JP4910131B2 - Surface information acquisition apparatus and surface information acquisition method - Google Patents

Description

  The present invention relates to a surface information acquisition device and a surface information acquisition method.

Conventionally, as a method for acquiring surface information of a sample, a method using an atomic force microscope (hereinafter referred to as AFM) or a method using a pair of microelectrodes is generally known (for example, a patent). Reference 1, Non-Patent Documents 1 and 2). In acquisition using AFM, the cantilever equipped with the probe scans the sample surface, and the surface information of the sample is acquired by measuring the displacement of the probe due to the atomic force between the sample surface and the probe. The In acquisition using a microelectrode, a pair of microelectrodes scans the sample surface, the potential distribution on the sample surface is obtained from the electrical conductivity obtained by the microelectrode, and the surface information of the sample is acquired.
US Pat. No. 4,724,318 G. Binning, et al., Atomic Force Microscope, Phys. Rev. Letters, 56,930 (1986) Koichi Aoki, Masao Morita, Tsutomu Horiuchi, Osamu Niwa, "Electrochemical measurement using microelectrodes" (edited by IEICE), 1998, Chapters 2 and 3

  However, in the acquisition of surface information using these, there is a problem that acquisition of information becomes difficult when a liquid or gas exists on the sample surface and is flowing. That is, when the AFM is used, the cantilever is subjected to a force due to the flow of liquid or the like, so that accurate measurement of the surface cannot be performed. In addition, when a microelectrode is used, the microelectrode is also subjected to a force due to a flow of liquid or the like, so that accurate measurement cannot be performed.

  The present invention has been made to solve the above problems, and a surface information acquisition apparatus and surface information capable of acquiring surface information even when a liquid or the like is flowing on the surface of the sample. The purpose is to provide an acquisition method.

  In order to achieve such an object, the surface information acquisition apparatus according to the present invention is based on a probe image obtained by floating a probe particle above the surface of a sample and capturing an image of the floating probe particle. A surface information acquisition apparatus for acquiring surface information of a sample, and a size specification value specifying means for obtaining a measurement value for specifying a size of the probe particle in the probe image, and a size of the probe particle in the probe image Position information that acquires position information from the reference surface of the probe particle based on calibration data indicating the relationship between the value and the distance of the probe particle from the reference surface, and the measured value obtained by the size specification value specifying means It comprises an acquisition means and a surface information acquisition means for acquiring the surface information of the sample based on the position information of the probe particles.

  On the other hand, the surface information acquisition method according to the present invention acquires probe surface information based on a probe image obtained by floating a probe particle above the surface of the sample and capturing an image of the floating probe particle. A method for obtaining surface information, comprising preparing calibration data indicating a relationship between a value defining the size of a probe particle in a probe image and a distance of the probe particle from a reference surface, and a probe particle in the probe image The position information from the reference plane of the probe particle is acquired based on the size specified value measurement step for obtaining the measured value that defines the size of the probe, the calibration data, and the measured value obtained by the size specified value specifying means. And a surface information acquisition step for acquiring surface information of the sample based on the position information of the probe particles. The features.

  In the surface information acquisition apparatus and the surface information acquisition method, the probe particles are organically related to the sample surface, for example, interact with the sample surface and distribute according to the shape of the sample surface. To position. Therefore, in the surface information acquisition apparatus and the surface information acquisition method, it is possible to acquire information on the sample surface from the position information of the probe particles. Even when a liquid or gas is present on the sample surface and the liquid or the like is flowing, the probe particles are floating, so that the surface information of the sample can be acquired without being hindered by the flow. It becomes possible. Moreover, it becomes possible to acquire surface information on a desired scale by changing the size of the probe particles. Further, unlike the measurement using an electrode such as a microelectrode, since it is not necessary to pass a current through the sample, the sample surface can be obtained without being affected by the current.

  The sample and the probe particle are preferably charged with the same sign. In this case, the probe particle is located under the influence of the interaction between the potential of the probe particle and the potential of the sample surface. Therefore, the surface potential of the sample surface can be acquired by obtaining the position information of the probe particles.

  The probe particles are preferably spheres. In this case, since the image of the probe particle obtained by the probe image is a circle, the diameter of the circle can be adopted as a value that defines the size of the probe particle. As a result, a value that defines the size of the probe particle can be obtained without depending on the direction in which the probe particle is imaged, and surface information can be easily acquired.

  The surface information acquisition apparatus may further include a light source that outputs light for irradiating the probe particles. Alternatively, in the surface information acquisition method, the probe particles may be irradiated with light output from the light source. In this case, it is possible to acquire surface information using probe particles that do not emit light by themselves.

  According to the present invention, it is possible to provide a surface information acquisition apparatus and a surface information acquisition method capable of acquiring surface information even when a liquid or the like is flowing on the sample surface.

  Hereinafter, preferred embodiments will be described in detail with reference to the accompanying drawings. In the description, the same reference numerals are used for the same elements or elements having the same function, and redundant description is omitted.

(First embodiment)
FIG. 1 is a diagram showing a configuration of a surface information acquisition apparatus 1A according to the first embodiment. As shown in FIG. 1, a surface information acquisition device 1A includes a light source 2, an imaging device (imaging means) 3, an imaging device (imaging means) 4, an image processing device (image processing means) 10, and a display device. (Display means) 5, and obtains surface information of a transparent substrate S as a sample by using a plurality of probe particles P. The surface information acquisition apparatus 1A is configured as a transmission microscope for the transparent substrate S. The transparent substrate S is a transparent glass plate, for example, and is placed on the sample stage 6.

  A solution is filled between the substrate S and the cover glass G, and the probe particle P is contained in the solution. Each of the probe particles P is a sphere, and is suspended in the solution at a predetermined distance from the surface of the sample. The substrate S does not coincide with the focal plane F of the imaging device 3, and in this embodiment, the substrate S is disposed so that the focal plane F of the imaging device 3 is located between the substrate S and the cover glass G. The For example, polystyrene latex fine particles having a diameter of about 1 μm are used as the probe particles P. The substrate S and the probe particles P may be charged with the same sign.

  The light source 2 is positioned below the sample stage 6, that is, on the side where the probe particles P of the substrate S are not disposed. The light source 2 outputs light l that irradiates the substrate S and the probe particles P.

  The imaging device 3 is located on the side of the substrate S where the probe particles P are arranged. The imaging device 3 has an imaging optical system, and connects an image (probe image) of the probe particle P irradiated with the light 1 output from the light source 2 on the light receiving surface of the imaging device 4.

  The imaging device 4 is a CCD camera that captures the probe image formed by the imaging device 3. The imaging device 4 outputs the probe image obtained by imaging the probe image to the image processing device 10.

  The image processing apparatus 10 inputs the captured probe image from the imaging apparatus 4. The image processing device 10 acquires the position information of the probe particles P from the probe image output from the imaging device 4, and acquires the surface information of the substrate S from the position information. The image processing apparatus 10 outputs the acquired surface information of the substrate S to the display device 5. Note that the image processing apparatus 10 may output a probe image to the display device 5 as necessary.

  The display device 5 displays the surface information or the probe image of the substrate S input from the image processing device 10. As the display device 5, for example, a CRT monitor, a liquid crystal display, or the like can be used.

  Next, functions of the image processing apparatus 10 will be described with reference to FIG. As illustrated in FIG. 2, the image processing apparatus 10 includes a calibration data storage unit 11, a size specification value specifying unit 12, a position information acquisition unit 13, and a surface information acquisition unit 14.

  The calibration data storage unit 11 stores calibration data indicating the relationship between the value that defines the size of the probe particle P in the probe image and the distance of the probe particle P from the surface of the substrate S. In this embodiment, since the probe particle P is a sphere, the value that defines the size of the probe particle P is defined as the diameter of the probe particle P on the image.

  FIG. 3 shows a graph obtained from the calibration data. The horizontal axis of the graph in FIG. 3 represents the distance Z (μm) from the focal plane F in the direction orthogonal to the surface of the substrate S as the sample (hereinafter referred to as the z direction), and the vertical axis represents the probe particle P in the probe image. Represents the diameter d (μm). The focal plane F of the imaging device 3 functions as a reference plane for specifying the position of the probe particle. The circle in the graph of FIG. 3 represents actual measurement data, and the curve is the result of fitting to these measurement data. The calibration data storage unit 11 stores, for example, a relational expression obtained by fitting as calibration data. Alternatively, the calibration data storage unit 11 stores, for example, actual measurement data as calibration data. FIG. 4 shows a table of calibration data when the calibration data storage unit 11 stores actual measurement data as calibration data.

The position of the focal plane F of the imaging device 3 from the surface of the substrate S is known. Therefore, as understood from the graph of FIG. 3, according to the calibration data, the distance Z from the surface of the substrate S of the probe particle P can be obtained by obtaining the diameter d of the probe particle P. Here, each distance _Z from the focal plane F _0, Z 1, _{Z 2} only separated probe particles _{P 0,} the P 1, _{P 2,} the distance _{Z 0,} Z 1, _{Z 2} and the diameter _d 0 of the probe image, The relationship between d ₁ and d ₂ will be described. In the description, reference is made to FIGS.

FIG. 5 is a diagram for explaining the relationship between the probe particles P ₀ , P ₁ , P ₂ and the focal plane F. FIG. As shown in FIG. 5, the probe particle P ₀ is located at the focal plane F and is separated from the focal plane F by Z ₀ (= 0). The probe particle P ₁ is at a position Z ₁ (<Z ₀ ) with respect to the focal plane F. The probe particle P ₂ is at a position Z ₂ (> Z ₀ ) with respect to the focal plane F.

FIG. 6 is an image of probe particles P ₀ , P ₁ , P _{2 in} the captured probe image. 6A is an image of the probe particle P ₀ , FIG. 6B is an image of the probe particle P ₁ , and FIG. 6C is an image of the probe particle P ₂ . As shown in FIG. 6, the size (diameter size) of the circle in the image of the probe particle P ₀ located in the focal plane F (FIG. 6B) is that of the other probe particles P ₁ and P ₂ . It becomes smaller than the circle (diameter) in the image (FIGS. 6A and 6C). The two-dot chain line T ₂ shown in FIG. 6A, the two-dot chain line T ₀ shown in FIG. 6B, and the two-dot chain line T ₁ shown in FIG. A line that passes through the center point of the particle and traverses the image of each probe particle. Further, the diameter d ₁₂ shown in FIG. 6A, the diameter d ₁₀ shown in FIG. 6B, and the diameter d ₁₁ shown in FIG. 6C are white circles of probe particles in the probe image, respectively. The diameter of the image represented by The diameter d ₂₂ shown in FIG. 6 (a), the diameter d ₂₀ shown in FIG. 6 (b), and the diameter d ₂₁ shown in FIG. 6 (c) are each represented by a black ring of probe particles in the probe image. Is the diameter of the image to be made.

FIG. 7 is a graph showing the intensity of each probe particle P ₀ , P ₁ , P ₂ in the probe image. 7A is a graph of the probe particle P ₀ , FIG. 7B is a graph of the probe particle P ₁ , and FIG. 7C is a graph of the probe particle P ₂ . The horizontal axis of each graph is the position T on the transverse lines T ₂ , T ₀ , and T ₁ shown in FIGS. 6A, 6B, and 6C, and the vertical axis is the intensity I (T) in the probe image. To express. The intensity I ₀ in each graph corresponds to the average intensity of the background image. The diameters d ₀ , d ₁ , d ₂ of each probe particle P ₀ , P ₁ , P _{2 in} the probe image are, for example, of the intensity I (T) located on each transverse line and within the circle of the probe particle on the image The distance between the vertices of the primary peak (convex portion) on both sides of the zero-order peak (convex portion) (the diameter of the white ring in each image of FIGS. 6A to 6C) d ₁₀ , d ₁₁ , and _{d 12.} Alternatively, the diameters d ₀ , d ₁ , d ₂ of the probe particles P ₀ , P ₁ , P ₂ in the probe image are, for example, the intensities I (T) located on the respective transverse lines and within the circle of the probe particles on the image. ) Between the vertices of the concave portions on both sides of the 0th-order peak (convex portion) (diameter of the black ring in each image of FIGS. 6A to 6C) d ₂₀ , d ₂₁ , d _22. May be.

Further, the case where the intensity of the zero-order peak is smaller than the average intensity I ₀ means that the probe particles are located above the focal plane F. On the other hand, the case where the intensity I (T = 0) of the zero-order peak is larger than the average intensity I ₀ means that the probe particles are located below the focal plane F. Thus, from FIG. 7 (a) ~ (c) , the probe particles _{P 2} is positioned above the focal plane F, probe particles _P 0, _{P 1} is shown to be positioned below the focal plane F.

  The description of the function of the image information processing apparatus 10 will be continued with reference to FIG. The size specification value specifying unit 12 inputs the captured probe image from the imaging device 4 and obtains a diameter d that is a measurement value of a value that specifies the size of each of the plurality of probe particles P in the probe image. That is, the diameter d is obtained as a distance between primary peaks based on a graph (see FIG. 7) obtained based on an image as shown in FIG. The size specification value specifying unit 12 outputs the diameter d to the position information acquisition unit 13.

  The position information acquisition unit 13 inputs the diameter d of the probe particle P from the size specification value specifying unit 12. The position information acquisition unit 13 acquires position information from the surface of the substrate S of the probe particles P based on the calibration data stored in the calibration data storage unit 11 and the diameter d of the probe particles P. That is, the position information acquisition unit 13 is based on the calibration data shown in FIG. 3 obtained from the calibration data storage unit 11 when the diameter of the probe particle P obtained by the size specification value specifying unit 12 is d. A distance (positional information) Z from the focal plane F is obtained. If the distance of the focal plane F from the surface of the substrate S is ZF, the ZF is known. Therefore, the position information acquisition unit 13 adds the ZF to the Z thus obtained, as necessary. A distance ZP from the surface of the substrate S is acquired.

  Furthermore, the position information acquisition unit 13 can also obtain position coordinates (x, y) when each probe particle is projected onto the surface of the substrate S, that is, on the surface of the substrate S, from a two-dimensional probe image. Therefore, based on the distance from the focal plane F, the position information acquisition unit 13 takes the desired position as the origin, and the three-dimensional coordinates (x, y, z) (for example, z = Z or z = ZP) of each probe particle P. ) Can be obtained. The position information acquisition unit 13 outputs the three-dimensional coordinates (x, y, z) that are the position information of each probe particle P thus obtained to the surface information acquisition unit 14.

  The surface information acquisition unit 14 inputs the position information (x, y, z) of the probe particle P from the position information acquisition unit 13. The surface information acquisition unit 14 acquires the surface information of the substrate S from the position information (x, y, z) of the plurality of probe particles P and outputs it to the display device 5. The specific example which acquires surface information below is shown.

A：定数
φ（ｘ、ｙ、ｚ）：静電的相互作用
ｎ_０：φ（ｘ、ｙ、ｚ）＝０におけるプローブ粒子の個数
ｋ：Boltzmann定数
Ｔ：絶対温度 For example, the case where the substrate S and the probe particle P are charged with the same sign and the surface information acquisition unit 14 acquires the surface potential of the substrate S as surface information will be described. In this case, first, the surface information acquisition unit 14 determines the position distribution function n (x, y) of the plurality of probe particles P floating on the surface of the substrate S from the position information (x, y, z) of the plurality of probe particles P. , Z). The position distribution function n (x, y, z) is a function that represents the distribution of the number of probe particles P on the surface of the substrate S at the coordinates (x, y, z). Furthermore, the surface information acquisition unit 14 calculates the electrostatic interaction φ (x, y, z) between the probe particle P and the substrate S from the following equation (1) based on the position distribution function of the probe particle P. Ask.

A: Constant φ (x, y, z): Electrostatic interaction n ₀ : Number of probe particles at φ (x, y, z) = 0 k: Boltzmann constant T: Absolute temperature

  In this case, the surface information acquisition unit 14 includes a position distribution calculation unit that obtains the position distribution of the floating probe particles P based on the position information of the probe particles, and the position distribution function n in the position distribution calculation unit. (X, y, z) may be calculated.

The surface information acquisition part 14 calculates | requires the surface potential which is the surface information of the board | substrate S surface by the following formula | equation (2) based on the electrostatic interaction calculated | required by Formula (1).

  In this way, the surface information acquisition unit 14 calculates and acquires the surface potential of the substrate S as the surface information.

  Alternatively, for example, a case where the substrate S and the probe particles P are not charged and the surface information acquisition unit 14 acquires the surface shape (unevenness in the z direction) of the substrate S as surface information will be described. The probe particles P are distributed according to the surface shape of the substrate S by being dispersed in the solution according to a certain probability distribution (density). Therefore, the surface information acquisition unit 14 calculates and acquires the surface shape of the substrate S based on the three-dimensional coordinates (x, y, z) of each probe particle P that is the position information of the probe particle P.

Alternatively, for example, another case where the substrate S and the probe particles P are not charged and the surface information acquisition unit 14 acquires the surface shape (unevenness in the z direction) of the substrate S as surface information will be described. First, the position information acquisition unit 13 acquires the position information of the probe particles P with a predetermined time interval. The surface information acquisition unit 14 obtains the flow velocity of the solution on the substrate S from the position information (three-dimensional coordinates (x, y, z) of each probe particle P) of the probe particles P with a predetermined time interval. This, for example, after a predetermined time has elapsed, if the z-coordinate was also changed not only x and y coordinates for a particular probe particles P _1, worked velocity of moving in the z-axis direction to the specific probe particles P ₁ As a result, it is possible to obtain the unevenness of the surface of the substrate S from the change of the z coordinate. Thus, the surface information acquisition unit 14 obtains the flow velocity distribution of the solution based on the position information of the probe particles P, and calculates and acquires the surface shape of the substrate S.

  Next, with reference to FIG.8 and FIG.9, the surface information acquisition method using 1 A of surface information acquisition apparatuses which concern on this embodiment is demonstrated, and operation | movement of 1 A of surface information acquisition apparatuses is also demonstrated collectively. FIG. 8 is a diagram illustrating a procedure of the surface information acquisition method.

  First, calibration data (relationship between the diameter d which is a value defining the size of the probe particle P in the image captured by the imaging device 4 and the distance Z of the probe particle P from the focal plane F which is the reference plane) ( 3 and 4) are prepared (calibration data preparation step S01). FIG. 9 is a diagram for explaining how to obtain calibration data. Calibration data is acquired by the following procedure.

  That is, as shown in FIG. 9, after the probe particles P sandwiched with the solution between the two cover glasses G1 and G2 are arranged on the sample stage 6, the sample stage 6 is moved in the z direction. That is, the case where it coincides with the focal plane F is set as a reference (Z = 0), and the stage 6 is moved together with the probe particles P in the vertical direction (Z <0, Z> 0) with respect to the reference. At that time, a probe image of the probe particle P at each position is picked up by the image pickup device 4, and the diameter d of the probe particle P in the probe image is obtained. Thus, the diameter d of the probe particle P in the probe image with respect to the distance from the focal plane F in the z direction is obtained, and calibration data is obtained. The obtained calibration data is stored in the calibration data storage unit 11 of the image processing apparatus 10.

  Returning to FIG. 8 again, the description of the surface information acquisition method will be continued. Next, a solution containing a plurality of probe particles P is filled between the substrate S and the cover glass G. Thereby, the substrate S and the probe particles P are arranged so that the probe particles P float at a predetermined distance from the surface of the substrate S (probe particle preparation step S02). The substrate S is disposed on the sample stage 6 of the surface information acquisition apparatus 1A.

  Subsequently, images of the plurality of probe particles P connected by the imaging device 3 are captured by the imaging device 4 (imaging step S03). A plurality of probe particles P having various position maps may be imaged in the probe image. The captured probe image is output from the imaging device 4 to the size specification value specifying unit 12 of the image processing device 10.

  The size specification value specifying unit 12 to which the probe image is input obtains a diameter d that is a measurement value that specifies the size of the probe particle P in the probe image captured by the imaging device 4 (size specification value specifying step). S04). When a plurality of probe particles P are imaged, the diameter d is obtained for them. The diameter d thus obtained is output from the size specification value specifying unit 12 to the position information acquiring unit 13.

  In the position information acquisition unit 13 to which the diameter d is input, based on the calibration data stored in the calibration data storage unit 11 and the diameter d of the probe particle P obtained in the size specification value specifying step S04, a reference is made. Position information (x, y, z) of the probe particle P from the focal plane F, which is a plane, is acquired (position information acquisition step S05). The position information (x, y, z) of each probe particle P thus obtained is output from the position information acquisition unit 13 to the surface information acquisition unit 14.

  The surface information acquisition unit 14 to which the position information (x, y, z) of each probe particle P is input acquires the surface information of the substrate S based on the position information (x, y, z) (surface information acquisition). Step S06). In the surface information acquisition step S06, for example, the position distribution function n (x, y, z) of the probe particles P floating in the solution is calculated from the position information (x, y, z) of each probe particle P to calculate the surface. You may use for acquisition of information. The acquired surface information is, for example, a surface potential or a surface shape. In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, the surface information of the substrate S is thus acquired.

  In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, the probe particles P are positioned in organic relation with the surface of the substrate S. Therefore, in the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, it is possible to obtain the surface information of the surface of the substrate S that is a sample from the position information (x, y, z) of the probe particles P.

  Specifically, for example, when the probe particle P and the substrate S are both charged with the same sign, the probe particle P and the surface of the substrate S interact with each other and affect the electrostatic interaction. In response, the probe particle P is positioned. Therefore, by obtaining the position distribution function n (x, y, z) of the probe particle P, the surface information acquisition device 1A and the surface information acquisition method according to the present embodiment can be obtained from the equations (1) and (2). It is possible to obtain the surface potential of the substrate S.

  In this case, the position distribution function of the probe particle has a sharp rising part, and this sharp rising part responds sensitively to a slight change in surface potential. It is possible to detect a minute change in the surface potential with high sensitivity, such as when adsorbed on the surface of the substrate S. In this case, since it is not necessary to chemically and physically bind the label molecule to the bio-related molecule, it is possible to omit the pretreatment for measurement. Furthermore, since it is based on the three-dimensional position distribution function n (x, y, z) of the probe particle P, the potential distribution can be obtained as the surface potential of the substrate S. Thus, by binding biomolecules corresponding to a plurality of different lesions on the substrate S, it is possible to inspect a plurality of items in one measurement, and the time required for biochemical examination in the medical field is reduced. Significant shortening is realized.

  Alternatively, for example, even when the probe particles P and the substrate S are not charged, the probe particles P dispersed with a certain probability distribution are distributed according to the shape of the surface of the substrate S. Thereby, in the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, the surface shape of the substrate S that is a sample can be obtained.

  Alternatively, for example, even when neither the probe particle P nor the substrate S is charged, the probe particle P moves at a flow velocity according to the shape of the surface of the substrate S. In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, the flow velocity distribution of the probe particles P can be obtained, and thereby the surface shape of the substrate S that is a sample can be obtained.

  In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, a solution that is a liquid exists on the surface of the substrate S, and probe particles P that are responsible for measurement float in the solution. Therefore, unlike conventional surface information acquisition devices (for example, AFM), acquisition of surface information is not hindered by the flow of the solution. Therefore, in the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, it is possible to acquire the surface information of the substrate S even when the solution is flowing on the surface of the substrate S.

  Furthermore, since the probe particle P is floating, it can respond to a weak force. Thereby, for example, the influence of a weak surface potential can be measured.

  In addition, since the probe particles P are floating, according to the surface information acquisition device 1A and the surface information acquisition method according to this embodiment, the probe particles P are located away from the focal plane F (for example, several tens of μm or more). It is also possible to acquire position information of the probe particle P. Therefore, for example, even when the surface of the substrate S has a high potential, the position information can be acquired without applying an external force for capturing the probe particles P.

  Further, by changing the size of the probe particle P, it is possible to acquire the surface information of the substrate S at a desired scale. That is, it is possible to acquire surface information on a very micro scale by selecting a smaller probe particle P, while acquiring surface information on a macro scale by selecting a larger probe particle P. Is possible.

  Further, in the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, it is not necessary to pass a current through the substrate S. Therefore, the surface information of the substrate S can be acquired without being affected by the current.

  In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, the position information (coordinates) of the probe particles can be acquired as long as the diameter of each probe particle in the probe image can be calculated. Therefore, even if there are a plurality of probe particles captured in the probe image, position information of the plurality of probe particles captured can be acquired. That is, it is possible to acquire the position information of a plurality of probe particles with one imaging, and it is possible to shorten the time required for acquiring the surface information.

  In this embodiment, since the probe particle P is a sphere, the diameter of the probe particle P obtained from the probe image (a value that defines the size of the probe particle P) does not depend on the direction in which the probe particle P is imaged. As a result, the probe particle P can be imaged from any direction, and surface information can be easily acquired.

  In the present embodiment, since the light source 2 that outputs the light 1 that irradiates the probe particle P is provided, it is possible to acquire surface information using the probe particle P that does not emit light by itself.

  In the surface information acquisition apparatus 1A and the surface information acquisition method according to the present embodiment, information other than the surface potential and the surface shape of the substrate S can be acquired as surface information based on the position information of the probe particles P.

(Second Embodiment)
With reference to FIG. 10, the structure of the surface information acquisition apparatus 1B which concerns on 2nd Embodiment is demonstrated. FIG. 10 is a diagram showing a configuration of a surface information acquisition apparatus 1B according to the second embodiment. The surface information acquisition apparatus 1B according to the second embodiment differs from the surface information acquisition apparatus 1A according to the first embodiment in that the sample is not a substrate but a transparent tube S.

  The transparent tube S is a glass tube, for example, and is placed on the sample stage 6 so that the longitudinal direction of the tube is perpendicular to the optical axis of the imaging device 3. The transparent tube S is filled with a solution, and the solution contains a plurality of probe particles P. The probe particles P are spherical and all float in the solution at a predetermined distance from the inner wall of the sample tube. The imaging device 3 is positioned such that its focal plane F crosses the inside of the transparent tube S. The transparent tube S and the probe particle P may be charged with the same sign.

  Similarly to the surface information acquisition method using the surface information acquisition device 1A, the surface information acquisition method using the surface information acquisition device 1B according to the present embodiment also has a calibration data preparation step S01, a probe particle preparation step S02, as shown in FIG. An imaging step S03, a size specification value specifying step S04, a position information acquisition step S05, and a surface information acquisition step S06 are provided.

  In the surface information acquisition apparatus 1B and the surface information acquisition method according to the present embodiment, the probe particles P are distributed by, for example, electrostatic interaction with the inner wall surface of the transparent tube S or the shape of the inner wall surface of the transparent tube S. It is located in an organically related manner with the inner wall surface of the transparent tube S, such as being distributed along the line. Therefore, in the surface information acquisition device 1B and the surface information acquisition method according to the present embodiment, the surface information (for example, surface potential, surface potential, etc.) of the inner surface of the transparent tube S that is the sample from the position information (x, y, z) of the probe particle P. Alternatively, the surface shape or the like can be obtained.

  In particular, since the transparent tube S is tubular, it is difficult to obtain surface information on the inner wall with a normal measurement method. However, in the surface information acquisition apparatus 1B and the surface information acquisition method according to the present embodiment, the surface information of the inner wall of the transparent tube S is acquired as long as the position information of the probe particles floating in the tubular transparent tube S is acquired. Can do. That is, in the surface information acquisition apparatus 1B and the surface information acquisition method according to the present embodiment, it is possible to acquire the surface information of a sample having a complicated shape.

  Moreover, in the surface information acquisition apparatus 1B and the surface information acquisition method according to the present embodiment, a liquid solution exists in the transparent tube S. In the surface information acquisition apparatus 1B according to the present embodiment, since the probe particles P are floating in the transparent tube S that is a sample, acquisition of surface information (surface information on the inner wall of the tube) is hindered by the flow of the solution. There is no. Therefore, in the surface information acquisition device 1B and the surface information acquisition method according to the present embodiment, it is possible to acquire the surface information of the inner wall of the transparent tube S even when the solution is flowing on the inner wall surface of the transparent tube S. It becomes.

  In addition, by changing the size of the probe particle P, it is possible to acquire the surface information of the inner wall of the transparent tube S at a desired scale.

  Moreover, in the surface information acquisition apparatus 1B and the surface information acquisition method according to the present embodiment, it is not necessary to pass a current through the transparent tube S. Therefore, it becomes possible to acquire the surface information of the inner wall of the transparent tube S without being affected by the current.

  In addition, since position information can be acquired for a plurality of probe particles with one imaging, actual measurement is facilitated, and the time required for acquiring surface information can be shortened.

  In this embodiment, since the probe particle P is a sphere, the probe particle P can be imaged from any direction, and actual measurement becomes easy. As a result, it is easy to obtain surface information.

  In the present embodiment, since the light source 2 that outputs the light 1 that irradiates the probe particle P is provided, it is possible to acquire surface information using the probe particle P that does not emit light by itself.

  In the surface information acquisition device 1B and the surface information acquisition method according to the present embodiment, information other than the surface potential and the surface shape of the inner wall of the transparent tube S may be acquired as surface information based on the position information of the probe particles P. it can.

(Third embodiment)
With reference to FIG. 11, the structure of 1 C of surface information acquisition apparatuses which concern on 3rd Embodiment is demonstrated. FIG. 11 is a diagram showing a configuration of a surface information acquisition apparatus 1C according to the third embodiment. The surface information acquisition apparatus 1C according to the third embodiment is a surface information acquisition apparatus 1A according to the first embodiment in that the sample is an opaque substrate S and is configured as a reflective microscope with respect to the opaque substrate S. And different.

  The opaque substrate S is, for example, a semiconductor device or a colored glass substrate, and is placed on the sample stage 6 so that the surface to be measured of the substrate S is orthogonal to the optical axis of the imaging device 3. A solution is filled between the substrate S and the cover glass G, and the probe particle P is contained in the solution. The probe particles P are all spherical and float in the solution at a predetermined distance from the surface of the substrate S. The imaging device 3 is arranged so that its focal plane F crosses the solution on the surface of the substrate S, that is, between the substrate S and the cover glass G. The substrate S and the probe particles P may be charged with the same sign.

  The surface information acquisition device 1C employs an ultra-illumination system, and has a predetermined angle with the optical axis of the imaging device 3 and outputs a light source 1 for irradiating the substrate S and the probe particles P (illustrated in FIG. 11). (Omitted). When using an ultra-illumination system, for example, there are a method using a high-power objective lens having a long working distance, a method combining a low-power objective lens with a zoom optical system, or a method using a dark-field objective lens.

  Similarly to the surface information acquisition method using the surface information acquisition device 1A, the surface information acquisition method using the surface information acquisition device 1C according to the present embodiment also has a calibration data preparation step S01, a probe particle preparation step S02, as shown in FIG. An imaging step S03, a size specification value specifying step S04, a position information acquisition step S05, and a surface information acquisition step S06 are provided.

  When a coaxial (bright field) illumination system is used in a reflection type microscope, background reflection is incident on the objective lens and the image of the probe particle becomes unclear. Since the surface information acquisition apparatus 1C uses an extreme (dark field) illumination system, an image of the probe particle P can be taken even when the opaque substrate S is measured. As a result, the diameter of the probe particle P in the probe image can be specified, so that the surface information of the substrate S can be acquired. As described above, the surface information acquisition apparatus 1 </ b> C and the surface information acquisition method according to the present embodiment can acquire the surface information of the opaque substrate S.

  In the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, the probe particles P are distributed by, for example, exerting an electrostatic interaction with the opaque substrate S surface, or distributed according to the shape of the substrate S surface. For example, it is organically related to the surface of the substrate S. Therefore, in the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, the surface information (for example, the surface potential or the surface shape) of the substrate S as the sample from the position information (x, y, z) of the probe particles P. Etc.) can be obtained.

  In the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, a liquid solution exists on the substrate S. In the surface information acquisition apparatus 1C according to the present embodiment, since the probe particles P are floating on the substrate S that is a sample, acquisition of surface information is not hindered by the flow of the solution. Therefore, in the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, it is possible to acquire the surface information of the substrate S even when the solution is flowing on the surface of the substrate S.

  Further, by changing the size of the probe particle P, it is possible to acquire the surface information of the substrate S at a desired scale.

  In the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, it is not necessary to pass a current through the substrate S. Therefore, it is possible to acquire the surface information of the substrate S without being affected by the current.

  In addition, since position information can be acquired for a plurality of probe particles with one imaging, actual measurement is facilitated, and the time required for acquiring surface information can be shortened.

  In this embodiment, since the probe particle P is a sphere, the probe particle P can be imaged from any direction, and actual measurement becomes easy. As a result, it is easy to obtain surface information.

  In the present embodiment, since the light source that outputs the light l for irradiating the probe particles P is provided, surface information can be acquired using the probe particles P that do not emit light by themselves.

  In the surface information acquisition apparatus 1C and the surface information acquisition method according to the present embodiment, information other than the surface potential and surface shape of the opaque substrate S can be acquired as surface information based on the position information of the probe particles P. .

  The preferred embodiments of the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the shape of the probe particle P is not limited to a spherical shape, and may be, for example, a rod shape. The value that defines the size of the probe particle P in the probe image when the rod-shaped probe particle is used is the length in the short direction at both ends in the longitudinal direction of the probe particle. That is, for example, when the lengths in the short direction at both ends of the probe particles in the longitudinal direction are the same in the probe image, the rod-like probe particles are distributed so that the longitudinal direction and the sample surface are parallel. ing. On the other hand, when the lengths in the short direction at both ends in the longitudinal direction of the probe particles are different in the probe image, the rod-like probe particles are distributed with the longitudinal direction having an angle with respect to the sample surface. Therefore, the inclination of the probe particle P with respect to the sample surface can be obtained as positional information of the probe particle P with respect to the sample surface by obtaining the length in the short direction at both ends in the longitudinal direction of the probe particle in the probe image. The inclination of the rod-like probe particle P is determined by being organically related to the sample surface. Therefore, based on the inclination of the probe particle P with respect to the sample surface, surface information such as surface potential or surface shape can be acquired.

The probe particles P are not limited to polystyrene latex, and may be made of other materials. However, when polystyrene latex fine particles are used, the probe particles P can be made into spheres with high sphericity, and the density (1.05 g / cm ³ ) is water (mixed of light water H ₂ O and heavy water D ₂ O). (Liquid) is almost the same as the density. Since the density of water is substantially the same as the density, the influence of gravity when the probe particles are suspended in water can be reduced.

  Further, information other than the surface potential and the surface shape can be acquired as the surface information of the sample. In addition, the surface information acquired based on the interaction between the probe particle and the sample surface is not limited to the surface potential of the sample obtained by electrostatic interaction. For example, the magnetic field strength distribution when the surface is magnetized. It may be information, electromagnetic wave oscillation information when the surface oscillates electromagnetic waves, acoustic wave oscillation information when the surface oscillates sound waves (dense wave), or surface temperature distribution. When the surface is magnetized, the probe particles are distributed according to the intensity distribution of the magnetism. Therefore, the intensity distribution of the magnetism (magnetic field) from the surface of the probe particles (position information) is the magnetic field strength. Obtained as distribution information. When electromagnetic waves are oscillated from the surface, the probe particles are distributed according to the wavelength distribution and intensity distribution of the oscillated electromagnetic waves. Therefore, the wavelength distribution or intensity distribution of the electromagnetic waves oscillated from the probe particle distribution (position information) is Acquired as electromagnetic wave oscillation information. When sound waves are oscillated from the surface, the probe particles are distributed according to the wavelength distribution and intensity distribution of the oscillated sound waves, so the wavelength distribution or intensity distribution of the sonic waves oscillated from the probe particle distribution (position information) is Acquired as sound wave oscillation information. The surface temperature distribution is obtained because the distribution of the probe particles changes according to the change in the density of the liquid or gas in contact with the surface due to the difference in the surface temperature. The probe particles are not limited to liquids, and may be suspended in a gas or in a vacuum, for example.

  Further, for example, the probe particle P itself may emit fluorescence, and an image thereof may be captured to acquire the surface information of the sample S without having a light source. The light source is not limited and may be a light source that emits light of any wavelength, such as visible light, ultraviolet light, infrared light, X-rays, electron beams, or neutron beams. The imaging device is not limited to an optical microscope, and may be, for example, a telescope, a camera, a magnifying glass, or an electron microscope.

It is a figure which shows the structure of the surface information acquisition apparatus which concerns on 1st Embodiment. It is a figure for demonstrating the function of an image process part. It is a figure of the graph obtained by calibration data. A table of calibration data when actual measurement data is used as calibration data is shown. It is a figure for demonstrating the relationship between probe particle | grains and the focal plane of an imaging device. It is a figure showing the image of the probe particle in the imaged probe image. It is a figure of the graph which shows the intensity | strength of each probe particle in a probe image. It is a figure which shows the procedure of the surface information acquisition method which concerns on 1st Embodiment. It is a figure for demonstrating calculating | requiring calibration data. It is a figure which shows the structure of the surface information acquisition apparatus which concerns on 2nd Embodiment. It is a figure which shows the structure of the surface information acquisition apparatus which concerns on 3rd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1A, 1B, 1C ... Surface information acquisition device, S ... Sample, P ... Probe particle, 2 ... Light source, 3 ... Imaging device, 4 ... Imaging device, 5 ... Display device, 10 ... Image processing device, 11 ... Calibration data Storage unit, 12 ... Size specification value specifying unit, 13 ... Position information acquisition unit, 14 ... Surface information acquisition unit

Claims (8)

A surface information acquisition device that floats probe particles above the surface of the sample and acquires surface information of the sample based on a probe image obtained by capturing an image of the floating probe particles,
A size specification value specifying means for obtaining a measurement value that specifies the size of the probe particle in the probe image;
Calibration data indicating a relationship between a value defining the size of the probe particle in the probe image and a distance of the probe particle from a reference plane, and the measured value obtained by the size defining value specifying means Position information acquisition means for acquiring position information from the reference surface of the probe particles based on,
A surface information acquisition device comprising: surface information acquisition means for acquiring the surface information of the sample based on positional information of the probe particles.   The surface information acquisition apparatus according to claim 1, wherein the sample and the probe particles are charged with the same sign.   The surface information acquisition apparatus according to claim 1, wherein the probe particle is a sphere.   The surface information acquisition apparatus according to claim 1, further comprising a light source that outputs light that irradiates the probe particles. A surface information acquisition method for obtaining surface information of the sample based on a probe image obtained by floating probe particles above the surface of the sample and capturing an image of the floating probe particles,
A calibration data preparation step of preparing calibration data indicating a relationship between a value defining a size of the probe particle in the probe image and a distance of the probe particle from a reference plane;
A size prescribed value measuring step for obtaining a measured value defining the size of the probe particle in the probe image;
A position information acquisition step of acquiring position information from the reference surface of the probe particles based on the calibration data and the measurement value obtained by the size prescribed value specifying means;
A surface information acquisition method comprising: a surface information acquisition step of acquiring the surface information of the sample based on the position information of the probe particles.   The surface information acquisition method according to claim 5, wherein the sample and the probe particles are charged with the same sign.   The surface information acquisition method according to claim 5 or 6, wherein the probe particles are spherical.   The surface information acquisition method according to claim 5, wherein the probe particles are irradiated with light output from a light source.

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