WO1999047910A2 - Verfahren zur kontrastverstärkung für ein transmissionselektronenmikroskop - Google Patents
Verfahren zur kontrastverstärkung für ein transmissionselektronenmikroskop Download PDFInfo
- Publication number
- WO1999047910A2 WO1999047910A2 PCT/DE1999/000727 DE9900727W WO9947910A2 WO 1999047910 A2 WO1999047910 A2 WO 1999047910A2 DE 9900727 W DE9900727 W DE 9900727W WO 9947910 A2 WO9947910 A2 WO 9947910A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- image
- contrast
- intensities
- enhancement method
- contrast enhancement
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 39
- 230000005540 biological transmission Effects 0.000 title claims abstract description 9
- 230000002708 enhancing effect Effects 0.000 title abstract description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 48
- 229910052737 gold Inorganic materials 0.000 claims abstract description 47
- 239000010931 gold Substances 0.000 claims abstract description 47
- 239000002245 particle Substances 0.000 claims abstract description 45
- 239000011159 matrix material Substances 0.000 claims description 10
- 239000000470 constituent Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 39
- 229910052770 Uranium Inorganic materials 0.000 description 15
- JFALSRSLKYAFGM-UHFFFAOYSA-N uranium(0) Chemical compound [U] JFALSRSLKYAFGM-UHFFFAOYSA-N 0.000 description 15
- 229910001385 heavy metal Inorganic materials 0.000 description 6
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 101100390736 Danio rerio fign gene Proteins 0.000 description 1
- 101100390738 Mus musculus Fign gene Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003126 immunogold labeling Methods 0.000 description 1
- 238000012886 linear function Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
Classifications
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
- H01J37/28—Electron or ion microscopes; Electron or ion diffraction tubes with scanning beams
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
- G01N23/06—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and measuring the absorption
Definitions
- the invention relates to a method for contrast enhancement for a specific particle in an image of a sample taken by a transmission electron microscope.
- One aspect of the present invention relates in particular to high-resolution structural analysis by transmission electron microscopy in biological and medical research using immunogold labeling.
- gold grains with a size between 1 nm and 20 nm are coupled to a specific biomolecule in the electron microscopic sample, so that this molecule can be detected indirectly via the position of the gold grains in the sample. It is assumed that gold as a heavy metal creates a particularly strong contrast in the electron microscope and is therefore clearly visible.
- the structure of the cell in which the gold-marked molecule is to be detected must also be contrasted, for example to make individual cell compartments visible. This is usually done with uranium and / or lead. Since these elements are also heavy metals, they create a similar contrast to gold, so that the gold grains cannot be clearly identified in many samples.
- the contrast for a particular particle can be increased by image processing.
- a calculated, high-contrast image is generated by subtracting background intensities calculated pixel by pixel from the intensities of a first image, the background intensities being calculated as a function of the intensities of a second image.
- An energy-filtering transmission electron microscope (EFTEM) is used to record the first image in an energy window in which there is an element-specific energy edge of the particular particle.
- the second picture is taken in an energy window, which is below the element-specific edge for energy values.
- a background intensity is determined as a linear representation of the intensities of the second image. The determination is made in such a way that this function, which is dependent on the intensities of the second image, is fitted to the intensities of the first image for a sub-area of both images in which none of the specific particles are present.
- 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 first image.
- the particular particle has an element-specific energy loss edge, such as the energy loss edge of uranium at 1 20 eV or the energy loss edge of phosphorus at 1 60 eV.
- not all particles have a specific energy loss edge, which provides a signal that can be selectively displayed in practice.
- gold has two specific energy loss edges, a first at 60 eV and another at over 2000 eV.
- the first edge, at 60 eV delivers a weak signal on a high element-unspecific background, so that this signal cannot be displayed selectively in practice.
- the second edge, at over 2000 eV is not detectable, since the inelastic signals become so weak already at around 1,000 eV that detection is virtually impossible.
- the present invention is therefore based on the object of providing a generic contrast enhancement method which has a broader field of application and is not necessarily dependent on the presence of specific energy loss edges.
- the method is intended to enable contrast enhancement for gold particles in the case of immunogold marking.
- the invention proposes a generic contrast enhancement method in which the first image is recorded under conditions in which the particle has the highest possible contrast, and in which the second image is recorded in a selected energy window, which is chosen such that the difference in contrast between the two images for the particle differs from the corresponding difference in contrast for at least one second sample component.
- This inventive method for contrast enhancement differs from the previously described contrast enhancement method by a fundamental difference in the property of that for image processing used images.
- the images are selected based on the specific energy loss edge of the element whose contrast is to be enhanced.
- the first image is recorded in an energy window that includes the specific energy loss edge of the element.
- the second image is taken in an energy window that lies below the specific energy loss edge.
- any specific energy loss edge of the particle, the contrast of which is to be enhanced is not used.
- this method is suitable both for particles which have no specific energy loss edge and for particles whose specific energy loss edge does not provide selectively representable signals.
- this method is particularly suitable for enhancing the contrast of gold.
- the first image is recorded under conditions in which the particle has the highest possible contrast.
- This can be, for example, an image of the sample with a transmission electron microscope without an energy filter. It is also possible, and in particular if the particle is a gold particle, it is advantageous to take the first image with an energy window of 0 eV. In the case of an energy-filtering transmission electron microscope, this means that the electrons are selected which have lost no energy.
- the sample contains a further sample component in addition to the specific particle and the second sample component, it may be advantageous if at least one further image is recorded in a further, selected energy window, which is selected such that the contrast difference between the first and the another image and / or the second and the further image for the particle and / or for the second Sample component differs from the corresponding contrast difference for the other sample component.
- This provides a set of images in which the second image and the further image each selectively contain information about two sample components.
- the background intensities can be calculated as a linear representation of the intensities of the second and the further image.
- the background intensities are represented as a polynomial of the first or higher (1st - 3rd) degree by the intensities of the second and the further image.
- the background intensity function is advantageously obtained by fitting the intensities of the first image. If the background intensity function comprises a linear mapping, the corresponding coefficients can be determined by any known statistical fitting method.
- the time required for this can also be reduced by fitting only for selected pixels arranged in a matrix.
- a pixel matrix 1,024 x 1,024 pixels thus result in approximately 1,000 image pixels which are used to determine the background intensity function.
- a pixel matrix to fit the background intensity function is also inventive independently of the other features of the contrast enhancement method according to the invention. It has been shown that individual pixels of the pixel matrix which happen to represent one of the specific particles, for example gold, are of no importance for the fitting carried out.
- the selected pixels arranged in the matrix can be selected that are based on an image of the particular one Particles lie.
- the background intensity function can then be fitted again to the intensities of the first image, the pixels selected in this way not being taken into account in the matrix or being replaced by other pixels.
- This second step in which the matrix is adapted to any special circumstances that may exist, can also be carried out automatically, so that this method is still suitable for automated particle evaluation.
- a particle of the sample has a specific energy loss edge which supplies a selectively representable signal
- the present contrast enhancement method according to the invention in this case uses the specific energy loss edge of another particle in order to increase the contrast for the particular particle. No use is made of any energy loss edge of the particular particle.
- the sample comprises uranium, for example, it is advantageous if an image is taken at an energy window of 1 20 eV, the specific energy loss edge of uranium.
- relative contrast denotes the manner in which a particle stands out from the background or from the background intensity in a specific one. Depending on the selected energy window and the particles, these may appear lighter or darker than the environment. In this respect, the concept of the sign of the relative contrast refers to whether a particle is shown lighter or darker than the background.
- gold grains appear darker than the surroundings, with these providing the strongest negative contrast at 0 eV. Above 70 eV, the gold grains are shown lighter.
- the energy window of the second image and / or a further image such that the relative contrast of a second sample component in either this image or the first image has the same sign as another sample component, while the relative contrast of the second sample component in the other image has an opposite sign as the other sample component.
- 1 a is an image of a sample with an energy window of 0 eV with a
- Fig. 1 c is an image of the sample with an energy window between 1 1 5 eV and
- 3a is a schematic sample sample in section
- FIG. 3b shows a schematic representation of measured intensities in images of the sample according to FIG. 3a
- 3c shows the gold signal calculated from the intensities according to FIG. 3b
- FIG. 5 shows the calculated gold distribution in superimposition with the 0 eV image according to FIG. 1a.
- the exemplary embodiment comprises a sample in which an ultrathin section of a cell with uranium and lead was made visible, in which the DNA was labeled with 6 nm gold grains.
- the image was chosen at 0 eV because it provides the strongest contrast for the gold grains.
- the window between 1 1 5 and 1 25 eV was chosen because the element-specific energy loss edge of uranium lies in this area and uranium can thus be displayed selectively in this window.
- the intensity of the background ⁇ ⁇ was calculated as a linear function of the intensities at 45 eV (l 45 ) and at 1 20 eV (l 120 ).
- the sample consists of a cut, which has a uniform thickness, but with which the density varies.
- the dark gray stands for a high-density area.
- a spot with uranium and a grain of gold is shown.
- the contrast for uranium in the 45 eV image is similar to that for gold.
- the difference between gold and uranium is clear in the 1 20 eV picture. While gold only creates a weak positive contrast, the intensity for uranium is much stronger.
- FIG. 4 The gold distribution calculated for each pixel after the fitting is shown as a binarized result image in FIG. 4, while FIG. 5 shows a superposition of the intensities at 0 eV and the binarized result image.
- a gold-labeled biomolecule used for a localization examination for example antibodies, DNA or RNA probe.
- a simultaneous consideration of the uranium and / or lead contrast also present in the sample is possible, which is essential for the structural recognition of the entire cell topology.
- the contrast of the gold particles appears to be strongly emphasized, while the contrast of the cell structure is lowered in a defined manner, so that the position and number of gold grains in relation to the underlying cell structure is clearly defined.
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000537054A JP2002506992A (ja) | 1998-03-16 | 1999-03-12 | 透過型電子顕微鏡のためのコントラスト増強方法 |
AU38095/99A AU3809599A (en) | 1998-03-16 | 1999-03-12 | Method for enhancing the contrast for a transmission electron microscope |
EP99920547A EP1064536A2 (de) | 1998-03-16 | 1999-03-12 | Verfahren zur kontrastverstärkung für ein transmissionselektronenmikroskop |
US09/623,933 US6563112B1 (en) | 1998-03-16 | 1999-03-12 | Method for enhancing the contrast for a transmission electron microscope |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19811395.1 | 1998-03-16 | ||
DE19811395A DE19811395A1 (de) | 1998-03-16 | 1998-03-16 | Verfahren zur Kontrastverstärkung für ein Transmissionselektronenmikroskop |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1999047910A2 true WO1999047910A2 (de) | 1999-09-23 |
WO1999047910A3 WO1999047910A3 (de) | 2000-01-13 |
Family
ID=7861083
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1999/000727 WO1999047910A2 (de) | 1998-03-16 | 1999-03-12 | Verfahren zur kontrastverstärkung für ein transmissionselektronenmikroskop |
Country Status (6)
Country | Link |
---|---|
US (1) | US6563112B1 (de) |
EP (1) | EP1064536A2 (de) |
JP (1) | JP2002506992A (de) |
AU (1) | AU3809599A (de) |
DE (1) | DE19811395A1 (de) |
WO (1) | WO1999047910A2 (de) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004212355A (ja) * | 2003-01-09 | 2004-07-29 | Hitachi Ltd | バイオ電子顕微鏡及び試料の観察方法 |
TWI753739B (zh) | 2021-01-08 | 2022-01-21 | 閎康科技股份有限公司 | 物性分析方法、物性分析試片及其製備方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5578823A (en) * | 1994-12-16 | 1996-11-26 | Hitachi, Ltd. | Transmission electron microscope and method of observing element distribution by using the same |
WO1999001753A2 (de) * | 1997-07-04 | 1999-01-14 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Verfahren zur detektion eines elementes in einer probe |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5713364A (en) * | 1995-08-01 | 1998-02-03 | Medispectra, Inc. | Spectral volume microprobe analysis of materials |
US5798524A (en) * | 1996-08-07 | 1998-08-25 | Gatan, Inc. | Automated adjustment of an energy filtering transmission electron microscope |
JP3439614B2 (ja) | 1997-03-03 | 2003-08-25 | 株式会社日立製作所 | 透過型電子顕微鏡及び元素分布観察方法 |
-
1998
- 1998-03-16 DE DE19811395A patent/DE19811395A1/de not_active Withdrawn
-
1999
- 1999-03-12 AU AU38095/99A patent/AU3809599A/en not_active Abandoned
- 1999-03-12 EP EP99920547A patent/EP1064536A2/de not_active Withdrawn
- 1999-03-12 US US09/623,933 patent/US6563112B1/en not_active Expired - Fee Related
- 1999-03-12 JP JP2000537054A patent/JP2002506992A/ja active Pending
- 1999-03-12 WO PCT/DE1999/000727 patent/WO1999047910A2/de not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5578823A (en) * | 1994-12-16 | 1996-11-26 | Hitachi, Ltd. | Transmission electron microscope and method of observing element distribution by using the same |
WO1999001753A2 (de) * | 1997-07-04 | 1999-01-14 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Verfahren zur detektion eines elementes in einer probe |
Non-Patent Citations (2)
Title |
---|
ANONYMOUS: "Spectroscopy Signal Processing Technique" IBM TECHNICAL DISCLOSURE BULLETIN,US,IBM CORP. NEW YORK, Bd. 28, Nr. 7, Dezember 1985 (1985-12), Seite 2903-2904 XP002088763 ISSN: 0018-8689 * |
H TENAILLEAU ET AL: "A new background substraction for low-energy EELS core edges" JOURNAL OF MICROSCOPY,GB,OXFORD, Bd. 166, Nr. 3, Juni 1992 (1992-06), Seite 297-306 XP002088762 ISSN: 0022-2720 * |
Also Published As
Publication number | Publication date |
---|---|
DE19811395A1 (de) | 1999-09-23 |
US6563112B1 (en) | 2003-05-13 |
AU3809599A (en) | 1999-10-11 |
EP1064536A2 (de) | 2001-01-03 |
WO1999047910A3 (de) | 2000-01-13 |
JP2002506992A (ja) | 2002-03-05 |
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