Radiant energy – Electron energy analysis
Reexamination Certificate
2000-10-27
2003-05-13
Lee, John R. (Department: 2881)
Radiant energy
Electron energy analysis
C250S311000, C250S310000
Reexamination Certificate
active
06563112
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a process for the contrast enhancement for a specific particle in an image of a specimen recorded by a transmission electron microscope.
One aspect of the present invention relates in particular to the high-resolution structure analysis by transmission electron microscopy in biological and medical research by means of immuno-gold marking. In this situation, gold grains of a size of between 1 nm and 20 nm are coupled to a specific biomolecule of the electron microscope specimen, with the result that this molecule can be indirectly demonstrated by means of the position of the gold grains in the specimen. It derives from this that gold, as a heavy metal, creates a particularly strong contrast and is therefore clearly visible.
However, the structure of the cell in which the gold-marked molecule is to be detected is likewise contrasted, in order, for example, to render individual cell compartments visible. This is done as a rile with uranium and/or lead. Because these elements are likewise heavy metals, they create a similar contrast to gold, so that the gold grains in many specimens cannot be unambiguously identified.
To facilitate the identification, larger gold grains can be used. With larger gold grains, however, the specificity of the coupling to the molecule is considerably reduced. It would also be possible to select a higher magnification, but this would still only show a relative small section of the cell, which is not always determinant alone. In addition, after the gold marking, it would be possible for silver to be deposited in a specific manner on the gold, so that the gold grains would be provided with a silver sheathing, and the contrast considerably increased. Such a silver depositing effect does not, however, occur with all gold grains, so that the contrast would not be uniformly distributed.
According to another formulation, it is possible for the contrast for a specific particle to be increased by image processing. In this case, a calculated contrast-rich image is created by calculated background intensities being derived pixel by pixel from the intensities of a first image, whereby the background intensities are calculated as a function of the intensities of a second image. In this situation the first image is recorded with an energy-filtering transmission electron microscope (EFTEM) in an energy window, in which an element-specific energy edge of the specific particle is located. The second image is recorded in an energy window which is located at energy values below the element-specific edge.
In an image-processing stage which follows this, a background intensity is determined as a linear representation of the intensities of the second image. The determination is effected in such a way that this function, which is dependent on the intensities of the second image in which none of the specific particles are present, is fitted to the intensities of the first image.
In view of the fact that in this manner the functional dependency of the background intensities of the first image was determined by the intensities of the second image, this background intensity is calculated for each pixel of the first image from the intensities of the second image, and subtracted from the intensity of the second image.
In this way a calculated image is acquired, in which the contrast for the specific particle is enhanced.
A process of this nature is disclosed, for example, in U.S. Pat. No. 5,578,823. In addition to this, H. TENAILLEAU ET AL. disclose in their article “A new background subtraction for low-energy EELS core edges”, JOURNAL OF MICROSCOPY, GB, OXFORD, Vol. 166, No. 3, June 1992, pages 297 to 306, an improved extrapolation function, which can be applied in the process applied in U.S. Pat. No. 5,578,823, depending on the application instance.
A precondition for the performance of this process is that the specific particle features an element-specific energy loss edge, such as, for example, the energy loss edge of uranium at 120 eV or the energy loss edge of phosphorous at 160 eV.
However, not all particles feature a specific energy loss edge which delivers a signal which can in practice be selectively demonstrated. For example, gold does indeed feature two specific energy loss edges, a first at 60 eV and another at over 2000 eV. The first edge, at 60 eV, however, delivers a weak signal on a high non-element-specific background, with the result that this signal does not in practice allow for selective representation. The second edge, at over 2000 eV, is not detectable, since as early as about 1000 eV the inelastic signals become so weak that detection is virtually impossible any longer.
SUMMARY OF THE INVENTION
The objective on which the present invention is based is therefore of providing a contrast enhancement process of generic type, which features a broad range of application, and is not mandatorily related to the present of specific energy loss edges. In particular, the process should allow for a contrast enhancement for gold particles with immuno-gold marking.
As a solution, the invention proposes a contrast enhancement process of generic nature, in which the first image is taken under conditions in which the particle features the highest contrast possible, and in which the second image is taken in a selected energy window which is selected in such a way that the contrast difference between the two images differs for the particle by the corresponding contrast difference for at least one second specimen constituent.
This process for contrast enhancement according to the invention differs from the contrast enhancement process described heretofore by a fundamental difference in the properties of the images used for the image processing. With the contrast enhancement processes functioning according to the prior art, the images are selected in accordance with the specific energy loss edge of the element of which the contrast is to be enhanced. The first image is in this case recorded in an energy window which encloses the specific energy loss edge of the element. The second image is recorded in an energy window which lies below the specific energy loss edge.
With the images according to the contrast enhancement process according to the invention, a specific energy loss edge which may be present of the particle of which the contrast is to be enhanced does not play any part. This process is accordingly well-suited both for particles which do not feature any specific energy loss edge, as well as for particles of which the specific energy loss edge does not deliver selectively detectable signals. It has transpired in particular that this process is particularly well-suited for the contrast enhancement of gold.
With the contrast enhancement process according to the invention, the first image is recorded under conditions in which the particle features the highest possible contrast. This can, for example, be an image of the specimen with a transmission electron microscope without energy filter. It is likewise possible and, in particular, of advantage if the particle is a gold particle, for the first image to be taken in an energy window of 0 eV. With an energy-filtering transmission electron microscope, this means that the electrons are selected which have not lost any energy.
If the specimen contains yet another specimen constituent, in addition to the specific particle and the second specimen constituent, it can be of advantage if at least one further image is taken in another selected energy window, which is chosen in such a way that the contrast difference between the first and the further image and/or the second and the further image for the particle and/or for the second specimen constituent differs from the corresponding contrast difference for the further specimen constituent.
As a result, a set of images is prepared in which the second image and the further image in each case contain selective information about two specimen constituents.
In this way it is possible for the background intensities to be calculated as a f
Haking Ansgar
Richter Karsten
Trendelenburg Michael
Tröster Helmut
Collard & Roe P.C.
Deutsches Krebsforschungszentrum Stiftung Des Öffentlichen Recht
Fernandez Kalimah
Lee John R.
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