WO2016103338A1 - 走査プローブ顕微鏡及びその試料ホルダ - Google Patents
走査プローブ顕微鏡及びその試料ホルダ Download PDFInfo
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- WO2016103338A1 WO2016103338A1 PCT/JP2014/084032 JP2014084032W WO2016103338A1 WO 2016103338 A1 WO2016103338 A1 WO 2016103338A1 JP 2014084032 W JP2014084032 W JP 2014084032W WO 2016103338 A1 WO2016103338 A1 WO 2016103338A1
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- 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
- G01Q30/20—Sample handling devices or methods
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- 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
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- 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
- G01Q60/18—SNOM [Scanning Near-Field Optical Microscopy] or apparatus therefor, e.g. SNOM probes
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- 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
Definitions
- the present invention relates to a scanning probe microscope and its sample holder.
- Hydration phenomena such as biomolecules, biological tissues, and biological substrate materials are important when measuring, evaluating, or controlling cell adhesion to biological substrate materials and subsequent biological reactions (elongation, differentiation, etc.) in the culture medium.
- Hydration structure consists of (1) the interaction between the sample surface and water molecules at the interface between the sample and the culture medium in the culture medium containing water as the main component, and (2) the interaction including hydrogen bonds between the water molecules. 3 shows a three-dimensional structure formed from the action. So-called biocompatibility represented by adhesion between the inner wall of an artificial blood vessel and erythrocytes is considered to be closely related to this hydration structure.
- unevenness of the sample surface in the culture medium, potential distribution, composition distribution and arrangement structure of molecules and proteins, etc. are particularly important characteristics regarding biological reactions such as biomolecules, biological tissues, and biological substrate materials in the culture medium. It is.
- Raman spectroscopy, second-harmonic generation, sum frequency generation (SFG: Sum Frequency) is used to observe or measure the interface between a sample such as biomolecules, biological tissue, and biological substrate materials in the culture solution and the culture solution.
- a linear or non-linear optical microscope such as the Generation method is used.
- the sum frequency generation method measures the intensity of scattered light from a region with no spatial symmetry of the sample by the scattering of infrared incident light (Raman scattering) due to molecular vibrations of molecules contained in the sample and the nonlinear optical phenomenon of visible incident light. Therefore, in particular, the arrangement structure of water molecules related to the hydration structure at the interface between the sample and the culture solution can be measured.
- Non-linear optical microscopes include, for example, a non-linear optical method in which the interaction between a probe and a target is surface-selectively observed by water molecules near the interface, solvent molecules, or second harmonic light or sum frequency light from a labeling substance.
- the spatial resolution in these optical microscopes is larger than 100 nm, typically about 1 ⁇ m.
- the scanning probe microscope is based on an atomic force microscope (AFM).
- An example of a scanning probe microscope is a scanning Kelvin probe microscope.
- the scanning Kelvin probe microscope is a method of mapping the electrostatic force distribution by scanning the probe surface on the sample surface while detecting the electrostatic force acting between the cantilever with the conductive probe and the sample as deflection of the cantilever. It is.
- interstitial force is added to the probe, and it is necessary to separate the electrostatic force 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 at the time of contact between the probe and the sample constant.
- the position of the sample surface in the height direction is determined, and the electrostatic force, which is a long-distance force, is detected from the phase change of the cantilever vibration while the probe is separated from the sample surface by a certain distance therefrom.
- the probe is sometimes called a probe.
- a scanning probe microscope can expect a spatial resolution of about 1 nm for unevenness measurement and a spatial resolution of about 10 nm for electrostatic force and optical measurement.
- the interaction area 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, particularly as in the nonlinear optical method. .
- the holder cover 11 and the spacer 15 provided on the sample surface make the depth of the liquid held on the sample table smaller than the length of the probe attached to the tip of the transducer, and the Q value due to viscous resistance.
- a probe microscope that suppresses the reduction has been proposed (Patent Document 1).
- a probe microscope is proposed that maximizes the probe detection efficiency by periodically displacing the probe position of the transducer and controlling the relative distance between the probe and the sample stage to synchronize with the irradiation of the laser pulse light. (Patent Document 2).
- Patent Document 1 performs a desired measurement by inserting a measurement probe into an opening provided on the bottom surface of the holder cover 11 (a portion defining the distance between the culture solution and the sample surface).
- the culture solution rises convexly from the opening due to the surface tension, and the length of the probe immersed in the culture solution is correspondingly increased.
- the distance is longer than the distance defined by the bottom surface of the holder cover 11 and the sample surface.
- Patent Document 2 synchronizes the irradiation of pulsed laser light, but does not consider the improvement of the spatial resolution by optimizing the depth of the culture solution between the tip of the short needle and the sample surface.
- the inventors have further reduced the depth of the liquid on the surface of the sample in which the probe is immersed, so that the physical information at the interface between the sample and the liquid in the liquid can be measured with a higher spatial resolution, and the scanning probe microscope The realization of a sample holder suitable for measurement was investigated.
- One of the typical inventions is (1) a container that holds the liquid, and (2) a flat upper cover that covers the upper opening of the container, and has an elongated slit above the sample placement position.
- the upper lid has a slit width that forms a thin film of the liquid having a film thickness smaller than the distance between the upper surface of the sample and the upper lid on the upper surface of the sample when the space between the container and the upper lid is filled with liquid.
- a sample holder for a scanning probe microscope is
- Another one of the representative inventions is (1) the sample holder, (2) ⁇ ⁇ probe, (3) a vibrator that displaces the probe in the vertical direction, and (4) a sample measured by the probe.
- a pulsed laser light source that irradiates a pulsed laser beam to the region of (5), and (5) a detector with a filter that measures the intensity of the output light generated on the sample by irradiation of the pulsed laser beam by energy spectroscopy, and (6)
- a scanning probe microscope having a scanning mechanism for moving the sample holder in the horizontal direction, (7) a vibrator, a pulsed laser light source, and a control device for controlling the scanning mechanism.
- another representative invention includes (1) the sample holder, (2) (probe, (3) a vibrator that displaces the probe in the vertical direction, and (4) alternating current with the probe.
- a probe power source for applying voltage and DC voltage, (5) a detection unit for detecting force applied to the probe, (6) a scanning mechanism for moving the sample holder in the horizontal direction, and (7) an oscillator. And a probe power supply and a control device for controlling the scanning mechanism.
- the liquid covering the surface of the sample can be made thinner, and physical information on the sample surface in the liquid can be measured with higher spatial resolution.
- FIG. 1 is a schematic configuration diagram of a scanning probe microscope (nonlinear optical scanning probe microscope) according to Example 1.
- FIG. 1 is a schematic configuration diagram of a sample holder used in Example 1.
- FIG. 1 is a schematic cross-sectional view of a sample holder used in Example 1.
- FIG. 5 is a schematic configuration diagram of a sample holder used in Example 2.
- FIG. 4 is a schematic cross-sectional view of a sample holder used in Example 2.
- FIG. 5 is a schematic configuration diagram of a scanning probe microscope according to a third embodiment.
- FIG. 10 is a schematic configuration diagram of a scanning probe microscope (scanning Kelvin probe microscope) according to Example 7.
- FIG. 10 is a schematic configuration diagram of a scanning probe microscope (submerged scanning probe microscope) according to Example 9.
- FIG. 10 is a schematic configuration diagram of a sample holder used in Example 9.
- 10 is a schematic cross-sectional view of a sample holder used in Example 9.
- FIG. FIG. 10 is a schematic configuration diagram of an in-liquid scanning probe microscope using the photothermal excitation method according to Example 10.
- FIG. 10 is a schematic configuration diagram of a scanning probe microscope (submerged scanning probe microscope) according to Example 11.
- FIG. 10 is a schematic configuration diagram of a sample holder and a sample position control mechanism used in Example 11.
- FIG. 14 is a schematic diagram of a graphical user interface according to a twelfth embodiment.
- a probe is arranged in near-field light (evanescent light) generated on the sample surface, and the electric field intensity of light near the sample surface is amplified by the near-field light from the probe and the near-field light from the sample.
- near-field light evanescent light
- SHG second harmonic generation
- Each example has a sufficiently thin film thickness on the surface of the sample by optimizing the surface tension of the liquid (culture medium, water, etc.) and the slit width provided on the top lid of the sample holder according to the wettability of the sample with respect to the liquid.
- the liquid thin film having a sufficiently thin film thickness is characterized in that the liquid thin film is generated by recessing the liquid in the slit portion of the upper lid.
- FIG. 1 discloses a probe enhanced scanning sum frequency microscope as a scanning probe microscope according to the present embodiment.
- the probe-enhanced scanning sum frequency microscope is a kind of nonlinear optical scanning probe microscope.
- the probe 1 is attached to the vibrator 2 and is displaced mainly in the vertical direction by the vibrator 2. By this displacement, the relative position (distance) between the probe 1 and the sample 3 is controlled.
- a material is selected that amplifies or concentrates the near-field light intensity in the vicinity of the tip when the probe 1 is placed in the incident light.
- a probe 1 is used for a metal such as gold, silver, copper, aluminum, or a compound thereof, which can effectively use surface-enhanced Raman scattering.
- a silicon probe deposited with a gold thin film having a thickness of 1 to 20 nm is a promising candidate for the probe 1.
- the vibrator 2 of this embodiment vibrates mainly in the vertical direction with respect to the surface of the sample 3, and controls the distance between the probe 1 and the sample 3 to 300 nm or less.
- the natural frequency of the vibrator 2 is, for example, 200 kHz to 2 MHz.
- a crystal resonator that expands and contracts in the longitudinal direction is used for the resonator 2.
- a tuning fork type crystal resonator, a resonator using a piezo element generally used in a scanning probe microscope such as an atomic force microscope, a resonator in which a piezo element is arranged on a cantilever, or the like can also be used.
- the vibration of the vibrator 2 causes the probe 1 to vibrate in a direction perpendicular to the surface of the sample 3 at a frequency close to 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, a phase difference occurs between the voltage applied to the vibrator 2 and the actual vibration amplitude of the vibrator 2. By 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 relative positions of the sample 3 and the probe 1 are perpendicular to the top surface of the sample 3 and on the top surface of the sample 3.
- an atomic force microscope which is one type of scanning probe microscope can be configured, and the unevenness of the sample surface can be measured.
- a scanning mechanism 31 is used for this scanning.
- the distance between the probe 1 and the sample 3 is generally 0 nm (contact) to 100 nm at the closest position, but the probe 1 can also be recessed into the sample 3.
- the distance between 1 and the sample 3 can be set to 0 nm at the closest position (tapping mode AFM).
- a probe power supply 5 is connected to the probe 1 through a wiring 4.
- the probe power source 5 can apply an AC voltage and a DC voltage between the probe 1 and the sample 3.
- no voltage is applied between the probe 1 and the sample 3.
- human liver cancer-derived cell line (HepG2) cells cultured on a mica substrate are fixed with formalin and used as sample 3.
- the sample 3 may be a surface-treated polycarbonate, a metal substrate such as gold, or another substrate.
- the sample holder 11 has a culture solution inlet 12 and a culture solution recovery port 13.
- a culture fluid circulation mechanism 19 (for example, a channel tube) is connected to the culture fluid inlet 12 and the culture fluid recovery port 13.
- the culture fluid circulation mechanism 19 is provided with a culture fluid circulation control mechanism 20, and the culture fluid circulation control mechanism 20 holds or replaces the culture fluid 14.
- water or a solvent can be used. In this embodiment, water is used instead of the culture solution 14.
- a pulsed laser beam generated by a pulsed laser oscillator 27 or a plurality of pulsed laser beams synchronized with each other are input.
- Output light 24 is generated from the sample 3 by the incidence of the pulsed laser beam.
- the output light 24 is received by a detector 25 with a filter.
- the detector with a filter 25 separates the output light 24 for each frequency and measures the intensity of each spectrum.
- a green pulsed laser beam having a wavelength of 532 nm (first pulsed laser beam 22) and an infrared pulsed laser beam having a wavelength of 2.3 to 10 microns and variable (second pulsed laser beam). 23) are synchronized and input to the sample 3.
- the detector with filter 25 measures the intensity of the output light 24 with respect to the sum frequency (sum frequency) of the frequency of the first pulsed laser light 22 and the frequency of the second pulsed laser light 23. By recording the intensity of the output light 24 having a sum frequency depending on the frequency of the second pulsed laser beam 23, sum frequency generation spectroscopy can be performed.
- the orientation of hydrocarbons contained in the cells of the sample 3 can be discussed based on the sum frequency intensity corresponding to the wave number of the second pulsed laser beam 23 corresponding to about 2900 Kaiser, and the wave number corresponds to about 3100 Kaiser.
- the orientation of the water molecules at the interface between the cells of the sample 3 and the mica substrate and the culture solution 14 can be discussed based on the sum frequency intensity.
- a pulsed laser having a higher intensity per pulse than a continuous wave laser For example, the number of photons per second of a CW laser having a wavelength of 532 nm and 50 mW used for the probe enhancement Raman is 1.34 ⁇ 10 17 .
- a pulsed laser having a wavelength of 532 nm and 0.1 to 1 mJ is used. Considering the peak output of the pulsed laser in the case of 1 mJ, the number of photons contained in one pulse is 2.68 ⁇ 10 15 .
- the pulse width of this pulsed laser is 20-30 ps.
- the number of photons in the previous CW laser included in this pulse time (20 ps) is 2.7 ⁇ 10 6 , and the number of photons is 9 digits or more in the pulsed laser as compared with the CW laser. Therefore, it is overwhelmingly advantageous to use a pulsed laser for mapping of nonlinear optical phenomena such as SHG and SFG.
- the Raman signal enhancement effect does not appear when the distance between the probe and the sample is 26.5 nm or more. Further, it is shown that the Raman signal enhancement effect increases exponentially when the distance is 26.5 nm or less.
- the distance between the surfaces of the probe 1 and the sample 3 is preferably about 2 nm or less.
- the distance between the surface of the probe 1 and the sample 3 is controlled and vibrated by the vibrator 2, but the vibration period is 200 kHz to 2 MHz, and is not necessarily synchronized with the oscillation period 50 Hz to 200 kHz of a general pulsed laser. .
- the control device 26 controls the vibration amplitude of the probe 1 to 1 nm or less, thereby realizing the Raman signal enhancement effect.
- This Raman signal enhancement effect is effective not only in the probe-enhanced scanning sum-frequency microscope, but also in the probe-enhanced scanning second harmonic microscope and the probe-enhanced scanning optical probe microscope shown in the following examples. It is.
- the localized plasmon polariton is excited at the tip of the probe 1 and the electric field intensity of the Raman signal is enhanced.
- the intensity of the output light 24 of the sum frequency is remarkably enhanced by the probe enhancement effect resulting from this (probe enhanced sum frequency generation spectroscopy).
- the intensity of 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.
- FIG. 2A shows a schematic configuration of the sample holder 11 used in this embodiment.
- FIG. 2A is a bird's-eye view seen from the incident direction of the pulsed laser beam (FIG. 1). For this reason, although the culture solution collection port 13 is shown, the culture solution injection port 12 is omitted.
- FIG. 2B is a schematic cross-sectional view of the sample holder 11 and shows an A-A ′ arrow in FIG. 2A.
- the sample holder 11 includes a rectangular container 11A that holds the culture solution 14 in a circulatory manner, a sample table 11B that is arranged in the container 11A, and a flat top lid 9 that covers the upper opening of the container 11A.
- a slit 15 is formed in the upper lid 9 along the flow path direction of the culture solution 14 (along the direction from the culture solution inlet 12 to the culture solution recovery port 13).
- the slit 15 is an elongated opening that extends from the culture solution inlet 12 side to the culture solution recovery port 13 side, and is provided above the sample stage 11B.
- the probe 1 and pulsed laser light are introduced into the sample holder 11 through the slit 15.
- the sample 3 is mounted on the upper surface of the sample table 11B.
- the sample holder 11 (the space surrounded by the container 11A and the upper lid 9) is filled with the culture solution 14.
- the liquid level of the culture solution 14 coincides with the height of the back surface of the upper lid 9.
- the liquid level of the culture solution 14 becomes higher than the upper surface of the sample 3 placed on the sample stage 11 ⁇ / b> B, and the flow path of the culture solution 14 is also formed on the upper surface of the sample 3.
- the thin film of the culture solution 14 (culture solution thin film 16) formed on the upper surface of the sample 3 is formed on the upper surface of the sample 3 and the upper lid 9 (slit by the capillary phenomenon caused by the presence of the slit 15.
- the film thickness T of the culture solution thin film 16 depends on the surface tension of the culture solution 14, the wettability of the culture solution 14 in the sample 3, the distance T ′ between the upper lid 9 of the sample holder and the upper surface of the sample 3, and the slit width W. Determined.
- the smaller the distance T ′ between the upper lid 9 of the sample holder 11 and the sample 3 the smaller the film thickness T of the culture solution thin film 16.
- the second pulsed laser beam 23 absorbed in the culture solution thin film 16 generated on the surface of the sample 3 depends on the wave numbers of the culture solution 14 and the second pulsed laser beam 23 (infrared light). . Therefore, in order for the second pulsed laser beam 23 to pass through the culture solution thin film 16 with sufficient intensity to reach the sample 3 and generate a sum frequency generation signal, the intensity of the second pulsed laser beam 23 is The film thickness T of the culture solution thin film 16 needs to be sufficiently small.
- the attenuation distance of water with infrared light having a wave number of about 3100 Kaiser is about 0.1 mm.
- the energy density of the second pulsed laser beam 23 is 120 ⁇ J / mm 2 / ps as a standard, the second pulsed laser beam 23 passes through the culture solution thin film 16 having a thickness of 100 ⁇ m.
- the sample 3 was reached and a sum frequency generation signal could be generated. Therefore, if the energy density of the second pulsed laser beam 23 is about 3 times (exactly, e ⁇ 2.7 times the base of natural logarithm), the film is 200 ⁇ m, and if it is about 9 times, the film is 300 ⁇ m.
- the culture solution thin film 16 having a thickness can be permeated.
- the film thickness T of the culture solution thin film is given by r ⁇ 100 ⁇ m
- the energy density per unit area of the second pulsed laser beam 23 is 60 for generating the sum frequency generation signal. It is a requirement to be given at ⁇ 480 ⁇ 3 r ⁇ 1 ⁇ J / mm 2 / ps.
- the upper lid 9 is interposed via a spacer (not shown) so that the distance T ′ between the upper lid 9 of the sample holder 11 and the sample 3 is 100 ⁇ m.
- the film thickness is 30 to 80 ⁇ m when the slit width W is 1.0 to 2.8 mm.
- a culture solution thin film 16 can be formed.
- FIG. 3A shows the SFG signal when the second pulsed laser beam 23 with sufficient intensity reaches the sample 3 (when the sufficiently thin culture solution thin film 16 is formed as described above) satisfying the following conditions.
- This is the measurement result.
- a sample 3 is a mica substrate and a cell of a human liver cancer cell line (HepG2) fixed on the mica substrate.
- HepG2 human liver cancer cell line
- the slit width W is set to 2.0 mm.
- the orientation of hydrocarbons contained in the cells of the sample 3 (corresponding to about 2900 Kaiser of the wave number of the second pulsed laser beam 23) and water molecules at the interface between the cells of the sample 3 and the mica substrate and water.
- SFG signal generated from the orientation (corresponding to about 3100 Kaiser of the wave number of the second pulsed laser beam 23) can be observed.
- FIG. 3B is a diagram showing the wave number dependency of the SFG light intensity when the intensity of the second pulsed laser beam 23 is not sufficient and no SFG signal is generated.
- a nonlinear optical signal is generated at the sample-culture medium interface, and the SFG spectrum is measured as shown in FIG. 3A. can do.
- physical information at the sample-culture solution interface in the culture solution can be measured with high spatial resolution. If the culture solution thin film 16 is too thick with respect to the intensity of the second pulsed laser beam 23, a sufficient nonlinear optical signal cannot be obtained as shown in FIG. 3B.
- Example 2 In this embodiment, a probe enhanced scanning sum frequency microscope that can replace the culture solution 14 will be described.
- the schematic configuration of the scanning probe microscope in the present embodiment is the same as that of the first embodiment (configuration shown in FIG. 1). The difference is the configuration of the sample holder 11.
- FIG. 4A shows a schematic configuration of the sample holder 11 used in this embodiment.
- 4A is a bird's-eye view seen from the incident direction of the pulsed laser beam (FIG. 1).
- the culture solution inlet 12 and the culture solution recovery port 13 are installed in directions different from those in the first embodiment.
- the sample holder 11 is provided with a slit 15 so as to be orthogonal to the flow of the culture solution 14.
- the slit 15 is disposed so as to cross the installation area of the sample 3.
- FIG. 4B is a schematic cross-sectional view of the sample holder 11 and shows a view along arrow B-B ′ of FIG. 4A.
- the partition plate 8 that does not exist on the line B-B ′ of FIG. 4A is indicated by a broken line.
- the probe 1 and the pulsed laser beam are introduced into the sample holder 11 through the slit 15.
- the slit width W is selected under the same conditions as in the first embodiment. For this reason, when the culture solution 14 is filled in the sample holder 11, the culture solution thin film 16 is generated on the upper surface of the sample 3.
- the film thickness T of the culture solution thin film 16 depends on the surface tension of the culture solution 14, the wettability of the culture solution 14 in the sample 3, the distance T ′ between the upper lid 9 of the sample holder and the upper surface of the sample 3 (the thickness of the spacer not shown). The corresponding distance) and the slit width W.
- the smaller the distance T ′ between the upper lid 9 of the sample holder and the sample 3 the smaller the film thickness T of the culture solution thin film 16.
- the culture medium partition 8 is installed along the slit 15 extending from one side of the container 11A to the other side so as to cross the flow path except for the opening above the sample 3. .
- the culture medium partition 8 divides the space in the sample holder 11 into two parts, the upstream side and the downstream side.
- the culture solution 14 flows from the upstream side to the downstream side through the peripheral region of the sample 3 where the culture solution partition 8 is not provided. That is, a channel is formed in the peripheral region of the sample 3.
- the culture medium partition 8 acts so as to prevent the flow of the culture medium 14
- the cross-sectional shape of the culture medium thin film 16 formed on the upper surface of the sample 3 is different between the upstream side and the downstream side as shown in FIG. 4B.
- Asymmetric (non-uniform) That is, as shown by the arrow in FIG. 4B, the upstream side of the culture solution 14 (the culture solution inlet 12 side) swells upward and becomes non-uniform (arrow in FIG. 4B). However, the swelling of the culture solution 14 does not affect the film thickness T of the culture solution thin film 16.
- the film thickness T of the culture solution thin film 16 is the same as in Example 1 in that the surface tension of the culture solution 14, the wettability of the culture solution 14 in the sample 3, and the distance T ′ between the upper lid 9 of the sample holder and the upper surface of the sample 3 And the slit width W.
- the culture solution partition 8 is arrange
- FIG. 5 shows a product example of the scanning probe microscope described above.
- the sample holder 11, the vibrator 2, the culture medium circulation mechanism 19, the culture medium circulation control mechanism 20, and the scanning mechanism 31 are built in the cell culture apparatus 32, and a pulse oscillation laser oscillator 27 and a detector 25 with a filter.
- the control device 26 is disposed outside the cell culture device 32.
- the cell culture device 32 is provided with windows 33 and 34.
- the first pulsed laser beams 22 and 23 pass through the window 33 and irradiate the sample 3.
- the output light 24 passes through the window 34 and is detected by the detector 25 with a filter. Therefore, the windows 33 and 34 are made of, for example, optical glass or quartz glass that transmits the wavelengths of the pulsed laser light 22 and 23 and the output light 24.
- Example 4 The scanning probe microscope described above can be applied to, for example, a probe enhanced scanning second harmonic microscope. Below, the said microscope is demonstrated using FIG.
- the first pulsed laser light 22 (infrared pulsed laser light) having a wavelength of 1064 nm is input near the region of the sample 3 to which the probe 1 is close.
- the probe-enhanced scanning second harmonic microscope detects the output light 24 generated by the sample 3 with a detector 25 with a filter.
- the detector with filter 25 measures the intensity of light having a frequency twice as high as that of the first pulsed laser light 22.
- a scanning second harmonic microscope is constructed by mapping the second harmonic intensity measured by the filter-detected detector 25 while measuring the unevenness of the nerve cell (while operating as an atomic force microscope AFM). The nerve activity intensity of the cell can be mapped.
- the probe enhancement scanning second harmonic microscope can be configured by optimizing the probe enhancement effect.
- Example 5 The scanning probe microscope described above can also be applied to, for example, a probe-enhanced scanning Raman microscope. Below, the said microscope is demonstrated using FIG.
- the first pulsed laser light 22 (green pulsed laser light) having a wavelength of 532 nm is input in the vicinity of the region of the sample 3 where the probe 1 is close.
- the probe-enhanced scanning Raman microscope inputs the output light 24 generated by the sample 3 to the detector 25 with a filter, and measures the light intensity of the Raman scattered light.
- cultured hepatocytes are used as sample 3. Mapping composition distribution of molecules, proteins, etc. in hepatocytes by measuring Raman scattering measured by detector 25 with filter while measuring irregularities of hepatocytes (operating as atomic force microscope AFM) Can do.
- the probe enhancement scanning Raman microscope can be configured by optimizing the probe enhancement effect as in the first embodiment.
- the scanning probe microscope described above can also be applied to, for example, a probe enhanced scanning CARS (Coherent Anti-Stokes Raman Scattering) microscope.
- This microscope uses coherent anti-Stokes Raman scattering.
- a first pulsed laser beam 22 (angular frequency ⁇ 1) and a second pulsed laser beam 23 (angular frequency) having different angular frequencies are located in the vicinity of the region of the sample 3 close to the probe 1.
- ⁇ 2 is input in a synchronized state.
- the probe-enhanced scanning CARS microscope inputs the output light 24 generated from the sample 3 into the detector 25 with a filter, and measures the light intensity of the CARS light.
- the probe enhancement scanning CARS microscope can be configured by optimizing the probe enhancement effect.
- Example 7 The scanning probe microscope described above can also be applied to a scanning Kelvin probe microscope that measures the electrostatic force distribution on the surface of the sample.
- the overall configuration of the scanning Kelvin probe microscope according to this example is the same as that of Example 1 (FIG. 1) and Example 3 (FIG. 5).
- FIG. 6 shows an electrode portion having a configuration unique to this embodiment.
- the bipotentiostat 51 controlled by the control device 26 controls the voltage applied to the probe electrode 52, the sample electrode 53, the working electrode 54, and the reference electrode 55.
- the bipotentiostat 51 measures the potential of the culture solution 14 with the reference electrode 55.
- the bipotentiostat 51 applies a first voltage (relative voltage of the probe 1 with respect to the potential of the culture solution 14) corresponding to the measured potential to the probe electrode 52.
- the bipotentiostat 51 applies a second voltage (relative voltage of the sample 3 with respect to the potential of the culture solution 14) corresponding to the measured potential to the sample electrode 53. At this time, the current flowing between the culture solution 14 and the reference electrode 55 is almost zero.
- the bipotentiostat 51 when a current is caused to flow between the culture solution 14 and the sample 3 to cause a chemical reaction, the bipotentiostat 51 causes a current to flow between the working electrode 54 and the sample electrode 53. When a voltage is applied between the probe 1 and the sample 3, the bipotentiostat 51 applies a voltage between the probe electrode 52 and the sample electrode 53.
- the voltage and electric current between the probe electrode 52 and the sample electrode 53 are used as an applied voltage and a tunnel current.
- the charge injection electrode 56 is used.
- the vibrator 2 is vibrated at a frequency near its natural frequency (within about ⁇ 1% of the natural frequency), and the probe 1 is vibrated in a direction perpendicular to the upper surface of the sample 3. .
- the interaction (force) between the probe 1 and the sample 3 is known from the phase difference between the AC voltage applied to the transducer 2 and the current flowing into the transducer 2, and the distance between the probe 1 and the sample 3. I understand.
- a voltage signal obtained by adding the AC voltage and the DC voltage is applied between the probe 1 and the sample 3.
- an electrostatic force F corresponding to the difference between this voltage signal and the work function of each surface 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 given by differentiation with respect to the voltage V of the electrostatic force F, it becomes dF / dV ⁇ V / z 2 , and if the distance z and the dielectric constant ⁇ are constant, the value is proportional to the potential difference. It becomes.
- the potential difference between the probe 1 and the sample 3 is always kept at zero.
- 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 output from the second lock-in amplifier is given by the second derivative 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 value is inversely proportional to the square of the distance z between the probe 1 and the sample 3.
- the scanning mechanism 31 scans the relative position of the sample 3 and the probe 1 in the direction perpendicular to the sample 3 and in the plane direction of the sample 3 while keeping the output signal from the second lock-in amplifier constant.
- the distance between the probe 1 and the sample 3 can be kept constant.
- an atomic force microscope which is one type of scanning probe microscope can be configured.
- the operation probe microscope of the present embodiment also uses the sample holder 11 described in the first embodiment, the culture solution thin film 16 is generated on the upper surface of the sample 3 at the opening of the slit 15, and the culture solution 14 and the probe 1 are formed.
- the contact area can be reduced. As a result, it is possible to suppress the current between the culture solution 14 and the probe 1 which is a main factor of sensitivity deterioration, and to measure the electrostatic field distribution of the sample 3 with high spatial resolution.
- Example 8 In this example, a nerve signal measurement method for nerve cells using the scanning probe microscope described in Example 7 will be described.
- the cultured nerve cells are set as the sample 3 in the culture solution 14.
- the probe 1 is attached to the vibrator 2 so as to face the upper surface of the sample 3.
- the probe 1 vibrates in the direction perpendicular to the upper 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 2 causes the probe 1 to vibrate in a direction perpendicular to the upper surface of the sample 1 at a frequency close to the natural frequency of the vibrator 2 (within about ⁇ 1% of the natural frequency).
- 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 1 and the sample 3 can be seen. .
- the scanning mechanism 31 scans the relative position of the sample 3 and the probe 1 in the vertical direction with respect to the sample 3 and in the plane direction with respect to the sample 3 while keeping this phase difference constant.
- an atomic force microscope (AFM) that is an equation can be constructed, and the unevenness of the sample surface can be measured.
- a nerve signal is a voltage pulse generated in a nerve cell when charge is injected into the nerve cell from the charge injection electrode 56 (FIG. 6).
- a predetermined charge is injected into the sample 3 to apply a voltage pulse to the nerve cells.
- the magnitude of the voltage pulse is about 50 ⁇ V to 100 mV.
- the probe 3 is brought into contact with or brought close to a desired position of the sample 3, and this voltage pulse is detected by the scanning probe microscope of the seventh embodiment.
- FIG. 7 shows a schematic configuration of an in-liquid scanning probe microscope according to the present embodiment.
- the cantilever 61 is installed in the excitation device 62.
- the probe 1 is formed near the free end of the cantilever 61, and the relative position between the probe 1 and the sample 3 is controlled by the excitation device 62 and the scanning mechanism 31.
- the cantilever 61 is mechanically vibrated by the excitation device 62.
- the vibration of the excitation device 62 is controlled by a control device (not shown).
- the vibration amplitude and phase of the cantilever 61 are detected based on a change in the angle of the detection laser 64 that is emitted from the detection laser light source 63 and then enters the back surface of the cantilever 61 and reflects.
- a divided photodiode 65 having a light receiving surface divided into a plurality of regions is used. Each light receiving region of the divided photodiode 65 generates a voltage corresponding to the incident area of the detection laser 64.
- a change in the angle of the detection laser 64 is detected by a control device (not shown) based on a difference in potential generated in each light receiving region in accordance with a change in the position of the detection laser 64.
- the measurement sensitivity decreases due to scattering of the detection laser 64, reflection from other than the back surface of the cantilever 61, and the like.
- the sensitivity decreases due to the viscous resistance that the cantilever 61 receives from the liquid. Is a problem.
- the probe height D of the cantilever needs to be larger than the film thickness T of the culture solution thin film 16.
- FIG. 8A is a schematic configuration of the sample holder 11 used in this embodiment.
- FIG. 8A is a bird's-eye view from the direction of the divided photodiode 65 (FIG. 7). For this reason, although the culture solution collection port 13 is shown, the culture solution injection port 12 is omitted.
- FIG. 8B is a schematic cross-sectional view of the sample holder 11 of the present embodiment, and shows an A-A ′ arrow in FIG. 8A.
- FIG. 8A shows the case where the sample holder 11 is provided with the slit 15 in parallel with the circulation direction of the culture solution 14 as in the first embodiment.
- a slit 15 is formed in the upper lid 9 of the sample holder 11.
- the slit 15 can be used for introducing the cantilever 61 as shown in FIG. 8A.
- a culture solution thin film 16 is generated between the opening of the slit 15 and the upper surface of the sample 3 as shown in FIG. 8B.
- the film thickness T of the culture solution thin film 16 is determined by the surface tension of the culture solution 14, the wettability of the culture solution 14 in the sample 3, the distance T ′ between the top lid 9 and the sample 3 of the sample holder, as described in the above-described embodiment. This corresponds to the thickness of the spacer inserted between the upper lid 9 and the sample 3) and the slit width W.
- the film thickness T of the culture solution thin film 16 becomes smaller.
- the slit width W needs to be set larger than the width B of the cantilever 61.
- FIG. 9 is a schematic configuration diagram of a submerged scanning probe microscope using the photothermal excitation method.
- a magnetic material is coated on the back surface of the cantilever 61.
- the photothermal excitation method the back surface of the cantilever 61 is coated with a metal thin film 66.
- contamination due to elution of the coating material becomes a problem.
- the back surface of the cantilever 61 coated with the metal thin film 66 is irradiated with the excitation laser 67, and the cantilever 61 is excited by the thermal gradient generated by the irradiation. For this reason, when the cantilever 61 is introduced into the liquid, there is a problem in that the sensitivity decreases due to scattering of the excitation laser 67 and thermal diffusion to the culture liquid 14.
- the back surface of the cantilever 61 can be exposed to the air by setting the film thickness T of the culture solution thin film 16 to be smaller than the mounting height D of the probe 1 attached to the cantilever 61.
- the film thickness T of the culture solution thin film 16 it is possible to prevent contamination due to scattering of the excitation laser 67 irradiating the back surface of the cantilever 61 and elution of the coating material. Accordingly, it is possible to realize a submerged scanning probe microscope that can detect the vibration amplitude and phase of the cantilever 61 with high sensitivity and is less invasive.
- the width of the slit 15 (slit width W) formed in the upper lid 9 of the sample holder 11 and the distance T ′ between the upper lid 9 of the sample holder 11 and the upper surface of the sample 3 are determined.
- the distance T ′ between the upper lid 9 of the sample holder and the sample 3 is the thickness of a spacer (not shown) sandwiched between the upper lid 9 of the sample holder and the upper surface of the sample 3. It can be decided by.
- the sample holder 11 capable of adjusting the distance T ′ between the upper lid 9 of the sample holder 11 and the upper surface of the sample 3 while observing the film thickness T of the culture solution thin film 16, and its control An in-liquid scanning probe microscope equipped with a mechanism will be described.
- FIG. 10A is a schematic configuration of the scanning probe microscope according to the present embodiment.
- a sample position control mechanism 71 is disposed below the sample holder 11, and the position of the sample 3 inside the sample holder 11 is adjusted so that the laser intensity detected by the detector 25 with a filter becomes a specified value. Control.
- FIG. 10B is a schematic configuration of the sample holder 11 and the sample position control mechanism 71. Similar to the previous embodiment, the sample holder 11 has a configuration in which the upper opening of the rectangular container 11A is closed by the upper lid 9. The upper lid 9 is provided with a slit 15. However, the bottom surface of the sample holder 11 of the present embodiment is cut out in a square shape, and is fitted so as to cover the upper part of the movable sample table 72 (corresponding to the sample table 11B) having the same shape.
- the movable sample stage 72 is attached to the sample holder 11 so that it can move up and down while maintaining watertightness with the bottom opening of the sample holder 11.
- two O-rings an O-ring 74 between the sample holder 11 and the fixture 73 and a movable member
- An O-ring 75 between the type sample stage 72 and the fixture 73 is provided. Thereby, the culture solution 14 is held in a space formed between the sample holder 11 and the movable sample stage 72.
- the sample holder 11 is fixed by a leaf spring 76 screwed to a convex member protruding upward from the main body of the fixture 73.
- the movable sample stage 72 can be moved up and down while maintaining watertightness by the O-ring 75.
- the vertical position of the movable sample stage 72 is controlled by the rotational movement of a stepping motor 78 and a vertical movement screw 79 attached to the lower part of the movable sample stage 72.
- the detection result of the detector with filter 25 is fed back to the control of the sample position control mechanism 71 (specifically, the stepping motor 78 that drives the movable sample stage 72). Specifically, the movable sample stage 72 is controlled to move up and down so that a sum frequency generation signal having a predetermined intensity is detected with reference to the laser intensity detected by the detector 25 with filter. That is, the distance T ′ between the upper lid 9 of the sample holder 11 and the upper surface of the sample 3 is adjusted so that the designated laser intensity can be obtained by the detector 25 with a filter. As a result, the relationship between the intensity of the pulsed laser beam emitted from the pulsed laser oscillator and the thickness T of the culture solution thin film 16 can be calibrated in advance. Thus, the submerged scanning probe microscope according to the present embodiment can reliably form the solution thin film 16 having the specified thickness T on the upper surface of the sample 3.
- the film thickness T of the culture solution thin film 16 formed on the upper surface of the sample 3 depends on the surface tension at the interface between the sample 3 and the culture solution 14, the upper lid 9 of the sample holder 11, and the culture solution 14.
- T ′ can be calculated.
- the following calculation function and display function are mounted on a computer (computer) independent of the control device 26 (FIG. 1) and the scanning probe microscope.
- the control device 26 of the present embodiment uses the input text box 81 on the user interface screen shown in FIG. 11 to (1) a contact angle that gives an index of the surface tension at the interface between the culture solution 14 and the sample 3 ( 2) An input of a contact angle giving an index of the magnitude of the surface tension at the interface between the culture solution 14 and the container (upper lid 9), and (3) a film thickness T of the culture solution thin film to be formed is received.
- the control device 26 When the program execution button 83 is clicked, the control device 26 is required to form the culture solution thin film 16 having the film thickness T based on the value input in the input text box 81 (1) Slit width W (2) The value of the distance T ′ between the upper lid 9 of the sample holder 11 and the upper surface of the sample 3 is calculated, and the calculation result is displayed in the output text 82.
- the operator By mounting the function, the operator can easily select an appropriate sample holder 11 (including the upper lid 9) when observing an arbitrary sample 3.
- the interface between the biomolecule, the biological tissue, the biological substrate material and the culture solution is mainly measured or evaluated, but the interface evaluation of the semiconductor device (for example, the PN junction interface), the interface evaluation of the battery It can also be used for (evaluation of interface between electrode and electrolyte).
- the interface between the battery electrode and the electrolytic solution is the interface between the surface and the liquid molecule, as is the case with the interface between the biomaterial and the culture solution.
- it may have an electrolytic solution circulating device as in FIG. 1, but when used only for the evaluation of the solid interface, the circulating device is not necessarily required. do not do.
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Abstract
Description
以下では、試料表面に生じた近接場光(エバネッセント光)中に探針を配置し、探針からの近接場光と試料からの近接場光とによって試料表面近傍の光の電場強度を増幅することにより、微弱なシグナル光を補償する探針増強検出法の走査プローブ顕微鏡について説明する。もっとも、各実施例に係る走査プローブ顕微鏡は、和周波発生(SFG)分光法、第二高調波発生(SHG)分光法、その他の線形又は非線形光学分光法に応じて様々な形態を採る。
図1に、本実施例に係る走査プローブ顕微鏡としての探針増強走査和周波顕微鏡を開示する。探針増強走査和周波顕微鏡は、非線形光学走査プローブ顕微鏡の一種である。探針1は、振動子2に取り付けられており、振動子2によって主に上下方向に変位される。この変位により、探針1と試料3の相対位置(距離)が制御される。
(1) 雲母基板と雲母基板上で固定したヒト肝癌由来細胞株(HepG2)の細胞を試料3とする。
(2) 培養液14の代わりに水を使用する。
(3) スリット幅Wを2.0mmとする。
本実施例も、引き続き、培養液14を交換することができる探針増強走査和周波顕微鏡について説明する。本実施例における走査プローブ顕微鏡の概略構成は、実施例1(図1に示す構成)と同じである。違いは、試料ホルダ11の構成である。
図5に、前述した走査プローブ顕微鏡の製品例を示す。本実施例の場合、試料ホルダ11、振動子2、培養液循環機構19、培養液循環制御機構20、走査機構31は細胞培養装置32に内蔵され、パルス発振レーザー発振器27、フィルター付検出器25、制御装置26は細胞培養装置32の外に配置される。細胞培養装置32には窓33及び34が設けられている。第一のパルス発振レーザー光22、23は、窓33を通過して試料3を照射する。出力光24は、窓34を通過してフィルター付検出器25で検出される。このため、窓33及び34は、パルス発振レーザー光22、23及び出力光24の波長を透過する例えば光学ガラス,石英ガラス等で構成される。
前述した走査プローブ顕微鏡は、例えば探針増強走査第二高調波顕微鏡にも適用することができる。以下では、図1を用いて当該顕微鏡を説明する。
前述した走査プローブ顕微鏡は、例えば探針増強走査ラマン顕微鏡にも適用することができる。以下では、図1を用いて当該顕微鏡を説明する。
前述した走査プローブ顕微鏡は、例えば探針増強走査CARS(Coherent Anti-Stokes Raman Scattering)顕微鏡にも適用することができる。この顕微鏡は、コヒーレントアンチストークスラマン散乱を使用する。この顕微鏡の場合、探針1が近接する試料3の領域近傍に、角振動数が異なる第一のパルス発振レーザー光22(角振動数ω1)と第二のパルス発振レーザー光23(角振動数ω2)が同期した状態で入力される。探針増強走査CARS顕微鏡は、試料3で発生した出力光24をフィルター付検出器25に入力し、CARS光の光強度を計測する。この際、試料3の凹凸を計測しながら(原子間力顕微鏡AFMとして動作させながら)、CARS光の光強度を調べることにより、試料3の分子、たんぱく質などの組成分布をマッピングすることができる。この際、実施例1と同様、探針増強効果を最適化することにより、探針増強走査CARS顕微鏡を構成することができる。
前述した走査プローブ顕微鏡は、試料の表面の静電気力分布を計測する走査ケルビンプローブ顕微鏡にも適用することができる。本実施例に係る走査ケルビンプローブ顕微鏡の全体構成は実施例1(図1)及び実施例3(図5)と同様である。図6に、本実施例に特有の構成である電極部分を示す。
本実施例では、実施例7で説明した走査プローブ顕微鏡を用いた神経細胞の神経シグナル計測方法について説明する。本実施例では、培養した神経細胞を試料3として培養液14中に設置する。また、探針1を、試料3の上面に対向するように振動子2に取り付ける。振動子2によって、探針1は、試料3の上面に対して垂直方向に振動する。
前述した走査プローブ顕微鏡は、例えば、カンチレバーを用いた液中走査プローブ顕微鏡にも適用することができる。図7に、本実施例に係る液中走査プローブ顕微鏡の概略構成を示す。カンチレバー61は、励振装置62に設置される。カンチレバー61の自由端近傍には探針1が形成されており、探針1と試料3との相対位置は、励振装置62及び走査機構31により制御される。カンチレバー61は、励振装置62により機械的に振動される。励振装置62の振動は不図示の制御装置により制御される。
実施例9で説明したカンチレバー61の励振機構には、機械的な方法以外にも、磁気励振法又は光熱励振法を用いることができる。図9は、光熱励振法を用いた液中走査プローブ顕微鏡の概略構成図である。磁気励振法では、カンチレバー61の背面に磁性体をコートするが、光熱励振法ではカンチレバー61の背面を金属の薄膜66でコートする。ところが、金属の薄膜66でコートされたカンチレバー61を液中に導入すると、コート材の溶出によるコンタミネーションが問題となる。
前述した実施例1における走査プローブ顕微鏡のように、試料ホルダ11の上蓋9に形成するスリット15の幅(スリット幅W)と、試料ホルダ11の上蓋9と試料3の上面との距離T′によって培養液薄膜16の膜厚Tを決定する場合、試料ホルダの上蓋9と試料3との距離T′は、試料ホルダの上蓋9と試料3の上面との間に挟むスペーサ(不図示)の厚みにより決めることができる。
実施例1で説明したように、試料3の上面に形成される培養液薄膜16の膜厚Tは、試料3と培養液14の界面における表面張力と、試料ホルダ11の上蓋9と培養液14の界面における表面張力と、スリット幅Wと、試料ホルダ11の上蓋9と試料3の上面との距離T’により決まる。従って、予め、各界面における表面張力が既知であれば、目的の培養液薄膜16の膜厚Tを得るために必要となるスリット幅W及び試料ホルダ11の上蓋9と試料3の上面との距離T’を計算することができる。
前述した実施例の採用により、液体中の生体分子、生体組織、生体基板材料等と液体との界面の物理上方を一段と高い精度で計測又は評価することができる。例えば培養基板、水浄化膜、培養細胞を評価できる。
T 培養液薄膜の膜厚
T′ 試料ホルダの上蓋と試料との距離
D カンチレバーの探針高さ
B カンチレバー幅
1 探針
2 振動子
3 試料
4 配線
5 探針用電源
8 培養液仕切り
9 上蓋
10 培養液の形状
11 試料ホルダ
11A 容器
11B 試料台
12 培養液注入口
13 培養液回収口
14 培養液(水、溶媒)
15 スリット
16 培養液薄膜
17 培養液イン
18 培養液アウト
19 培養液循環機構
20 培養液循環制御機構
21 プリズム
22 第一のパルス発振レーザー光
23 第二のパルス発振レーザー光
24 出力光
25 フィルター付検出器
26 制御装置
27 パルス発振レーザー発振器
31 走査機構
32 細胞培養装置または半導体検査装置等
33 窓
34 窓
51 バイポテンショスタット
52 探針電極
53 試料電極
54 作用電極
55 参照電極
56 電荷注入電極
61 カンチレバー
62 励振装置
63 検出用レーザー光源
64 検出用レーザー
65 分割フォトダイオード
66 磁性体または金属の薄膜
67 励振用レーザー
68 励振用レーザー光源
71 試料位置制御機構
72 可動式試料台
73 固定具
74 試料ホルダ-固定具間Oリング
75 可動式試料台-固定具間Oリング
76 板バネ
77 固定ネジ
78 ステッピングモーター
79 上下移動用ネジ
81 入力用テキストボックス
82 出力用テキスト
83 プログラム実行ボタン
Claims (14)
- 液体を保持する容器と、
前記容器の上部開口を覆う平板状の上蓋であって、前記試料の載置位置の上方に細長いスリットを有する上蓋であり、前記スリットは、前記容器と前記上蓋の間を液体で満たしたとき、前記試料の上面に、前記試料の上面と前記上蓋との距離よりも小さい膜厚を有する前記液体の薄膜を形成するスリット幅を有する、上蓋と、
を有する走査プローブ顕微鏡用の試料ホルダ。 - 請求項1に記載の走査プローブ顕微鏡用の試料ホルダにおいて、
前記膜厚は、パルス発振レーザー光で前記液体中の試料を照射した場合に、前記試料に非線形光学シグナルが発生する膜厚である
ことを特徴とする走査プローブ顕微鏡用の試料ホルダ。 - 請求項1に記載の走査プローブ顕微鏡用の試料ホルダにおいて、
前記容器は、前記液体の注入口と回収口を有し、
上記上蓋には、前記スリットが前記液体の循環方向に対して平行に形成されている
ことを特徴とする走査プローブ顕微鏡用の試料ホルダ。 - 請求項1に記載の走査プローブ顕微鏡用の試料ホルダにおいて、
前記容器は、前記液体の注入口と回収口を有し、
上記上蓋には、前記スリットが前記液体の循環方向に対して直交するように形成されている
ことを特徴とする走査プローブ顕微鏡用の試料ホルダ。 - 請求項4に記載の走査プローブ顕微鏡用の試料ホルダにおいて、
前記液体の流れを制限して、前記液体を前記試料の上面に誘導する仕切りを更に有する
ことを特徴とする走査プローブ顕微鏡用の試料ホルダ。 - 液体を保持する容器と、前記容器の上部開口を覆う平板状の上蓋であって、試料の載置位置の上方に細長いスリットを有し、前記スリットは、前記容器と前記上蓋の間を液体で満たしたとき、前記試料の上面に、前記試料の上面と前記上蓋との距離よりも小さい膜厚を有する前記液体の薄膜を形成する、上蓋とを有する試料ホルダと、
探針と、
前記探針を上下方向に変位させる振動子と、
前記探針により計測する前記試料の領域にパルス発振レーザー光を照射するパルス発振レーザー光源と、
前記パルス発振レーザー光の照射により前記試料に発生する出力光の強度をエネルギー分光して計測するフィルター付検出器と、
前記試料ホルダを水平方向に移動する走査機構と、
前記振動子と、前記パルス発振レーザー光源と、前記走査機構を制御する制御装置と、
を有する走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記薄膜の膜厚をr×100μmとするとき、前記パルス発振レーザー光の単位面積当たり単位時間当たりのエネルギー密度は、60~480×3r-1μJ/mm2/psである
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記試料ホルダの下部に、前記試料の高さ位置を調整する試料位置制御機構を更に有し、
前記制御装置は、前記フィルター付検出器の計測結果に基づいて前記試料位置制御機構を制御して前記試料の高さ位置を調整し、前記薄膜の膜厚を調整する
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記制御装置は、ユーザインターフェースを通じて受け付けた、前記試料と前記液体の界面における表面張力と、前記試料ホルダの前記上蓋と前記液体の界面における表面張力に基づいて、パルス発振レーザー光で前記液体中の試料を照射した場合に、前記試料に非線形光学シグナルを発生させる前記試料ホルダの前記上蓋と前記試料の上面との距離、及び、前記スリットの幅を計算して前記ユーザインターフェースに表示する
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記パルス発振レーザー光源は、固定波長のパルス発振レーザー光を照射する第一の光源と、可変波長のパルス発振レーザー光を照射する第二の光源を有し、
前記前記フィルター付検出器は、前記出力光として和周波光を検出する
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記出力光は、前記パルス発振レーザー光の第二高調波である
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記出力光は、前記パルス発振レーザー光のラマン散乱光である
ことを特徴とする走査プローブ顕微鏡。 - 請求項6に記載の走査プローブ顕微鏡において、
前記振動子は、探針を先端に取り付けたカンチレバーと、前記カンチレバーを上下方向に変位させる励振装置で構成される、
ことを特徴とする走査プローブ顕微鏡。 - 液体を保持する容器と、前記容器の上部開口を覆う平板状の上蓋であって、試料の載置位置の上方に細長いスリットを有し、前記スリットは、前記容器と前記上蓋の間を液体で満たしたとき、前記試料の上面に、前記試料の上面と前記上蓋との距離よりも小さい膜厚を有する前記液体の薄膜を形成するスリット幅を有する、上蓋とを有する試料ホルダと、
探針と、
前記探針を上下方向に変位させる振動子と、
前記探針に交流電圧と直流電圧を印加する探針用電源と、
前記探針に印加される力を検出する検出部と、
前記試料ホルダを水平方向に移動する走査機構と、
前記振動子と、前記探針用電源と、前記走査機構を制御する制御装置と、
を有する走査プローブ顕微鏡。
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