavity  44  is defined by opposing side enclosure panels  103   a  and  103   b  ( 103   b  visible in  FIG. 2D ), a light-tight partition  52  on the bottom, a top panel (not shown), a back enclosure panel  47 , and a front wall  48  defining a cavity opening  49  into the interior cavity  44 . 
     Below the cavity  44  is a smaller compartment separated therefrom by the light-tight partition  52 , the upper surface of which serves as a floor for the cavity  44 . In one embodiment, the smaller compartment provides a housing space which is adapted to slideably receive a drawer  54  though a front opening  55  formed in the body  14 . The drawer  54  houses electronic components  56  which are in electrical communication with the computer  28  ( FIG. 1A ) and control various components and functions of the box  14 . In a specific embodiment, the imaging box  12  has a body  14  made of a suitable metal such as steel. 
     A latchable door  18  is pivotally attached to box body  14  by way of hinges  46  which permit the door  18  to be moved from the closed position as shown in  FIG. 1A  to the open position as shown in  FIG. 1B . In the open position, door  18  enables user access to the cavity  44  through the opening  49 . In the closed position, door  18  prevents access to the cavity interior  44  through the cavity opening  49 . 
     Referring now primarily to  FIGS. 2A-E , various internal components of box  12  (shown in broken lines) will now be described in accordance with one embodiment of the present invention.  FIG. 2A  is a top perspective view of the components in box  12  with the exterior walls removed showing stage  204  directly below fixed datum  107 .  FIG. 2B  is a top perspective view of the components in box  12  with the exterior walls removed showing stage  204  below and off-center from fixed datum  107 .  FIG. 2C  is a top perspective view of the components in box  12  with the exterior walls removed showing stage  204  above and off center from fixed datum  107 .  FIG. 2D  is an internal side view of box  12  showing side wall  103   b  and housing  16  without light transmission device  111 .  FIG. 2E  is an internal top perspective view of side wall  103   b  and housing  16  without light transmission device  111 .  FIGS. 2A-E  are all shown with door  18  and exterior walls removed for illustration. 
     Referring to  FIGS. 2C-2E , camera  20  is mounted to side housing  16  with the camera lens  100  in view of interior cavity  44  through a port  101  formed in side wall  103   b  of box  12 . The camera lens  100  is optically coupled to camera  20  of  FIG. 1A  and includes a user controlled aperture or F-stop ring  102  for adjusting the F-stop or aperture of lens  100 , thereby modulating the amount of light passing through the lens. A Navitar, f 0.95, 50 mm TV lens is suitable for use as camera lens  100 . The F-stop ring  102  includes circumferentially disposed teeth that engage a gear  104  driven by an F-stop motor  105 . The F-stop motor  105  is in electrical communication with the electrical components  56  and controlled by computer  28 . Collectively, the motor  105  and a processor in computer  28  cooperate to position the f-stop of lens  100 . 
     A focusing mechanism  160  ( FIG. 2E ) provides reciprocal movement of the lens for focusing thereof. The focusing mechanism includes a lens support  162  showing a stationary portion mounted to upper housing  16  and a movable portion that includes a threaded bore  113 . A bolt  108 , operably engageable with bore  113 , includes a wheel that is driven by a toothed belt  110  through a corresponding drive wheel  112  of a camera lens focus motor  114  to move camera lens  100  into focus. The camera lens focus motor  114  is in electrical communication with the electrical components  56  and controlled by a processor included in computer  28  of  FIG. 1A . 
     A fixed datum represents a fixed region along the line of site of the camera lens  100  into the interior cavity  44  of the box  12 . Thus, the fixed datum  107  extends from the interior cavity in a direction substantially perpendicular to side wall  103   b  and through the center of camera lens  100  ( FIGS. 2A-E ). This datum  107 , for clarity, is represented by a stationary axis that provides a reference line of site upon which the transport mechanism  202  and the light transmission device  111  cooperate therebetween to direct light reflected or emitted from sample  106  towards and into the camera lens  100  to capture images by camera  20 . 
     As shown in  FIG. 2C , a camera mount  109  is attached to side housing  16  of side wall  103   b . Camera mount  109  is adapted to receive and position camera  20  relative to fixed datum  107  for viewing of sample  106  within cavity  44  by camera  20 . While camera  20  is capable of capturing photographic images (i.e., reflection based images) of sample  106 , it is also sensitive enough to capture luminescence images thereof. Camera  20  may employ a charge coupled device (CCD), a photodiode array, a photogate array, or similar image capture device. 
     A moveable stage apparatus  200  is disposed in interior cavity  44 , and includes a transport mechanism  202  and a stage  204  to support the light-emitting sample  106 . Moveable stage apparatus  200  is capable of two degrees of freedom movement to reposition the stage  204  (and sample  106 ) to a plurality of positions within interior cavity  44 . Any one position therebetween may be retained for image capture. 
     As shown in  FIGS. 2A-C , the transport mechanism  202  in the embodiment comprises two linear actuators  206  and  208  oriented at substantially perpendicular to one another. Each linear actuator  206  and  208  is capable of positioning stage  204  linearly along the respective actuator. Linear actuator  206  provides vertical positioning for stage  204  while linear actuator  208  provides horizontal positioning for stage  204 . Linear actuator  206  has a stationary portion attached to box  12  and a mobile portion attached to linear actuator  208 . Linear actuator  208  has a relatively stationary portion attached to linear actuator  206  and a mobile portion attached to stage  204 . An example of one such linear actuator suitable for use in the transport mechanism  202  is a LC-33 produced by Thomson Industries of Port Washington, N.Y. Each linear actuator  206  and  208  also includes displacement limiting devices on either end to restrict motion along their respective mobile portions. 
     The transport mechanism  202  preferably includes a set of position sensors that are operably coupled to the computer  28  to provide position feedback to control the position of stage  204 . In this case, the position sensors include a string or thin string  144  having one end attached to the stage  204  while the other end is attached to a take-up reel  212   a  ( FIG. 2A ). Based on the amount of string  144  wound on the reel and the total length of the string  144 , computer  28  can determine the length of string between the stage  204  and the sensor  142 , (i.e., based on changing resistance of the string with length and by using a look-up table in computer  28  to carry out the conversion). In another embodiment, the position sensor is provided by a laser positioned in interior cavity  44  to intercept the moveable stage  204  at a starting vertical or horizontal position. The laser may then be used to calibrate the position of the moveable stage  58  to a common vertical or horizontal position. 
     Linear actuators  206  and  208 , position sensors  212 , and computer  28  combine to provide closed loop position control for stage  204  within interior cavity  44 . More specifically, a user, via computer  28 , may input one or more positions for stage  204  along a substantially circular path about fixed datum  107 . In one embodiment, a user provides a viewing angle for stage  204  relative to fixed datum  107 . Software included in computer  28  then converts the viewing angle into control signals for moving each of the linear actuators  206  and  208 . Motors included in each of the two linear actuators  206  and  208  then receive the control signals provided by computer  28  and position stage  204  accordingly. The motion of stage  204  between image capture positions may be accomplished by simultaneous motion of actuators  206  and  208  or by stepwise sequential activation of each of the actuators  206  and  208 . 
     Light transmission device  111 , as best reviewed in  FIGS. 2A-2C , directs light reflected or emitted from sample  106  along the direction of fixed datum  107  and into lens  100  for image capture by camera  20 . Light transmission device  111  is mounted to housing  16  using stationary bracket  119  ( FIG. 2A ), which includes circumferentially disposed bearings between stationary bracket  119  and moving bracket  126  that allow mirror assembly  120  to rotate freely relative to stationary bracket  119 . Mirror assembly  120  is thus rotably coupled to housing  16  and rotates about an axis co-axially aligned with the stationary axis of the fixed datum  107 . 
     Referring to  FIG. 2C , mirror assembly  120  comprises an angled mirror  121  that reflects light from sample  106  on stage  204  in a direction along fixed datum  107 . Outer wall  123  is substantially cylindrical and includes aperture  122  that enables light to pass between stage  204  and mirror  121 . Outer wall  123  of mirror assembly  120  also prevents residual light in interior cavity  44  not directly associated with the current viewing angle of stage  204  from reaching lens  100 . This is partially performed by configuring mirror  121  to be sufficiently long to span the length of stage  204 . As the stage is positioned along the circular path about the stationary axis, outer wall  123  and mirror  121  cooperate to collect light primarily from the angular direction of stage  204  which is then reflected along fixed datum  107  for reception by lens  100 . 
       FIG. 2F  illustrates a simplified view of light transmission within box  12  using light transmission device  111 . As shown in  FIG. 2F , for the position of stage  204  as shown in  FIG. 2A , light is emitted from sample  106 , reflected off mirror  121 , and transmitted along fixed datum  107 . 
     In one embodiment, a light source is provided within the barrel of mirror assembly  120  to illuminate the sample or specimen in the imaging box  12 . The light source may be continuously illuminated or flashed to capture photographic images of the sample and is turned off when capturing luminescence images. In a specific embodiment, the light source comprises a ring of low-wattage lights disposed circumferentially around the camera lens  100 . In another embodiment, the light source comprises four pairs of white-light emitting diodes (LEDs), one pair mounted in each of four corners around the camera lens  100 . One advantage of using LEDs is that the spectral emission thereof may be contained to visible light while excluding infrared light. Wires (not shown) may extend from the lights to the electronic components  56  and computer  28  to allow light levels to be controlled externally through the computer  28 . 
       FIG. 2F  also illustrates the use of structured light source  170 . As shown, structured light  175 , emitted from structured light source  170 , reflects off a mirror  173 , passes through partially transparent mirror  121 , and onto sample  106 . In one embodiment, the partial transparence of mirror  121  is achieved using a half-silvered or partially silvered mirror. In another embodiment, a dichroic mirror having wavelength specific transparency properties is used. The structured light  175  may then be captured by camera  20 . 
     In the embodiment shown in  FIGS. 2A-2E , light transmission device  111  employs computer  28  to control and position mirror assembly  120  relative to fixed datum  107 . Mirror assembly  120  includes circumferentially disposed teeth on the inside of moving bracket  126  (teeth not shown) that engage a belt driven by a mirror assembly motor  128  ( FIG. 2E ). Moving bracket  126  then provides rotational motion relative to stationary bracket  119  for motor  128  input. Motor  128  is in electrical communication with the electrical components  56  and controlled by computer  28 . Together, motor  128  and a processor in computer  28  cooperated to control the rotary position of mirror assembly  120 . 
     The two degrees of freedom movement provided by transport mechanism  202  allow stage  204  and sample  106  to be positioned at multiple angles relative to fixed datum  107  for image capture by camera  20 . Thus, based on user input via computer  28 , transport mechanism  202  and light transmission device  111  cooperate to direct light from sample  106  on stage  204  to fixed datum  107  and lens  100  to capture image using camera  20 . In addition to providing full 360 degree angular viewing of sample  106  about the circular path, transport mechanism  202  is capable of varying the image depth for a given angle of stage  204  relative to fixed datum  107 . Together, transport mechanism  202  and light transmission device  111  cooperate to provide a field of view for camera  20  in the range of about 7.5 cm to about 16.5 cm. In a specific embodiment, light transmission device  111  cooperate to provide a field of view for camera  20  in the range of about 13 cm to about 16.5 cm. Similar to the user initiated angular position control described above, a user may input a desired focal depth and viewing angle for stage  204 . Software included in computer  28  and linear actuators  206  and  208  would then combine to position stage  204  at the desired angle and depth relative to fixed datum  107 . 
     To prevent undesirable contact between stage  204  and mirror assembly  120  during operation, transport mechanism  202  may incorporate crash protection measures. In one embodiment, the crash protection measures are software based and controlled by a processor in computer  28 . Thus, based on position feedback of stage  204  and known position of mirror assembly  120 , computer  28  generates control signals that insure that stage  204  does not undesirably contact with mirror assembly  120 . This may be advantageous for movement of stage  204  between a position such as that shown in  FIG. 2C  and a position 180 degrees away. In this case, the processor of computer  28  transmits control signals to linear actuators  206  and  208  which move stage  204  orbitally around mirror assembly  120 , e.g., by maintaining a minimum radius from fixed datum  107 . 
     Referring now to  FIGS. 3A and 3B , a top and side view, respectively, of stage  204  is illustrated in accordance with one embodiment of the present invention. 
     In one embodiment, stage  204  includes hardware based crash protection measures that prevent undesirable contact between stage  204  and other components within box  12 . In a specific embodiment, crash pin  250  is placed on the side of stage  204  closest to the camera  20 , as shown in  FIG. 3A . Crash pin  250  prevents contact between stage  204  and components within cavity  44 . To prevent contact between stage  204  and light transmission device  111 , camera  20  or wall  103   b , a metal ring  260  is perimetrically disposed around light transmission device  111  on stationary bracket  119 . Since metal crash pin  250  is ground and metal ring  260  is maintained at 5V, inadvertent contact between crash pin  250  and metal ring  260  acts as a limit switch and provides immediate electrical communication with computer  28  that contact has been made with stage  204 . Movement of stage  204  is then stopped. Together, crash pin  250  and metal ring  260  provide a circular crash protection boundary around light transmission device  111  during movement of linear actuators  206  and  208 . 
     In another embodiment, software based crash protection may be implemented for preventing undesirable stage  204  contact with components within cavity  44 . Based on position feedback of stage  204  using position sensors  212  and known position of mirror assembly  120 , computer  28  provides control signals that ensure stage  204  does not overlap with mirror assembly  120 , thus minimizing the risk of undesirable contact between sample  106  and components within cavity  44 . 
     As shown in  FIG. 3A , stage  204  comprises a frame  252  and a transparent portion  254 . Transparent portion  254  allows light emitted or reflected from sample  106  to be transmitted to light transmission device  111  with substantially no interference and minimal distortion for any position of stage  204  about fixed datum  107 . Transparent portion  254  preferably, comprises a transparent wire array  256  that supports sample  106 . In a specific embodiment, transparent wire array  256  is a single transparent nylon line interwoven through holes  258  on opposing edges of frame  252  and secured in a taut manner to support sample  106 . In another embodiment, array  256  is a mesh that resembles a cross pattern grid similar to a tennis racket mesh. 
     Box  12  may also include other components to facilitate image capture of a sample within box  12 . In addition to automated focus control of the camera lens  100 , the system  10  also includes an automated filter select device  117  capable of selectively providing multiple filters  118  at least partially between the camera  20  and light passing along fixed datum  107 . The filters  118  may each facilitate image capture for one or more particular imaging applications. As shown in  FIG. 2D , the optical filter select device  117  includes a circular filter select wheel  116  adapted to carry a plurality of optical filters  118  around its perimeter. The wheel  116  is rotatably mounted at its center to a mounting bracket  130  attached to side housing  16 . The filter wheel  116  is mounted off-center from lens  100  such that the individual filters  118  can each be rotated into position to intersect light emitted from the sample and reflected by mirror  121  before reaching the camera lens  100 . Filter wheel  116  has a groove along its perimeter edge in which a toothed belt  131  is seated. The toothed belt  131  is also engaged with a drive wheel  134  on a filter wheel motor  136 . The filter wheel motor  136  is in electrical communication with the electrical components  56  and controlled by a processor included in computer  28 . The plurality of optical filters  118  carried by filter wheel  116  may include any of a variety of optical filters for facilitating image capture such as a neutral density filter for bright samples, one or more wavelength cutoff filters for restricting specific wavelengths, a fluorescent filter for fluorescence applications in which the excitation light differs from the detected light, etc. 
     Other components used to facilitate image capture of a sample within box  12  may also include a gas manifold to anesthetize one or more mammalian samples. In one embodiment, the gas manifold is detachably coupled to stage  204  and includes a plurality of interfaces. Each interface is adapted to provide a gas to a mammalian sample resting on the stage  204 . An exemplary gas manifold suitable for use with the present invention is described in commonly owned co-pending U.S. patent Ser. No. 09/795,056 by Nelson et al. filed on Feb. 21, 2001, the entire disclosure of which is incorporated herein by reference for all purposes. 
     Referring now to  FIG. 3C , there is shown a top perspective view of drawer  54  and electronic components  56  housed therein. As previously noted, these components interface with computer  28  and are used to control the various motors and other components of imaging system  10 . A 3 V power supply  137  provides electrical power to the various active components in the drawer  54 . A motor control board  146  has four motor controllers  148 ,  150 ,  152 ,  154  mounted thereon. The motor controllers  148 ,  150 ,  152 ,  154  are in communication with each of the F-stop motor  109 , lens focus motor  114 , filter wheel motor  136 , mirror assembly motor  128 , and stage motor  138 , respectively. Suitable control boards include the TMG control board as provided by TMG of Mountain View, Calif. Each motor controller interfaces, via cable  34 , with computer  28  where the motor controllers and motors are controlled by user input and appropriate software running on computer  28 . Drawer  54  also houses a data acquisition board (DAB)  156 . On the face of drawer  54  is a knob  155  which is in communication with an interior cavity  44  light source and allows a user to manually to control the light intensity in the interior cavity  44 . 
     The F-stop motor  109 , lens focus motor  114 , mirror assembly motor  128 , and filter wheel motor  136  are each stepper motors capable of suitable position control of their respective components. By way of example, a model number SST 39D 1010 (1.8 deg/step, 4.3V, 0.85 A), manufactured by Shinano Kenshi Co., Ltd, Japan, is suitable for use with any of the motors  109 ,  114 ,  128  and  136 . Each of the motors is in electrical communication with one or more electronic components  56  housed in drawer  54 . The electronic components  56  are, in turn, in communication with the computer  28  where the motors  109 ,  114 ,  128  and  136  may be controlled by appropriate software and/or by user input. 
     Referring now primarily to  FIGS. 4A-C , an imaging apparatus for capturing an image of sample  306  with camera  20  is illustrated in accordance with another embodiment of the present invention.  FIG. 4A  is a top perspective view of the components in box  12  with the exterior walls removed showing stage  304  directly above fixed datum  307 .  FIG. 4B  is a top perspective view of the components in box  12  with the exterior walls removed showing stage  304  below and off-center from fixed datum  307 .  FIG. 4C  illustrates a top perspective view of the components in box  12  with the exterior walls removed showing the moveable stage above and off center from fixed datum  307 . 
     Box  12  includes a camera lens  100  mounted on side housing  16  and coupled to camera  20 , similar to that as described with respect to  FIGS. 2C-2E . This datum  107 , for clarity, is represented by a stationary axis that provides a reference line of site upon which the transport mechanism  202  and the light transmission device  111  cooperate therebetween to direct light reflected or emitted from sample  106  towards and into the camera lens  100  to capture images by camera  20 . 
     A moveable stage apparatus  300  is disposed in interior cavity  44 , and includes a transport mechanism  302  and a stage  304  to support the light-emitting sample  306 . Moveable stage apparatus  300  is capable of two degrees of freedom movement to reposition the stage  304  (and sample  306 ) to a plurality of positions within interior cavity  44 . Any one position therebetween may be retained for image capture. 
     As shown in  FIGS. 4A-C , transport mechanism  302  rotates about main axis  320  which passes through side wall  103   a . The center of rotation for main axis  320  which is co-axially aligned with the stationary axis of fixed datum  307 . Bearings are included between main axis  320  and sidewall  103   a  which allow main axis  320  to rotate freely relative to side wall  103   a . A proximal end  320   a  of main axis  320  is fixed to worm gear  325 , which is operably driven by motor  324 . A distal end  320   b  of main axis  320  is fixed to a positioning arm  322  which supports the moveable stage apparatus  300 . As motor  324  rotates worm gear  325 , this rotational motion is transmitted to the positioning arm  322  and the movable stage apparatus  300  for rotation about fixed datum  307 . A S23T as provided by Industrial Devices Corp. of Petaluma, Calif. is suitable for use as motor  324 . 
     Motor  324  is in electrical communication with electrical components  56  and controlled by computer  28 . Together, the motor  324  and the processor in computer  28  position movable stage apparatus  300  along the circular path about fixed datum  307 . An electrical slip ring  323  is provided to electrically couple the components of box  12  to the stage mechanism to maintain continuous electrical communication regardless of the rotation of positioning of main axis  320  without risk of wrapping. A AC4831-18 as provided by Industrial Devices Corp. of Petaluma, Calif. is suitable for use as electrical slip ring  323 . 
     Positioning arm  322  provides the main structural support for movable stage apparatus  300  upon which stage  304  is rotably and slideably coupled. Stage  304  is coupled to positioning arm  322  in a manner such that, as positioning arm  322  rotates via main axis  320  about fixed datum  307 , stage  304  remains substantially horizontal relative the bottom of cavity  44 . This allows a sample  306 , which is supported atop stage  304 , to be viewed from multiple positions and angles without falling off stage  304 . To maintain the stage  304  in this horizontal position as the positioning arm  322  rotates about main axis  320 , a set of bevel gears  350   a  and  350   b  are disposed between main axis  320  and a rod  330  that rotably couples stage  304  to main support  322  ( FIG. 4D ). The bevel gears  350   a  and  350   b  thus rotably couple main axis  320  to stage  304 . The bevel gears  350   a  and  350   b  reverse rotation received by rod  330  for rotation provided by main axis  320  in a 1:1 reverse gear ratio. For example, as main axis  320  rotates clockwise 30 degrees, rod  330  rotates counterclockwise 30 degrees via bevel gears  350   a  and  350   b , thus keeping stage  304  horizontal. In this manner, stage  304  remains substantially horizontal for any rotation position of positioning arm  322  relative to box  12 , as shown in  FIGS. 4B and 4C . 
     Centrally attached to main support  322  is light transmission device  311 . Light transmission device  311  rotates with main support  322  about fixed datum  307  and directs light reflected or emitted from sample  106  along fixed datum  307  and towards lens  100  for image capture by camera  20 . Light transmission device  311  includes two mirrors  335  and  336 . Each mirror  335  and  336  is attached to mirror support  339 , which is fixed to and extends perpendicularly from positioning arm  322 . Mirrors  335  and  336  rotate with positioning arm  322  about fixed datum  307 . Each mirror  335  and  336  is configured to reflect light emitted or reflected from sample  306  at least partially along fixed datum  307  and towards lens  100 . 
     Rotation about main axis  320  using motor  324  provides a first rotational degree freedom for movable stage apparatus  300 . Movable stage apparatus  300  also includes a second degree of freedom. More specifically, stage  304  may translate linearly along positioning arm  322  towards and away from mirrors  335  and  336  to vary the field of view for viewing of sample  306  on stage  304 . To allow linear translation of stage  304  along positioning arm  322 , positioning arm  322  includes a linear slide  342  which includes two cylindrical holes for receiving slide bars  342   a  and  342   b  therethrough. Sliding mount  346  allows attachment by stage  304  to linear slide  342 . Sliding mount  346  is rotably coupled to linear slide  342  via rod  330  and bearings disposed therebetween. Thus, stage  304  is orthogonally fixed to sliding mount  346 , which rotates via rod  330  and translates via linear slide  342 . 
     Motor  340  is capable of moving sliding mount  346  along slide bars  342   a  and  342   b  using a worm gear  349  operably coupled to motor  340  and linear slide  342 . A SSD55D5C0D0 as provided by Shinano Kenski Co. of Japan is suitable for use as motor  340 . Together, motor  340  and a processor in computer  28  act to position stage  304  relative to mirrors  335  and  336  to control the field of view for viewing of sample  306  on stage  304 . 
     In operation, movable stage apparatus  300  and light transmission device  311  may be used as follows. A user, via computer  28 , inputs one or more positions or angles for stage  304  relative to fixed datum  307 . For example, the user may provide two viewing angles for stage  304  relative to fixed datum  307 , both having the same field of view. For the first viewing angle, software included in computer  28  then converts the viewing angle into control signals for controlling motor  324 . Motor  324  then receives the control signals provided by computer  28  and positions stage  304  at the first position having a first angle relative to fixed axis  307 . After imaging is complete from the first viewing angle, software included in computer  28  then sends control signals to motor  324 , which re-positions stage  304  at the second position having a second angle relative to fixed axis  307 . 
     Each mirror  335  and  336  is designed to provide a different field of view for imaging within cavity  44 . Coupled with the ability to move stage  304  towards and away from mirrors  335  and  336 , mirror  335  provides a field a view in the range of about 15 cm to 25 cm. Similarly, mirror  336  a field a view in the range of about 9 cm to 11 cm. 
     Similar to the stage embodiment in  FIG. 3A , stage  304  comprises a transparent portion that allows light emitted or reflected from sample  306  to be transmitted to light transmission device  311  with substantially no interference and minimal distortion for any position of stage  304  about fixed datum  307 . In addition, movable stage apparatus  300  includes hardware based crash protection devices that prevent undesirable contact between stage  304  and other components within box  12 . For example, slide  342  includes a hard stop at each end to prevent movement of stage  304  to undesirable positions along positioning arm  322 . Further, main axis  320  also includes a hard stop at the top center thereof that prevents movable stage apparatus  300  from continually circling about main axis  320 . Upon reaching the hard stop at top center from a first direction, movement to the other side of the hard stop at top center may be accomplished by rotating the movable stage apparatus about main axis  320  360 degrees in the opposite direction. 
     III. Operation of the Imaging System 
     The present invention may be employed in a wide variety of imaging applications. Generally, the present invention may be applied with any non-invasive methods and compositions for detecting, localizing and tracking light-emitting entities and biological events in a mammalian subject. For example, the imaging system  10  may be implemented with intensified Charge-Coupled Device (CCD) cameras to detect the localization of light-producing cells (e.g., certain bacteria or tumor cells made bioluminescent by transforming them with luciferase DNA constructs) inside of living animals, such as mice. In such applications, an animal containing the bioluminescent cells is placed inside of box  12  and on stage  204 . Camera  20  is then activated to detect the emitted photons. The photon signal may then be used to construct a luminescent image of photon emission. The luminescent image is constructed without using light sources other than the luminescence from the sample itself. This luminescence is recorded as a function of position to produce the luminescence image. The photographic image may also be taken of the same sample to aid in position visualization of the luminescent image. One approach to generating such composite photographic/luminescence images is described in U.S. Pat. No. 5,650,135 issued to Contag et al. on Jul. 22, 1997. The entire disclosure of that patent was previously incorporated herein by reference. 
     Turning now to  FIG. 5 , process flow  500  illustrates a method of capturing photographic and luminescent images using the imaging system  10  in accordance with one embodiment of the present invention. Process flow  500  begins by placing a specimen or assay to be imaged for light emission on stage  204  within imaging box  12  ( 202 ). Using computer  28 , a user inputs a desired position for stage  204 . Based on the input, transport mechanism  202  moves stage  204  to the corresponding position according to a control signal provided by computer  28  ( 504 ). Light transmission device  111  also re-positions according to a control signal provided by computer  28 . The imaging box  12  and associated image components are then prepared for photographic image capture of the sample ( 506 ). Preparation may include launching imaging and acquisition software (e.g., “LivingImage” as provided by Xenogen Corporation of Alameda, Calif.) on the computer  28  and initializing camera  20 . Further preparations may include closing door  18 , activating the photographic capture option in the software, focusing camera  20  to a specific depth of the sample or animal, and turning on the lights in box  12 . Preparations may also include focusing lens  100 , selectively positioning an appropriate lens filter  118 , setting the f-stop, etc. 
     A photographic image is then captured ( 508 ). In one embodiment, a “live mode” is used during photographic imaging of the sample to observe the sample in real time. The live mode includes a sequence of photographic images taken frequently enough to simulate live video. Upon completion of photographic capture, the photographic image data is transferred to an image processing unit  26  and/or a processor in computer system  28  ( 510 ). These may be used to manipulate and store the photographic image data as well as process the data for display on computer monitor  38 . 
     Subsequently, with stage  204  at the same position, the imaging apparatus  10  is prepared for luminescence image capture ( 512 ). Such preparation may include selecting luminescent exposure time and binning level using the computer  28 , and turning off the lights in interior cavity  44 . When ready, the CCD camera  20  then captures ( 514 ) the luminescence image over a set period of time (up to several minutes). The luminescence image data are transferred to the image processing unit  26  and/or a processor in computer  28  ( 516 ). 
     At this point, a user may manipulate and store the luminescence image data as well as process it for display on the computer display  38 . The manipulation may also include overlaying the luminescent image with the photographic image and displaying the two images together as a 2-D “overlay” image, with the luminescence data typically shown in pseudocolor to show intensity. This overlay image may then be the basis for user analysis and may be analyzed and manipulated as desired. In particular, an analysis may include a summation of the illumination magnitudes over the pixels within a portion of the luminescence representation. Note that although the discussion will focus on a single luminescence representation for the overlay image, the process flow  500  may include taking multiple luminescence representations from the same position of stage  204 , e.g., at the same time or a later time ( 518 ). 
     If desired, stage  204  may then be moved to a second position ( 520 ). While the stage is at the second position, one or more photographic and/or luminescence images of the sample may be captured as described above. Upon completion of each image capture, a processor in computer  28  then receives the image data. Image collection may further continue by capturing images of the sample from alternate positions and views of the sample. 
     As mentioned, the photon emission data may represent the specific pixels on the CCD camera  20  that detect photons over the duration of the image capture period. Together, a structured light photographic representation of the sample and a luminescence representation of the sample may be combined to form a structured light superposition or overlay image. Because the imaging apparatus  100  is typically used to measure the entire sample  106 , the data in the luminescence representation typically has one or more distinct luminescent portions of interest. 
     In one embodiment, the present invention includes the use of structured light during image capture. In this case, imaging apparatus  100  provides a sequence of images of a small animal containing a bioluminescent source. This sequence of images is taken at different viewing angles and provides the information necessary to reconstruct the location, brightness, and size of the bioluminescent source within the animal. Once the images are received by processor  28 , one suitable reconstruction algorithm (or inversion algorithm) suitable for use with the present invention is diffuse optical tomography. In order to apply diffuse optical tomography, it is necessary to determine the 3D surface topology of the animal and to map the bioluminescent emission onto this surface. In one embodiment, 3D surface topology is accomplished using a structured light projection system. 
     Structured light uses a series of lines of light that are projected down on an object at an angle (at about 30 degrees, for example) to the surface normal. The lines bend as they pass over the object, and the bend in the lines can be used to determine the height of the surface at all locations that are illuminated by a structured light projector  170 . As shown in  FIG. 2A , structured light projector  170  is attached to and rotates with light transmission device  111 . In this case, structured light projector  170  consists of a Kohler illumination system where a slide is illuminated by a light source and then an image of the slide is projected onto the animal. The projection angle is large enough to get sufficient “bend” in the lines to achieve spatial resolution, but small enough that large shadows are not present. 
     An image of the structured light is taken with camera  20 . After the 2-D structured light images have been captured and stored, computer  28  may then process the structured light data to generate a structured light representation ( 522 ). As one of skill in the art will appreciate, there are numerous conventional algorithms for reconstructing a surface from structured light images. For example, the phase shift of each line at all points on the image can be determined from a computationally-efficient 2D Fourier transform. The actual surface height is then computed by “unwrapping” the phase map. 
     Each structured light image provides the surface topology for approximately the facing half of the animal only. By taking images from several viewing angles, e.g., about every 45 degrees, the entire 3D surface of the animal can be reconstructed by “stitching” together the partial surface reconstructions obtained from each view. 
     Although the present invention has been discussed primarily in the context of a moveable stage useful for in-vivo imaging applications, the present invention is suitable for other imaging applications and may be tailored correspondingly. In addition, although the present invention has been described with respect to an isolated box  12  and separate computer  28 , one embodiment of the present invention relates to a stand-alone cabinet unit housing all imaging components and computer processing components therein. Further, the present invention is scalable and may be adapted in size to fit to needs of a particular application. Although various details have been omitted for brevity&#39;s sake, obvious design alternatives may be implemented. Therefore, the present examples are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope of the appended claims.