JP2011158331A - Identification method of film-like element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an observation method for identifying a fine particle set to which a heterogeneous element has been added using: a method of determining observation conditions of SEM reflection electron measurement capable of giving a maximum luminance contrast and performing measurement with high reproducibility with respect to the kind and thickness of a film-like element; and a method of predicting a combination of element kinds that can be identified by SEM reflection electron measurement. <P>SOLUTION: The invention provides a fine particle label in which different regions of the surface of a carrier are film-coated with three or more kinds of elements. Different regions of the surface of the carrier are film-coated using a combination of three or more kinds of elements such that, when the elements are arranged in ascending or descending order based on the value of total reflectivity coefficient η of each element, a difference between total reflectivity coefficient η of adjacent elements is 0.02 or more. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for inspecting a metal, a semiconductor or the like configured in a thin film shape.

  As one of methods for examining in detail the morphological characteristics and molecular expression state of individual cells, an observation method using an electron microscope is widely used. Measurement with an electron microscope is mainly classified into transmission electron microscope (TEM) measurement and scanning electron microscope (SEM) measurement. In the SEM measurement, secondary electrons, reflected electrons, characteristic X-rays, cathodoluminescence, sample current, and transmission generated from the sample surface by irradiating the sample surface with an electron beam with a diameter of several nanometers by an electromagnetic coil. By detecting electrons and the like with a detector, shape information and element configuration information on the sample surface are obtained. The shape of the sample surface can be examined mainly by detecting secondary electrons, and the surface element composition information can be examined by detecting reflected electrons and characteristic X-rays.

  Backscattered electron measurement, which is one of the methods for examining elemental composition information, uses the fact that when an electron beam is irradiated on the sample surface, elements with a larger atomic number generally reflect more electrons and thus appear brighter on the image. This is a measurement method for examining element distribution information on the sample surface, and has features such as higher spatial resolution than characteristic X-ray measurement.

The luminance of each element obtained at the time of reflected electron measurement depends on the total reflection coefficient η. η depends on the atomic number Z and the energy E ₀ of the electron of the incident electron beam, and there are various theoretical and experimental descriptions of the relationship between η, Z, and E ₀ , but they have been proposed and widely accepted by Neubert et al. A typical relationship obtained by experimentally verifying E _{0 in} the range of 5 keV to 60 keV is as shown in the following formula (1) (Neubert & Rogaschewski (Non-patent Document 1)).

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)] ···· ... (1)

  In SEM measurement in cell research, the element brightness contrast at the time of backscattered electron measurement is used, biomolecules to be detected are labeled with fine particles composed of heavy elements such as gold, and organic light elements such as carbon are used. The spatial distribution information in the cell of the detection target molecule is accurately examined by adding a luminance contrast to the other intracellular microstructures configured (Deharven (Non-patent Document 2)). Soligo & Lambertenghi-Deliliers (Non-Patent Document 3), Scala et al. (Non-Patent Document 4)).

  The inventors of the present application have developed a biomolecule detection method by SEM as described above, a method of supplying a microscopic body composed of various elements for labeling a molecule to be detected in a cell, and a microscopic measurement of a plurality of elements by SEM measurement. The principle of the test method for simultaneously identifying and detecting biomolecules labeled with the body was proposed. In Takei (Non-patent Document 5), a method of arranging fine particles such as commercially available polystyrene spheres in a single layer with high density on the surface of the substrate, and coating the surface of the particles with various elements using a vacuum deposition method Thus, the principle of a method for producing various microscopic bodies for labeling a molecule to be detected is specified. In Kim et al. (Non-patent Document 6), an example of a method for identifying the produced fine particles of various elements as a difference in luminance of the particles by SEM reflected electron measurement is specified.

G. Neubert and S. Rogaschewski, Physica Status Solidi A Appl. Res. 59, 35 (1980) E. Deharven, Ultrastruct. Pathol. 11, 711 (1987) D. Soligo and G. Lambertenghi-Deliliers, Scanning 9, 95 (1987) C. Scala, G. Cenacchi, P. Preda, M. Vici, R. P. Apkarian, and G. Pasquinelli, Scanning Microsc. 5, 135 (1991) H. Takei, J. Vac. Sci. Technol. B 17, 1906 (1999) H. Kim, K. Yasuda, and H. Takei, Sens. Actuators B: Chem. 142, 1 (2009)

  However, in the above document, the optimum SEM measurement conditions for identifying the various fine metal particles produced and the analysis method for deriving the conditions, the number of identifiable elements and their combinations at the same time, the optimum SEM measurement conditions The method of deriving from is not specified. In addition, the previous reports on SEM backscattered electron measurement specify element identification methods for specimens with a sufficient thickness of micron or more in the electron beam incident direction, but thin film elements from several nanometers to microns in thickness. A method for efficiently and reproducibly discriminating is not specified.

  Therefore, in order to use a microscopic object coated with a thin film element of several nanometers to a thickness of micron as a label for visual identification of a biomolecule in a cell, the type and thickness of the thin film element to be used In contrast, it is desirable to disclose a method for determining observation conditions for SEM backscattered electron measurement that can measure with maximum brightness contrast and good reproducibility, and a method for predicting the optimum combination of element types to be used as a label. It is.

  In view of the above situation, the present invention identifies a set of labeled microparticles having a function of specifically binding to each living body object, which can be used to simultaneously identify a plurality of in vivo molecules and molecular assemblies, for example. In order to achieve this, SEM backscattered electron measurement can be measured with maximum brightness contrast and reproducibility with respect to the type and thickness of thin-film elements added to each labeled particle surface with a certain thickness. The present invention provides an observation method for identifying each fine particle by using a method for determining conditions and a method for predicting an optimum combination of element types to be used as a label.

That is, the present invention provides the following optimum acceleration voltage determination method, fine particle labeling, and biomolecule identification method.
(1) A method for determining an optimum acceleration voltage value of an SEM electron beam used for a method of identifying fine particles covered with different elemental thin films by reflection electron measurement using a scanning electron microscope (SEM),
(i) preparing a sample in which three or more kinds of elements are deposited in a thin film on different regions of the support surface;
(ii) irradiating the element of the sample with a SEM electron beam over a predetermined acceleration voltage range;
(iii) obtaining a reflected electron image of the sample at each acceleration voltage value;
(iv) Based on the above image, the following formula for each of the above elements:
[Where I _Z is the brightness of the reflected electron image of the thin film region composed of the element with atomic number Z, and I _max is composed of the element with the largest atomic number Z of the brightness of the reflected electron image of the observed element. The luminance of the reflected electron image of the thin film region, and I _min is the luminance of the reflected electron image of the thin film region composed of the element having the smallest atomic number Z among the luminances of the reflected electron images of the observed elements. ] Calibration brightness I ~ _Z [where I ~ is above I ~. The same applies hereinafter. ] For each acceleration voltage value,
(v) calculating a ratio of I to _Z between each of the elements at each acceleration voltage value,
(vi) selecting a combination of elements that minimizes the ratio of I to _Z at each acceleration voltage value, and
(vii) including a step of selecting an acceleration voltage value when the ratio of I to _Z of the combination of the selected elements becomes a maximum within the range of the predetermined acceleration voltage value as the optimum acceleration voltage value of the SEM electron beam. ,Method.
(2) The method according to (1) above, wherein in the sample, three or more kinds of the elements are deposited in a thin film in different regions on the surface of the support with substantially the same thickness.
(3) The method according to (2) above, wherein the thickness is in the range of 1 to 500 nm.
(4) The above elements are
(I) Transition metals up to 79 excluding atomic number 43,
(II) metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, 83, and
(III) The method according to any one of (1) to (3) above, which is selected from among semiconductors having atomic numbers 14, 34, and 52.
(5) The method according to any one of (1) to (4), wherein the element is selected from Au, Ag, Ge, Cu, Fe, Si, Eu, Y, Ti, and Al.
(6) In the step (v) of calculating the ratio of I to _Z between each element at each acceleration voltage value,
Following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . Denominator I ~ _Z elements of the total reflection coefficient total reflection coefficient between adjacent elements when arranged in ascending or descending order based on the value of eta eta small side of each element represented by, the total reflection coefficient eta There calculates a ratio of the I ~ _Z a _I-Z of a large side element as molecules, the method according to any one of the above (1) to (5).
(7) Select the elements so that the difference in total reflection coefficient η between adjacent elements when the elements are arranged in ascending or descending order based on the value of the total reflection coefficient η of each of the elements is 0.02 or more. The method according to (6) above.
(8) The method according to any one of (1) to (8), wherein the range of the predetermined acceleration voltage value is in the range of 0.1 kV to 30 kV.
(9) A fine particle label formed by coating different regions of the support surface with a thin film with three or more elements,
Following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . ] Three or more types of elements such that when arranged in ascending or descending order based on the value of the total reflection coefficient η of each element, the difference in total reflection coefficient η between adjacent elements is 0.02 or more The particulate label | marker by which the different area | regions of the said support body surface are coat | covered in thin film form using the combination of these.
(10) The fine particle label according to (9), wherein the size of the support is in the range of 1 nm to 1 mm.
(11) The fine particle label according to (9) or (10), wherein in the sample, the three or more kinds of elements are deposited in a thin film in different regions on the surface of the support with substantially the same thickness.
(12) The fine particle label according to (11), wherein the thickness is in the range of 1 to 500 nm.
(13) The above element is
(I) Transition metals up to 79 excluding atomic number 43,
(II) metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, 83, and
(III) The fine particle label according to any one of (9) to (12), which is selected from the semiconductors having atomic numbers 14, 34, and 52.
(14) The particulate label according to any one of (9) to (13), wherein the element is selected from Au, Ag, Ge, Cu, Fe, Si, Eu, Y, Ti, and Al. .
(15) The fine particle label according to any one of (9) to (14), wherein the support is made of a material such as silicon, mica, polystyrene, polypropylene, or glass.
(16) The fine particle label according to any one of (9) to (15), wherein a probe molecule capable of binding to a biomolecule is bonded to the surface of the fine particle label.
(17) The fine particle label according to any one of (9) to (16), wherein the probe molecule has a thiol group, and a surface of the fine particle label is coated with a gold element.
(18) A step of labeling a biomolecule by binding the fine particle label according to any one of (9) to (17) above,
Irradiating the labeled biomolecule with an electron beam under SEM to obtain a reflected electron beam image from the particulate label;
Analyzing the brightness of the reflected electron image and identifying the biomolecule by identifying the type of particulate label,
A biomolecule identification method comprising:

In one exemplary embodiment of the present invention, SEM electron beam incidence is performed on a sample in which three or more elements are deposited in a thin film with a specific film thickness in a range of 1 to 500 nm in different regions of a support. Electrons are accelerated at a voltage in the range of 0.1 kV to 30 kV and sequentially irradiated, and the reflected electrons generated thereby are detected. After obtaining a backscattered electron image of the sample at each acceleration voltage value, calibration luminances I to _Z of the thin film sample composed of the element of atomic number _Z are calculated by the following equation (2).
In formula (2), I _Z is the brightness in the backscattered electron image of the thin film sample composed of the element with atomic number Z, I _max is the brightness of the element thin film with the largest Z observed, and I _min is observed Among them, Z is the brightness of the elemental thin film. After calculating I to _Z at all acceleration voltage values for all the observed elements, the ratio of I to _Z between each element is calculated. The acceleration voltage value at which the ratio of the element combination that minimizes the ratio of I to _Z is maximized is determined to be the optimum acceleration voltage value, that is, the luminance ratio is the maximum for the sample of the specific film thickness measured. To do.

  In addition, the combination of elements used as a label is calculated by using formula (1) to calculate the total reflection coefficient η using the optimum acceleration voltage value for a specific film thickness, and the element whose η value difference is 0.02 or more is calculated. select.

  In addition, in order to modify probe molecules that bind to biomolecules on the surface of the labeled fine particle set, for example, a thiol group is added to the end of the probe molecule, and different elements are added to the surface of the labeled fine particles uniformly. Add elements that have the ability to bond directly with thiol groups such as gold elements by S-Au bonds.

  According to the present invention, for a thin film-like element having a specific thickness, a method for determining observation conditions for SEM backscattered electron measurement that maximizes brightness contrast, and a combination of element types that are optimal for use as labels are determined. Since the method is provided, in the SEM measurement method using a heavy element microparticle as a label, a microbody set made of different elements can be used as a label set that can be used simultaneously. This makes it possible to label a large number of target molecules at once with microelement sets of different elements when measuring intracellular biomolecules using SEM. Develops.

(a) is a schematic diagram of a sample in which Au, Ag, Ge, Cu, and Fe are deposited in a thin film on a Si substrate. (b) is an example of an image obtained by depositing Au, Ag, Ge, Cu, Fe on a Si substrate in a 50 nm thin film and performing SEM backscattered electron measurement with an incident electron beam acceleration voltage of 5 kV. In (a), the brightness of Au is I _max and the brightness of the Si substrate is I _min for a sample in which five elements of Au, Ag, Ge, Cu, and Fe are deposited on a Si substrate support by 20 nm. In the above example, the relationship between the calibration brightness I to _Z of each element and the acceleration voltage value V is shown for a range of V from 3 kV to 30 kV. In the graph, the brightness of Au corresponds to I to _Z = 100, and the brightness of the Si substrate corresponds to I to _Z = 0. (b) is an example in which the same measurement as in (a) was performed with a film thickness of 50 nm. (a) shows the relationship between the ratio of calibration brightness I to _Z of each element and the acceleration voltage value V for a sample in which five elements of Au, Ag, Ge, Cu, and Fe are deposited on a Si substrate support by 20 nm. , V is an example shown for a range of 3 kV to 30 kV. (b) is an example in which the same measurement as in (a) was performed with a film thickness of 50 nm.

1. A method for determining an optimum acceleration voltage value of an SEM electron beam used in a method for identifying fine particles coated with different elemental thin films by backscattered electron measurement using SEM. In one embodiment, the present invention is a reflection using SEM. Provided is a method for determining an optimum acceleration voltage value of an SEM electron beam used in a method for identifying fine particles coated with different elemental thin films by electronic measurement. This method typically includes the following steps (i) to (vii).
(i) preparing a sample in which three or more kinds of elements are deposited in a thin film on different regions of the support surface;
(ii) irradiating the element of the sample with a SEM electron beam over a predetermined acceleration voltage range;
(iii) obtaining a reflected electron image of the sample at each acceleration voltage value;
(iv) Based on the above image, the following formula for each of the above elements:
[Where I _Z is the brightness of the reflected electron image of the thin film region composed of the element with atomic number Z, and I _max is composed of the element with the largest atomic number Z of the brightness of the reflected electron image of the observed element. The luminance of the reflected electron image of the thin film region, and I _min is the luminance of the reflected electron image of the thin film region composed of the element having the smallest atomic number Z among the luminances of the reflected electron images of the observed elements. ] Calculating the calibration luminances I to _Z represented by the above acceleration voltage values,
(v) calculating a ratio of I to _Z between each of the elements at each acceleration voltage value,
(vi) selecting a combination of elements that minimizes the ratio of each of the above I to _Z at each of the acceleration voltage values; and
(vii) a step of selecting an acceleration voltage value when the ratio of I to _Z of the selected combination of elements becomes a maximum within the range of the predetermined acceleration voltage value as the optimum acceleration voltage value of the SEM electron beam.

  In this specification, “SEM” means a scanning electron microscope. In the reflected electron measurement using the SEM of the present invention, the sample is observed by irradiating the sample to be examined with the electron beam and detecting the reflected electrons emitted from the sample. A signal emitted from the sample is detected by a detector, and is displayed on a display as an image through signal processing such as amplification and modulation. The acceleration voltage of the electron beam of the SEM used in the present invention is typically in the range of 0.1 kV to 30 kV, but is not limited thereto.

  If the thicknesses of the thin films of three or more elements deposited in different regions on the surface of the support are coated with substantially the same thickness, it is convenient for comparing the luminance. However, as long as the degree of contribution of the thickness of the element thin film to the luminance can be calibrated, it is not always necessary to coat with the same thickness. The thickness of the elemental thin film is typically in the range of 1 to 500 nm, but is not limited to this range. The elements used are typically (I) transition metals up to 79 excluding atomic number 43, (II) atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, You may choose arbitrarily from the 83rd metal and the semiconductor of (III) atomic number 14,34,52. For example, elements such as Au, Ag, Ge, Cu, Fe, Si, Eu, Y, Ti, and Al can be used.

In the step (v) in the method of the present invention, typically, the following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . Denominator I ~ _Z elements of the total reflection coefficient total reflection coefficient between adjacent elements when arranged in ascending or descending order based on the value of eta eta small side of each element represented by, the total reflection coefficient eta There calculates a ratio of the I ~ _Z a I ~ _Z large side element as molecules. Here, when the above elements are selected so that the difference in total reflection coefficient η between adjacent elements is 0.02 or more when arranged in ascending or descending order based on the value of total reflection coefficient η of each element, This is advantageous in identifying a given sample by the luminance ratio of the reflected electron signal.

2. Fine particle label In a further embodiment of the present invention, a fine particle label is provided in which different regions of the support surface are coated in a thin film using a combination of three or more elements. This particulate label typically has the following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . ] Three or more types of elements such that when arranged in ascending or descending order based on the value of the total reflection coefficient η of each element, the difference in total reflection coefficient η between adjacent elements is 0.02 or more The support surface is coated with a combination of the above. The size of the support is typically in the range of 1 nm to 10 μm, but is not limited to this range. The support may be composed of a material such as silicon, mica, polystyrene, polypropylene, or glass.

  The thickness of the elemental thin film coated on the support surface is typically in the range of 1 to 500 nm. The thickness of the element thin film is preferable for comparison of the brightness of the reflected electron image of the SEM electron beam as long as it is uniform among the different elements to be used, but is not necessarily uniform. The elements used are the same as in the method for determining the optimum acceleration voltage value of the EM electron beam.

  The particulate label of the present invention can typically be used for labeling and detecting biomolecules and the like. In that case, a probe molecule that can bind to a biomolecule may be bound to the surface of the particulate label. The probe molecule may have a thiol group, and the surface of the fine particle label may be coated with a gold element reactive with the thiol group.

3. Biomolecule Identification Method In a further embodiment, the present invention provides a biomolecule identification method using the fine particle label described above. This method typically includes a step of labeling a biomolecule by binding the fine particle label, and irradiating the labeled biomolecule with an electron beam under SEM, and a reflected electron beam image from the fine particle label. And a step of analyzing the brightness of the reflected electron image and identifying the biomolecule by identifying the type of the particulate label.

  Hereinafter, embodiments of the present invention will be described more specifically with reference to the drawings. However, these are merely examples, and the scope of the present invention is not limited to these examples.

  FIG. 1 is a schematic diagram of a thin film element sample deposited on a support and an example of an SEM backscattered electron measurement image for identification in the present invention. Here, an example is shown in which five kinds of elements of Au, Ag, Ge, Cu, and Fe are deposited on different regions by 50 nm each on a Si substrate support. The type of the support on which the thin film is deposited is a flat substrate such as silicon, mica, polystyrene, or polypropylene, polystyrene or glass, silicon spherical, prismatic, cylindrical, or conical minute bodies, and the minute body is the substrate. It is recommended to select an appropriate one from those arranged above. The size of the microparticles used as the support ranges from about 1 nm to about 1 mm, preferably from about 1 nm to about 500 μm, more preferably from about 5 nm to about 100 μm, and most preferably from about 5 nm to 1 μm. Without being limited to these ranges, the lower limit can be appropriately set in accordance with the purpose in an arbitrary range from about 1 nm to an upper limit of about 1 mm. The size of the micro object is specified by performing observation with a microscope capable of observing a nanometer-sized structure such as a scanning electron microscope, a transmission electron microscope, or an atomic force microscope, A micro object having a size suitable for the purpose may be selected based on the observation result. The thickness of the thin film is appropriately selected from the range of 1 to 500 nm, more preferably 1 to 200 nm. In addition, as a method for depositing the element in a thin film, an appropriate method may be selected from generally widely used thin film construction methods such as a vacuum evaporation method and a sputtering method. For example, in FIG. 1, a thin film sample is manufactured using a vacuum deposition method.

Examples of elements that can be used as elements for forming a thin film in the present invention are listed in the periodic table as follows.
(1) Transition metals up to 79 excluding atomic number 43,
(2) Metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, and 83, and (3) Semiconductors with atomic numbers 14, 34, and 52

  Any three or more kinds of elements are selected from the above elements and deposited on the support in a thin film shape so as to have a specific film thickness.

  In order to determine the optimum SEM measurement conditions for identifying the thin film sample, the prepared sample is set in the SEM sample chamber. Electron beam acceleration voltage values for observation are sequentially irradiated with a step size of 1 kV or less in the range of 0.1 kV to 30 kV, and a reflected electron image at each acceleration voltage value is acquired.

Next, the brightness value of each element thin film region of the acquired SEM reflected electron image is acquired. Luminance measurement uses various image analysis software such as Image J (http://rsbweb.nih.gov/ij/) distributed free of charge via the Internet from the National Institutes of Health (NIH). This is convenient, and after displaying the total pixel luminance of the obtained reflected electronic image as a black and white gradation having 256 levels or more, the value is obtained. The average value of the black and white gradation values of each pixel in the thin film region composed of the element having the atomic number Z is calculated, and this is defined as the reflected electron luminance I _Z of the atomic thin film having the atomic number Z. Also, the luminance of the thin film region of the element with the largest Z among the elements observed is defined as I _max , and the luminance of the thin film region of the element with the smallest Z is defined as I _min . For example, in the example of FIG. 1, the luminance of Au having the largest Z among the observed elements is I _max , and the luminance of Si as the support is I _min . After obtaining I _Z , I _max , and I _min of each element, calibration luminances I to _Z of each element are calculated using the above equation (2). The above calculation is performed for all elements in the image at all acquired acceleration voltage values. FIG. 2 shows the brightness of Au as I _max and the brightness of the Si substrate as I _max for a sample in which five elements of Au, Ag, Ge, Cu, and Fe are deposited on a Si substrate support at 20 nm or 50 nm. _In this example, the relationship between the calibration brightness I to _Z of each element and the acceleration voltage value V is shown for a range of V from 3 kV to 30 kV. In the graph, the brightness of Au corresponds to I to _Z = 100, and the brightness of the Si substrate corresponds to I to _Z = 0. The error range in Fig. 2 shows the error when four measurements are performed under the same conditions. When the calibration luminances I to _Z of each element are calculated using equation (2), the coefficient of variation is 3kV. To less than 4% for the range of 30 kV. As shown in FIG. 2, the relationship between I to _Z and V shows different behavior depending on the film thickness of the thin film, and therefore it is necessary to measure every film thickness.

Next, the ratio of calibration luminances I to _Z between different elements is calculated. Fig. 3 shows the ratio of calibration brightness I to _Z of each element and acceleration voltage value V for a sample in which five elements of Au, Ag, Ge, Cu, and Fe are deposited on a Si substrate support at 20 nm or 50 nm. Is an example in which V is in the range of 3 kV to 30 kV. The acceleration voltage value that maximizes the ratio of the element combination that minimizes the ratio of I to _Z is maximized. Determine the voltage value.

For example, in the example of FIG. 3 (b), the element combination that minimizes the ratio of I to _Z at each acceleration voltage value V is Ge and Cu when V is in the range of 3 kV to 9 kV, and Ag and Ge when 9 V to 30 kV. The acceleration voltage value at which the minimum I to _Z ratio value is maximized is 9 kV, and the luminance ratio between all elements is 1.3 times or more. In other words, the optimum acceleration voltage value for discrimination can be determined to be 9 kV for a sample in which five kinds of elements of Au, Ag, Ge, Cu, and Fe are deposited on a Si substrate support by 50 nm. The optimum acceleration voltage value varies depending on the thickness of the thin film, and can be determined to be 3 kV for 10 nm, 4 kV for 20 nm, and 9 kV for 50 nm, for example.

  In the example of FIG. 3B, when the total reflection coefficient η is calculated using the above equation (1), η of each element with respect to incident electrons having an energy of 9 keV accelerated by an acceleration voltage of 9 kV is Au. Is 0.497, Ag is 0.433, Ge is 0.349, Cu is 0.329, Fe is 0.305, and Si is 0.186. That is, the example of FIG. 3B shows that when a 50 nm thin film sample is observed at an acceleration voltage value of 9 kV, an element having a difference of η of 0.02 can be identified as a luminance ratio of about 1.3 times. As shown in FIG. 3, the reflected electron luminance of the element thin film greatly depends on the film thickness. For example, when forming a thin film by the vacuum evaporation method, an error of about 10% occurs in the film thickness. It is desirable that the luminance ratio between each elemental thin film is 1.3 times or more.

  From the above, when selecting a combination of elements to be used as labels, in order to expect that each element can be identified as a luminance ratio of 1.3 times or more, total reflection using the optimum acceleration voltage value for a specific film thickness The coefficient η is calculated using the above formula (1), and it is necessary to select an element having a difference in value of η of at least 0.02. For example, when a thin film is formed by a vacuum deposition method or a sputtering method, 10 types of Au, Eu, Ag, Y, Ge, Cu, Fe, Si, Ti, and Al are selected in consideration of ease of process.

As described above, the present invention performs reflected electron measurement in the range of acceleration voltage of SEM electron beam from 0.1 kV to 30 kV on a sample in which three or more kinds of different elements are deposited in a thin film shape. By calculating the calibration brightness I to _Z of the thin film sample composed of the element of Z, and determining the acceleration voltage value at which the ratio of the element combination that minimizes the ratio of I to _Z between the elements is maximized, It is possible to determine the optimum acceleration voltage value for thin film identification by SEM backscattered electron measurement.

  Further, by calculating the total reflection coefficient η using the formula (1) and selecting an element having a difference in value of η of 0.02 or more, it is possible to predict an optimal combination of elements for identification. If this is used, a set of microscopic bodies composed of different elements can be prepared and identified by SEM backscattered electron measurement. Therefore, in the measurement of intracellular biomolecules by SEM, a set of microscopic bodies of different elements It becomes possible to label all at once.

  Furthermore, in order to find a probe molecule that binds to a biomolecule on the surface of the labeled fine particle set, for example, a thiol group is added to the end of the probe molecule, and a different element is added to the surface of the labeled fine particle uniformly. It is also possible to add an element capable of directly bonding to a thiol group such as a gold element through an S-Au bond. In this case, when the acceleration voltage intensity is sufficiently large, the reflected electron intensity reflecting the different element thin films under the commonly added gold element thin film can be measured. Measurements can be made by similar means.

  INDUSTRIAL APPLICABILITY The present invention is useful as a method for enabling the simultaneous use of a microbody set made of different elements, a microparticle labeling, and the like in an SEM measurement method using a heavy element microbody as a label. The present invention is also useful as a tool or the like in intracellular biomolecule interactions, spatial colocalization studies, and the like.

Claims (18)

A method for determining an optimum acceleration voltage value of an SEM electron beam used in a method for identifying fine particles coated with different elemental thin films by backscattered electron measurement using a scanning electron microscope (SEM),
(i) preparing a sample in which three or more kinds of elements are deposited in a thin film on different regions of the support surface;
(ii) irradiating the element of the sample with a SEM electron beam over a predetermined acceleration voltage range;
(iii) obtaining a reflected electron image of the sample at each acceleration voltage value;
(iv) Based on the image, for each of the elements:
[Where I _Z is the brightness of the reflected electron image of the thin film region composed of the element with atomic number Z, and I _max is composed of the element with the largest atomic number Z of the brightness of the reflected electron image of the observed element. The luminance of the reflected electron image of the thin film region, and I _min is the luminance of the reflected electron image of the thin film region composed of the element having the smallest atomic number Z among the luminances of the reflected electron images of the observed elements. ] Calculating the calibration luminances I to _Z represented by
(v) calculating a ratio of I to _Z between each of the elements at each acceleration voltage value;
(vi) selecting a combination of elements having a minimum ratio of I to _Z at each acceleration voltage value; and
(vii) including a step of selecting an acceleration voltage value when the ratio of I to _Z of the selected combination of elements becomes a maximum within the range of the predetermined acceleration voltage value as the optimum acceleration voltage value of the SEM electron beam. ,Method.   The method according to claim 1, wherein in the sample, three or more kinds of the elements are deposited in a thin film in different regions of the support surface with substantially equal thickness.   The method of claim 2, wherein the thickness is in the range of 1 to 500 nm. The element is
(I) Transition metals up to 79 excluding atomic number 43,
(II) metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, 83, and
(III) The method according to any one of claims 1 to 3, wherein the semiconductor is selected from among semiconductors having atomic numbers 14, 34 and 52.   The method according to claim 1, wherein the element is selected from Au, Ag, Ge, Cu, Fe, Si, Eu, Y, Ti, and Al. In the step (v) of calculating the ratio of I to _Z between each element at each acceleration voltage value,
Following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . Denominator I ~ _Z elements of the total reflection coefficient total reflection coefficient between adjacent elements when arranged in ascending or descending order based on the value of eta eta small side of each element represented by, the total reflection coefficient eta There calculates a ratio of the I ~ _Z a I ~ _Z large side element as molecules a method according to any of claims 1 to 5.   The element is selected so that a difference in total reflection coefficient η between adjacent elements when the elements are arranged in ascending or descending order based on the value of the total reflection coefficient η of each of the elements is 0.02 or more. The method described in 1.   The method according to claim 1, wherein the range of the predetermined acceleration voltage value is in a range of 0.1 kV to 30 kV. A fine particle label formed by coating different regions of the support surface with a thin film with three or more elements,
Following formula:

_{η (Z, E 0) =} (-272.5 + 168.6Z - 1.925Z 2 + 0.008225Z 3) × 10 -4 [1 + (0.2043 - 0.6543Z -0.3) ln (E 0/20000)]

[Where η is the total reflection coefficient of the element with atomic number Z when irradiated with an electron beam having energy E ₀ . ] Three or more types of elements such that when arranged in ascending or descending order based on the value of the total reflection coefficient η of each element, the difference in total reflection coefficient η between adjacent elements is 0.02 or more The particulate label | marker by which the different area | region of the said support body surface is coat | covered in thin film form using the combination of these.   The fine particle label according to claim 9, wherein the size of the support is in the range of 1 nm to 1 mm.   The fine particle label according to claim 9 or 10, wherein in the sample, the three or more kinds of elements are deposited in a thin film shape in different regions of the support surface with a substantially equal thickness.   12. The particulate label according to claim 11, wherein the thickness is in the range of 1 to 500 nm. The element is
(I) Transition metals up to 79 excluding atomic number 43,
(II) metals with atomic numbers 13, 31, 32, 33, 49, 50, 51, 81, 82, 83, and
(III) The particulate label according to any one of claims 9 to 12, which is selected from semiconductors having atomic numbers 14, 34 and 52.   The particulate label according to any one of claims 9 to 13, wherein the element is selected from Au, Ag, Ge, Cu, Fe, Si, Eu, Y, Ti, and Al.   The fine particle label according to any one of claims 9 to 14, wherein the support is made of a material such as silicon, mica, polystyrene, polypropylene, or glass.   The microparticle label according to any one of claims 9 to 15, wherein a probe molecule capable of binding to a biomolecule is bound to the surface of the microparticle label.   The fine particle label according to any one of claims 9 to 16, wherein the probe molecule has a thiol group, and a surface of the fine particle label is coated with a gold element. A step of labeling a biomolecule by binding the fine particle label according to claim 9,
Irradiating the labeled biomolecule with an electron beam under SEM to obtain a reflected electron beam image from the particulate label;
Analyzing the brightness of the reflected electron image and identifying the biomolecule by identifying the type of particulate label;
A biomolecule identification method comprising: