Radiant energy – Inspection of solids or liquids by charged particles – Methods
Reexamination Certificate
2000-01-03
2003-07-01
Lee, John R. (Department: 2881)
Radiant energy
Inspection of solids or liquids by charged particles
Methods
C280S300000, C280S300000, C280S300000, C280S300000
Reexamination Certificate
active
06586735
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a procedure for the detection of an element in a sample. A transmission electronic microscope is used to measure a first image of the intensities of the sample for an energy-loss domain before the element edge. A second image of the intensities of the sample for an energy loss is measured in the area of the element edge.
2. The Prior Art
This procedure is also termed the “two-windows difference method.” The difficulty of this method is that the intensity of the background is a function of the energy loss and, therefore, various background intensities need to be determined within the energy window. The background intensity essentially depends on the thickness of the sample point and decreases as energy loss increases. In order to determine the purely element-specific signal, i.e. the element-specific intensity, initially the intensity is measured in the energy-loss domain specific to the element to be detected.
Therefore, a background value in the energy-loss domain is subtracted from these measured values, where the background in the element-specific energy window is a function of the background outside the element-specific energy window. In the well known method, the assumption is made that the intensity of the background in the domain of the energy window is a linear function of the intensity before the energy window.
For many applications, this assumption is approximately justified. However, the way in which the parameters of the linear function are to be calculated is not satisfactorily described in the state of the art. Furthermore, in many cases a linear function does not correspond to the natural facts.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to further develop a generic procedure so that the function described can be determined and reproduced in order to calculate a more precise image of the element-specific intensities.
The invention comprises a procedure for the detection of an element in a sample comprising the steps of measuring a first image of the intensities I
1p
of the sample for an energy loss before the element edge using a transmission electronic microscope. Then a second image of the intensities I
2′p
is measured for an energy loss in the area of the element edge. The next step is determining the non-element-specific intensities I
1
of the sample for an energy loss before the element edge for various points at a reference sample point that does not contain the element. Then the non-element-specific intensities I
2
for an energy loss are determined in the area of the element edge are determined. An approximation function I
2
(I
1
) and an image of the element-specific intensities I
E
is then calculated from these values. The corresponding intensity I
2p
with the approximation function for every point in the first image with the intensity I
1p
is calculated, and the difference between the measured intensity I
2′p
and the calculated intensity I
2p
as the element-specific intensity I
E
for the corresponding point in the second image is determined.
In the present invention, the intensity I
1
for an energy loss at various points of a reference sample not containing the element is determined before the element edge. The non-element-specific intensities I
2
for an energy loss are determined in the area of the element edge. From these values an approximation function I
2
(I
1
) is calculated and an image of the element-specific intensities I
E
is calculated. Therefore, for every point in the first image with the intensity I
1p
, the corresponding intensity I
2p
is calculated from the approximation function and, for the corresponding point in the second image, the difference between the measured intensity I
2′p
and the calculated intensity I
2p
is determined as the element-specific intensity I
E
.
The procedure of the invention allows the function I
2
(I
1
) to be calculated experimentally. This is useful because it allows the removal of the non-element-specific background from the image of the intensities measured in the area of the element edge for an energy loss. Therefore, systematic error is markedly reduced and the background subtraction can be matched to each sample and to all variable parameters that can be set at the microscope.
The intensity pair I
1
/I
2
can be graphically represented so that it can also be easily determined visually whether the calculated approximation function corresponds with the measured values.
It is advantageous for the reference sample to display a course of different sample thicknesses. Since sample thickness affects the background signal most strongly, a course of different sample thicknesses permits the measurement of various background signals and thus the determination of various intensity pairs I
1
/I
2
. This course should display no steps and preferably takes the shape of a ramp.
Preferably, the reference sample displays at least the thickness of the sample. This ensures that, for all background signals occurring in the area of the sample, there is a corresponding background signal to be determined from the reference sample.
Reference samples consisting of pure carbon have shown themselves to be suitable. Such reference samples can be produced without difficulty in all electron-microscope oriented laboratories and are mainly suitable for biological samples if they have not subsequently been treated with heavy metals.
A more detailed determination of the background signal may be required. The reason is that, as well as a purely mass-thickness contrast, a further contrast mechanism plays a role in the emergence of an image. It may, for example, be an inhomogeneous concentration of various elements. A contrast resulting from this is termed a “compositional contrast” (CC). For this reason, it can be advantageous for the reference sample to contain at least two elements. Because of the high carbon content in biological samples, carbon is recommended as one of the elements, while the other element should be a heavier element than carbon, e.g. nitrogen, oxygen or sulphur.
However, the preparation of suitable reference samples is difficult. In a reference sample intended to contain oxygen in addition to carbon, for example, almost all the oxygen is lost by evaporation. Furthermore, a heavier element is more suitable for a reference sample. Sulphur can be prepared in different thicknesses only with great difficulty, if at all.
For this reason, dithiouracil (DTU) was used. In DTU, both of the oxygen atoms of the uracil, which is one of the four bases of DNA, are replaced by sulphur atoms. Compounds containing oxygen may, however, also be considered.
In order to reduce noise in the graphs derived from the images, it is suggested that the intensity of each point be measured as the mean value of its environment. Each of the images, for example, can consist of 0141×1024 image points, the intensity value of each image point being replaced by the mean value of the environment of 10×10 image points. After the calculated function is obtained, however, the original images are used in further work.
In order to determine the quality of the approximation function and, where appropriate, replace the approximation function with a further approximated function, it is suggested that the quality of the function I
2
(I
1
) be determined by statistical functions. This also permits the reproducible determination of error in the procedure.
The procedure of the invention was tried with great success on the element phosphorus. The procedure can also, however, be used for the detection of other elements, e.g. iron. Since DNA contains phosphorus, the procedure of the invention permits the course of DNA in, for example, viruses or other DNA protein complexes to be shown.
If a residual contrast remains after the procedure of the invention has been carried out, at least a third image of the intensities I
3′p
of the sample for a third energy loss can be recorded. This energy loss should also lie befo
Haking Ansgar
Richter Karsten
Collard & Roe P.C.
Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Recht
Lee John R.
Vanore Dav A.
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