WO2013051094A1 - Microscope-sonde à balayage - Google Patents

Microscope-sonde à balayage Download PDF

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Publication number
WO2013051094A1
WO2013051094A1 PCT/JP2011/072812 JP2011072812W WO2013051094A1 WO 2013051094 A1 WO2013051094 A1 WO 2013051094A1 JP 2011072812 W JP2011072812 W JP 2011072812W WO 2013051094 A1 WO2013051094 A1 WO 2013051094A1
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Prior art keywords
probe
sample
scanning
probe microscope
scanning probe
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PCT/JP2011/072812
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English (en)
Japanese (ja)
Inventor
富博 橋詰
誠嗣 平家
山本　剛
小泉　英明
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株式会社日立製作所
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Priority to JP2013537300A priority Critical patent/JP5820886B2/ja
Priority to PCT/JP2011/072812 priority patent/WO2013051094A1/fr
Publication of WO2013051094A1 publication Critical patent/WO2013051094A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q30/00—Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
    • G01Q30/02—Non-SPM analysing devices, e.g. SEM [Scanning Electron Microscope], spectrometer or optical microscope
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q30/00—Auxiliary means serving to assist or improve the scanning probe techniques or apparatus, e.g. display or data processing devices
    • G01Q30/08—Means for establishing or regulating a desired environmental condition within a sample chamber
    • G01Q30/12—Fluid environment
    • G01Q30/14—Liquid environment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/02—Multiple-type SPM, i.e. involving more than one SPM techniques
    • G01Q60/06—SNOM [Scanning Near-field Optical Microscopy] combined with AFM [Atomic Force Microscopy]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
    • G01Q60/30—Scanning potential microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]
    • G01N2021/656—Raman microprobe

Definitions

  • the present invention enhances physical information such as the orientation distribution of water molecules at the sample-culture solution interface in the culture solution, and the surface irregularities, potential distribution, composition distribution of molecules and proteins, and the array structure in the culture solution.
  • the present invention relates to a scanning probe microscope for measuring with spatial resolution.
  • Hydration phenomena such as biomolecules, biological tissues, and biological substrate materials are important when measuring, evaluating, and controlling biological reactions such as cell adhesion to the biological substrate material in the culture medium and subsequent extension / differentiation.
  • the hydration structure is formed from the interaction between the sample surface and water molecules and the interaction including hydrogen bonds between the water molecules at the sample-culture solution interface in the culture solution containing water as a main component. 3D structure is shown. It is considered that so-called biocompatibility typified by adhesion between the inner wall of an artificial blood vessel and erythrocytes is closely related to this hydration structure (for example, Non-Patent Document 1).
  • unevenness of the sample surface in the culture medium, potential distribution, composition distribution and arrangement structure of molecules and proteins, etc. are particularly important for biological reactions such as biomolecules, biological tissues, and biological substrate materials in the culture medium. It is a characteristic.
  • optical microscopes, Raman spectroscopy, second harmonic method, sum frequency spectroscopy are methods for observing and measuring the sample-culture solution interface such as biomolecules, biological tissues, and biological substrate materials in the culture solution.
  • Nonlinear optical microscopes such as are used.
  • sum frequency spectroscopy can measure the arrangement of water molecules related to the hydration structure at the sample-culture liquid interface.
  • a non-linear optical microscope for example, in Patent Document 1, the interaction between a probe and a target is expressed by water selectivity near the interface, solvent molecules, or surface selectivity by second harmonic light or sum frequency light by a labeling substance.
  • Non-linear optical methods are disclosed. However, the spatial resolution in these optical microscopes and nonlinear optical microscopes is greater than 100 nm, typically about 1 ⁇ m.
  • the scanning probe microscope is based on an atomic force microscope (AFM).
  • the scanning Kelvin probe microscope which is an example of a scanning probe microscope, scans the probe surface on the sample surface while detecting the electrostatic field force acting between the cantilever with the conductive probe and the sample as the deflection of the cantilever. This is a technique for mapping the electrostatic field force distribution.
  • the probe also includes interatomic forces and the like, and the electrostatic field forces need to be separated from other interactions. For this purpose, first, the cantilever is vibrated, and the distance between the probe and the sample is adjusted so as to keep the vibration amplitude reduced by the atomic force acting when the probe and the sample are in contact with each other.
  • the position of the sample surface in the height direction is determined, and the electrostatic field force, which is a long-distance force, is detected from the phase change of the cantilever vibration in a state where the probe is separated from the sample surface by a certain distance therefrom (for example, Patent Document 2).
  • the probe is sometimes called a probe.
  • a scanning probe microscope can be expected to have a spatial resolution of about 1 nm for unevenness measurement, an electrostatic field, and a spatial resolution of about 10 nm for optical measurement.
  • the interaction region between the probe and the sample is limited to the diameter of the tip of the probe, it is generally difficult to realize a scanning probe microscope that uses a physical quantity with a weak signal like the nonlinear optical method. It is.
  • Patent Document 3 a uniform metal that efficiently induces surface-enhanced Raman scattering in a near-field microscope that inserts a probe into an evanescent field generated on the surface of a sample and scatters the evanescent field at the probe tip to detect scattered light.
  • a near-field microscope probe in which particles are reproducibly coated is disclosed.
  • Non-Patent Document 2 light can be confined in the nanospace of the tip by irradiating light to a metal probe having a tip of nanometer diameter, and the molecule can be illuminated from the molecule by illuminating the molecule as a nano light source. It has been shown that Raman scattered light can be detected with nano-spatial resolution. In particular, the localized plasmon polariton, which is a resonance phenomenon, is excited at the tip of the probe, so that the electric field intensity of light is enhanced and the scattering cross section of Raman scattering is effectively increased, thereby compensating for weak scattering ( It has been shown that a 15 nm spatial resolution is achieved.
  • a laser beam is focused on a sample to generate Raman scattered light, and the Raman scattered light is enhanced and scattered by a probe that is close or in contact with the sample, and a Raman spectrum is detected from the scattered light scattered.
  • An ultraviolet near-field optical microscope using a probe-enhanced Raman detection method is disclosed.
  • the excitation laser light is ultraviolet / deep ultraviolet laser light
  • the material at the tip of the probe is a metal having a dielectric constant of ⁇ 2 or less at the wavelength of the excitation laser.
  • vacuum evaporation is performed on the surface of a silicon probe. Therefore, an aluminum thin film having a thickness of about 25 nm and a metal particle diameter of 10 to 20 nm is preferred.
  • the object of the present invention is to measure the arrangement structure of water molecules related to the hydration structure at the sample-culture solution interface such as biomolecules, biological tissues, and biological substrate materials in the culture solution with high spatial resolution.
  • An object of the present invention is to provide a scanning probe microscope that measures the unevenness of the sample surface, the potential distribution, the composition distribution of molecules and proteins, the arrangement structure, and the like with high spatial resolution.
  • the scanning probe microscope of the present invention includes a probe, a sample holder on which a sample is placed, a vibrator for displacing the position of the probe, and a pulsed laser beam incident on a region of the sample measured by the probe.
  • the control device periodically displaces the probe position of the vibrator and maximizes the output light intensity depending on the distance between the probe and the sample.
  • the probe enhancement detection efficiency is optimized.
  • the scanning probe microscope of the present invention includes a probe, a sample holder on which a sample is placed, a vibrator that displaces the position of the probe, a detection unit that detects a force applied to the probe,
  • a scanning probe microscope comprising a probe power supply for applying an AC voltage and a DC voltage to the probe, a scanning mechanism for moving the sample holder, and a control device, and measuring electrostatic field force distribution on the surface of the sample
  • the control device periodically displaces the probe position of the vibrator and controls the timing of the AC voltage applied to the probe position and the probe to control the electrostatic field distribution measurement in the culture medium. It is characterized by optimizing.
  • the present invention it is possible to measure the interaction between water and molecules at the interface between a biomolecule, a biological tissue, a biological substrate material, and the like and the culture solution in the culture solution with high spatial resolution.
  • a probe is arranged in near-field light (evanescent light) generated on a sample surface, and the electric field intensity of light near the sample surface is determined by the near-field light from the probe and the near-field light from the sample.
  • near-field light evanescent light
  • SHG sum frequency spectroscopy
  • SHG second harmonic method
  • other linear / nonlinear optical spectroscopy is used.
  • the probe enhancement effect strongly depends on the probe-sample distance
  • the probe enhancement effect can be optimized by measuring the detected light intensity that depends on the probe-sample distance. It is based on the new knowledge that there is.
  • the electrostatic force acting on the probe strongly depends on the distance between the probe and the sample. This is based on the new finding that by measuring the electrostatic field force intensity depending on the probe-sample distance, the sensitivity of the electrostatic field distribution measurement can be optimized even in the culture solution.
  • FIG. 1 is a schematic configuration diagram of a scanning probe microscope according to Embodiment 1 of the present invention.
  • the probe 1 is installed on the vibrator 2, and the relative position to the sample 3 is controlled by the vibrator 2.
  • the probe 1 is selected from a material that amplifies and concentrates near-field light intensity near the tip when placed in incident light.
  • Raman scattering such as Raman spectroscopy or sum frequency spectroscopy
  • metals such as gold, silver, copper, and aluminum, and compounds thereof, which can effectively use surface-enhanced Raman scattering, are used. .
  • a probe obtained by depositing a gold thin film with a thickness of 1 to 20 nm on a silicon probe is used as an effective probe candidate.
  • the vibrator 2 vibrates mainly in the vertical direction of the sample 3, the distance between the probe 1 and the sample 3 is controlled to 300 nm or less, and the natural frequency of the vibrator 2 is 200 kHz to 2 MHz is used.
  • a quartz crystal vibrator that expands and contracts in the longitudinal direction is used as the vibrator 2.
  • a tuning fork type crystal vibrator that is generally used in a scanning probe microscope such as an atomic force microscope, or vibration caused by a piezo element.
  • a vibrator in which a piezo element is arranged on a child or a cantilever can be used.
  • the probe 1 is vibrated in a direction perpendicular to the surface of the sample 3 by the vibrator 2 at a frequency near the natural frequency of the vibrator 2 (within about ⁇ 1% of the natural frequency). Due to the interaction (force) between the probe 1 and the sample 3, there is a phase difference between the voltage applied to the vibrator 2 and the actual vibration amplitude of the vibrator 2.
  • the phase difference in this embodiment From the phase difference between the AC voltage applied to the vibrator 2 and the current flowing into the vibrator 2, the interaction (force) between the probe and the sample is known, and the distance between the probe and the sample is known.
  • the scanning mechanism 31 scans the relative position between the sample 3 and the probe 1 in the direction perpendicular to the sample and the plane direction of the sample.
  • a force microscope A force microscope
  • the distance between the probe 1 and the sample 3 is generally close to a distance of 0 nm (contact) to 100 nm at the closest position, but the probe 1 can be embedded into the sample 3.
  • the scanning mechanism 31 scans the relative position between the sample 3 and the probe 1 in the vertical direction and the plane direction of the sample while reducing the vibration amplitude of the vibrator 2 by a certain amount.
  • the distance between the needle 1 and the sample 3 can be set to 0 nm at the closest position (tapping mode AFM).
  • the probe 1 is connected to a probe power source 5 by wiring 4, and an AC voltage and a DC voltage can be applied between the probe 1 and the sample 3.
  • the surface-treated polycarbonate is used as the sample 3, and the voltage applied between the probe 1 and the sample 3 is not used.
  • the sample holder 11 includes a culture solution inlet 12 and a culture solution recovery port 13 and can hold or replace the culture solution 14. Instead of the culture solution 14, water or a solvent can be used.
  • a pulsed laser beam or a plurality of pulsed laser beams that are input in synchronism are input near the region of the sample 3 where the probe 1 is close, and the intensity of the output beam 24 is measured by the detector 25 with a filter.
  • a first pulse laser beam 22 that is a green pulse laser beam having a wavelength of 532 nm and a second pulse laser beam 23 that is a variable infrared pulse laser beam having a wavelength of 2.3 to 10 microns are provided.
  • Input synchronously.
  • the output light 24 is input to the detector 25 with a filter, and the intensity of the sum (frequency) of the frequency of the first pulse laser light 22 and the frequency of the second pulse laser light 23 is measured.
  • sum frequency spectroscopy can be performed.
  • the peak of the wave number 3200 Kaiser and the peak of the wave number 3400 Kaiser are compared, and the ratio of the orientation of the water molecules asymmetrically bonded to the tetrahedrally coordinated water molecules at the interface between the polycarbonate and the culture solution 14 is calculated. Discuss.
  • the probe When the AFM is configured and the probe 1 and the sample 3 are sufficiently close to each other, the probe is caused by the localized plasmon polariton being excited at the tip of the probe 1 and the electric field intensity of light being enhanced. Due to the enhancement effect, the intensity of the output light 24 of the sum frequency is dramatically enhanced (probe enhanced sum frequency spectroscopy). Furthermore, by measuring the intensity of the sum frequency output light 24 at a specific wave number while scanning a part of the surface of the sample 3 with the probe 1, the orientation of water molecules at the interface between the sample 3 and the culture solution 14 is measured. The spatial distribution can be mapped with high spatial resolution (probe enhanced scanning sum frequency microscope). In this embodiment, the sum frequency output light 24 is enhanced 10,000 times by the probe enhancement effect, and the spatial resolution of the scanning sum frequency microscope is 10 nm.
  • the probe enhancement effect occurs when the distance between the probe 1 and the sample 3 is 20 nm or less.
  • the probe 1 is microvibrated at a distance of about 1 nm by the vibrator 2 to measure the distance between the probe 1 and the sample 3 (probe-sample distance).
  • the controller 26 controls the scanning mechanism 31 to measure the output of the detector 25 with the filter while changing the closest position of the probe 1 and the sample 3.
  • FIGS. 2A to 2C are plots showing the probe-sample distance dependence of the detector output measured by the scanning probe microscope of this example.
  • FIG. 2A shows a case where the distance between the probe 1 and the sample 3 is relatively long and there is no effect of enhancing the probe, and when the distance between the probe and the sample is large, the pulse laser beam blocked by the probe 1 is reduced.
  • the output light maximum 41 is shown under the geometric condition where the probe does not block the light.
  • FIG. 2B shows a case where the distance between the probe 1 and the sample 3 is sufficiently close and has a probe enhancement effect.
  • Pulse laser beam incident position, incident angle, output light angle, synchronization conditions of multiple pulse laser beams, probe 1 material and shape, vibrator 2 vibration frequency, amplitude, vibrator 2 vibration and pulse laser light may be set so as to optimize the output light maximum 42 due to the probe enhancement effect. Further, in FIG.
  • the vibration of the probe 1 and the repetition of the pulse laser beam are synchronized to optimize the probe enhancement effect.
  • the repetition frequency of the pulse laser beam is 1 / the frequency of the probe 1.
  • the case of 3 is shown.
  • the value of the phase difference is shown in FIGS. 2A and 2B. Setting by measurement is essentially important. Depending on the distance between the probe and the sample, the detector output changes as shown in FIGS.
  • the experiment parameters for changing the probe enhancement effect, such as the amplitude and the synchronization condition between the vibration of the vibrator 2 and the pulse laser beam, may be adjusted.
  • FIG. 3 is a schematic configuration diagram of a scanning probe microscope according to the second embodiment of the present invention. A description will be given centering on differences from the scanning probe microscope of the first embodiment.
  • the sample 3 is installed on the upper surface of the prism 21.
  • the sample holder 11 is made of a ring-shaped shape having no bottom or a material that has a very thin portion in contact with the prism 21 and that transmits pulse laser light well.
  • the sample 3 is limited to a material having a small thickness and a material that can transmit pulsed laser light well.
  • the pulsed laser light that is input substantially perpendicularly to the cylindrical surface of the prism 21 or the plurality of pulsed laser beams that are input in synchronization with each other are totally reflected on the upper surface of the prism or the sample surface and scattered as output light 24.
  • the intensity of the output light 24 is measured by the detector 25 with a filter.
  • a first pulse laser beam 22 that is a green pulse laser beam having a wavelength of 532 nm and a second pulse laser beam 23 that is a variable infrared pulse laser beam having a wavelength of 2.3 to 10 microns are provided. Input synchronously.
  • the output light 24 is input to the detector 25 with a filter, and the intensity of the sum (frequency) of the frequency of the first pulse laser light 22 and the frequency of the second pulse laser light 23 is measured.
  • sum frequency spectroscopy can be performed.
  • the peak of the wave number 3200 Kaiser and the peak of the wave number 3400 Kaiser are compared, and the ratio of the orientation of the water molecules asymmetrically bonded to the tetrahedrally coordinated water molecules at the interface between the polycarbonate and the culture solution 14 is calculated. taking measurement.
  • a probe-enhanced scanning second harmonic microscope and a probe-enhanced scanning optical probe microscope with other linear / nonlinear optical characteristics are disclosed as an embodiment of the scanning probe microscope. This embodiment will be described with reference to FIG. 1 as in the first embodiment.
  • the first pulsed laser beam 22 that is an infrared pulsed laser beam having a wavelength of 1064 nm is input in the vicinity of the region of the sample 3 to which the probe 1 is close.
  • the output light 24 is input to the detector 25 with a filter, and the light intensity at a frequency twice the frequency of the first pulse laser light 22 is measured.
  • a scanning second harmonic microscope can be constructed, and the nerve activity intensity of the nerve cell is determined. Can be mapped.
  • the probe enhancement effect can be optimized as in the first embodiment, and a probe enhanced scanning second harmonic microscope can be configured.
  • the first pulse laser beam 22 that is a green pulse laser beam having a wavelength of 532 nm is input in the vicinity of the region of the sample 3 to which the probe 1 is close.
  • the output light 24 is input to the detector 25 with a filter, and the light intensity of the Raman scattered light is measured.
  • cultured hepatocytes as the sample 3 and measuring Raman scattering while measuring the unevenness of hepatocytes by AFM, it is possible to map the composition distribution of molecules and proteins in the hepatocytes.
  • the probe enhancement effect can be optimized similarly to the first embodiment, and a probe enhancement scanning Raman microscope can be configured.
  • the probe-enhanced scanning CARS microscope in this example uses coherent anti-Stokes Raman scattering (CARS).
  • a first pulsed laser beam 22 (angular frequency ⁇ 1) having a different angular frequency and a second pulsed laser beam 23 (angular frequency ⁇ 2) are input in synchronism with each other in the vicinity of the region of the sample 3 close to the probe 1.
  • the output light 24 is input to the detector 25 with a filter, and the light intensity of the CARS light is measured.
  • AFM By measuring the light intensity of the CARS light while measuring the unevenness of the sample 3 by AFM, it is possible to map the composition distribution of the sample 3 such as molecules and proteins.
  • the probe enhancement effect can be optimized as in the first embodiment, and a probe enhancement scanning CARS microscope can be configured.
  • a scanning probe microscope (scanning Kelvin probe microscope) that measures the electrostatic field force distribution on the surface of the sample is disclosed as one form of the scanning probe microscope.
  • FIG. 4 which is the same as that in Embodiment 1
  • FIG. 5 which shows an example of an electrode portion.
  • FIG. 5 is a schematic configuration diagram illustrating an example of an electrode portion in the scanning probe microscope according to the fourth embodiment of the present invention.
  • a bipotentiostat 51 controlled by the control device 26 controls the probe electrode 52, the sample electrode 53, the working electrode 54, and the reference electrode 55.
  • the potential of the culture solution 14 is measured by the reference electrode 55, the voltage of the probe 1 with respect to the culture solution 14 is applied by the probe electrode 52, and the voltage of the sample 3 with respect to the culture solution 14 is applied by the sample electrode 53. .
  • the current flowing between the culture solution 14 and the reference electrode 55 is almost zero.
  • an electric current between the working electrode 54 and the sample electrode 53 is used.
  • a voltage is applied between the probe 1 and the sample 3
  • a voltage is applied between the probe electrode 52 and the sample electrode 53.
  • the voltage and current between the probe electrode 52 and the sample electrode 53 are used as the applied voltage and the tunnel current.
  • the charge injection electrode 56 is used.
  • the vibrator 2 is vibrated at a frequency near the natural frequency (within about ⁇ 1% of the natural frequency), and the probe 1 is vibrated in a direction perpendicular to the surface of the sample 3.
  • the interaction (force) between the probe 1 and the sample 3 can be found from the phase difference between the AC voltage applied to the vibrator 2 and the current flowing into the vibrator 2, and the distance between the probe and the sample can be found.
  • a voltage signal obtained by adding an AC voltage and a DC voltage is applied between the probe 1 and the sample 3.
  • an electrostatic force F corresponding to the difference between the voltage signal and the work function of the surfaces of the probe 1 and the sample 3 is applied between the sample 3 and the probe 1.
  • the amplitude of the AC voltage is a preset value, but the value of the DC voltage is determined as follows.
  • the interaction (force) (force signal) between the probe 1 and the sample 3 is measured by the vibrator 2.
  • the intensity of the same frequency component as the AC voltage of the force signal is detected by a lock-in amplifier.
  • the electrostatic force F applied to the probe 1 is F ⁇ V 2 / z 2 . Since the signal output from the lock-in amplifier is differentiated with respect to the voltage V of the electrostatic force F, it becomes dF / dV ⁇ V / z 2 , and is a value proportional to the potential difference if the distance z and the dielectric constant ⁇ are constant. . Therefore, the potential difference between the probe 1 and the sample 3 is always kept at zero by adjusting the DC voltage so that the output signal from the lock-in amplifier becomes zero. Thereby, the electrostatic force F applied to the probe 1 can be made zero regardless of the surface potential of the sample 3. That is, the potential difference between the probe 1 and the sample 3 can be measured by a DC voltage adjusted so that the electrostatic force F is zero.
  • the force signal f is input to the second lock-in amplifier, and the intensity of the double frequency component of the force signal f is detected using a double frequency signal synchronized with the alternating voltage of the voltage signal as a reference signal. Since the signal outputted from the lock-in amplifier is differentiated twice with respect to the voltage V of the force signal f, it becomes d 2 f / dV 2 ⁇ / z 2 , and if the dielectric constant ⁇ is constant, the probe 1 and the sample 3 It becomes a value inversely proportional to the square of the distance z between.
  • the scanning mechanism 31 scans the relative position between the sample 3 and the probe 1 in the direction perpendicular to the sample and in the plane direction of the sample.
  • the distance between the sample 3 and the sample 3 can be kept constant, and an atomic force microscope (AFM) which is one type of scanning probe microscope can be configured.
  • AFM atomic force microscope
  • the control device 26 can control the AC voltage timing of the voltage signal with respect to the distance between the sample 3 and the probe 1 to optimize the sensitivity of the electrostatic field distribution measurement in the culture solution. It was. It was also found that the sensitivity of electrostatic field distribution measurement can be optimized with respect to the frequency of the AC voltage. By these, the scanning probe microscope characterized by measuring the electrostatic field distribution of the sample 3 with high spatial resolution can be configured.
  • the nerve cells cultured as the sample 3 are placed in the culture solution 14.
  • a probe 1 installed on the vibrator 2 is provided so as to face the surface of the sample 3.
  • the probe 1 is vibrated in a direction perpendicular to the surface of the sample 3 by the vibrator 2.
  • the sample 3 is fixed on the scanning mechanism 31 via the sample holder 11 and can be moved in the three-dimensional azimuth direction with respect to the probe 1.
  • the probe 1 is vibrated in the direction perpendicular to the surface of the sample 1 by the vibrator 2 at a frequency near the natural frequency of the vibrator 2 (within about ⁇ 1% of the natural frequency).
  • the scanning mechanism 31 scans the relative position between the sample 3 and the probe 1 in the direction perpendicular to the sample and the plane direction of the sample.
  • a force microscope can be constructed, and irregularities on the sample surface can be measured.
  • the nerve signal is a voltage pulse generated in the nerve cell by injecting a charge into the nerve cell by the charge injection electrode 56.
  • a predetermined charge is injected into the sample 3 to apply a voltage pulse to the nerve cell.
  • the magnitude of the voltage pulse is about 50 ⁇ V to 100 mV.
  • the probe 3 is brought into contact with or close to a desired position of the sample 3, and this voltage pulse is detected by the scanning probe microscope of the fifth embodiment.

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Abstract

Selon la présente invention, afin de mesurer la structure d'agencement de molécules d'eau à une interface de fluide d'échantillon de culture dans un fluide de culture, l'irrégularité de la surface d'un échantillon dans le fluide de culture, la distribution de potentiel et les distributions de composition, les structures d'agencement et similaires pour des molécules, protéine et similaire, avec une résolution spatiale élevée, un microscope-sonde à balayage comporte une sonde (1), un porte-échantillon (11) dans lequel un échantillon (3) est monté, un oscillateur (2) qui déplace la position de la sonde, des faisceaux lasers à impulsion (22, 23) qui sont incidents sur une région mesurée par la sonde dans l'échantillon, un détecteur equipé d'un filtre (25) qui mesure l'intensité de la lumière de sortie (24) délivrée en sortie depuis l'échantillon par spectroscopie d'énergie, un mécanisme de balayage (31) qui déplace le porte-échantillon et un dispositif de commande (26), et mesure la forme de la surface de l'échantillon et des caractéristiques optiques linéaires/non linéaires. L'efficacité de détection améliorée de la sonde est optimisée par déplacement périodique de la position de la sonde de l'oscillateur et par le fait de rendre maximale l'intensité de lumière de sortie dépendant de la distance entre la sonde et l'échantillon par le dispositif de commande.
PCT/JP2011/072812 2011-10-03 2011-10-03 Microscope-sonde à balayage WO2013051094A1 (fr)

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JP2013537300A JP5820886B2 (ja) 2011-10-03 2011-10-03 走査プローブ顕微鏡
PCT/JP2011/072812 WO2013051094A1 (fr) 2011-10-03 2011-10-03 Microscope-sonde à balayage

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WO2016185518A1 (fr) * 2015-05-15 2016-11-24 オリンパス株式会社 Procédé d'acquisition d'informations pour microscope à force atomique
JPWO2016185518A1 (ja) * 2015-05-15 2018-03-01 オリンパス株式会社 原子間力顕微鏡の情報取得方法
WO2017012927A1 (fr) * 2015-07-22 2017-01-26 Vmicro Sonde pour microscopie a force atomique a faible encombrement et microscope a force atomique comprenant une telle sonde
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US10495665B2 (en) 2016-09-19 2019-12-03 Zyvex Labs, Llc Methods, devices and systems for scanning tunneling microscopy control system design
CN112513648A (zh) * 2018-05-25 2021-03-16 分子前景公司 用于针对样本改善光透导力的使用传感器分子的扫描探针显微镜
KR102619577B1 (ko) * 2022-12-22 2023-12-29 포항공과대학교 산학협력단 탐침 증강 현미경의 분석 방법 및 탐침 증강 현미경

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