WO1992005427A1

WO1992005427A1 - Method and device for the scanning of the distribution of sources of radiation in a medium, for example the distribution of proteins in a gel

Info

Publication number: WO1992005427A1
Application number: PCT/DK1991/000287
Authority: WO
Inventors: Peter Mose Larsen; Stephen John Fey; Palle Rasmus Jensen
Original assignee: Jydsk Telefon A/S
Priority date: 1990-09-25
Filing date: 1991-09-24
Publication date: 1992-04-02
Also published as: AU8650191A; DK232090A; DK232090D0

Abstract

A method for the scanning of radiation source distribution, e.g. protein distribution in a medium (1), e.g. a 2-D electrophoresis gel which is placed in contact with a phosphor-containing plate (2), is executed by scanning with punctiform laser light (3) from a laser (5). The blue light (16) thus generated is scanned with a photomultiplier tube (4), after which the scanned data is used for electronic image processing. The scanning of the same medium and the same plate can be repeated. The application also relates to an apparatus for the execution of the method.

Description

METHOD AND DEVICE FOR THE SCANNING OF THE DISTRIBUTION OF SOURCES OF RADIATION IN A MEDIUM, FOR EXAMPLE THE DISTRIBU¬ TION OF PROTEINS IN A GEL

Background of the invention

The invention relates to a method of the kind disclosed in the preamble to claim 1, and to an apparatus for use in the execution of the method as disclosed in the preamble to claim 7.

In connection herewith, a detection is effected of dis¬ tributed and/or separate atoms which are able to emit radiation, e.g. beta, gamma, x-ray, chemiluminescent and/or fluorescent emission. In order to illustrate the use of the method and the apparatus according to the invention, a two- dimensional gel-electrophoresis of (³⁵S)-methionine labeled proteins has been used.

2-D gel-electrophoresis is a technique which permits a very selective and sensitive analysis of the quantititive amount of the protein or the various proteins in a sample, e.g. a sample of tissue.

Several variations of the method exist. One of the most sensitive and selective methods involves that via a cul¬ tivation of a tissue or cell structure, all of the proteins which contain methionine are labeled with (³⁵S)-methionine. This isotope has a half-life of approx. 87 days and decays by emitting soft β-rays (electrons) with an energy of 0.17 MeV.

The first dimension consists in exposing the sample, which is of a few microlitres, to electrophoresis through a pH- gradient with isoelectric focussing. The sample is thus dispersed along the pH-gradient, and the proteins are depo- sited in positions in which their electrical character¬ istics match those of the gel.

The second dimension involves encapsulating the proteins in a negatively-charged medium SDS (sodiumdodecylsulphate) which deposits itself like a jacket around each individual protein. At the same time, the protein is stretched out to an elongated structure, the length of which depends on its size (molecular weight).

The gel with the pH-gradient (1-dimensional gel) is placed along the one edge of a gel plate with a suitably open structure, e.g. polyacrylamide, and an electric current is sent lengthwise along the plate. The SDS-encapsulated pro- teins are hereby drawn through the gel at speeds which depend on their size.

After an appropriate time, the proteins lie distributed over the whole of the gel, which is typically plate-formed and in the size range of 20 x 20 cm, but strongly varying, and the proteins lie like stains whose relative positioning is specific for the individual protein and the electro- phoretic conditions under which the sample is analysed. The extent and the activity of the stains indicate the amount of the relevant protein, which is very decisive information in the diagnosis of many sicknesses.

By a process called blotting, e.g. electro-, contact or suction blotting, the proteins distributed in the gel can be transferred to paper or film, without any significant change in the distribution in the plane of the proteins.

Today, the analysis of the distribution and amount of pro¬ teins is effected by giving the gel an application of flourescent material which emits blue light at a wavelength of approx. 400 nm, which by long-time exposure is capable of printing an X-ray film, so that a picture is provided of the amount of proteins. By placing the film under a video camera or a scanner, various data can be fed into a com¬ puter for later data processing.

This known technique is extremely unsatisfactory, the reason being that the sensitivity of the film is so low that it must be exposed for many days. This time extends the period of time a patient must wait, with subsequently high human and financial costs. Moreover, the X-ray film is not linear, which means that the amount of data must go through an extra processing stage in order for it to be us¬ able. Furthermore, the dynamic range of the X-ray film is far smaller than the difference between the contents of the information of the strongest and the weakest stain. This means that several films with different exposure times must be developed, after which the information contents of the individual films are added together, which further complic¬ ates the scanning and increases the costs and time consump- tion.

From, for example, USA patents nos. 4,777,597 and 4,852,050, it is known to scan a phosphor-containing sheet after it has been in contact with, for example, a 2-D electrophoresis gel before exposure, and that a finished "electrical" image is formed on the sheet. The scanning is effected by punctiformed scanning with laser beams, which make the sheet release emission which is scanned with, for example, a photomultiplier tube. This type of scanning of the information from, e.g. 2-D electrophoresis gels, has the great disadvantage that the scanning of the contents of the information on the sheet cannot begin until its expos¬ ure by contact with the gel is completed, which lasts sev¬ eral hours. The patient from whom the sample has been taken must thus wait maybe several days for the result, and even longer if the result shows that the sample must be taken again. Since these forms of investigations are often used for the examination of very sick people, it will easily be appreciated that the possibility of being able to present a safe diagnosis is of very great significance.

The phosphor-containing sheet has the known characteristic that phosphor absorbs energy at one wavelength and can emit energy at another wavelength, when that area (the atoms) which are influenced are stimulated with a suitable source of emission, e.g. a light source. If, for example, a phos¬ phor sheet is exposed to β-radiation, the atoms stimulated will be raised to a higher energy level which is stable. If these atoms are stimulated with, for example, laser beams with a wavelength of 632 nm, the atoms will be raised to an unstable energy level, and will immediately decay to their original stable level during the emission of blue light.

From- European patent application no. 159,523 it is known to place a medium, e.g. with isotope labeled molecules, in permanent contact with a stimulable phosphor sheet. When the exposure, cf. above, is finished, the scanning of the contents of the information on the phosphor sheet can com¬ mence without the sheet first being separated from the medium. Therefore, the sheet and the medium are placed in a holder in which they are held between glass plates, so that the scanning becomes possible without first having to re¬ move the stimulable phosphor sheet.

Advantages of the invention

By proceeding as disclosed and characterized in claim 1, e.g. while using an apparatus as disclosed and character¬ ized in claim 6, it is possible to immediately undertake a provisional scanning of the emission source distribution, e.g. the distribution of protein in a medium, e.g. a gel after the 2-D electrophoresis method. The expression "shortly after" in claim 1 means that it is not necessary to wait until after the many hours' long normal exposure time, but that the scanning is commenced immediately after the medium has been placed in contact with the sheet in a suitable holder. Particularly in the examination of very sick patients, it is important that a correct diagnosis on the basis of a sample of tissue or the like can be presented as quickly as possible.

The known methods scan the whole of the medium when it has been completely exposed. But the invention introduces "intelligence" into the scanning, in that, for example, it is utilized that one knows where in the medium the informa- tion being sought is lying, whereby the time consumption is reduced. In some investigations, the medium has character¬ istic, punctiformed exposures which can be used as labels for electronic alignment of an askew gel, or to delimit the area which is to be scanned. By knowing where the informa- tion being sought is to be found, this area of the gel can be determinitive for how long the exposure should last. In this way, it is thus often possible to scan information which cannot be scanned at all by the known technique.

In this manner the medium can be scanned after a shorter period of exposure than by the X-ray film method, and also much earlier than with the known apparatus and methods for the scanning of emission energy stored in an radiation- sensitive sheet, for example a stimulable phosphor sheet. It is also possible to scan the medium's data several times during the exposure. The data collected is shown on a data monitor or is stored on a suitable storage medium gradually as it is collected. Some very important provisional results can thus be obtained, which can perhaps make further expos- ures superfluous. Not only are the results of the invest¬ igation obtained much more quickly than by the known methods, but the costs are also considerably reduced.

Special investigations can be carried out very quickly, e.g. of proteins which are known beforehand to be distrib- uted in certain places in the medium. The scanning can also be effected in "stripes", e.g. with the view to the ana¬ lysis of the DNA content after so-called genetic screening. By a quick and more coarse provisional scanning, it is also possible to have scanned an area which requires closer in- vestigation, after which a more detailed scanning of a limited area can be carried out.

The phosphor-containing material is activated very effect¬ ively by, for example, β-radiation, i.e. almost no decay goes to waste, and the material has proven to be linear over a very wide dynamic range. The active material main¬ tains its condition until the scanning is effected, and can be re-used innumerable times.

By proceeding as disclosed and characterized in claim 2, the possibility is provided of addition, e.g. of a first and a second scanning. Each time the phosphor sheet or a part hereof is scanned, at the same time there occurs an erasure of a part of the information it contains. Since the phosphor sheet is in permanent contact with the gel with the sources of emission, this is not normally of any sig¬ nificance, in that the "image" on the sheet is under con¬ stant exposure. Moreover, under certain conditions it can be of importance that the results of several scannings can be added together.

By proceeding as disclosed and characterized in claim 3, the costs and the time taken to carry out the investiga¬ tions can be further reduced, in that several media are scanned by the same scanner, since there is sufficient time for this to be effected. By proceeding as disclosed and characterized in claim 4 or 5, other purely practical advantages can be achieved, and with this method a very thin phosphor sheet is used, through which the irradiation takes place. Alone the use of very thin phosphor sheets has shown to provide the desired results.

The invention has been developed especially for use in con- nection with a method as disclosed and characterized in claim 6, which has great practical application in the diag¬ nosis of sicknesses, inheritable conditions and character¬ istics etc.

By configuring the apparatus according to the invention as disclosed and characterized in claims 7-13, a simple and herewith inexpensive apparatus is achieved, and which in a fully automatic and reproduceable manner and at reasonable costs can scan one or several media at any time and in accordance with requirements. The apparatus is simple to operate, and the results of the investigations are quickly obtained.

By letting the scanning be controlled by the electronic circuit, which for example is a suitably programmed com¬ puter or other program-controlled apparatus, "intelligence" is introduced into the scanning. The programming will norm¬ ally be such that by inter-active control the apparatus is made to scan desired areas. Together with the circuit, the user controls which part and which item among several in the apparatus shall be examined first. If desired, it is also possible to return to a scanned item and, for example, scan a certain area in more detail, or after shorter or longer periods of further exposure. The drawing

The invention will now be described in more detail with reference to the drawing, in that

fig. 1 shows the principle according to the invention with a first embodiment,

fig. 2 shows the principle according to the invention with a second embodiment,

fig. 3 shows, on a larger scale, what takes place in the scanning of a medium, e.g. a 2-D gel,

fig. 4 shows an example of a practical embodiment of the apparatus according to the invention,

fig. 5 shows an example of an embodiment of that part of the apparatus which fixes the medium and the sheet,

fig. 6-8 show another example of an embodiment of a hol¬ der for the medium and the sheet,

fig. 9 shows a second example of a practical embodi¬ ment of the apparatus according to the inven¬ tion.

Description of embodiment examples

The reference number 1 in fig. 1 indicates a 2-D gel which is produced by a commonly-known electrophoresis process so that the proteins are distributed over the area of the plate, and have been labeled with, e.g., 35S atoms or sim- ilar radio-active material. The β-radiation 15 from the protein molecules in the sheet 1 is intercepted by a phos- phor-containing plate 2, and those atoms in the plate 2 which receive the β-radiation are raised to a stable energy level which is higher than the non-stimulated atoms in the plate. Hereafter, the contents of the information in the plate 2 are scanned by means of a light source 5 which emits a very thin light beam 3. The light source can, for example, be a HeNe laser which emits light 3 at a wave¬ length of 632 nm. The laser beam 3 is scanned over the sheet line by line, and each time the laser beam hits an atom which has been raised to a higher energy level by the β-radiation, so much further energy is imparted to such an atom that it is raised to an even higher but unstable en¬ ergy level, and immediately decays to the basic level while emitting the excess energy in the form of blue light 16. This light 16 can be scanned with a suitable scanning ap¬ paratus 4 which, for example, is a photomultiplier tube which is sensitive only to blue light, and which scans the sheet synchronously with the laser beam 3. The sensitivity of the photomultiplier tube to various forms of light (wavelengths) can be regulated by inserting filters in the emission path. The signals from the photomultiplier tube 4 are fed to suitable equipment for further processing.

In fig. 2 is seen another embodiment where instead of mak- ing use of a phosphor sheet 2 in direct contact with the electrophoresis gel, a phosphor sheet in contact with a film 6 is used, to which the protein distribution from the gel is transferred by commonly-known blotting. The scanning of the protein distribution in the film 6 is carried out in a manner corresponding to that explained in connection with fig. 1. Since a film 6 is used, it is possible to place the laser 5 on the one side of the film, and the photomulti¬ plier tube 4 on the same side or on the opposite side as shown by the stippled line. If the laser 5 and the photo- multiplier tube 4 are placed directly opposite each other, it will presumably be necessary also to place a means of shielding, e.g. a "blind" spot, on the photomultiplier tube where the laser beam 3 hits the tube.

Fig. 3 shows in greater detail how the scanning in fig. 1 is effected in practice. The laser beam 3 is conducted to the scanning area by means of a light conductor 7, and the light emitted from the phosphor plate 2 is detected with one or more light conductors 8 and fed to the multiplier tube 4.

The plate 2, which contains phosphorus or is phosphorus coated, can, for example, be of the type Fuji Imaging Plates, type CR ST-3. Such plates of so-called Storage Phospher can be re-used more than a thousand times, in that the information on the plate can be erased by irradiation. The material can be activated linearly over a dynamic range in the magnitude of 1:100,000, and the activated material maintains its condition until it is scanned, and no great demands are placed on the storing of the plates.

In fig. 4 is seen an example of a practical arrangement for the scanning of the information in 2-D electrophoresis gels 1, each covered by a phophorus plate 2. A scanner 11 is placed on lengthwise guides 12, and comprises an arm 13 or similar support element for the movable holder 14 for ir¬ radiation and scanning of the phosphorus plate 2.

A large number of gels 1 with phosphor plates 2 are placed in one row e.g. on a horizontal shelf ^'9, as shown, or pos- sibly in several rows so that the information in the indi¬ vidual gels is scanned successively, and the information which is scanned with the photomultiplier tube 4 via a cable is fed to a data processing unit 10, this unit com¬ prising means for the conversion of the signals to digital signals, a data store, a screen for the displaying of the results, means for image data processing etc. The data pro- cessing unit 10 also comprises means for controlling the scanner 11 on the basis of keyed-in data concerning which part of the gel is to be scanned. These means can be a pro¬ gram installed in the data processing unit.

The apparatus shown in fig. 4 is only one example of a practical embodiment for the invention. It will be obvious to those familiar with the art that the scanner can be con¬ figured in innumerous different ways, and be used with a variety of mechanical and electronic equipment without de¬ viating from the principles according to the invention.

The apparatus shown in figs. 9A and 9B is more compact that the apparatus shown in fig. 4. Fig. 9A shows an apparatus seen from the side and with the side wall removed, and fig. 9B shows the same apparatus seen from the front. The ap¬ paratus comprises a cabinet 30 in which gel 1 with, for example, sets of phosphor plates 2, are inserted in drawers or on shelves vertically over one another. New items for investigation can be introduced through doors 26 while others are being scanned. A part 23 which can be moved ver¬ tically comprises a laser source and a photomultiplier tube, both of which are coupled possibly via light con¬ ductors in a movable arm 24 to that part 25 where the actual scanning takes place. A computer 29 supervises and controls the scanning and the storing of the scanned in¬ formation. A vertical row of control lamps 27 indicates which phosphor plate 27' is presently being scanned for in¬ formation. The computer is also programmed and arranged in such a manner that it controls the scanner 23, 24, 25, and such that the scanner can be controlled by input data.

In figs. 4 and 9 is shown direct scanning simultaneously as the phosphor plates 2 are stimulated by the gel 1, hereby providing immediate results, possibly provisional results.

It will be obvious to those familiar with the art that it is possible to scan fully exposed phosphor plates 2 which are no longer lying in contact with the gel, e.g. in con¬ nection with later investigations or comparison investiga¬ tions.

In fig. 5 is seen a cross-section of an example of a prac¬ tical configuration of a suction arrangement with a housing 19, whereby the medium, e.g. a gel 1 or a film 6 containing the emission sources which decay during the emission, are placed on a porous plate 17 which can be evacuated from below or secured by pressure. The plate 2, which contains an emission-sensitive material in which the emission gives rise to a storage of energy, e.g. a phosphor-coated plate or a plate containing phosphorus, is held in a sealing ar- rangement 18, and very tight contact is achieved with the medium 1 or 6. A short distance is hereby achieved between the source of emission and the plate, whereby a more cor¬ rect scanning is obtained; less absorption of the emission; and this will prevent the plate and the medium from moving in relation to each other and in relation to the scanner.

Figs. 6-8 show a second example of a holder for a medium 1 or 6 and a phosphor plate 2 comprising two slightly curved, cylindrical or double-arched plates 20,21, of which at least one is of glass or another material which permits the passage of the necessary emission. By applying pressure at the corners and/or along the edges, the two relatively thin layers, e.g. a gel 1 or a film 6 and a phosphor plate 2, are held pressed together without hindering or disturbing the optical scanning. The pressing-together is shown with adjustable clamps, but can naturally be effected with many different kinds of clamping or tightening elements.

It will be obvious to those familiar with the art that the fixing of a medium and the plate in relation to each other and in relation to the scanner can be carried out in in- numerous different ways, and while being used with various mechanical and electronic equipment, without deviating from the principles according to the invention.

Example

Three women (A. Andersen, B. Berthelsen and C. Christensen) are sent for examination at a gynaecological/obstetrical department because atypical cells have been found in a smear sample. A colposcopic examination of the three women (using a microscope, the gynaecologist applies a thin acetic acid solution to the neck of the uterus), reveals that all three women show signs of changes in the trans¬ formation zone - signs which can be the intitial stages of cancer of the neck of the uterus. Cancer of the neck of the uterus is due to infection with a virus - human papilloma virus (HPV)- where in the neck of the uterus there can be found four types, namely the types 6, 11, 16 and 18. Infec¬ tion with the types 6 and 11 cannot lead to the induction of cancer of the uterus, whereas the types 16 and 18 must be considered as being very dangerous. The various types of HPV contain different proteins, which means that an exam¬ ination of possible HPV proteins in a tissue sample will be able to reveal which HPV type exists in the tissue.

A very small (approx. 1 x 1 mm) biopsy is taken from each of the patients. Half of the biopsy is sent for normal pathological examination. The other half is sent for exam¬ ination by means of 2-dimensional gel electrophoresis.

The three small amounts of biopsy are labeled with ( 35S)- methionine overnight, whereby all of the proteins which are synthetisized during the course of the night are radio- actively labeled. Hereafter, the tissue is homogenized and then dissolved in a buffer. The sample is now ready to receive the 1st dimension part which separates the protein content in accordance with the proteins' pH character¬ istics. After the 1st dimension gel is made ready, this gel is applied over the 2nd dimension gel which separates the proteins according to their size, and the 2nd dimension gel is run. After final electrophoresis, the gels are dried and the dried gels are ready to be examined.

The gels from the three patients, one from each, which in size are approx. 20 x 20 cm, are each placed together with their respective phosphor plates, and these "sandwiches" are placed in the scanner. Each plate is scanned only in the area (approx. 2 x 2 cm) where there will be markers for the HPV type.

The following are the findings after a short scanning:

- for A. Andersen, that there are no markers for HPV. The scanning is continued to determine whether or not there should be other micro organisms which can give rise to the found atypic cells. The subsequent total scanning of the gel shows that the patient is infected with Chlamydia, and a treatment with an antibiotic can commence very quickly. As a rule, this would involve the patient being called in again, the taking of a sample and cultivation for the various micro organisms which have been suspected; a method which can take several days or weeks. By 2-D gel electro¬ phoresis, it is not necessary to have any suspicion, since each living micro organism will show itself in the gel, in that the proteins from the micro organism will be able to be found here.

- for B. Berthelsen, that markers are found for HPV type 11

- the non-dangerous type. The scanning can be stopped, and a local treatment can commence - cryo or laser surgery - of the infected area. - for C. Christensen, that markers are found for HPV type 18. Since this is one of the dangerous types of HPV, the scanning is moved over to another area where markers are to be found for the onco genes (the cancer-promoting genes). After a very short scanning of this small area, it is found that the one of the onco genes is activated. Normally, this information could not have been obtained by a traditional investigation. Therefore, the conclusion must be that it is necessary to undertake a conic section operation and remove a large part of the neck of the uterus, since the cancer process has already commenced.

The corresponding pathological investigation would not have been able to distinguish between B. Berthelsen's and C. Christensen¹s conditions. Furthermore, it would require a very skilled pathologist to find the cause of the condi¬ tions found in A. Andersen's case.

Claims

C A I M S

1. Method for the scanning of a medium containing distrib¬ uted and/or separate atoms which are able to emit radia- tion, e.g. β-, gamma-, X-ray, chemiluminescence and/or fluorescent radiation, said medium being placed facing and permanently secured in contact with a plate which contains a material which is sensitive to the radiation, and in which the radiation gives rise to a storing of energy, in that the plate is scanned line-by-line by punctiformed ir¬ radiation with monochromatic light, while at the same time herewith the contents of the information on the plate are read by scanning the emission released from the plate, this information being converted to digital signals for use in image processing, c h a r a c t e r i z e d in that the scanning and the collection of information commences shortly after the radiation-sensitive plate is brought into contact with the medium, and in that the scanning is ef¬ fected over a predetermined section of the plate.

2. Method according to claim 1, c h a r a c t e r i z e d in that the scanned information is stored in a data store.

3. Method according to claim 1 or 2, c h a r a c t e r - i z e d in that a number of media, each with a radiation- sensitive plate, are placed in one common scanning appar¬ atus, after which the scanning is carried out.

4. Method according to any of the claims 1-3, c h a r - a c t e r i z e d in that the plate is irradiated on the one side, and the released emission is scanned on the opposite side.

5. Method according to any of the claims 1-3, c h a r - a c t e r i z e d in that the plate is irradiated through a gel or through a membrane onto which the distributed and/or separate atoms are transferred, and that the re¬ leased emission is scanned from the same side as that on which the irradiation takes place (fig. 2).

6. Method according to any of the claims 1-5, c h a r ¬ a c t e r i z e d in that the medium is a 2-D electro¬ phoresis gel with proteins which are labeled with atoms which can emit radiation, e.g. β-radiation, and that use is made of a plate which contains phosphor or is phosphor- coated.

7. Apparatus for use in the execution of the method accord¬ ing to claim 1 for the scanning of the radiation source distribution in a medium (1) or a membrane or a sheet (6) containing distributed and/or separate atoms which are able to emit radiation, e.g. β-radiation, after which the medium, the film or the sheet is placed opposite to and permanently secured in contact with a plate (2) containing a material which is sensitive to radiation, and in which radiation gives rise to a storage of energy, said apparatus comprising a scanner (14) with an irradiation element (5) having an organ for the generation of monochromatic light (3) and a scanning element (4) for released emission (16), said scanner comprising means (11,12,13) for scanning the plate (2) line-by-line by punctiformed irradiation, and an electronic circuit (10) for the conversion of the contents of the information in the scanned emission to digital signals or numerical values, c h a r a c t e r i z e d in that the apparatus is controlled by the electronic circuit (10) to carry out the scanning and the detection of the in¬ formation contents shortly after the radiation-sensitive plate (2) is placed in contact with the medium (1), that the apparatus is controlled in such a manner that the scan¬ ning is effected over a predetermined section of the plate (2), and in that it comprises means for securing the plate and the medium in contact with each other until the scan- ning of the information has been finally concluded.

8. Apparatus in accordance with claim 7, c h a r a c ¬ t e r i z e d in that the scanner's irradiation element is a laser (5), that the element used for detecting the in¬ formation is a photomultiplier tube (4), and in that the scanner (11,23) comprises a movable holder (14,25) which with flexible light-conductor cables (7,8) is coupled to the laser (5) and the photomultiplier tube (4).

9. Apparatus according to claim 7 or 8, c h a r a c t e r ¬ i z e d in that it comprsies a data store for digital image data.

10. Apparatus according to claim 7, 8 or 9, c h a r a c ¬ t e r i z e d in that it comprises a supporting element (12,28) for the scanner (11,23) so that scanning can be effected on more than one plate (2) at a time or immediate¬ ly after one another.

11. Apparatus according to any of the claims 8-10, c h a r a c t e r i z e d in that the scanner is arranged in such a manner that the laser (5), or the light conductor (7) coming from the laser, irradiates the one side of the plate (2), and that the photomultiplier tube (4), or the or those light conductors (8) leading to it, are arranged so that the emission (16) is scanned from the opposite side of the plate.

12. Apparatus according to claim 7, c h a r a c t e r ¬ i z e d in that the means (18,19) for securing the medium (1,6) and the plate (2) with a material sensitive to the radiation in close contact with each other comprise a sup¬ porting element (17), and functions with the use of vacuum or overpressure.

13. Apparatus according to claim 7, c h a r a c t e r ¬ i z e d in that the means comprise two plates (20,21) arranged to surround the medium (1,6) and the radiation- sensitive plate (2), and means (22) for clamping the plates together at their edges, and in that at least one of the plates is not plane, and at least one of the plates is of a material which allows the necessary radiation to pass.