CA2157755A1 - Camera system for imaging at low light levels - Google Patents
Camera system for imaging at low light levelsInfo
- Publication number
- CA2157755A1 CA2157755A1 CA002157755A CA2157755A CA2157755A1 CA 2157755 A1 CA2157755 A1 CA 2157755A1 CA 002157755 A CA002157755 A CA 002157755A CA 2157755 A CA2157755 A CA 2157755A CA 2157755 A1 CA2157755 A1 CA 2157755A1
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- Prior art keywords
- image
- camera
- converting
- charge
- representation
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J31/00—Cathode ray tubes; Electron beam tubes
- H01J31/08—Cathode ray tubes; Electron beam tubes having a screen on or from which an image or pattern is formed, picked up, converted, or stored
- H01J31/50—Image-conversion or image-amplification tubes, i.e. having optical, X-ray, or analogous input, and optical output
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J31/00—Cathode ray tubes; Electron beam tubes
- H01J31/08—Cathode ray tubes; Electron beam tubes having a screen on or from which an image or pattern is formed, picked up, converted, or stored
- H01J31/50—Image-conversion or image-amplification tubes, i.e. having optical, X-ray, or analogous input, and optical output
- H01J31/506—Image-conversion or image-amplification tubes, i.e. having optical, X-ray, or analogous input, and optical output tubes using secondary emission effect
- H01J31/507—Image-conversion or image-amplification tubes, i.e. having optical, X-ray, or analogous input, and optical output tubes using secondary emission effect using a large number of channels, e.g. microchannel plates
-
- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N25/00—Circuitry of solid-state image sensors [SSIS]; Control thereof
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J2231/00—Cathode ray tubes or electron beam tubes
- H01J2231/50—Imaging and conversion tubes
Abstract
An electronic imaging system is disclosed which provides a very high level of sensitivity to enable imaging of biological and chemical specimens at low light levels.
The system includes an integrating cooled CCD camera which has an image intensifier coupled thereto. With the present invention, light from the specimen is amplified by the intensifier, and the amplified light is integrated onto the cooled CCD camera over a period of time lasting for at least one second. At the end of the integration period, the camera is read out to a dedicated controller or imaging apparatus to create a digital image of the specimen. Frame averaging can be used within the imaging apparatus or controller, to improve the signal to noise ratio and increase the dynamic range of the camera. In addition, shading correction can be applied to remove spatial variations in camera sensitivity, to provide a homogeneous image background.
The system includes an integrating cooled CCD camera which has an image intensifier coupled thereto. With the present invention, light from the specimen is amplified by the intensifier, and the amplified light is integrated onto the cooled CCD camera over a period of time lasting for at least one second. At the end of the integration period, the camera is read out to a dedicated controller or imaging apparatus to create a digital image of the specimen. Frame averaging can be used within the imaging apparatus or controller, to improve the signal to noise ratio and increase the dynamic range of the camera. In addition, shading correction can be applied to remove spatial variations in camera sensitivity, to provide a homogeneous image background.
Description
215775~
SCOPE OF THE INVENTION
The present invention relates to an imaging system for creating digital images of very faint or low light specimens. More particularly the imaging system includes an image intensifier coupled to an integrating cooled charge coupled device (CCD) camera, and in which the output of the intensifier is integrated onto the CCD camera for relatively long periods of time.
BACKGROUND OF THE INVENTION
Standard solid state and tube-type cameras are excellent for imaging well-illuminated biological specimens. However, standard cameras lack the sensitivity to image specimens under low light conditions, as for example, those which result in an irradiance of less than about 20 picowatts/cm2 at 500 nm. By way of comparison of the sensitivity desired, the detection limit for the human eye is about 17 picowatts/cm2 (10-3 foot candles) at 500 nm.
Traditionally, low light specimens are imaged by one of three approaches, intensification, integration or photon counting.
Intensification, as for example, is disclosed in U.S. Patent No. 5,204,533 to Simonet, involves the coupling of an image intensifier to a CCD camera. The image intensifier typically includes a photocathode, a phosphor screen and a multichannel plate lMCP) connected between the photocathode and phosphor screen and provides an enhanced image of a specimen. Amplification factors of up to about 90,000 are possible with this type of device.
In the image intensified CCD camera, the image is created at three or four planes. At each of these planes, there is some loss of quantum efficiency. Therefore, the 21~77 `~
-image intensifier is operated at high gain to overcome signal losses within the optical chain. High gain shortens intensifier life, and increases noise. At very high gain factors, noise and ionic feedback through the MCP become so severe that further improvement of sensitivity is impossible. Even when run at maximum gain, conventional image intensified CCD cameras are not sensitive enough to image the dimmest specimens.
Faced with a typical very dim specimen, most image intensified CCD cameras will fail to produce an image, or will produce a very poor image, in which the target will be difficult to discriminate from background, and the image will not reflect the true range of target intensities. In the worst cases, the target will be indiscriminable from background.
Conventional image intensified CCD cameras use short integration periods and, in most cases, the integration period is equal to a single television frame.
The short integration period allows the intensifier to be used with standard, low-cost video cameras, as for example, are used in the television industry. In other cases, the intensifier is gated, to use very short integration periods (e.g. 1 msec). The use of gating allows the intensifier to be used with specimens that would be too bright for a standard intensifier, and can also be used to run the intensifier in a photon counting mode.
It is possible to construct image intensified CCD
cameras with higher sensitivity, as for example, to increase the gain of MCPs by mounting two or more MCPs in serial fashion. This can result in much higher levels of gain, though linearity of response and dynamic range are compromised. In addition, multi-stage MCPs add greatly to the cost of a device. Other than very costly and - 21577~
-specialized multistage cameras, there are no image intensified CCDs that can image very dim specimens.
While all cameras perform some integration in that they operate by accumulating light over a period of time, integrating cameras generally refer to cameras which integrate for periods of longer than a video frame (i.e. 33 msec.) In this regard, most intensified CCD cameras operate at video frame rates and would not be considered to include integration capabilities.
U.S. Patent No. 4,922,092 to Rushbrooke et al.
discloses the use of an image intensified CCD camera which is coupled to a special fibre optic lens. The fibre optic lens provides an efficient light pipe between spatially invariant specimens and the input of the intensifier.
While Rushbrooke suggests the use of integration on a CCD
camera for periods of up to one second, it is disclosed as being preferable that the image be read out at television frame rates. This short integration period might be sufficient for some specimens connected via the efficient light pipe, however, the short integration period does not provide sufficient sensitivity to allow imaging of spatially variable specimens.
Conventional integrating cameras have a broader dynamic range (up to four orders of magnitude vs. 1.5 for an image intensified CCD), higher quantum efficiency (40%
is typical) and better contrast transfer than image intensified CCDs. However, they require more photons to overcome noise inherent to the camera.
In addition, while the invention disclosed by Rushbrooke may be suitable for biochemical specimens in well plates, it would be, however, incapable of imaging most biological specimens. In particular, biological 21~7~
specimens are spatially variable, and cannot be coupled to the intensifier by a light pipe. Instead, they must be imaged using a lens. The use of a lens is less efficient than a light pipe, and lens-based cameras present special difficulties in that they require much higher sensitivity than the light pipe cameras disclosed in Rushbrooke.
A photon-counting camera uses a selected image intensifier operating at very high levels of gain. The intensifier transforms incident photons on the input window into somewhat diffused spots of light on the output window.
The spots of light from the intensifier which are bright enough to be detected by a low-lag video camera form the basis of photon-counting imaging. Sensitivity can be high enough to detect single photons, but without processing of the spots of light, resolution is poor because of the diffusion inherent to the amplification process.
Resolution recovery circuitry (a digital discriminator in the video camera or an imaging system) selects the brightest part and/or center of gravity of the light spot, to remove some of the diffusion and regenerate some of the resolution lost by the amplification process.
Now, the incident light is present as a spatially localized event within a frame buffer. It is this resolution recovery which is a critical aspect of the photon counting camera. The camera is exposed to the specimen until enough counts are accumulated to form a usable image. In a sense, the photon counting camera uses both intensification and integration. However, in photon counting mode, the integration is quantal. Suprathreshold scintillations are detected as counts assigned to a specific XY location within the image. The camera sensor is periodically sampled for the presence of detected events (at rapid rates, e.g. 1 millisecond). There is no image integration in the camera sensor, before readout, but rather, image - 21577S~
integration occurs within a memory buffer as sequential readouts are summed.
In practice, the photon counting camera has two disadvantages. It requires longer exposures than the present invention. It also forms images with poor spatial resolution. However, it does have much broader dynamic range, because counts can be accumulated over long periods of time without saturating the detector. Photon counting cameras are best suited to imaging the very dimmest specimens when broad dynamic range is more important than image quality and speed. Their major disadvantages are high cost, and that they produce images with relatively poor resolution. The present invention is best suited to imaging somewhat less dim specimens, when reasonable cost, superior image quality, and speed of operation are more important than dynamic range.
SUMMARY OF THE INVENTION
Accordingly, it is an objective of the present invention to provide a system for use in imaging very low-light specimens using intensification in combination with extended periods of integration, lasting from one or more seconds to one minute or more.
Another object of the invention is to provide an apparatus which can image very dim specimens, and which may be built at a relatively low cost.
A further-object of the invention is to provide an electronic camera having a very high level of sensitivity which is readily adaptable for imaging a variety of different samples, and without the need of physically coupling the sample to a fixed sample holder.
- 21 ~'155 Another object of the invention is to provide a highly sensitive imaging apparatus which includes an integrating CCD camera for recording the image, and which is adapted to perform integration within the camera sensor and before readout.
A further object of the invention is to provide an imaging system particularly suited for imaging low-light biological specimens which may be spatially variable.
Another object of the invention is to provide a camera for imaging very faint specimens which can be used in either integration or photon counting mode.
To achieve at least some of the foregoing objects the present invention provides a camera which has an enhanced level of sensitivity by using a synergistic combination of image intensification and integration. The applicant has appreciated that by coupling the output of an image intensifier to an integrating camera capable of long periods of integration the disadvantages of prior art low light imaging cameras may be overcome. Instead of running the intensifier at very high gain, or using multi-stage intensifiers, the intensifier may thereby be operated at optimal gain levels and accumulate its output over relatively long periods of time. The integration time period might be from a few seconds to minutes. As any integration period may be selected, even very faint specimens can be seen, without excessive intensifier gain.
With the present invention, high sensitivity is achieved by engineering the camera to use long integration periods. For example, the present invention can image a typical chemiluminescent southern blot in about 10 seconds using an fl.2 lens. By way of comparison, the same specimen would typically require about a 3 minute exposure ~77S5 with a cooled CCD camera, a two minute exposure on film, and would be invisible to a standard image intensified CCD
camera.
In the integrating camera, the signal is accumulated onto a CCD sensor for a period of time. Both video integrating cameras and asynchronous integrating cameras may be used with the present invention. Video integrating cameras accumulate signal over a number of video frames, directly onto the CCD element and before the camera is read out into a frame buffer. That is, one might integrate two or more video frame (each being 1/30 sec), before an image is output from the camera. Asynchronous integrating cameras integrate and read out in the same way as video integrating cameras, but do not have fixed frame rates. They can accumulate an image onto the chip over any time period. Integration periods might be 100 msec, or any other time interval.
A preferred embodiment of the imaging system may be obtained by the use of long-term integration of the output of an image intensifier onto a cooled CCD camera.
With such a system components may be selected at reasonable cost to provide a system having low noise, flat field response, and high inherent contrast, which does not require direct coupling to specimens (is lens-based), and which can be used to image a broad variety of specimens over a very broad range of specimen intensities. At the user's option, frame averaging can be added to reduce noise, and shading correction can be added to remove background inhomogeneities.
The imaging system therefore is provided with an image intensifier placed in front of a CCD camera. The image intensifier has relatively low quantum efficiency (typically less than 20~), but can provide very high levels - 21~77~i5 of gain. The intensifier may be coupled to the CCD camera by a relay lens, or alternatively, by a fibre optic coupling. A relay lens allows the intensifier to be used with any camera, is not subject to the "chicken wire"
pattern that tends to appear in fibre optic images. In addition, a relay lens will not delaminate as fibre optic couplers tend to. Fibre optic couplers are advantageous as they transfer light more efficiently than relay lens couplers, and allow the intensifier to be used at lower gain.
of the intensifier, three major types (i.e. GEN
I (generation 1), GEN II (generation 2), and GEN III
(generation 3)) are in common use, each differing in component organization and in the materials from which the components are constructed. Most preferably an Extended Blue GEN III image intensifier is used fibre optically coupled to the CCD camera. The applicant has discovered that the intensified camera unit of the present invention is well-suited to imaging dim specimens. When used at video frame rates, this intensifier has a detection limit of about 4 x 10-7 foot candles. Used within the present invention, the intensifier becomes much more sensitive.
The synergistic combination of integration and intensification of the present system provides several advantages over integration alone. First, images can be formed in a much shorter time period. An exposure of 10 seconds with the present invention is roughly equivalent to an exposure of 3 to 4 minutes with a thermionically cooled integrating camera, and 1 to 2 minutes with a cryogenically cooled camera. Second, the present invention can be lower in cost than a high-quality cooled integrating camera.
In use of the integrating camera, the signal is accumulated onto a CCD sensor or element for a period of time lasting for two or more seconds up to several minutes.
Both video integrating cameras and asynchronous integrating cameras may be used with the present invention. Video integrating cameras accumulate the signal over a number of video frames, directly onto the CCD element and before the camera is read out into a frame buffer.
In biological research, cooled integrating cameras may be used if exposure time, convenience, and cost are not major factors. Preferably the integrating camera of the present invention incorporates a cooling element to maintain the CCD element at a cooler temperature and permit integration over a longer period of time.
A liquid, cryogenically or thermionically cooled camera, such as one incorporating a Peltier cooler element has, for example, been found to be suited for use with the present invention. In contrast to the 18 bit precision of cryogenically cooled camera, liquid or thermionically cooled cameras typically function to only 12 or 14 bit precision, are not as sensitive as cryogenic cameras, and need rather long exposures (typically 2 to 10 minutes) to image dim fluorescence, chemiluminescence or bioluminescence. One must sit with the imaging system while it exposes, and any number of lengthy test exposures are necessary before the best integration period is found for a particular specimen. The combination of integration and intensification of the present invention, however, permits faster imaging than when a conventional cooled camera is used alone, and operates conveniently and at lower cost than a cryogenically cooled integrating camera, but with the same sensitivity.
Accordingly, in one aspect the invention resides in an electronic imaging system which provides a very high level of sensitivity to enable imaging of biological and - 21~7~
chemical specimens at low light levels. The system includes an integrating cooled CCD camera which has coupled thereto an image intensifier. Incident illumination from the specimen is amplified by the intensifier, and the amplified light is accumulated onto the integrating camera over an integration period which typically lasts for at least one second. At the end of the integration period, the camera is read out to a dedicated controller or imaging apparatus to reproduce the light image. Frame averaging is used within the imaging apparatus or controller, to reduce noise and improve the dynamic range of the camera.
In addition, shading correction is applied to remove spatial variations in camera sensitivity and provide enhanced imaging of the light images.
In another aspect, the present invention resides in an image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device coupled to said output surface and being responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions to produce charge values representative of the intensified electron representation at each CCD region, integration means for integrating the charge values over an integration period to produce an adjusted charge for each said CCD
region and - 21~77'j~
provide an integrated signal representative of the light image, wherein said integration period is selected greater than one second and less than five minutes.
In a further aspect the present invention resides in an image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device and a fibre optic minifier for optically coupling the output surface to the charge coupled device, cooling means thermally coupled to the charge coupled device for dissipating heat therefrom, the charge coupled device responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions over a period of time to produce localized charge values representative of the intensified electron representation at each CCD region, integration means for integrating the localized charge values over an integration period to produce an adjusted charge for each said CCD region and provide an integrated signal representative of the light image, said integration period being selected greater than five seconds and less than one minute, and - ~1 577~S
output means for outputting said integrated signal.
BRIEF DESCRIPTION OF THE DRAWINGS
Further objects and advantages of the invention will appear from the following description taken together with accompanying drawings in which:
Figure 1 is a schematic illustration of a computerized system for imaging low-light specimens in accordance with a first preferred embodiment of the invention;
Figure 2 schematically illustrates a front view of the CCD camera used in the system of Figure 1;
Figure 3 schematically illustrates a side view of the CCD camera of Figure 2;
Figure 4 is a schematic illustration of the intensifier, fibre optic coupler, and CCD sensor used in the system shown in Figures 1 and 2; and Figure 5 is a schematic illustration of a stand-alone system for imaging low-light specimens in accordance with a second embodiment of the invention.
DETAILED DESCRIPTION OF THE DRAWINGS
Reference is made first to Figure 1, which shows an image intensifier system 10 in accordance with a first embodiment of the invention. The system 10 includes an integrating video camera 12 which incorporates a CCD
element 14, an image intensifier 16 and a fibre optic coupler 18 optically coupling the intensifier 16 to the - ~57755 front of the camera 12. An external camera control 20 (ECC) contains circuitry for controlling the camera and conditioning the signal. A computer 22 is also provided coupled to the input and output of the camera control unit 20 for software-driven control of the system operation and storing of output images.
Figures 2 and 3 show best the camera 12 for use with the system 10. The camera 12 includes a CCD element 14 positioned behind a camera aperture 25. To reduce dark noise produced by electrons within the CCD element, the CCD
element 14 is mounted to a heat sink 26, which in turn is thermally coupled to a Peltier cooling element 28 for providing enhanced heat dissipation. An electronic shutter mechanism 32 is additionally provided within the camera 12 for limiting the exposure of the image on the CCD element 14. Preferably the camera 12 is a high resolution 768H x 482V pixel black and white full frame shutter camera, with asynchronous reset capability and uniform modulation transfer characteristic functions. The camera 12 provides an 8-bit digital signal output via a standard digital interface protocol 34 such as EIA-422. An interlaced or progressive scan analog output is available from an integral frame buffer 36. The flexible provision of both digital and analog output permits the camera 12 to be used as a stand alone unit, with a display monitor, or as part of a computer image analysis system.
The camera 12 operation is controlled externally by the camera control unit 20. The control unit 20 controls the integration time of the camera 12, adjusts the intensifier gain, and adjusts the video gain and black level of the camera 12. The camera control unit 20 includes the asynchronous reset circuitry which operates to accentuate edges in the image. This circuit can be useful in restoring some of the edge degradation produced when 21~7 ~5~
using the system 10 at high gain factors.
Image averaging circuits 38 are also provided in the camera 12 to decrease noise. Data from the CCD element 14 are digitized and fed to the internal frame buffer 36.
As each frame arrives at the buffer 36, it is passed through an arithmetic logic unit which performs iterative averaging of the incoming frame with previous frames.
Although most image analyzers report data with 8 bit (256 level) precision, video cameras provide only about 45-50 dB
of signal-to-noise ratio for any individual pixel.
Intensifiers are much worse, particularly at high gain.
The present system 10 therefore is additionally provided with noise reduction circuitry to provide noise reduction by frame averaging. With such circuitry, as the number of frames imaged by the camera 12 increases, the root mean square noise amplitude decreases by a factor of 1 / V~~~
(where n is the number of frames).
In addition to controlling the operation of the camera 12 and the intensifier 16, the camera control unit 20 outputs the integrated data to the computer 22 where it may be stored, output to a video terminal 58 or a printer (not shown) or the like.
The asynchronous reset capability of the camera 12 is flexible, and takes external horizontal synchronization for phase locking to a trigger. When an initialization pulse is applied by the camera control unit 20, it resets the camera's scanning, and purges the CCD
element 14. At that point the image is transferred from the CCD element 14 to the internal frame buffer 36.
The shutter mechanism 32 is of a substrate drain type and provides rapid clearing of the CCD element charges, so that the camera 12 may be used at high shutter speeds without smearing of images. This feature is - 21~775~
particularly useful if the camera 12 is operated in photon counting mode.
Figure 4 shows best the intensifier 16 as being of the GEN III type and including a lens 40, a photosensitive cathode 42, a microchannel plate (MCP) 44, a phosphor screen 46, and a vacuum sealed body 48 or enclosure. The lens 40 is a high-numerical aperture design, which captures light very efficiently. The lens 40 focuses to within 30 cm, and permits system 10 to be used to image almost any specimen. At its output, the lens 40 is focused on an input window of the cathode 42 so as to transfer the specimen image thereto. The photosensitive cathode 44 is selected to emit electrons in proportion to the intensity of light falling upon it. The microchannel plate (MCP) 44 is positioned within the vacuum sealed body 48, between and coupled at each end to the cathode 42, and the phosphor screen 46. The MCP 44 is provided with an array of small diameter MCP channels 50, each of the MCP
channels 50 are coated with gallium arsenide. The electrons emitted from the cathode 42 are accelerated along the MCP channels 50 to the phosphor screen 46. As the electrons from the cathode 42 are accelerated along the small diameter channels 50, they strike the coated channel walls to produce additional electrons. As the multiplied electrons leave the MCP channels 50, they strike the phosphor screen 46 and produce an intensified image of the specimen on an output screen 52.
It has been found that the use of the Extended Blue GEN 3 image intensifier 16 is advantageous over other types of intensifiers in that the image provided on the output screen 52 is sharper, has less shading error, and has less noise than those produced by GEN 1 and GEN 2 intensifiers. It is to be appreciated, however that as better intensifier technologies are developed, they may 21577~
equally be incorporated into the present system 10.
The fibre optic coupler 18 is preferably a bonded fibre optic coupler used to couple the output screen 52 of the intensifier 16 directly to the CCD element 14 of the video camera 12. Direct fibre optic coupling of the intensifier 16 to the camera CCD element 14 is four to eight times more efficient than a lens in transferring light from the output screen 52 to the camera 12. In the present case, the fibre optic coupler 18 includes a fibre optic input window 60 and a fibre optic minifier 62. The minifier 62 provides a 1.5:1 fibre optic taper to a smaller fibre optic output window 64 which is bonded to the CCD
element 14. The 1.5:1 reduction ratio of the minifier 62 has been found to allow full use of the highest resolution obtainable from the image intensifier 16. It will present an image having over 76 linepairs/mm to the CCD element 14, with distortion of less than 3% and overall light transmission of >75~.
In use of the system 10, the intensifier lens 40 is adjusted to focus a bioluminescent or fluorescent specimen on the input window of the intensifier photocathode 42. The photocathode 42 emits electrons in proportion to the intensity of light from the specimen falling upon it. A voltage is applied to the emitted electrons as they move from the photocathode 42 into the MCP 44, thereby accelerating the electrodes as they enter the small diameter MCP channels 50. Within the thousands of MCP channels 50, the electrons collide with the coated channel wall surfaces and dislodge additional secondary electrons. In this manner, one input electron can generate thousands of secondary electrons to provide light amplification. The electrons are accelerated again as they leave the MCP 44, and then strike the phosphor screen 46 on its inner surface, to generate a visible image on the ~5~75~
output window 52. The visible image is then transferred from the output window 52 to the camera 12 by the fibre optic coupler 18.
The integrating camera is configured so that the highly amplified image generated on the output window 52 is focused by the lens 40 and coupler 18 onto the CCD element 14. To image low light specimens, the CCD element 14 integrates for a period of at least one second, accepting a trigger pulse from the camera control unit 20 to initiate the operation of the electronic shutter 32. The electronic shutter clears the CCD element 14, from which point the image is transferred to the internal frame buffer. For very dim specimens, a period lasting from two or more seconds upwards to more than one minute may be used.
During the integration period, photons from the output window 52 incident to the CCD element 14 are stored as negative charges (the Signal) in numerous discrete regions of the CCD element 14. The amount of the charge in each discrete region of the CCD element 14 is accumulated as follows.
Signal = Incident light x Quantum efficiency x Integration time The greater the relative intensity of the incident light coming from the intensifier 16, the greater the signal stored in the corresponding region of the CCD element 14.
Upon completion of the integration period, a signal representative of the specimen image is exported from the camera 12 to the computer 22 for output or further image averaging externally by the computer 22.
As the present invention is designed for imaging very dim specimens, and as the gain available from an - 215775~
intensifier is limited, relatively long periods of integration of the intensifier output onto the cooled integrating camera 12 are provided. For chemiluminescent blots, integration periods of 10 sec to 1 minute can be used, equivalent to a film exposure of about 1 minute to 4 minutes. For more dim specimens, multiple 10 sec to 1 minute exposures are necessary, and frame averaging by the control unit 20 (typically four frames) may be used to improve signal to noise ratio in a final output image.
Figure 1 shows the CCD camera 12 as being provided without an image averaging circuit with image averaging done externally on a personal computer 22. If desired, image averaging may equally be accomplished within the camera 12 itself or within the camera control unit 20, by the addition of image averaging circuitry. Figure 5 illustrates schematically a modified system 10 wherein like reference numerals are used to identify like components and in which the camera~unit 20 incorporates image averaging circuitry and outputs the final image as an analog signal sent to a display monitor 70.
Although the camera 12 could be used without cooling the CCD element 14, extended periods of integration are achieved by using a CCD camera with an integral cooling element. The effectiveness of integration is limited by the degree of cooling. With thermionic cooling using a standard Peltier cooling device, sensor temperatures of about -20C can be achieved. This allows integration for periods of up to about two minutes before background noise becomes bothersome. Thermionic cooling has the advantage of low cost and easy implementation.
It is to be appreciated, however, that longer periods of integration are possible if multistage thermionic, liquid, or cryogenic cooling are employed.
These more effective cooling methods would be combined with ~157;7~
..
a more sophisticated camera, capable of higher precision (10-16 bits is typical). Using a high precision liquid cooled camera does achieve better image quality and higher sensitivity, but with a corresponding increase in the cost of the camera.
In addition, with the system 10 shown in Figure 1, only the CCD element 14 is cooled. This is sufficient for routine imaging. It is to be appreciated however, that for more demanding tasks, the photocathode 42 could also be cooled, thereby improving the signal to noise ratio of the intensifier 16. Similarly, the entire photosensitive apparatus (i.e. intensifier and CCD) can be cooled.
However, cooling the entire photosensitive apparatus has the disadvantage that the efficiency of the phosphor on the fibre optic output window is decreased.
In the preferred embodiment of the invention shown in Figure 1, the system 10 incorporates an Extended Blue type of GEN 3 image intensifier 16. Other types of intensifiers, although less preferred, may also be used.
The three major types of intensifier (GEN 1, GEN 2 and GEN
3) differ in the organization of their components and in the materials of which the components are constructed. In a GEN 1 intensifier, illumination incident to a photocathode results in emissions at a rate proportional to the intensity of the incident signal. The electrons emitted from the photocathode are then accelerated through a high potential electric field, and focused onto a phosphor screen using electrostatic or proximity focusing.
The phosphor screen can be the input window to a video camera (as in the silicon intensified target camera), or can be viewed directly. GEN 1 intensifiers, however, suffer from bothersome geometric distortion, and have relatively low quantum efficiency (about 10~).
21~775~
The GEN 2 intensifiers, like the GEN 3, incorporate a MCP into an image tube, between the cathode and an anode. The GEN 2 intensifiers are smaller, lower in noise, and have higher gain than the GEN 1 intensifiers.
However, their quantum efficiency is fairly low (typically <20%), and they tend to suffer from poor contrast transfer characteristics. In contrast, the GEN 3 intensifier tube has a quantum efficiency of about 30% or higher (needs less gain), and very high intrinsic contrast transfer. With recent versions of the GEN 3, gain levels are about equal to those of a GEN 2 (ultimate gain level available is about 90,000). Therefore, a GEN 3 intensifier will tend to yield better images than a GEN 2. Where necessary for reasons of cost or specific design features, other forms of intensifier could be used. Similarly devices with high intrinsic gain (such as electron bombarded back-illuminated CCD sensors) could be used in place of image intensifiers.
The CCD camera 12 of the present invention uses an asynchronous reset which takes an external drive signal from the control unit 20 for phase locking. When the signal is applied, it resets the camera 12, also sc~nn;ng and purging the CCD element 14. As the CCD camera 12 incorporates mechanisms that provide very low lag, short integration periods (e.g. 1/16,000 second) can be used. If desired, these integration periods can be locked to a gated power supply (not shown) in the image intensifier 16, with the result that the camera 12 can be read out at very short intervals. Using the gating and fast readout feature, and with the intensifier 16 run at highest gain or with a multistage intensifier 16, the present invention can thereby be operated as a conventional photon counting light imaging system. Thus, the present system 10 can advantageously be used for both direct imaging of faint specimens, or as a standard photon counting camera by changing its mode of operation from integration to gating.
21~i77~i~
While the preferred embodiment of the invention discloses the use of a cooled CCD element 14, for relatively bright specimens, if cost is a major factor, the present invention could be constructed without cooling element 28. In this case, a camera 12 could be constructed at quite a low cost and integration periods of about 5 seconds can be achieved, albeit with a reduction in image quality and ultimate sensitivity.
Although the preferred embodiment of the invention illustrates a bonded fibre optic coupler 18 with a minifier 62 for coupling the intensifier 16 to the video camera 12, the invention is not so limited. If desired the image intensifier 16 could also be coupled to the integrating camera using a lens, a conventional fibre optic coupler or any other suitable optical coupling device.
Although the detailed description describes and illustrates preferred embodiments of the present apparatus, the invention is not so limited. Modifications and variations will now appear to persons skilled in this art.
For a definition of the invention reference may be had to the appended claims.
SCOPE OF THE INVENTION
The present invention relates to an imaging system for creating digital images of very faint or low light specimens. More particularly the imaging system includes an image intensifier coupled to an integrating cooled charge coupled device (CCD) camera, and in which the output of the intensifier is integrated onto the CCD camera for relatively long periods of time.
BACKGROUND OF THE INVENTION
Standard solid state and tube-type cameras are excellent for imaging well-illuminated biological specimens. However, standard cameras lack the sensitivity to image specimens under low light conditions, as for example, those which result in an irradiance of less than about 20 picowatts/cm2 at 500 nm. By way of comparison of the sensitivity desired, the detection limit for the human eye is about 17 picowatts/cm2 (10-3 foot candles) at 500 nm.
Traditionally, low light specimens are imaged by one of three approaches, intensification, integration or photon counting.
Intensification, as for example, is disclosed in U.S. Patent No. 5,204,533 to Simonet, involves the coupling of an image intensifier to a CCD camera. The image intensifier typically includes a photocathode, a phosphor screen and a multichannel plate lMCP) connected between the photocathode and phosphor screen and provides an enhanced image of a specimen. Amplification factors of up to about 90,000 are possible with this type of device.
In the image intensified CCD camera, the image is created at three or four planes. At each of these planes, there is some loss of quantum efficiency. Therefore, the 21~77 `~
-image intensifier is operated at high gain to overcome signal losses within the optical chain. High gain shortens intensifier life, and increases noise. At very high gain factors, noise and ionic feedback through the MCP become so severe that further improvement of sensitivity is impossible. Even when run at maximum gain, conventional image intensified CCD cameras are not sensitive enough to image the dimmest specimens.
Faced with a typical very dim specimen, most image intensified CCD cameras will fail to produce an image, or will produce a very poor image, in which the target will be difficult to discriminate from background, and the image will not reflect the true range of target intensities. In the worst cases, the target will be indiscriminable from background.
Conventional image intensified CCD cameras use short integration periods and, in most cases, the integration period is equal to a single television frame.
The short integration period allows the intensifier to be used with standard, low-cost video cameras, as for example, are used in the television industry. In other cases, the intensifier is gated, to use very short integration periods (e.g. 1 msec). The use of gating allows the intensifier to be used with specimens that would be too bright for a standard intensifier, and can also be used to run the intensifier in a photon counting mode.
It is possible to construct image intensified CCD
cameras with higher sensitivity, as for example, to increase the gain of MCPs by mounting two or more MCPs in serial fashion. This can result in much higher levels of gain, though linearity of response and dynamic range are compromised. In addition, multi-stage MCPs add greatly to the cost of a device. Other than very costly and - 21577~
-specialized multistage cameras, there are no image intensified CCDs that can image very dim specimens.
While all cameras perform some integration in that they operate by accumulating light over a period of time, integrating cameras generally refer to cameras which integrate for periods of longer than a video frame (i.e. 33 msec.) In this regard, most intensified CCD cameras operate at video frame rates and would not be considered to include integration capabilities.
U.S. Patent No. 4,922,092 to Rushbrooke et al.
discloses the use of an image intensified CCD camera which is coupled to a special fibre optic lens. The fibre optic lens provides an efficient light pipe between spatially invariant specimens and the input of the intensifier.
While Rushbrooke suggests the use of integration on a CCD
camera for periods of up to one second, it is disclosed as being preferable that the image be read out at television frame rates. This short integration period might be sufficient for some specimens connected via the efficient light pipe, however, the short integration period does not provide sufficient sensitivity to allow imaging of spatially variable specimens.
Conventional integrating cameras have a broader dynamic range (up to four orders of magnitude vs. 1.5 for an image intensified CCD), higher quantum efficiency (40%
is typical) and better contrast transfer than image intensified CCDs. However, they require more photons to overcome noise inherent to the camera.
In addition, while the invention disclosed by Rushbrooke may be suitable for biochemical specimens in well plates, it would be, however, incapable of imaging most biological specimens. In particular, biological 21~7~
specimens are spatially variable, and cannot be coupled to the intensifier by a light pipe. Instead, they must be imaged using a lens. The use of a lens is less efficient than a light pipe, and lens-based cameras present special difficulties in that they require much higher sensitivity than the light pipe cameras disclosed in Rushbrooke.
A photon-counting camera uses a selected image intensifier operating at very high levels of gain. The intensifier transforms incident photons on the input window into somewhat diffused spots of light on the output window.
The spots of light from the intensifier which are bright enough to be detected by a low-lag video camera form the basis of photon-counting imaging. Sensitivity can be high enough to detect single photons, but without processing of the spots of light, resolution is poor because of the diffusion inherent to the amplification process.
Resolution recovery circuitry (a digital discriminator in the video camera or an imaging system) selects the brightest part and/or center of gravity of the light spot, to remove some of the diffusion and regenerate some of the resolution lost by the amplification process.
Now, the incident light is present as a spatially localized event within a frame buffer. It is this resolution recovery which is a critical aspect of the photon counting camera. The camera is exposed to the specimen until enough counts are accumulated to form a usable image. In a sense, the photon counting camera uses both intensification and integration. However, in photon counting mode, the integration is quantal. Suprathreshold scintillations are detected as counts assigned to a specific XY location within the image. The camera sensor is periodically sampled for the presence of detected events (at rapid rates, e.g. 1 millisecond). There is no image integration in the camera sensor, before readout, but rather, image - 21577S~
integration occurs within a memory buffer as sequential readouts are summed.
In practice, the photon counting camera has two disadvantages. It requires longer exposures than the present invention. It also forms images with poor spatial resolution. However, it does have much broader dynamic range, because counts can be accumulated over long periods of time without saturating the detector. Photon counting cameras are best suited to imaging the very dimmest specimens when broad dynamic range is more important than image quality and speed. Their major disadvantages are high cost, and that they produce images with relatively poor resolution. The present invention is best suited to imaging somewhat less dim specimens, when reasonable cost, superior image quality, and speed of operation are more important than dynamic range.
SUMMARY OF THE INVENTION
Accordingly, it is an objective of the present invention to provide a system for use in imaging very low-light specimens using intensification in combination with extended periods of integration, lasting from one or more seconds to one minute or more.
Another object of the invention is to provide an apparatus which can image very dim specimens, and which may be built at a relatively low cost.
A further-object of the invention is to provide an electronic camera having a very high level of sensitivity which is readily adaptable for imaging a variety of different samples, and without the need of physically coupling the sample to a fixed sample holder.
- 21 ~'155 Another object of the invention is to provide a highly sensitive imaging apparatus which includes an integrating CCD camera for recording the image, and which is adapted to perform integration within the camera sensor and before readout.
A further object of the invention is to provide an imaging system particularly suited for imaging low-light biological specimens which may be spatially variable.
Another object of the invention is to provide a camera for imaging very faint specimens which can be used in either integration or photon counting mode.
To achieve at least some of the foregoing objects the present invention provides a camera which has an enhanced level of sensitivity by using a synergistic combination of image intensification and integration. The applicant has appreciated that by coupling the output of an image intensifier to an integrating camera capable of long periods of integration the disadvantages of prior art low light imaging cameras may be overcome. Instead of running the intensifier at very high gain, or using multi-stage intensifiers, the intensifier may thereby be operated at optimal gain levels and accumulate its output over relatively long periods of time. The integration time period might be from a few seconds to minutes. As any integration period may be selected, even very faint specimens can be seen, without excessive intensifier gain.
With the present invention, high sensitivity is achieved by engineering the camera to use long integration periods. For example, the present invention can image a typical chemiluminescent southern blot in about 10 seconds using an fl.2 lens. By way of comparison, the same specimen would typically require about a 3 minute exposure ~77S5 with a cooled CCD camera, a two minute exposure on film, and would be invisible to a standard image intensified CCD
camera.
In the integrating camera, the signal is accumulated onto a CCD sensor for a period of time. Both video integrating cameras and asynchronous integrating cameras may be used with the present invention. Video integrating cameras accumulate signal over a number of video frames, directly onto the CCD element and before the camera is read out into a frame buffer. That is, one might integrate two or more video frame (each being 1/30 sec), before an image is output from the camera. Asynchronous integrating cameras integrate and read out in the same way as video integrating cameras, but do not have fixed frame rates. They can accumulate an image onto the chip over any time period. Integration periods might be 100 msec, or any other time interval.
A preferred embodiment of the imaging system may be obtained by the use of long-term integration of the output of an image intensifier onto a cooled CCD camera.
With such a system components may be selected at reasonable cost to provide a system having low noise, flat field response, and high inherent contrast, which does not require direct coupling to specimens (is lens-based), and which can be used to image a broad variety of specimens over a very broad range of specimen intensities. At the user's option, frame averaging can be added to reduce noise, and shading correction can be added to remove background inhomogeneities.
The imaging system therefore is provided with an image intensifier placed in front of a CCD camera. The image intensifier has relatively low quantum efficiency (typically less than 20~), but can provide very high levels - 21~77~i5 of gain. The intensifier may be coupled to the CCD camera by a relay lens, or alternatively, by a fibre optic coupling. A relay lens allows the intensifier to be used with any camera, is not subject to the "chicken wire"
pattern that tends to appear in fibre optic images. In addition, a relay lens will not delaminate as fibre optic couplers tend to. Fibre optic couplers are advantageous as they transfer light more efficiently than relay lens couplers, and allow the intensifier to be used at lower gain.
of the intensifier, three major types (i.e. GEN
I (generation 1), GEN II (generation 2), and GEN III
(generation 3)) are in common use, each differing in component organization and in the materials from which the components are constructed. Most preferably an Extended Blue GEN III image intensifier is used fibre optically coupled to the CCD camera. The applicant has discovered that the intensified camera unit of the present invention is well-suited to imaging dim specimens. When used at video frame rates, this intensifier has a detection limit of about 4 x 10-7 foot candles. Used within the present invention, the intensifier becomes much more sensitive.
The synergistic combination of integration and intensification of the present system provides several advantages over integration alone. First, images can be formed in a much shorter time period. An exposure of 10 seconds with the present invention is roughly equivalent to an exposure of 3 to 4 minutes with a thermionically cooled integrating camera, and 1 to 2 minutes with a cryogenically cooled camera. Second, the present invention can be lower in cost than a high-quality cooled integrating camera.
In use of the integrating camera, the signal is accumulated onto a CCD sensor or element for a period of time lasting for two or more seconds up to several minutes.
Both video integrating cameras and asynchronous integrating cameras may be used with the present invention. Video integrating cameras accumulate the signal over a number of video frames, directly onto the CCD element and before the camera is read out into a frame buffer.
In biological research, cooled integrating cameras may be used if exposure time, convenience, and cost are not major factors. Preferably the integrating camera of the present invention incorporates a cooling element to maintain the CCD element at a cooler temperature and permit integration over a longer period of time.
A liquid, cryogenically or thermionically cooled camera, such as one incorporating a Peltier cooler element has, for example, been found to be suited for use with the present invention. In contrast to the 18 bit precision of cryogenically cooled camera, liquid or thermionically cooled cameras typically function to only 12 or 14 bit precision, are not as sensitive as cryogenic cameras, and need rather long exposures (typically 2 to 10 minutes) to image dim fluorescence, chemiluminescence or bioluminescence. One must sit with the imaging system while it exposes, and any number of lengthy test exposures are necessary before the best integration period is found for a particular specimen. The combination of integration and intensification of the present invention, however, permits faster imaging than when a conventional cooled camera is used alone, and operates conveniently and at lower cost than a cryogenically cooled integrating camera, but with the same sensitivity.
Accordingly, in one aspect the invention resides in an electronic imaging system which provides a very high level of sensitivity to enable imaging of biological and - 21~7~
chemical specimens at low light levels. The system includes an integrating cooled CCD camera which has coupled thereto an image intensifier. Incident illumination from the specimen is amplified by the intensifier, and the amplified light is accumulated onto the integrating camera over an integration period which typically lasts for at least one second. At the end of the integration period, the camera is read out to a dedicated controller or imaging apparatus to reproduce the light image. Frame averaging is used within the imaging apparatus or controller, to reduce noise and improve the dynamic range of the camera.
In addition, shading correction is applied to remove spatial variations in camera sensitivity and provide enhanced imaging of the light images.
In another aspect, the present invention resides in an image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device coupled to said output surface and being responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions to produce charge values representative of the intensified electron representation at each CCD region, integration means for integrating the charge values over an integration period to produce an adjusted charge for each said CCD
region and - 21~77'j~
provide an integrated signal representative of the light image, wherein said integration period is selected greater than one second and less than five minutes.
In a further aspect the present invention resides in an image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device and a fibre optic minifier for optically coupling the output surface to the charge coupled device, cooling means thermally coupled to the charge coupled device for dissipating heat therefrom, the charge coupled device responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions over a period of time to produce localized charge values representative of the intensified electron representation at each CCD region, integration means for integrating the localized charge values over an integration period to produce an adjusted charge for each said CCD region and provide an integrated signal representative of the light image, said integration period being selected greater than five seconds and less than one minute, and - ~1 577~S
output means for outputting said integrated signal.
BRIEF DESCRIPTION OF THE DRAWINGS
Further objects and advantages of the invention will appear from the following description taken together with accompanying drawings in which:
Figure 1 is a schematic illustration of a computerized system for imaging low-light specimens in accordance with a first preferred embodiment of the invention;
Figure 2 schematically illustrates a front view of the CCD camera used in the system of Figure 1;
Figure 3 schematically illustrates a side view of the CCD camera of Figure 2;
Figure 4 is a schematic illustration of the intensifier, fibre optic coupler, and CCD sensor used in the system shown in Figures 1 and 2; and Figure 5 is a schematic illustration of a stand-alone system for imaging low-light specimens in accordance with a second embodiment of the invention.
DETAILED DESCRIPTION OF THE DRAWINGS
Reference is made first to Figure 1, which shows an image intensifier system 10 in accordance with a first embodiment of the invention. The system 10 includes an integrating video camera 12 which incorporates a CCD
element 14, an image intensifier 16 and a fibre optic coupler 18 optically coupling the intensifier 16 to the - ~57755 front of the camera 12. An external camera control 20 (ECC) contains circuitry for controlling the camera and conditioning the signal. A computer 22 is also provided coupled to the input and output of the camera control unit 20 for software-driven control of the system operation and storing of output images.
Figures 2 and 3 show best the camera 12 for use with the system 10. The camera 12 includes a CCD element 14 positioned behind a camera aperture 25. To reduce dark noise produced by electrons within the CCD element, the CCD
element 14 is mounted to a heat sink 26, which in turn is thermally coupled to a Peltier cooling element 28 for providing enhanced heat dissipation. An electronic shutter mechanism 32 is additionally provided within the camera 12 for limiting the exposure of the image on the CCD element 14. Preferably the camera 12 is a high resolution 768H x 482V pixel black and white full frame shutter camera, with asynchronous reset capability and uniform modulation transfer characteristic functions. The camera 12 provides an 8-bit digital signal output via a standard digital interface protocol 34 such as EIA-422. An interlaced or progressive scan analog output is available from an integral frame buffer 36. The flexible provision of both digital and analog output permits the camera 12 to be used as a stand alone unit, with a display monitor, or as part of a computer image analysis system.
The camera 12 operation is controlled externally by the camera control unit 20. The control unit 20 controls the integration time of the camera 12, adjusts the intensifier gain, and adjusts the video gain and black level of the camera 12. The camera control unit 20 includes the asynchronous reset circuitry which operates to accentuate edges in the image. This circuit can be useful in restoring some of the edge degradation produced when 21~7 ~5~
using the system 10 at high gain factors.
Image averaging circuits 38 are also provided in the camera 12 to decrease noise. Data from the CCD element 14 are digitized and fed to the internal frame buffer 36.
As each frame arrives at the buffer 36, it is passed through an arithmetic logic unit which performs iterative averaging of the incoming frame with previous frames.
Although most image analyzers report data with 8 bit (256 level) precision, video cameras provide only about 45-50 dB
of signal-to-noise ratio for any individual pixel.
Intensifiers are much worse, particularly at high gain.
The present system 10 therefore is additionally provided with noise reduction circuitry to provide noise reduction by frame averaging. With such circuitry, as the number of frames imaged by the camera 12 increases, the root mean square noise amplitude decreases by a factor of 1 / V~~~
(where n is the number of frames).
In addition to controlling the operation of the camera 12 and the intensifier 16, the camera control unit 20 outputs the integrated data to the computer 22 where it may be stored, output to a video terminal 58 or a printer (not shown) or the like.
The asynchronous reset capability of the camera 12 is flexible, and takes external horizontal synchronization for phase locking to a trigger. When an initialization pulse is applied by the camera control unit 20, it resets the camera's scanning, and purges the CCD
element 14. At that point the image is transferred from the CCD element 14 to the internal frame buffer 36.
The shutter mechanism 32 is of a substrate drain type and provides rapid clearing of the CCD element charges, so that the camera 12 may be used at high shutter speeds without smearing of images. This feature is - 21~775~
particularly useful if the camera 12 is operated in photon counting mode.
Figure 4 shows best the intensifier 16 as being of the GEN III type and including a lens 40, a photosensitive cathode 42, a microchannel plate (MCP) 44, a phosphor screen 46, and a vacuum sealed body 48 or enclosure. The lens 40 is a high-numerical aperture design, which captures light very efficiently. The lens 40 focuses to within 30 cm, and permits system 10 to be used to image almost any specimen. At its output, the lens 40 is focused on an input window of the cathode 42 so as to transfer the specimen image thereto. The photosensitive cathode 44 is selected to emit electrons in proportion to the intensity of light falling upon it. The microchannel plate (MCP) 44 is positioned within the vacuum sealed body 48, between and coupled at each end to the cathode 42, and the phosphor screen 46. The MCP 44 is provided with an array of small diameter MCP channels 50, each of the MCP
channels 50 are coated with gallium arsenide. The electrons emitted from the cathode 42 are accelerated along the MCP channels 50 to the phosphor screen 46. As the electrons from the cathode 42 are accelerated along the small diameter channels 50, they strike the coated channel walls to produce additional electrons. As the multiplied electrons leave the MCP channels 50, they strike the phosphor screen 46 and produce an intensified image of the specimen on an output screen 52.
It has been found that the use of the Extended Blue GEN 3 image intensifier 16 is advantageous over other types of intensifiers in that the image provided on the output screen 52 is sharper, has less shading error, and has less noise than those produced by GEN 1 and GEN 2 intensifiers. It is to be appreciated, however that as better intensifier technologies are developed, they may 21577~
equally be incorporated into the present system 10.
The fibre optic coupler 18 is preferably a bonded fibre optic coupler used to couple the output screen 52 of the intensifier 16 directly to the CCD element 14 of the video camera 12. Direct fibre optic coupling of the intensifier 16 to the camera CCD element 14 is four to eight times more efficient than a lens in transferring light from the output screen 52 to the camera 12. In the present case, the fibre optic coupler 18 includes a fibre optic input window 60 and a fibre optic minifier 62. The minifier 62 provides a 1.5:1 fibre optic taper to a smaller fibre optic output window 64 which is bonded to the CCD
element 14. The 1.5:1 reduction ratio of the minifier 62 has been found to allow full use of the highest resolution obtainable from the image intensifier 16. It will present an image having over 76 linepairs/mm to the CCD element 14, with distortion of less than 3% and overall light transmission of >75~.
In use of the system 10, the intensifier lens 40 is adjusted to focus a bioluminescent or fluorescent specimen on the input window of the intensifier photocathode 42. The photocathode 42 emits electrons in proportion to the intensity of light from the specimen falling upon it. A voltage is applied to the emitted electrons as they move from the photocathode 42 into the MCP 44, thereby accelerating the electrodes as they enter the small diameter MCP channels 50. Within the thousands of MCP channels 50, the electrons collide with the coated channel wall surfaces and dislodge additional secondary electrons. In this manner, one input electron can generate thousands of secondary electrons to provide light amplification. The electrons are accelerated again as they leave the MCP 44, and then strike the phosphor screen 46 on its inner surface, to generate a visible image on the ~5~75~
output window 52. The visible image is then transferred from the output window 52 to the camera 12 by the fibre optic coupler 18.
The integrating camera is configured so that the highly amplified image generated on the output window 52 is focused by the lens 40 and coupler 18 onto the CCD element 14. To image low light specimens, the CCD element 14 integrates for a period of at least one second, accepting a trigger pulse from the camera control unit 20 to initiate the operation of the electronic shutter 32. The electronic shutter clears the CCD element 14, from which point the image is transferred to the internal frame buffer. For very dim specimens, a period lasting from two or more seconds upwards to more than one minute may be used.
During the integration period, photons from the output window 52 incident to the CCD element 14 are stored as negative charges (the Signal) in numerous discrete regions of the CCD element 14. The amount of the charge in each discrete region of the CCD element 14 is accumulated as follows.
Signal = Incident light x Quantum efficiency x Integration time The greater the relative intensity of the incident light coming from the intensifier 16, the greater the signal stored in the corresponding region of the CCD element 14.
Upon completion of the integration period, a signal representative of the specimen image is exported from the camera 12 to the computer 22 for output or further image averaging externally by the computer 22.
As the present invention is designed for imaging very dim specimens, and as the gain available from an - 215775~
intensifier is limited, relatively long periods of integration of the intensifier output onto the cooled integrating camera 12 are provided. For chemiluminescent blots, integration periods of 10 sec to 1 minute can be used, equivalent to a film exposure of about 1 minute to 4 minutes. For more dim specimens, multiple 10 sec to 1 minute exposures are necessary, and frame averaging by the control unit 20 (typically four frames) may be used to improve signal to noise ratio in a final output image.
Figure 1 shows the CCD camera 12 as being provided without an image averaging circuit with image averaging done externally on a personal computer 22. If desired, image averaging may equally be accomplished within the camera 12 itself or within the camera control unit 20, by the addition of image averaging circuitry. Figure 5 illustrates schematically a modified system 10 wherein like reference numerals are used to identify like components and in which the camera~unit 20 incorporates image averaging circuitry and outputs the final image as an analog signal sent to a display monitor 70.
Although the camera 12 could be used without cooling the CCD element 14, extended periods of integration are achieved by using a CCD camera with an integral cooling element. The effectiveness of integration is limited by the degree of cooling. With thermionic cooling using a standard Peltier cooling device, sensor temperatures of about -20C can be achieved. This allows integration for periods of up to about two minutes before background noise becomes bothersome. Thermionic cooling has the advantage of low cost and easy implementation.
It is to be appreciated, however, that longer periods of integration are possible if multistage thermionic, liquid, or cryogenic cooling are employed.
These more effective cooling methods would be combined with ~157;7~
..
a more sophisticated camera, capable of higher precision (10-16 bits is typical). Using a high precision liquid cooled camera does achieve better image quality and higher sensitivity, but with a corresponding increase in the cost of the camera.
In addition, with the system 10 shown in Figure 1, only the CCD element 14 is cooled. This is sufficient for routine imaging. It is to be appreciated however, that for more demanding tasks, the photocathode 42 could also be cooled, thereby improving the signal to noise ratio of the intensifier 16. Similarly, the entire photosensitive apparatus (i.e. intensifier and CCD) can be cooled.
However, cooling the entire photosensitive apparatus has the disadvantage that the efficiency of the phosphor on the fibre optic output window is decreased.
In the preferred embodiment of the invention shown in Figure 1, the system 10 incorporates an Extended Blue type of GEN 3 image intensifier 16. Other types of intensifiers, although less preferred, may also be used.
The three major types of intensifier (GEN 1, GEN 2 and GEN
3) differ in the organization of their components and in the materials of which the components are constructed. In a GEN 1 intensifier, illumination incident to a photocathode results in emissions at a rate proportional to the intensity of the incident signal. The electrons emitted from the photocathode are then accelerated through a high potential electric field, and focused onto a phosphor screen using electrostatic or proximity focusing.
The phosphor screen can be the input window to a video camera (as in the silicon intensified target camera), or can be viewed directly. GEN 1 intensifiers, however, suffer from bothersome geometric distortion, and have relatively low quantum efficiency (about 10~).
21~775~
The GEN 2 intensifiers, like the GEN 3, incorporate a MCP into an image tube, between the cathode and an anode. The GEN 2 intensifiers are smaller, lower in noise, and have higher gain than the GEN 1 intensifiers.
However, their quantum efficiency is fairly low (typically <20%), and they tend to suffer from poor contrast transfer characteristics. In contrast, the GEN 3 intensifier tube has a quantum efficiency of about 30% or higher (needs less gain), and very high intrinsic contrast transfer. With recent versions of the GEN 3, gain levels are about equal to those of a GEN 2 (ultimate gain level available is about 90,000). Therefore, a GEN 3 intensifier will tend to yield better images than a GEN 2. Where necessary for reasons of cost or specific design features, other forms of intensifier could be used. Similarly devices with high intrinsic gain (such as electron bombarded back-illuminated CCD sensors) could be used in place of image intensifiers.
The CCD camera 12 of the present invention uses an asynchronous reset which takes an external drive signal from the control unit 20 for phase locking. When the signal is applied, it resets the camera 12, also sc~nn;ng and purging the CCD element 14. As the CCD camera 12 incorporates mechanisms that provide very low lag, short integration periods (e.g. 1/16,000 second) can be used. If desired, these integration periods can be locked to a gated power supply (not shown) in the image intensifier 16, with the result that the camera 12 can be read out at very short intervals. Using the gating and fast readout feature, and with the intensifier 16 run at highest gain or with a multistage intensifier 16, the present invention can thereby be operated as a conventional photon counting light imaging system. Thus, the present system 10 can advantageously be used for both direct imaging of faint specimens, or as a standard photon counting camera by changing its mode of operation from integration to gating.
21~i77~i~
While the preferred embodiment of the invention discloses the use of a cooled CCD element 14, for relatively bright specimens, if cost is a major factor, the present invention could be constructed without cooling element 28. In this case, a camera 12 could be constructed at quite a low cost and integration periods of about 5 seconds can be achieved, albeit with a reduction in image quality and ultimate sensitivity.
Although the preferred embodiment of the invention illustrates a bonded fibre optic coupler 18 with a minifier 62 for coupling the intensifier 16 to the video camera 12, the invention is not so limited. If desired the image intensifier 16 could also be coupled to the integrating camera using a lens, a conventional fibre optic coupler or any other suitable optical coupling device.
Although the detailed description describes and illustrates preferred embodiments of the present apparatus, the invention is not so limited. Modifications and variations will now appear to persons skilled in this art.
For a definition of the invention reference may be had to the appended claims.
Claims (16)
1. An image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device coupled to said output surface and being responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions to produce charge values representative of the intensified electron representation at each CCD region, integration means for integrating the charge values over an integration period to produce an adjusted charge for each said CCD region and provide an integrated signal representative of the light image, wherein said integration period is selected greater than one second and less than five minutes.
2. An apparatus as claimed in claim 1 wherein said converting means for converting photons to said electron representation comprises a phosphor screen.
3. An apparatus as claimed in claim 2 wherein said output surface comprises a phosphor screen for converting said intensified electron representation into visible light representation of said image.
4. An apparatus as claimed in claim 1 further including fibre optic coupling means for optically coupling said output surface to said charge coupled device.
5. An apparatus as claimed in claim 4 wherein said converting means comprises a photocathode.
6. An apparatus as claimed in claim 4 further including cooling means for cooling said charge coupled device.
7. An apparatus as claimed in claim 6 wherein said cooling means comprises a thermionic cooling device.
8. An apparatus as claimed in claim 1 further including read out means coupled to said integration means for reading out and receiving the integrated signal.
9. An apparatus as claimed in claim 7 wherein said charge coupled device is coupled to said output surface by fibre optic coupling means.
10. An apparatus as claimed in claim 4 wherein said fibre optic coupling means comprises a fibre optic minifier coupled directly to said charge coupled device.
11. An image receiving and converting apparatus comprising, converting means responsive to a light image for converting photons from said image to an electron representation of said image, electron multiplier means for increasing the intensity of said electron representation of said image, the electron multiplier means having an input surface coupled to the converting means, and an output surface for outputting the intensified electron representation of the image thereon, a charge coupled device and a fibre optic minifier for optically coupling the output surface to the charge coupled device, cooling means thermally coupled to the charge coupled device for dissipating heat therefrom, the charge coupled device responsive to said intensified electron representation to produce a signal representative of said image, the charge coupled device including, a plurality of CCD regions charged on exposure of said intensified electron representation, controller means for cyclically reading the charge in each of said CCD regions over a period of time to produce localized charge values representative of the intensified electron representation at each CCD region, integration means for integrating the localized charge values over an integration period to produce an adjusted charge for each said CCD region and provide an integrated signal representative of the light image, said integration period being selected greater than five seconds and less than one minute, and output means for outputting said integrated signal.
12. An apparatus as claimed in claim 11 wherein said converting means for converting photons to said electron representation comprises a phosphor screen.
13. An apparatus as claimed in claim 12 wherein said cooling means comprises a heat sink thermally coupled to a cooling element.
14. An apparatus as claimed in claim 13 wherein said output surface comprises a phosphor screen for converting said intensified electron representation into visible light representation of said image, and said converting means comprises a gallium arsenide photocathode.
15. An apparatus as claimed in claim 14 wherein said electron multiplier means includes an array of MCP
channels.
channels.
16. An apparatus as claimed in claim 14 further including electronic shutter means for clearing said charge values from said CCD regions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002157755A CA2157755A1 (en) | 1995-09-07 | 1995-09-07 | Camera system for imaging at low light levels |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002157755A CA2157755A1 (en) | 1995-09-07 | 1995-09-07 | Camera system for imaging at low light levels |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2157755A1 true CA2157755A1 (en) | 1997-03-08 |
Family
ID=4156568
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002157755A Abandoned CA2157755A1 (en) | 1995-09-07 | 1995-09-07 | Camera system for imaging at low light levels |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2157755A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8278114B2 (en) * | 1999-07-21 | 2012-10-02 | Applied Biosystems, Llc | Method for measuring luminescence at a luminescence detection workstation |
| EP3002620A1 (en) | 2014-10-03 | 2016-04-06 | Thales | Method for producing a coupling optic for a low-light image sensing system and associated coupling optic |
| EP3024011A1 (en) | 2014-11-21 | 2016-05-25 | Thales | System for collecting low-light images comprising a lens having a phase and/or amplitude filter |
-
1995
- 1995-09-07 CA CA002157755A patent/CA2157755A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8278114B2 (en) * | 1999-07-21 | 2012-10-02 | Applied Biosystems, Llc | Method for measuring luminescence at a luminescence detection workstation |
| US20120309103A1 (en) * | 1999-07-21 | 2012-12-06 | Life Technologies Corporation | Method for measuring luminescence at a luminescence detection workstation |
| US8865473B2 (en) * | 1999-07-21 | 2014-10-21 | Applied Biosystems, Llc | Luminescence detecting apparatuses and methods |
| EP3002620A1 (en) | 2014-10-03 | 2016-04-06 | Thales | Method for producing a coupling optic for a low-light image sensing system and associated coupling optic |
| EP3024011A1 (en) | 2014-11-21 | 2016-05-25 | Thales | System for collecting low-light images comprising a lens having a phase and/or amplitude filter |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |
Effective date: 19980908 |