Abstract:
A method of autoradiography imaging includes steps of: (a) forming a subject having at least first and second markers, each marker providing radiation having a characteristic energy distribution; (b) detecting radiation from the marked subject using a semiconductor radiation detector having an array of cells, each cell recording a charge value dependent on the energy of incident radiation; (c) processing the output from the cells including discriminating charge values within at least two charge value ranges and allocating a display color value to each pixel cell position in the array dependent upon the recorded charge value; and (d) forming an image for display with individual cell positions having a color representative of the color values. The method enables multiple label or multiple marker imaging in autoradiography to be performed by energy discriminating imaging, thus enhancing experimental accuracy and reproducibility.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to a method and apparatus for autoradiography imaging. 
     Autoradiography is a technique which is widely used for imaging in various different application. Typically, imaging is performed by detecting beta rays using isotopes such as 3H, 35S, 32P, 33P and 14C and 125I (for X-rays). These isotopes are used as labels or markers for marking a subject to be imaged. Examples of subjects to be imaged can be slices of tissue taken from a human or animal body which has been marked by the radioactive isotope, or by another radiation emitting marker, or, a blot formed as a part of DNA, RNA, etc., analysis. 
     Where the sample to be imaged is a slice of tissue from a human or animal body, this will typically result from the injection of the body with radioactive markers, the sample then being taken after the marker has been dispersed within the tissue to be examined. 
     Where the sample is a “blot”, this can result from the conventional techniques such as “Western blotting”, “Southern blotting”, “Northern blotting”, etc. The technique most widely used for separating DNA, RNA or protein molecules of useful size is electrophoresis on an agarose gel which separates the DNA, RNA or protein molecules into discrete bands dependent on their size. The position of the bands on the gel is shown by a fluorescent ethidium bromide dye, or by autoradiography. This technique is carried out by denaturing and transferring the fragments using the so-called Southern, Northern or Western blotting techniques onto a matrix which can be probed with a radioactive DNA, RNA, protein or carbohydrate “probe” (a molecule that attaches to a specific location on the fragment). After the unbounded probe is washed off, the amount and position of the DNA, RNA, protein carbohydrate fragments which hybridized with the probe can be detected by counting the radioactivity or by autoradiography. 
     DNA sequence analysis is based on high-resolution electrophoresis on denaturing (SDS) polyacrylamide gels. Samples of label fragments are treated under four different conditions with chemical reagents that cause cleavage in known positions along the molecules. The pattern of the tracks and the resulting four “lanes” of sequences are used to read the sequence. Western blotting techniques are generally similar to the Southern blotting of DNA and are applied to separating and analyzing proteins as in the screening of antisera and antigens and DNA or RNA binding proteins. 
     RNA can be characterized (that is its base sequence or protein amino acids sequence determined) by an adaptation of the Southern blotting transfer technique, for example by so-called “Northern blotting” where RNA is transferred from the gel to nitro-cellulose under high salt conditions. The fractionated RNA characterization is by hybridization to specific probes usually labelled with radioactive markers. The process involves running a sample and then running a reference under hopefully identical conditions. 
     Most widely used today in the above methods is detection by film. This is a non-digital imaging technique with the radiated beta rays being recorded on the film. The image resolution is better than 50 μm, with a sensitivity for the isotope 14 C. (this isotope is used here as a reference) less than 0.015% and a dynamic range of two orders of magnitude. There is no possibility for real time imaging, although after image accumulation, digitization is possible. 
     Digital imaging is offered by a digital imaging plate operating on a photoluminescence principle. In this, beta rays are accumulated on the digital imaging plate which is later on scanned with a laser to produce a digital image. Image resolution with this technique is about 100 μm, the sensitivity to 14 C. is less than 1% and the dynamic range is about four orders of magnitude. Real time imaging is not a possibility for this type of autoradiography imaging. The whole image is first digitally accumulated and then displayed after the laser scan. 
     A further digital imaging technique is provided by wire gas chambers. Image accumulation and display is in real time, but the image resolution is at best 300 μm. The sensitivity to 14 C. is 1.5% and the dynamic range is 5 to 6 orders of magnitude. 
     A yet further imaging technique has been brought to the Applicant&#39;s attention which helps put the present invention in context. Nuclear Instruments &amp; Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, Vol. A228, No. 1, Mar. 1, 1990, pages 250to 253, XP000100349, Schooneveld E M et al; “a Silicon Strip Detector for Radiochromatography” discloses an imaging technique utilising a silicon strip detector, sensitive to β-rays and a single radioactive isotope marker. The analogue signal output from the detector is digitised by way of threshold circuitry and R/S flip flops. The image resolution for this detector is given as being better than 500 μm. 
     None of the above methods and systems provide an optimal combination of performance characteristics for use in autoradiography. Moreover, the conventional methods of performing autoradiography suffer from reproducibility difficulties. In other words, if a comparison is to be made between various markers, the process needs to be repeated at different distinct times. This has the disadvantage that conditions may change between the tests, and there are opportunities for errors to occur. 
     SUMMARY OF THE INVENTION 
     The present invention seeks to address and to mitigate the above-mentioned problems. 
     In accordance with a first aspect of the invention, there is provided a method of autoradiography imaging comprising: 
     a) forming a subject ( 11 ) having a first marker for providing radiation having a characteristic energy distribution; and 
     b) detecting radiation from said marked subject using a semiconductor radiation detector ( 20 ) characterised by: 
     c) forming the subject having at least a further second marker, wherein each marker provides radiation having a characteristic energy distribution; 
     d) detecting radiation from said marked subject using a semi conductor radiation detector having an array of cells, each cell of which records a charge value dependent on the energy of radiation incident thereon; 
     e) processing the output from said cells including discriminating charge values within at least two charge value ranges and allocating a display colour value to each cell position in said array dependent upon the recorded charge value; and 
     f) forming an image for display with individual cell positions having a colour representative of said colour values. 
     Thus, the invention provides a technique for performing multiple label or multiple marker imaging in autoradiography based on an energy discriminating imaging technique and the use of two, or more, markers, each providing a respective distinct radiation energy distribution. By simultaneously performing imaging for different markers, enhanced accuracy and reproducibility of the results is possible. 
     The colour values can be respective grey scale values for a predetermined colour or each colour can be a respective, distinct colour. 
     Preferably, the markers comprise radioactive markers, for example radioactive isotopes chosen from the following list: 3H, 35S, 32P, 14C and 125I. Preferably also, the markers emit high energy radiation having an energy in excess of 1 keV. More preferably the markers emit beta-rays and each provides a different energy distribution. 
     The invention finds application to a method where step (a) comprises forming a subject in the form of a DNA, RNA or protein blot by using first and second probes, each of which has a different radioactive marker. 
     The invention also finds application to a method where the method comprises marking a tissue sample with at least two markers. 
     In one embodiment step (b) comprises detecting radiation from the marked subject using a semiconductor radiation detector having a one-dimensional array of strip cells. 
     In another embodiment step (b) comprises detecting radiation from the marked subject using a semiconductor radiation detector having first and second one-dimensional arrays of strips arranged orthogonally to one another to define a two-dimensional array of pixel cells. 
     Step (b) can also comprise detecting radiation from the marked subject using a semiconductor radiation detector having a two-dimensional array of pixel cells. 
     In accordance with another aspect of the invention, there is provided autoradiography apparatus for performing the method as defined above, comprising a semiconductor radiation detector having an array of cells for recording a charge value dependent on the energy of radiation incident thereon from a marked subject and processing means for processing the output from the cells, the processing means being arranged to discriminate charge values within at least two charge value ranges and to allocate a display colour value to each pixel cell position in the array dependent upon the recorded charge value for forming an image for display with individual cell positions having a colour representative of the colour values. 
     Embodiments of the invention will be described hereinafter with reference to the accompanying drawings in which: 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is an overview of an example of an autoradiography imaging system in accordance with the present invention; 
     FIG. 2 is a schematic representation of one form of radiation detector for use in the imaging system of FIG. 1; 
     FIG. 3 is a schematic cross-section of the detector of FIG. 2; 
     FIG. 4 is a second schematic cross-section of the detector of FIG. 2; 
     FIG. 5 is a schematic cross-section of a second type of detector; 
     FIG. 6 is a schematic representation of part of the imaging system of FIG. 1; 
     FIG. 7 is a schematic representation showing the juxtaposition of two detectors as described with reference to FIG. 5; 
     FIG. 8 is a schematic representation of the processing of detected image signals; and 
     FIG. 9 is a flow diagram illustrating the processing of detected image signals. 
    
    
     DETAILED DESCRIPTION 
     FIG. 1 is an overall schematic representation of an example of an autoradiography system in accordance with the present invention. The system of claim  1  includes an radiation detector system  10  on which a sample to be imaged  11  is clamped, an image processing system  12  and a display  14 . The image processing system  12  can be implemented using a conventional personal computer suitably programmed to provide the necessary image processing. The personal computer  12  can be provided with a conventional hardware configuration including a processor, memory, background storage devices, keyboard and other input devices, input and output ports and a graphical user interface for interfacing with the display  14 . The computer can be programmed suitably to interact with the display and the user input devices and to receive and process electrical signals from the detector system  10 . 
     FIG. 2 is a schematic perspective representation of a double-sided silicon strip detector  20 . A schematic cross-section along line A—A is shown in FIG. 3. A schematic cross-section along line B—B is shown in FIG.  4 . The double-sided silicon strip detector will now be described with reference to FIGS. 2-4. It should be noted that FIGS. 2-4 are merely schematic. In practice, a silicon radiation detector includes a large number of parallel strips, rather than the two parallel strips in each direction illustrated in FIGS. 2-4. Also, FIGS. 2-4 do not show the edge details for providing connection to read-out circuits to be described later with reference to FIG.  6 . The edge connections can be provided in a conventional manner. 
     The body of the silicon strip detector  20  forms a depletion layer  31  represented in FIGS. 3 and 4. Parallel P + strips  25  are formed at the upper surface of the depletion layer  31 . Each P + strip  25  is covered with an aluminum strip  24  to achieve electrical contact for charge collection. A field oxide layer  23  extends between the aluminium strips  24 . A passivation layer  22  of silicon dioxide (SiO 2 ) is formed over the aluminium strips  24  and the field oxide layer  23 . The top surface of the detector  20  is provided with a thin MYLAR™ layer  21  to protect the underlying layers. In use the sample to be examined is clamped to the MYLAR™ layer  21  by a suitable clamping arrangement (not shown). The layers described above are best seen in FIG. 3 as the strips  25  run perpendicular to the plain of the cross-section. 
     At the lower side of the depletion layer  31 , parallel N + strip implantations  29  are provided, with corresponding aluminum strips  28  to provide good electrical connection. Electrical separation of the N + strips  29  is achieved with a P + layer  30 . A field oxide layer  26  is provided between the aluminium strips  28 . Also, a passivation layer  27  of SiO 2  is provided covering the aluminium strips  28  and the field oxide layer  26 . The structure of the layers and strips below the depletion layer  31  is best seen in FIG. 4 as the cross-section on line B—B runs perpendicular to the line of the strips  29 . 
     It will, accordingly, be appreciated that the strips  25  run perpendicular to the strips  29 . Accordingly, with strips running in perpendicular directions, a two-dimensional detector grid can be provided. With the example of a double-sided silicon strip detector as shown in FIGS. 2-4, beta particles must penetrate through the layers  21 ,  22  and  23  in order to reach the depletion layer  31 . The thickness of this entrance window (that is the combination of the layers  21 ,  22  and  23 ) can be as small as 1-2 μm. As mentioned above, as result of the perpendicularly running strips above and below the depletion layer  31 , two-dimensional detection of the location of the incident ray can be detected. This is achieved by detecting the charge which is caused by the incident ray on the closest adjacent strips  25  and  29 . The size of the charge created depends upon the energy of the incident ray. 
     FIG. 5 illustrates an alternative strip detector, this time a single-sided strip detector. As with FIGS. 2-4, FIG. 5 is merely schematic, and in practice the detector will include many parallel strips rather than the two shown in FIG. 5. A central depletion layer  39  and parallel P + strips  36  are formed at the lower surface of the depletion layer  39 . The P + strips  36  have adjoining aluminum strips  35  to achieve electrical contact for charge collection. A field oxide layer  34  extends between the aluminium strips  35 . A passivation layer  33  of silicon dioxide is formed over the aluminium strips  35  and the field oxide layer  34 . 
     At the upper surface of the depletion layer  39 , a pattern of N + strips  38  is provided in order to achieve good electrical contact to the read-out circuitry in order to provide good reliability, while at the same time minimizing the thickness of the entrance window which is formed by the field oxide layer  37  and MYLAR™ film  32 . A bias is provided on the N + strips  38  through a common bus line (not shown in FIG.  5 ). As shown in FIG. 7, several detectors of this type can be connected together to form a larger detection area without any wire bonds on the entrance surface of the detector. The composite detector can thus have a flat entrance surface with a smooth contact with the beta-ray active sample. 
     The MYLAR™ layer on the contact surface of the semiconductor substrate should be kept as thin as possible in order to allow the radiation reach the depletion layer of the detectors described above. How thin the layer needs to be is dependent upon the energy of the radiation it is intended to detect. Preferably the MYLAR™ layer is in the form of a film having a thickness of 10 μm of less, more preferably 5 μm or less, and yet more preferably 2 μm or less. As the MYLAR™ film forms substantially the thickness of the radiation entrance window, the radiation entrance window to a cell would thus have a thickness of 10 μm of less, preferably 5 μm or less, and more preferably 2 μm or less. 
     FIG. 6 illustrates bottom and cross-sectional schematic views of a double-sided strip detector module for the radiation detector system  10  of FIG.  1 . The detector module includes a strip detector  20  as illustrated schematically in FIGS. 2-4 mounted onto hybrid boards  42  and  43 . The detector strips are wire bonded  47  to readout chips  41  and  48 . Cables  40  and  46  provide connection to control and data acquisition electronics (not shown in FIG.  6 ). The detector module is supported by means of a structure  45  which is glued or otherwise attached on the side (that is the lower surface as shown in FIGS. 2-4) opposite to that on which the sample is to be placed. As shown in a schematic cross-section on line C—C, readout chips  41  for the strips  29  (see FIG. 4) at the lower side of the detector  20  are provided in a recess  44  in the supporting structure  45 . As shown in a cross-section along lines D—D a cover structure  49  provides protection for the readout chips  48  and associated wire bonds to the strips  25  (see FIG. 3) on the entrance (upper) side of the detector  20 . The readout chips  41  and  48  provide energy resolution based on charge accumulated for each and every impinging beta-ray. Typical beta-ray energy used for labelling, or marking, vary from 5 keV to about 1700 keV. Charge and consequently energy resolution which can be achieved with commercially available charge amplifiers is of the order of 5%. Preferably, readout chips  48  are operated in a self-triggering mode, where for each signal detected, which is above the minimum threshold, the value of the charge is then recorded. 
     Single detector modules made of silicon can have an active imaging surface of up to 10 cm by 10 cm. If larger areas are needed, this is possible by combining together individual silicon modules, for example as shown in FIG.  7 . FIG. 7 shows the reverse (lower) side of two single-sided strip detectors  51  and  53  connected together to form a larger detection area. The N + implantation  52  is patterned in a strip-like configuration to minimise the thickness of the entrance window as described above. The front side bias is provided through bus-lines  50 . Electrical contact between the detectors  51  and  53  is provided by conventional wire bonding or a thin layer of conductive glue or conductive polymers  54 . 
     FIG. 8 is a schematic representation of the processing performed by the apparatus as described above. In particular, control and data acquisition electronics  56  are connected via the cables  40  and  46  to the self-triggering readout chips  41  and  48 . The readout chips  41  and  48  include charge amplifiers, for example operable at 100 kHz providing 100,000 samples per second. Typical counting rates encountered in autoradiography extend from 0.01 counts/(min.mm 2 ) to 1000 counts/(min.mm 2 ), so that 100 kHz is normally sufficient. The charge amplifiers can be arranged to signal an incident beta-ray for charge values detected which are representative of an energy above a predetermined threshold, for example for energies in excess of 1 keV, or another preferred value, of, 4 keV. When the readout chips  48  detect a charge value greater than a predetermined threshold and supply this charge value to the control and data acquisition electronics  56 , the latter responds to this by supplying address information  57  to a digital signal processor  58 , which can be implemented by means of the personal computer as described above, for providing imaging processing. The analogue charge value is also supplied to an analogue to digital converter  59  which converts the charge value into a digital number for processing by the digital signal processor  58 . 
     FIG. 9 is a flow diagram illustrating the processing of an incident beta-ray hit. 
     At  60 , if a charge is registered representative of a radiation hit in excess of a predetermined energy (for example 1 keV or 4 keV), then the analogue charge value is supplied via the electronics  56  to the analogue to digital converter  59  and the address indicating the position on the detector at which the radiation was detected is supplied to the digital signal processor  58 . The digital value received from the analogue to digital converter  59  is stored  64  in the digital signal processor  58  in an appropriate location in a pixel map. If the detected charge value is above the selected marker threshold  66  then a first colour value  68  is allocated to the pixel position, otherwise a second colour value  70  is allocated to the pixel position. The resulting image can be displayed  72  in real time as the image is collected in the display  14 . The pixel map for controlling the display  14  can be stored in the digital signal processor in conventional memory. The pixel map can be arranged to accumulate intensity values for each marker type for each pixel. In other words, for each marker type which is being recorded for each pixel on the display, the number of hits for that marker type is recorded, thus providing the intensity of the specific marker type for each pixel on the display. 
     As well as discriminating between energy ranges, the digital signal processor can be arranged to count radiation hits within respective ranges and to allocate intensity values to each colour value, thereby permitting a display of the number and energy of the radiation hits. The digital signal processor is arranged to update the displayed image at user selectable or predetermined intervals. 
     Using the apparatus as described above, therefore, it is possible to use two separate markers for a sample to form the subject of autoradiography. For example, for toxicological and pharmacokinetic investigations using samples of human or animal tissue, drugs can be labelled with two different markers which emit different beta-ray radiation characteristics. Then, when the tissue sample is attached to the surface of the detector, the respect distributions of the markers in the sample can be measured at the same time to give a direct comparison of the distribution of the radioactive markers through the sample. That is, the detector detects radiation from both markers, but, through the use of the marker threshold at 66 in the process described in FIG. 9, different colours are allocated the charge values recorded depending on whether those charge values are above or below the threshold in question. The “colours” can be distinct colours, or alternatively can be grey-scales of a particular colour. Accordingly, it will be appreciated that it is desirable that the markers chosen have different radiation emission distribution characteristics (spectra) so that the use of a threshold will be able readily to discriminate emissions from the first and from the second marker. In practice, there will be some overlap between the emissions from the two markers, as the beta-ray emission characteristics are spread over a range rather than being restricted to a single energy value. 
     The same basic approach can be used, for example, for autoradiography analysis of “blots”. Thus, by applying two different RNA/DNA probes with different radioactive markers, a direct, immediate, and real time comparison under identical conditions can be achieved of the resulting blots. In other words, the blot will include different bands for each of the two markers so that the marker thresholding described above can be used to discriminate the bands resulting from each of the markers. It will be appreciated that, by the use of markers having different radiation emission distribution characteristics (spectra), concurrent examination using different probes can be achieved under identical conditions, thereby increasing accuracy and reproducibility of the autoradiography testing procedure. 
     Thus, there has been described a new technique for performing multiple label or marker imaging in autoradiography based on an energy discriminating imaging technique. Impinging beta-rays originating from different isotopes within the same sample are registered/coloured according to their energy. With currently available charge amplifiers offering a resolution in energy of the order of 5% or better, a high level of discrimination is possible. In the preferred embodiments, a strip semiconductor detector (double or single-sided) can be used. The inactive depth in the semiconductor entrance phase is typically of a few microns and since a sample can be brought and pressed into contact with the semiconductor, the efficiency is extremely high compared with any other method used today for beta-ray imaging and autoradiography. For 14 C., an efficiency of greater than 70% with a position resolution of better than 50 μm can be achieved. Large imaging areas can be constructed by combining single semiconductor strip detector modules in the manner described with reference to FIG. 7, or in other ways (e.g. tiling). As mentioned above, the imaging can be performed in real time with user defined image display updates. 
     Although particular embodiments of the inveniton have been described, it will be appreciated that many modifications and/or additions may be made within the scope of the invention. 
     For example, although silicon has been described as the preferred semiconductor, other options may include cadmium telluride (CdTe), cadmium zinc telluride (CdZnTe), mercury iodide (Hgl), lead iodide (Pbl), and so on. 
     Although strip detectors have been described, other detector configurations, for example based on individually addressable pixel cells, could be used. 
     Also, although in the preferred embodiments a personal computer has been employed for implementing the digital signal processor, it will be appreciated that some of all of the functions performed in the present invention may be implemented by means of special purpose hardware, using, for example, ASIC, or like technology. 
     Moreover, although the use of two markers having respective energy distributions is described, the invention includes the use of three or more markers and discrimination of three or more energy ranges with respect to appropriate thresholds to provide multiple label radiography imaging.