Radiant energy – Invisible radiant energy responsive electric signalling – Semiconductor system
Reexamination Certificate
1999-02-26
2001-07-31
Hannaher, Constantine (Department: 2878)
Radiant energy
Invisible radiant energy responsive electric signalling
Semiconductor system
C250S370110
Reexamination Certificate
active
06268605
ABSTRACT:
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 &mgr;m, 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 &mgr;m, 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 &mgr;m. 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's attention which helps put the present invention in context. Nuclear Instruments & 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 &bgr;-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 &mgr;m.
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 (
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) 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
Orava Risto O.
Pyyhtia Jouni I.
Sarakinos Miltiadis E.
Schulman Tom G.
Spartiotis Konstantinos E.
Gabor Otilia
Hannaher Constantine
Simage Oy
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