JP2009282012A - Non-contact scanning probe microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a non-contact scanning probe microscope capable of measuring an internal structure of extremely fragile samples such as biological and organic materials at high resolution without damaging the internal structure. <P>SOLUTION: A sample 13 is placed on a sample stage 11. A first probe 14 is disposed on a front surface of the sample stage and a second probe 15 is disposed on a rear surface of the sample stage. An alternating current signal with the same frequency as a mechanical resonance frequency of a cantilever, i.e., the second probe 15, is applied with an amplitude of several volts to the first probe 14 from an FM detection module 18, a signal which has been modulated by the sample 13 is sensed (detected) by a leading end of the second probe, and the cantilever resonates by the signal. Resonant oscillation based on such interaction is detected as an electrical signal by the cantilever 15, an amplitude change and frequency change of the detected signal are processed by a control PC 20, and image information (mapping image) is formed based on amplitude change information and frequency change information. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a non-contact scanning probe microscope. More specifically, the form and structure of a non-contact scanning probe microscope can be quickly observed with high resolution without damaging even extremely fragile samples such as biological materials and organic materials. The present invention relates to a scanning probe microscope capable of measuring.

  Microscopes such as optical microscopes, electron microscopes, or probe microscopes such as AFM and SPM have been developed as means for morphological observation and structural analysis of various samples. Optical microscopes are capable of observing cells in a solution, and are used for various functional analyzes in combination with various fluorescent dyes. Since the electron microscope measures reflected electrons and transmitted electrons when the sample is irradiated with an electron beam, the resolution is extremely high, such as several millimeters, and it is possible to specify the atomic position in the sample to be observed. In addition, the probe microscope enables observation of the surface structure of various samples, and its resolution is extremely high at 10 mm or less.

  In addition to these methods, X-ray crystal structure analysis that derives a three-dimensional structure by preparing a protein crystal sample and measuring the fraction of X-ray irradiation as a biological sample, particularly protein structure measurement technology Method and NMR method.

  However, these conventional measurement techniques have several difficulties. Specifically, in an optical microscope, since light is used as a probe, its resolution is as low as about 200 nm, and it is impossible to directly observe proteins and viruses in cells.

  In an electron microscope, there is a possibility that proteins, viruses, and the like may be damaged and destroyed by irradiation with an electron beam, so it is necessary to observe with a low irradiation electron dose. For this reason, the obtained captured image tends to be extremely noisy and low in contrast, and various image processing techniques must be used in order to analyze an accurate sample structure from the electron microscope image.

  Although the X-ray crystal structure analysis method has an extremely high resolution of several tens of meters, the preparation of a crystallized sample is indispensable, so that a sample that is difficult to crystallize cannot be analyzed. Further, in the analysis by NMR, although it is not necessary to crystallize the sample, it is difficult to analyze a sample having a relatively large size. In addition to these difficulties, the information obtained, whether by X-ray crystal structure analysis or NMR analysis, is only the average structure of a large number of particles present in the sample. It is impossible to analyze.

The scanning probe microscope (SPM) is a technique capable of observing the shape and shape of a sample surface with a high resolution by scanning a probe with a sharp tip. At present, an atomic force microscope (AFM) It has been developed as various methods such as a scanning near-field optical microscope (SNOM) (Non-Patent Document 1), and its resolution is extremely high at 10 mm or less, and it can be used for surface measurement of various samples such as metals, organic materials, and organisms. Has been. However, probe microscopes such as AFM and SPM can only obtain information on the shape of the sample surface, and cannot obtain information on the internal structure.
JP 11-160333 A JP 2005-172571 A “Surface analysis technology selection, scanning probe microscope for nanotechnology” published by Maruzen Co., Ltd. (2002) H. Takahashi et al., "Self-sensing piezoresistive cantilever and its magnetic force microscopy applications" Ultramicroscopy 91 (2002) 63-72.

  The present invention has been made in view of the disadvantages of the conventional measurement techniques as described above, and its object is to increase the internal structure of extremely fragile samples such as biological materials and organic materials without damage. It is an object of the present invention to provide a non-contact scanning probe microscope capable of measuring at a resolution and extremely rapidly and obtaining a contrast and an S / N ratio that can clearly analyze individual proteins.

  In order to solve such a problem, a non-contact scanning probe microscope according to the present invention has a first probe arranged in a non-contact manner so as to face one of the front and back sides of the sample and the other of the front and back sides of the sample. A second probe arranged in a non-contact manner, wherein the tip of the probe is arranged in the vicinity of the tip of the first probe, and the tip of the first probe A signal processing unit that forms a mapping image based on at least one of amplitude change information and frequency change information of a signal detected by the tip of the second probe after being input and modulated by the sample. It is characterized by.

  For example, the second probe is a cantilever, and the signal detected by the tip of the second probe is a change in amplitude or resonance frequency of the cantilever.

  Preferably, an AC signal having the same frequency as the mechanical resonance frequency of the cantilever is input from the tip of the first probe.

The signal detected by the tip of the second probe is, for example, a Coulomb force given by the following equation. Q ₁ is the charge at the tip of the first probe, Q ₂ is the charge at the tip of the second probe, d is the distance between the tips of the first and second probes, and ε (x, y) is the sample The relative dielectric constant at the measurement position (x, y), ε ₀ is the dielectric constant of the measurement environment.

  For example, the first probe is a tungsten probe, the second probe is a self-sensing cantilever, and the tips of the first and second probes both have a radius of curvature of 10 nm or less. Has been sharpened.

Preferably, the signal processing unit, the position within the XY plane of the front end portion of the said first position in the XY plane of the tip of the probe and (x _1, y ₁₎ second probe (x ₂ , Y ₂ ) based on a plurality of mapping images obtained under a plurality of relative positional relationships between the probe position control means capable of independently controlling the tip portions of the first and second probes. Image processing means for imaging the three-dimensional structure is provided.

  Preferably, the signal processing unit includes means for automatically changing a plurality of relative positional relationships between the distal end portions of the first and second probes within a predetermined range.

  More preferably, the image processing means defines a reference point in the plurality of mapping images, corrects a positional shift between the mapping images from the reference point, and images a three-dimensional structure.

  Moreover, it is preferable that the first and second probes are provided in an apparatus in which the positional relationship between the tip of the probe and the sample can be visually confirmed.

  The apparatus is, for example, a scanning electron microscope.

  All of the conventional scanning probe microscopes have a configuration in which the probe is arranged only on the surface side of the sample (for example, JP-A-11-160333 (Patent Document 1) and JP-A-2005-172571 (Patent Document). In the present invention, the mapping image is formed on the basis of the signal modulated by the sample by arranging the tip portions of the two probes to face both sides of the sample. Therefore, it is possible to obtain physical characteristic information inside the sample.

  In the microscope of the present invention, the signal applied to measure the weak physical quantity between the probes is as weak as about several volts and is non-contact, so there is no damage to the sample. For this reason, biological samples such as viruses and proteins can be measured with high resolution without damage.

Furthermore, in the microscope of the present invention, the positions ((x ₁ , y ₁ ) and (x ₂ , y ₂ )) of the tip portions of the first and second probes in the XY plane can be controlled independently. Therefore, it is possible to obtain image information (mapping image) corresponding to that observed when the sample is observed from an oblique direction, and the three-dimensional structure of the sample can be imaged.

  Thus, according to the present invention, the internal structure of a very fragile sample such as a biological material or an organic material can be measured at a high resolution without damage and at a very high speed, and further, the individual protein can be clearly analyzed. It is possible to provide a non-contact scanning probe microscope that can obtain contrast and SN ratio.

  The non-contact scanning probe microscope of the present invention will be described below with reference to the drawings.

  FIG. 1 is a block diagram for explaining a configuration example of a non-contact scanning probe microscope according to the present invention. In this figure, the probe of the non-contact scanning probe microscope is connected to the tip of the probe and a sample. An example is shown that is provided in a scanning electron microscope (SEM), which is a device that can visually recognize the positional relationship, and is capable of measuring a sample while observing the SEM.

  In the following description, for the sake of convenience, a signal detection method based on a so-called “FM detection method” in which the degree of interaction between the distal ends of the first probe 14 and the second probe 15 is obtained from a change in the resonance frequency of the cantilever. Although the present invention will be described as a microscope, the adoption of the FM detection method itself is not an essential condition for the present invention.

  A sample 13 supported by a support film 12 is placed on a sample stage (XY stage) 11 that enables sample movement in the XY plane. A first probe 14 (here, a tungsten probe) is disposed in a non-contact manner so as to face one of the front and back surfaces of the sample (here, the front surface). A probe 15 (here, a self-detecting cantilever) is arranged. In any of these probes, the position of the tip can be moved in the Z direction (vertical direction) as well as in the XY plane. The manipulators 16 and 17 can precisely position the tip. The tip portions of both probes are directly opposed with a gap of submicron order through the sample 12.

  It should be noted that these probe tips may be arranged in the vicinity as long as the signal input from the tip of the first probe and modulated by the sample is detected with sufficient sensitivity by the tip of the second probe. It is not always necessary that both tip portions are arranged at the same position (x, y) in the XY plane.

  The tips of these probes are all sharpened (for example, the radius of curvature of the tip is 10 nm or less). The tungsten probe, which is the first probe 14, is supplied from the FM detection module 18 to the second probe 15. An AC signal having the same frequency as the mechanical resonance frequency of a cantilever is applied with an amplitude of several volts. On the other hand, a voltage of 6 V is applied to the cantilever which is the second probe 15 by the DC voltage source 19, and the reference side is connected to, for example, the ground.

  Under such conditions, at the tip of the second probe, the signal input from the tip of the first probe 14 and modulated by the sample 13 is sensed (detected) to be the second probe 15. The cantilever resonates with the signal. Note that the higher the sharpness of the tip of the probe and the higher the aspect ratio (ratio of the length and diameter of the probe tip), the higher the sensitivity of the microscope.

Here, the signal sensed (detected) by the tip portion of the second probe 15 is, for example, a minute Coulomb force (the following formula) acting between the probe tip portions via the sample 13. Where Q ₁ is the charge at the tip of the first probe 14, Q ₂ is the charge at the tip of the second probe 15, d is the distance between the first and second probe tips, and ε (x, y ) Is the relative dielectric constant at the measurement position (x, y) of the sample 13, and ε ₀ is the dielectric constant of the measurement environment (here, vacuum).

  The resonance vibration based on such interaction is detected as an electric signal by the cantilever 15, and both the input signal from the first probe 14 and the detection signal from the second probe are controlled by the FM detection module 18. . Then, the amplitude change and frequency change of the detection signal are sent from the FM detection module 18 to the control PC 20.

  Control of the measurement position of the sample (movement of the sample stage 11) is also performed by the control PC 20, and the control signal from the control PC 20 is amplified by the high voltage amplifier module 21 and sent to the sample stage 11.

  The detection signal associated with the measurement position (x, y) collected in this way is processed by the control PC 20, and image information (mapping image) is formed based on the amplitude change information and the frequency change information.

  Here, a probe (and a manipulator and a sample stage) of a non-contact scanning probe microscope is provided in the SEM, and the probe and the cantilever are moved to a target position in the sample by operating the manipulator while observing the sample with the SEM. However, it is also possible to use an optical microscope instead of the SEM. Further, it is possible to adopt a mode in which the position is automatically determined by measuring the distance between the probes and the distance between the sample and the probe with a sensor.

  FIG. 2 is an enlarged view of the vicinity of the probe and the sample. A sample 13 on a sample stage (not shown) is supported on a support film 12, and is arranged in a non-contact manner on the back surface of the sample and the first probe 14 arranged in a non-contact manner on the sample surface as the sample stage moves. The probe tip position (x, y) on the sample of the second probe 15 thus changed is changed.

  A signal (an AC signal having the same frequency as the mechanical resonance frequency of the cantilever) input from the distal end portion of the first probe 14 is detected as a Coulomb force by the distal end portion of the second probe 15 after being modulated by the sample 13. Thus, a detection signal is generated as a change in the resonant frequency of the cantilever. Information extracted based on such amplitude change information and frequency change information of the detection signal is mapped in association with the measurement position (X, Y).

  Since the sample 13 is supported on the support film 12, the signal detected by the second probe 15 is affected not only by the sample 13 but also by the presence of the support film 12. In order to remove the influence of the support film 12, a signal obtained by measuring a region where the sample 13 does not exist may be handled as a blank.

  FIG. 3 is a block diagram for explaining a detailed circuit configuration from the FM detection module to the probe used in an embodiment described later. A signal from the FM detection module 18 is amplified about 50 times by the OP amplifier 22 and then input to the tungsten probe 14. This input signal is set to be the same as the mechanical resonance frequency of the self-detecting cantilever 15.

  On the other hand, the self-detecting cantilever 15 that receives this input signal has a DC voltage of 6 V applied to the lever tip side and the reference side connected to the ground. In addition, H. Takahashi et al., “Self-sensing piezoresistive cantilever and its magnetic force microscopy applications” Ultramicroscopy 91 (2002) 63-72 (Non-Patent Document 2) and the like are available as technical documents relating to self-detecting cantilevers.

  The cantilever 15 vibrates by receiving only the Coulomb force from the tungsten probe 14, and this vibration changes the resistance of the cantilever connecting portion to cause a potential change. The detection signal generated in this way passes through the high-pass filter, is amplified by 6 times by the OP amplifier 23, and is input to the FM detection module 18. The FM detection module 18 compares the input signal and the detection signal, and measures changes in frequency and amplitude.

  Note that the sample stage 11 on which the sample 13 is placed is connected to the ground via a 10 MΩ resistor. Thereby, charging and discharging from the probe can be prevented.

  The measurement sample of this example is a carbon thin film mesh. The carbon thin film was dotted with holes of several micrometers, and such a carbon thin film was fixed to a copper frame.

  FIGS. 4A to 4D are diagrams for explaining the observation results. FIG. 4A is a diagram in which the tip of the tungsten probe is opposed to the upper side of the carbon mesh and the tip of the self-sensing cantilever is located below. It is a SEM photograph which shows a mode that it is opposing. The measurement region of the carbon mesh by the non-contact scanning probe microscope of the present invention is a region indicated by a square in the SEM photograph of FIG.

  FIG. 4C shows a mapping image (frequency image) obtained by extracting frequency change information (frequency signal) included in the detection signal when the region is measured by the non-contact scanning probe microscope having the above-described configuration. The outline of the carbon mesh is observed very clearly. On the other hand, in the SEM image of the same region, the outline of the carbon mesh is blurred, and the superiority of the image obtained by the microscope of the present invention can be confirmed.

  The measurement sample of this example is obtained by attaching a gold colloid solution having a diameter of approximately 30 nm to a carbon thin film.

  FIGS. 5A to 5C are diagrams for explaining the observation results, and FIG. 5A is an image obtained by observing the gold colloid adhesion site on the carbon film with an SEM, and is a cloud shape in a white square. Is the gold colloid deposition site.

  FIG. 5 shows a mapping image (amplitude image) obtained by extracting (amplitude signal) amplitude change information contained in the detection signal when the gold colloid adhesion site is measured by the non-contact scanning probe microscope having the above-described configuration. FIG. 5B shows the same region observed by SEM. Although the shapes of the gold particles that can be recognized from both images are similar, the amplitude image of FIG. 5B is excellent in terms of contrast and resolution.

  The measurement sample of this example is an example of a biological sample, in which T4 phage which is a kind of virus is attached on a carbon thin film. Note that no treatment such as osmium staining performed during normal SEM observation is performed.

  FIGS. 6A and 6B are diagrams for explaining the measurement results by the non-contact scanning probe microscope having the above-described configuration, and FIG. 6A shows the amplitude change information of the T4 phage on the carbon thin film. This is a result of imaging based on the image, and it can be confirmed that match-stick-shaped T4 phages are scattered.

  T4 phage is present in the black square shown in FIG. 6 (A), and an enlarged view of this is FIG. 6 (B). The right side of FIG. 6B schematically shows T4 phage that can be confirmed from each image, and it can be confirmed that virus observation is possible without any treatment such as staining.

  The non-contact scanning probe microscope according to the present embodiment has a configuration capable of observing a three-dimensional structure inside an observation sample such as a living organism.

  FIG. 7 is a block diagram for explaining a configuration example of the non-contact scanning probe microscope of the present embodiment. Also in the example shown in this figure, the probe (tip is sharply polished) of the non-contact scanning probe microscope. A tungsten probe) is provided in a scanning electron microscope (SEM), which is a device that can visually recognize the positional relationship between the tip of the probe and the sample, and the sample can be measured while observing the SEM. Has been.

  For convenience, a microscope based on a signal detection technique based on a so-called “FM detection method” in which the degree of interaction between the tips of both probes (the first probe 14 and the second probe 15) is obtained from a change in the resonance frequency of the cantilever. This embodiment will be described as follows, but the adoption of the FM detection method itself is not an essential condition for the present invention, as described above.

The difference between the configuration of the non-contact scanning probe microscope of the present embodiment and the configuration shown in FIG. 1 is that the position (x ₁ , y ₁ ) of the distal end portion of the first probe 14 in the XY plane is different from the second configuration. A manipulator control device 24 for enabling the position (x ₂ , y ₂ ) in the XY plane of the tip of the probe 15 to be independently controlled, and for three-dimensional analysis for imaging the three-dimensional structure of the observation sample The PC 25 is provided.

In the configuration shown in FIG. 7, the positions ((x ₁ , y ₁ ) and (x ₂ , y ₂ )) in the XY plane of the tip portions of the first and second probes (14, 15) are independent. Since control is possible, when the positions of the tips of the two probes in the XY plane are different, the positional relationship between these tips can be inclined (angle θ ≠ 0) with respect to the Z axis.

  In the embodiment shown in FIG. 1, the straight line connecting the tips of both probes is the Z-axis direction (θ = 0), and the detection signal processed by the control PC 20 is associated with the measurement position (x, y). there were. On the other hand, in the configuration shown in FIG. 7, measurement can be performed in a state where θ ≠ 0 is an angle formed by the straight line connecting the tip portions of both probes with the Z axis, and therefore the measurement angle θ is included as information. The detection signal associated with the measurement position (x, y, θ) is processed by the control PC 20.

  When mapping measurement is executed under the condition of θ ≠ 0, image information (mapping image) corresponding to the sample 13 observed from an oblique direction is obtained, and a plurality of θ values (measurement angles) are obtained. If the mapping images are collected, the three-dimensional structure of the sample 13 can be imaged by the PC 25 for three-dimensional analysis. As a signal processing method for obtaining an image showing such a three-dimensional structure, a back projection method or the like often used for CT or the like can be used.

FIG. 8 is a diagram for conceptually explaining a procedure for imaging the three-dimensional structure of the sample 13. The position (x ₁ , y ₁ ) of the tip of the tungsten probe 14 in the XY plane is fixed, The position (x ₂ , y ₂ ) in the xy plane of the tip of the cantilever 15 is changed.

  The left figure of FIG. 8 is a state in which the position of the tip of the cantilever 15 is made coincident with the position of the tip of the tungsten probe 14 (measurement position (x, y, θ = ± 0)), and the center view of FIG. FIG. 8 shows the position of the tip of the cantilever 15 in a state where the position of the part is positioned on the left side of the tip of the tungsten probe 14 (measurement position (x, y, θ = −30 °)). The state (measurement position (x, y, θ = + 30 °)) positioned on the right side with respect to the position of the tip of the tungsten probe 14 is shown.

  When measurement is performed under the relative positional relationship between these probes and the measurement is executed (mapped) in a predetermined observation region, a vertical image, a left tilt image, and a right tilt image can be obtained. Based on these mapping images, the three-dimensional structure of the sample 13 is reconstructed as an image. The PC 25 for three-dimensional analysis can also have a function of automatically changing a plurality of relative positional relationships between the tip portions of the first and second probes within a range designated in advance.

  9A to 9C are views showing the positional relationship between the probe 14 and 15 and the sample 13 from the side. FIG. 9A shows the position of the tip of the cantilever 15 and the tip of the tungsten probe 14. FIG. 9B shows a state where the tip of the cantilever 15 is positioned on the left side of the tip of the tungsten probe 14 (θ = −30 °). FIG. 9C shows a state where the tip position of the cantilever 15 is positioned on the right side with respect to the tip position of the tungsten probe 14 (θ = + 30 °). This corresponds to the probe arrangement in the central view and the right view.

  FIG. 8 shows an example in which the position of the tungsten probe 14 is fixed and the cantilever 15 is moved relative to the tungsten probe 14. On the contrary, the position of the cantilever 15 is fixed. The tungsten probe 14 may be moved relative to the cantilever 15. It is also possible to automatically determine the movement of the tungsten probe 14 and the cantilever 15 by specifying the observation region (measurement range) and the number of images to be acquired in advance.

  FIGS. 10A to 10F are diagrams for explaining changes in the detected image when bacteria as observation samples are attached to the carbon thin film and the position of the tungsten probe is changed.

  FIG. 10A is a scanning electron microscope image (SEM image) at the time of observing a bacterial sample. In this SEM image, a tungsten probe (the front end portion) located above the carbon film can be confirmed. The tip of the cantilever is positioned directly below the tip of the tungsten probe (θ = ± 0 °) below the carbon film.

  FIG. 10B shows a mapping image acquired with this probe position relationship, and FIG. 10C schematically shows the mapping image of FIG. 10B. From FIG. 10C, it can be seen that two rod-shaped bacteria are arranged in a V shape.

  FIG. 10D is an SEM image in a state where the tungsten probe is moved relative to the cantilever (3 μm) while the position of the cantilever is fixed. Note that the white circle in FIG. 10D indicates the position of the tip of the tungsten probe in FIG.

  FIG. 10E shows a mapping image acquired based on the probe positional relationship, and FIG. 10F schematically shows the mapping image of FIG. 10E.

  Compared to the image obtained with the probe positional relationship shown in FIG. 10A, the image of the lower bacteria is slightly shorter, and it can be read that the thickness of the upper bacteria is increased.

  Such a difference corresponds to a change in the relative positional relationship of the probes. That is, by changing the relative positional relationship of the probes, the measurement angle changes and the obtained image also changes. As described above, it is possible to reconstruct the three-dimensional structure by acquiring images under the relative positional relationship of various probes.

  In FIG. 7 as well, the probe (and manipulator and sample stage) of the non-contact scanning probe microscope is provided in the SEM, and the manipulator is operated while observing the sample with the SEM. Although the mode of movement to the position is illustrated, an optical microscope can be used instead of the SEM.

  Further, it is possible to adopt a mode in which the position is automatically determined by measuring the distance between the probes and the distance between the sample and the probe with a sensor.

  Although the microscope of the present invention has been specifically described above, the present invention is not limited to the above-described microscope. For example, the PC for three-dimensional analysis may have a function of defining a reference point in a plurality of obtained mapping images and correcting a positional deviation between the mapping images from the reference point to image a three-dimensional structure. it can. Further, the interprobe signal may be due to a magnetic force in addition to the coulomb force, or a tunnel current flowing between the probes can be used as the interprobe signal. Furthermore, there may be a mode in which a carbon nanotube or the like is provided at the tip of the probe and the field emission current is used as an interlobe signal.

  The present invention measures the internal structure of extremely fragile samples such as biological materials and organic materials with high resolution without damage, and further provides non-contact scanning that provides a level of contrast and SN ratio that can clearly analyze individual proteins. A probe microscope is provided.

It is a block diagram for demonstrating the structural example of the non-contact-type scanning probe microscope of this invention. It is the figure which expanded and showed the vicinity area | region of the probe and the sample. It is a block diagram for demonstrating the detailed circuit structure from the FM detection module used in the Example to a probe. It is a figure which shows the result of having measured the carbon film mesh with the non-contact type scanning probe microscope of this invention. It is a figure which shows the result of having measured the adhesion site | part of the gold colloid by the non-contact type scanning probe microscope of this invention. It is a figure which shows the result of having measured the sample which adhered T4 phage to the carbon thin film with the non-contact-type scanning probe microscope of this invention. It is a block diagram for demonstrating the other structural example of the non-contact-type scanning probe microscope of this invention. It is a figure for demonstrating notionally the procedure which images the three-dimensional structure of a sample. It is the figure which showed the positional relationship of a probe and a sample from the side. It is a figure for demonstrating the change of a detection image when the bacteria which are observation samples are made to adhere on a carbon thin film, and the position of a tungsten probe is changed.

Explanation of symbols

11 Sample stage (XY stage)
DESCRIPTION OF SYMBOLS 12 Support film 13 Sample 14 1st probe 15 2nd probe 16, 17 Manipulator 18 FM detection module 19 DC voltage source 20 Control PC
21 High voltage amplifier module 22, 23 OP amplifier 24 Manipulator control device 25 PC for 3D analysis

Claims (10)

A first probe disposed in a non-contact manner opposite one of the front and back of the sample;
A second probe disposed in a non-contact manner opposite to the other of the front and back of the sample, wherein the tip of the probe is disposed in the vicinity of the tip of the first probe;
A mapping image is input based on at least one of amplitude change information and frequency change information of a signal input from the tip of the first probe and modulated by the sample and detected by the tip of the second probe. A non-contact type scanning probe microscope, comprising: a signal processing unit to be formed.   2. The non-contact type according to claim 1, wherein the second probe is a cantilever, and a signal detected by a tip portion of the second probe is a change in amplitude or resonance frequency of the cantilever. Scanning probe microscope.   3. The non-contact scanning probe microscope according to claim 2, wherein an AC signal having the same frequency as the mechanical resonance frequency of the cantilever is input from the tip of the first probe. 4. The non-contact scanning probe microscope according to claim 1, wherein the signal detected by the tip of the second probe is a Coulomb force given by the following equation.
Here, Q ₁ : charge at the tip of the first probe, Q ₂ : charge at the tip of the second probe, d: distance between the first and second probe tips, ε (x, y): The relative dielectric constant at the measurement position (x, y) of the sample, ε ₀ : the dielectric constant of the measurement environment.   The first probe is a tungsten probe, the second probe is a self-sensing cantilever, and the tip of each of the first and second probes is sharp so that the radius of curvature is 10 nm or less. The non-contact scanning probe microscope according to any one of claims 1 to 4, wherein the non-contact scanning probe microscope is formed. The signal processing unit
The position (x ₁ , y ₁ ) of the tip of the first probe in the XY plane and the position (x ₂ , y ₂ ) of the tip of the second probe in the XY plane can be controlled independently. Probe position control means,
Image processing means for imaging the three-dimensional structure of the sample based on a plurality of mapping images obtained under a plurality of relative positional relationships between the tip portions of the first and second probes. ,
The non-contact scanning probe microscope according to any one of claims 1 to 5.   7. The non-transitory device according to claim 6, wherein the signal processing unit includes means for automatically changing a plurality of relative positional relationships between the tip portions of the first and second probes within a predetermined range. Contact scanning probe microscope.   The non-contact according to claim 6 or 7, wherein the image processing means defines a reference point in the plurality of mapping images, and corrects a positional shift between the mapping images from the reference point to image a three-dimensional structure. Scanning probe microscope.   The said 1st and 2nd probe is provided in the apparatus which can visually recognize the positional relationship of the front-end | tip part of this probe and a sample, The any one of Claim 1 thru | or 8 characterized by the above-mentioned. Non-contact scanning probe microscope.   The non-contact scanning probe microscope according to claim 9, wherein the device is a scanning electron microscope.