ENHANCED AUTORADIOGRAPHY

Abstract: The invention comprises a method of more efficient detecting of radioactivity from a low energy radioactive source and apparatus therefor. The method comprises providing a sample prepared for presentation in close readable contact with a detector, providing a scintillator material in sheet form adapted for application to the prepared sample, contacting the prepared sample with said sheet to provide a layered composite, providing heating means and means for evenly distributing heat to the layered composite, applying heat to the layered composite for a sufficient period to permit the scintillator material to be introduced intimately to the sample. Then, after a cooling, the sample is then exposed to a detector means capable of detecting emissions from the scintillator material arising from interaction with the radioactivity in the sample and thereby determining the radioactivity of the sample.

Description: Enhanced Autoradiography This invention relates to a method of detecting radioactivity and in particular to a method for enhanced and quicker detection of radioactivity. Prior art liquid scintillation counters and gamma counters have enabled those involved in scientific research to utilise the quantitative accuracy of radioisotopes in biological experiments. Other methods of detecting radioactivity are autoradiography with X-ray film, storage phosphor screens with laser scanners to replace the X-ray film, and instant imaging wherein direct, reasonable resolution imaging and quantitation of radioactivity in flat samples occurs. However, these are often slow to produce results for low energy radionuclide such as Tritium( 3 H) , in the case of the Instantlmager this is not possible at all. Autoradiography in many cases may take as long as eight to ten weeks, or even longer, to produce an image. There are Phosphor Imagers on the market at present wherein the sample is held in close contact with a screen containing phosphors, this allows the radio decay events from the sample to strike the phosphors. These phosphors store the energy of the emitted beta particle. The screen is then taken and placed in a laser reader in order to release the stored energy as light, to obtain an image. These screens are very expensive for Tritium( 3 H) , and can be easily damaged by contamination, moisture, or by handling. There are also Instant Imagers on the market which produce the results much more quickly. The samples are loaded directly into the detection machine which is connected to a computer via a fibre optical link. Data is transmitted at high speed through the optical cable to the computer as the image is acquired. The detector machine allows for direct nuclear counting of the entire sample area simultaneously without scanning or stepping. The image can be observed by the operator, on the computer VDU, as the image file is acquired by detection of the decay events. Direct beta counting ensures accurate and reproducible sample measurement. The above prior art systems can be used for radio isotopes such as 14 C, 35 S, 32 P, 33 P, etc., but none of these systems have proved successful in routine detection of radioactive tritium( 3 H) contained in samples, at a reasonable cost and time. Aims of one or more aspects of the present invention include providing a process which is cost efficient, quick to provide results, can be used effectively for samples containing most forms of radioactivity and in particular radioactive tritium( 3 H) , is in general, non destructive of the sample, and which obviate or mitigate the aforementioned disadvantages of the prior art. According to one aspect of the present invention there is provided a method for the detection of radioactivity in a biological sample which comprises providing a sample prepared for presentation in close readable contact with a detector e.g. as a smooth planar surface, providing a scintillator material in a sheet form adapted for application to the prepared sample, contacting the prepared sample with said sheet to provide a layered composite, providing heating means, providing means for evenly distributing heat to the layered composite, applying heat to the layered composite for a sufficient period to permit the scintillator material to be introduced intimately to the sample, and after a cooling period, providing a detector means capable of detecting emissions from the scintillation material arising from interaction with the radioactivity in the sample and thereby determining the radioactivity of the sample. According to another aspect of the invention there is provided an apparatus for the detection of radioactivity in a biological sample which comprises a permeable substrate containing or treated with the sample, a meltable sheet containing a scintillation material, means for providing heat sufficient to cause melting of said sheet, means for evenly distributing heat to said sheet, optionally including means for cooling said sheet, and including means for detecting emissions from the scintillation material responsive to the radioactivity in the sample. The invention thus defined is of particular benefit in the examination of samples labelled by weakly radioactive sources such as tritium( 3 H) which presents difficulty for detection. The invention will now be further described by way of examples herein described below. A method for the detection of the radioactive isotope Tritium( 3 H) is provided wherein samples such as macromolecules are radiolabelled with radioactive tritium( 3 H) directly on a flat matrix such as paper, cellulose or glass fiber filter, polyester or aluminium thin layer chromatographic plate, electrophoreses gels which have been electroblotted or filtered on to membranes, and other flat samples. The flat membrane can be of any suitable size for example 20 cm by 20 cm and can be of a commercially available type which is suitable for the purpose such as Whatman DE81, GFB or GFC, etc. A wax scintillator sheet is then placed on top of the sample. A flat glass sheet of substantially similar dimensions to the flat membrane is taken and covered on its top surface with a piece of double sided silicone release material . It is essential that the silicone release material lies flat against the surface of the glass and does not wrinkle or crease. The sample as prepared above is placed on top of the double sided silicone release material and then covered with another piece of double sided silicone release material again of similar dimensions as the flat membrane. Again care must be taken to ensure that the double sided silicone release material is smoothed flat against the sample before putting a further piece of glass on the top. The glass should be 4mm in thickness. The so packaged sample is then placed in a heated cabinet at between 50°C - 55°C for a period of 5 to 10 minutes. This allows the heat to be evenly distributed throughout the sample ensuring that the wax scintillator sheet melts evenly throughout the sample. It is advantageous to preheat the glass plates to the working temperature before use, and to clamp the completed assembly before placing in the oven. The packaged sample once it has been in the heated cabinet for the desired length of time is then removed and cooled by placing the package in the refrigerator. The sample which is now embedded in wax, is removed from the assembly, placed in a light-tight autoradiographic cassette with X-ray film which has been preflashed. The cassette should have both inside surfaces lined with aluminium foil, which will act as reflectors and increase the efficiency of detection, is placed in a -70°C deep freeze. The procedure when using polyester or aluminium thin layer chromatographic (TLC)plates is slightly different. The wax scintillator should not be applied as a single sheet but cut into strips 2 cm wide, and * placed 1 cm apart. This ensures that air bubbles are not trapped in the silica. The sample is then left in contact with the film for the required period of time generally 2 to 3 days. The film can then be developed and the image visualised. The advantages of Enhanced Autoradiography are that : The time interval between an experiment or procedure being performed and the results being obtained can be substantially reduced. Samples which normally take 8 to 10 weeks, can now be ready in 2 to 3 days. The process is in general non-destructive of the sample, so further analysis can be performed on that sample. The invention allows the accurate quantitation of radioactivity particularly that of tritium( 3 H) which currently is not possible. Tritium( 3 H) has always been the radioisotope of choice for both research, and routine laboratory use, but it has always been the most difficult to detect. Enhanced Autoradiography solves the detection problem. High cost equipment, with high cost consumables, and maintenance are not required. Enhanced Autoradiography will benefit research and routine science, in the Third World countries as well as developed countries. There are plastic Scintillators commercially available, FlexiScint from Packard Instrument Ltd. Holland, and MeltiLex from Wallac Oy, Finland. Both these products were designed to be specifically used with "dot blots", and Liquid Scintillation counters as the final detectors. The invention will now be further described by way of Experiments 1 and 6. However, a novel and inventive wax scintillator material has been developed for the purposes of this invention. The Wax Scintillator which has been designed specifically and optimised for Enhanced Autoradiography, with X-ray film as the final detector, has the following characteristics. A melting point of 48°C which ensures that neither the sample nor the membranes used are heat damaged. The wavelength of the emitted light is matched to the optimum response of the X-ray film (364nm) . The solvent should have a flash point well above the melting point of the wax. The Wax Scintillator should be able to be washed completely from the " sample by use of a suitable solvent such as toluene. The components and formulae for the wax are: Paraffin wax 5205. <1000g.) Liquid Diisopropylnaphthalene. {500g.) 2,5-diphenyloxazole(PPO) . (3g) The PPO is dissolved in the liquid Diisopropylnaphthalene at a temperature of 95°C and the wax added. The temperature is maintained for a period of 30 minutes while the mixture is continually stirred. The mixture is then formed into sheets of the required thickness. The materials are available from Shearer Ltd. Ibrox, Glasgow: Rutgers Kureha Solvents GmbH, Germany: Packard Instrument Ltd. Groningen, The Netherlands. Two sided Silicone release material grade RB/90/11 88GSM is available from Cotek Paper Ltd. Glos. England. Experiment 1. Conventional autoradiographic detection of Tritiated( 3 H) and Carbon-14 ( 14 C) sugars on paper is compared to the enhanced autoradiographic procedure. Mixtures containing D- [ 14 C]glucose, D- [ 3 H]mannose, L- [ 3 H]arabinose and L[ 3 H] fucose were separated by descending paper chromatography on Whatman 3MM paper in ethyl acetate / pyridine / water (8:2:1) for 24 hours. Samples applied were (i) InCi of each monosaccharide, (ii) lOnCi of each monosaccharide, (iii) lOOnCi of each monosaccharide. This procedure was carried out on three separate sheets of paper. (1) . Chromatographic paper was placed in a cassette with X- ray film, placed in a -70° C freezer and exposed for 7 days, then developed. Figure 1(a). (2) . Chromatographic paper, before exposure to the film, was treated by a process called Fluorography. This is a process which improves the detection of Radio Isotopes on X- ray film, by saturation of the paper with a fluor(PPO) . When the radioactivity on the surface of the paper interacts with the fluor, light is produced. Signal enhancement results because it is easier to detect light than to detect the beta particle directly. All other conditions were as described in (1) above. Figure Kb). (3) . Chromatographic paper, before exposure to the film, was treated by the process called Enhanced Autoradiography. A wax scintillator was melted at low temperature (55°C) into the chromatographic paper. The chromatographic paper where the wax was applied became translucent. Almost all of the radioactive decays , throughout the paper, were converted to light, and more easily be detected. All other conditions were as described in (1) above. Figure 1( c ). Data on the detection of radiolabelled sugars on paper chromatography Figure 1 shows the difference between the 3 methods used. ( a ) Autoradiography. ( b ) Conventional Fluorography. ( c ) Enhanced Autoradiography. The superior detection efficiency of Enhanced Autoradiography, over the other methods is obvious, by visual inspection. Further enhancement could be achieved, by placing sample (3) and the X-ray film between two sheets of aluminium foil prior to loading them into the cassette. The advantages of Enhanced Autoradiography in this experiment are: 1. The separated components, D- [ 14 C]glucose, D- [ 3 H]mannose, L- [ 3 H]arabinose and L[ 3 H] fucose shown on figure 1( c ) (iii) may be cut out from the chromatographic paper, placed in a vial, and counted in a Liquid Scintillation Counter. In this way the amount of each component can be quantified. 2. There is no requirement for a liquid scintillation cocktail. 3. After quantification as described in 1. above, the sample can be washed with an appropriate solvent, such as Toluene, to remove the Wax Scintillator. The sample is left intact for further investigation. 4. Enhanced Autoradiography is so efficient that results that would normally take between 8 to 10 weeks to obtain can now be achieved in 3-4 days. 5. Due to the long exposure times required in the past, researchers increased the amount of radioactivity to try and speedup the process. The amount of activity now required can be reduced considerably, with great financial and material savings, and reduce the problem of disposal of radioactivity. Experiment 2. Detection of tritiated( 3 H) labelled compounds after Thin Layer Chromatography(TLC) . Samples of tritiated( 3 H) labelled CTP, UTP, GDP, and TTP were separated using thin layer chromatography(TLC) . All compounds were spotted onto the TLC plate as a 2μl sample of stock solution of lμM, and the specific activities of the stock were as follows: CTP: 1.7kBq; UTP: 2.7kBq; GDP:2.5kBq; and TTP:6.2kBq. Standard autoradiography was used in an attempt to visualise the spatial distribution of the various tritiated( 3 H) labelled compounds. The TLC plate was exposed to preflashed X-ray film , in a cassette, which was placed in a -70 C freezer . After 8 weeks the film was developed and was found to be blank. The same TLC plate was then prepared for Enhanced Autoradiography. The wax scintillator was melted into it at a temperature of 55 C for a period of 10 minutes. After the wax scintillator was cooled, the prepared plate was again placed in a cassette with preflashed X-ray film, and placed in a -70°C freezer. The film was developed after a period of 10 days. The resultant image is shown. Figure 6. The advantage of Enhanced Autoradiography in this experiment is: The superior detection efficiency of Enhanced Autoradiography over Standard Autoradiography is obvious, by visual inspection. The tritiated( 3 H) labelled compounds are all clearly visible. A usable image could well be available in a shorter time. Standard Autoradiography produced no result after 8 weeks. It could be more than double this period of time before a usable image was produced.

Claims: CLAIMS 1. A method for the more efficient detection of radioactivity from a low energy radioactive source in a biological sample which comprises providing a sample prepared for presentation in close readable contact with a detector, providing a scintillator material in a sheet form adapted for application to the prepared sample, contacting the prepared sample with said sheet to provide a layered composite, providing heating means, providing means for evenly distributing heat to the layered composite, applying heat to the layered composite for a sufficient period to permit the scintillator material to be introduced intimately to the sample, and after a cooling or freezing period, providing a detector means capable of detecting emissions from the scintillation material arising from interaction with the radioactivity in the sample and thereby determining the radioactivity of the sample. 2. A method as claimed in claim l, in which the radioactive source is tritium ( H) . 3. A method as claimed in claim 1 or 2 , in which the detector is provided as a smooth planar surface. 4. A method as claimed in claim 1,2 or 3, in which the sample is provided on flat membrane. 5. A method as claimed in claim 1,2,3 or 4, in which the scintillator material is formed from wax. * 6. A method as claimed in claim 5, in which the forming of the wax scintillator material includes dissolving 2,5- diphenyloxazole in liquid Diisopropylnaphthalene at a temperature of about 95°C, subsequently adding the wax and maintaining the temperature for about 30 minutes whilst stirring continuously. 6. A method as claimed in any one of claims 1 to 5, in which a wax scintillator is contacted with the prepared sample to provide a layered composite. 7. A method as claimed in any one of the preceding claims, in which the layered composite is heated to a temperature of between 50°C and 55°C for a period of 5 to 10 minutes to permit the wax scintillation material to be introduced intimately to the sample. 8. A method as claimed in claim 1, in which the sample is provided on; the scintillator material in sheet form is placed on top of the sample; the sample is then placed on a piece of double sided silicone release material smoothed flat over a piece of glass and then covered with a second piece of double sided silicone release material and a further piece of glass; heating the sample so that the heat is evenly distributed throughout the sample and thereby melting the sheet of scintillator material to provide a layered coposite; cooling the layered composite to allow the melted material to set; subsequently removing the second piece of double sided silicone release material and further piece of glass; and contacting the sample with X-ray film. 9. A method as claimed in Claim 1, in which the layered composite along with the now intimately introduced scintillator material is placed in contact with X-ray film in a light - tight autoradiographic cassette and subjected to freezing at about -70°C. 10. A method according to Claim 8 or 9 in which the glass for the covering step is about 4mm in thickness. 11. Apparatus for the detection of radioactivity from a low energy radioactive source in a biological sample which comprises a permeable substrate containing or treated with the sample, a meltable sheet containing a scintillator material, means for providing heat sufficient to cause melting of said sheet, means for evenly distributing heat to said sheet, optionally including means for cooling said sheet, and including means for detecting emissions for the scintillator material responsive to the radioactivity in the sample. 12. Apparatus as claimed in claim 11, wherein the meltable sheet containing a scintillator material is formed from wax. 13. Apparatus as claimed in claim 12, in which the wax scintillator material comprises about lOOOg paraffin wax 5205, about 500g liquid Diisopropylnaphthalene, and about 3g 2, 5-diphenyloxazole. 15. Apparatus as claimed in claim 13 in which the wax scintillator is in the form of planar sheets. 16. Apparatus as claimed in any one of claims 11 to 15, wherein the permeable substrate is a flat membrane.

Inventors: FOX FRANCIS JOSEPH, FOX, FRANCIS, JOSEPH
Applicants: FOX FRANCIS J, FOX, FRANCIS, JOSEPH
Application year: 1997
Main classification: G01T1
IPC codes: G01T1