Stage for scanning probe microscopy and sample observation method

It is an object of the invention to provide a stage for scanning probe microscopy that can be used in any kind of SPM and can effectively irradiate light to a sample and a solution near the sample without irradiated light blocked by a cantilever. The stage for scanning probe microscopy of the invention is a stage for scanning probe microscopy for fixing a sample substrate that mounts a sample to be observed thereon and has optical transparency and includes an opening that is provided below a portion where the sample substrate is fixed and that has an opening area included within the sample substrate in plan view. Light is irradiated from a bottom surface of the sample substrate onto the sample through the opening.

TECHNICAL FIELD

The present invention relates to a stage for scanning probe microscopy used to fix a sample substrate for mounting a sample thereon in a scanning probe microscopy and a sample observation method using the stage.

BACKGROUND ART

A scanning probe microscopy (hereinafter, referred to as an ‘SPM’) is a general term for microscopes that observe the three-dimensional shape of a sample surface as physical information of the sample surface with high magnification of nanometer size by scanning the sample surface using a fine probe (cantilever) as a probe. SPMs may be classified into various types of SPMs according to which kind of physical quantity of a sample the probe detects, as shown inFIG. 10.

In addition, the SPMs are very advantageous particularly in the evaluation of a semiconductor device, the evaluation of the quantum structure, and the observation of a biomolecule and are mentioned as observation apparatuses which will develop hereafter. That is, for the semiconductor device, high vertical resolution of the SPM is expected since observation of irregularities of a thin film is required as a thin film technique is developed. In addition, the SPM is used since the electrical property of a semiconductor material in which impurities (dopant atoms) are mixed can be measured with high precision and a distribution situation of the dopant atoms in the semiconductor material can be evaluated.

Furthermore, in evaluation of the quantum structure, the SPM is used as almost a sole observation means for evaluating the quantum structure of each shape of for example, a quantum dot or quantum wire having a size of about 10 nm with high spatial resolution regarding electrical and optical properties. Moreover, in observation of a biomolecule, the SPM can obtain high spatial resolution and does not damage the biomolecule because electron irradiation or observation under vacuum is not performed and accordingly, the biomolecule can be observed in a live state. For the observation of biomolecules, an atomic force microscope (hereinafter, referred to as an ‘AFM’) and a near-field scanning optical microscope (hereinafter, referred to as an ‘NSOM’) are especially used, and a range of utilization of these microscopes is expected to extend.

Regarding the observation of biomolecules, by the use of the AFM or NSOM, it became possible to observe how protein moves in an antigen-antibody reaction to change the bond or to observe generation of a force or movement of myosin concerned with muscular contraction and a process of transport of an ATP (adenosine triphosphate) molecule, which is fluorescently labeled, by using protein in a live state. On the other hand, in the case of the AFM, not only two-dimensional imaging indicating the height and hardness of a sample can be performed by contact, dynamic, and phase modes, but also controls of force modulation, a magnetic force (MFM), a current, a surface potential (KFM), nanoindentation for making a hole in a sample in a nanometer size, the atmosphere can be comparatively easily performed. In addition, since there is no limitation in a sample, a biological sample can also be measured.

However, even in the case of SPMs, a large apparatus needs to be remodeled in order to change the environment around a sample, especially an optical environment. This was a big problem in terms of both cost and remodeling time. For this reason, there was almost no technique allowing light to be irradiated onto a sample in the SPM. On the other hand, since irradiation of light onto a sample has been generally performed in an optical microscope until now, a function of light irradiation for dissociating a caged compound is typically mounted as an additional function of a microscope commercially available.

Here, the caged compound refers to a compound which confines a material, which gives the activity, with another molecule, which reacts to light (light having a specific wavelength), as a method of controlling the activity in a biomolecule, for example. Specifically, the caged compound indicates a compound obtained by bonding a photodissociable protecting group, such as a nitrobenzene group, to a bioactive material in order to control the activity of the bioactive material. That is, the bioactive material is in a physiologically inactive state when the bioactive material is bonded to the photodissociable protecting group. However, the state of the bioactive material can be changed to an active state by dissociating the bonding between the bioactive material and the photodissociable protecting group by irradiation of ultraviolet rays and the like, and drug stimulation can be given to a biomolecule by the bioactive material.

That is, the composition of molecules included in a solution can be changed by irradiating light onto the caged compound. Accordingly, for example, an operation of giving drug stimulation can be performed on a cell at a required timing. Since a laser beam is irradiated as the light described above, it is necessary to dispose optical components, such as an optical path, a condensing lens, and an iris with high precision.

Furthermore, since a device of the SPM is expensive, many researchers share one SPM in many cases. For this reason, the device should be simply installed and detached, but there have been no such optical devices. A method of directly irradiating light from the outside, for example, with a laser using an optical fiber may also be considered. Also in this case, an adjustment of an optical axis of a laser beam is complicated and the system size becomes large. Accordingly, it is not possible to easily detach the device from the SPM.

Regarding irradiation of light onto the sample described above, a high-speed atomic force microscope has been newly developed, being provided as an SPM capable of performing irradiation of light onto a sample (for example, refer to JP-A-2005-106790). The technique of irradiation of light onto a sample disclosed in JP-A-2005-106790 is applied to a case where an optical microscope is combined to the SPM. Accordingly, the technique is based on a light irradiation technique using an optical microscope in the related art, and reaction caused by the light irradiation is restricted to a case of using a caged compound.

In the SPM disclosed in JP-A-2005-106790, a scanning mechanism that relatively scans a cantilever existing in a solution and a substrate, on which a sample is mounted, in a state where the sample is dipped in the solution and an irradiation mechanism that irradiates release light to a photodissociable protecting group of a caged compound existing in the solution or in the sample are provided. However, the irradiation mechanism in the SPM is formed integrally with the device of the SPM and has a large-scale, expensive, and complicated system configuration. Since the irradiation mechanism is united with the device of the SPM, the irradiation mechanism cannot be shared for other SPMs, which is a disadvantage. Furthermore, in the case of the SPM described above, since a cantilever exists between a light source and a sample when irradiating light onto a solution near the sample, the light is blocked by the cantilever. As a result, irradiation of light cannot be effectively performed on the sample or the solution, onto which the light needs to be irradiated most, located below the cantilever.

DISCLOSURE OF INVENTION

The invention has been finalized in view of the drawbacks inherent in the related art, and it is an object of the invention to provide a stage for scanning probe microscopy that can be used in any kind of SPM and can effectively irradiate light to a sample and a solution near the sample without irradiated light being blocked by a cantilever.

According to an aspect of the invention, a stage for scanning probe microscopy for fixing a sample substrate that mounts a sample to be observed thereon and has optical transparency includes an opening that is provided below a portion where the sample substrate is fixed and that has an opening area included within the sample substrate in plan view. Light is irradiated from a bottom surface of the sample substrate onto the sample through the opening.

In the stage for scanning probe microscopy according to the aspect of the invention, preferably, a light irradiating portion that irradiates the light having a wavelength which affects the sample is provided within the opening.

In the stage for scanning probe microscopy according to the aspect of the invention, preferably, the light irradiating portion has a light source.

In the stage for scanning probe microscopy according to the aspect of the invention, it is preferable to further include: a light source portion configured to include a plurality of light sources that emit light components having different wavelengths; and a waveguide corresponding to light having each wavelength from the light source portion. Each waveguide is combined with the light irradiating portion, and the light sources are ON/OFF controlled to irradiate the light having a desired wavelength onto the sample.

In the stage for scanning probe microscopy according to the aspect of the invention, preferably, light is irradiated from the light irradiating portion using an LED as a light source.

In the stage for scanning probe microscopy according to the aspect of the invention, preferably, a battery connected to the light source by a wiring line is provided, as a power source of the light source, at the position distant from the stage for scanning probe microscopy.

In the stage for scanning probe microscopy according to the aspect of the invention, preferably, the sample substrate is a mica substrate

According to another aspect of the invention, a sample observation method of fixing a sample substrate, which mounts a sample to be observed thereon and has optical transparency, to a stage for scanning probe microscopy and observing the sample with a scanning probe microscopy includes irradiating light from a bottom surface of the sample substrate onto the sample through an opening that is provided below a portion where the sample substrate is fixed and that has an opening area included within the sample substrate in plan view.

In general, observation of a sample is performed by mounting biomolecules on a sample substrate formed of for example, mica, which is flat at an atomic level, on slide glass serving as a sample stage. According to the invention, the stage for scanning probe microscopy, the sample, and the sample substrate (platform) are configured to be unified by the configuration described above. Accordingly, since a function of light irradiation can be given to the sample stage itself, it becomes possible to directly fix the sample substrate on a light irradiation base. This allows light to be easily irradiated from the bottom surface of the sample substrate.

Furthermore, as described above, since the stage for scanning probe microscopy, the sample, and the sample substrate (platform) are configured to be unified in the stage for scanning probe microscopy according to the invention, the stage for scanning probe microscopy can be easily attached to or detached from a SPM. As a result, the stage for scanning probe microscopy can be commonly used in a plurality of SPMs. Furthermore, according to the invention, since light irradiation from the bottom surface of the sample substrate is performed, it is possible to perform SPM observation in the related art by placing the sample substrate below the cantilever of the SPM. Furthermore, according to the invention, the shape and material (in addition to glass, a change to plastic or metal is easy) which were difficult in the related art can be used. For example, a cylindrical plate or a plate with a hole opened may be used as a sample stage. Accordingly, versatility and operability are improved.

Furthermore, according to the invention, by providing the stage for scanning probe microscopy instead of a slide glass generally used as a stage for scanning probe microscopy of an SPM currently used, a function of performing light irradiation from a lower portion of the sample substrate can be provided with a simple and cheap configuration (the SPM is very expensive and it imposes a large burden on a purchaser to buy the SPM again. In addition, attachment of the SPM is also expensive).

In addition, according to the invention, since a plurality of light sources (LEDs) corresponding to light components with wavelengths required for an experiment are provided, light (from an infrared ray to ultraviolet light) with a desired wavelength used for a sample to be observed can be easily irradiated by changing a light source. Furthermore, according to the invention, the versatility is improved since a simple structure is used without using various optical components, unlike the related art. In addition, since the structure is simple as compared with the related art, it does not easily break. Furthermore, according to the invention, unlike a laser light source in the related art, a battery, such as a dry cell or a secondary battery, is used without generating a DC voltage from an AC power source as power of a light source. Accordingly, since an electromagnetic noise originating from a power source can be suppressed, an increase in a measurement noise in the SPM can be prevented by suppressing the measurement noise to a minimum.

BEST MODE FOR CARRYING OUT THE INVENTION

First Embodiment

Hereinafter, a stage for scanning probe microscopy according to a first embodiment of the invention will be described with reference to the accompanying drawings.FIG. 1is a block diagram illustrating an example of the configuration of a stage for scanning probe microscopy according to the first embodiment. Referring toFIG. 1, a stage for scanning probe microscopy1is configured to include: a container11that has a cylindrical shape with a hole H11and has an opening in an upper portion (surface on a side where a sample substrate12to be described later is disposed) of a tube; the sample substrate12that is a planar substrate which is provided on the upper portion of the hole H11in the container11in the axial direction of the tube such that the substrate surface is perpendicular to the axial direction, serves to fix a sample100to be observed on an upper surface thereof, and is formed of mica, for example; a light source13that irradiates light, which has a wavelength set beforehand, from the bottom surface direction of the sample substrate12to the sample; a power source14of the light source13; and a switch15that switches between ON (irradiation of light) and OFF (no irradiation of light) of the light source13.

The light source13is an LED, for example, is provided in a lower portion of the sample substrate12within the container11, and irradiates light from a bottom surface of the sample substrate12to the sample100on a top surface of the sample substrate12. The power source14(for example, a battery) of the light source13and the switch15which performs an ON/OFF control of the light source13are disposed at positions outside the container11, which are distant from a measurement place, and are connected to the light source13in the container11by wiring lines16(for example, thin electric wires), such that vibration and noises when turning on or off the switch15are not given to a measurement system of the SPM. The sample100is dipped in a solution102adhered on the sample substrate12due to the surface tension. In addition, when this sample100is observed, a cantilever101which is a probe of the SPM is provided in a state close to the sample100in the solution102.

In a sample observation method of the SPM in the present embodiment, a change of the sample100is measured by fixing the sample substrate12which fixes the sample100thereon to an upper opening of the container11and irradiating light of the light source13from the bottom surface side of the sample substrate12. Thus, according to the present embodiment, the light is irradiated onto the sample100from a side opposite the cantilever101. Therefore, unlike the related art, light from the light source13is not blocked and a sufficient amount of light can be irradiated onto the sample100.

In addition, a smaller stage for scanning probe microscopy than that in the related art can be configured by using an LED as the light source13and using a battery (small battery, such as a button battery) as the power source14. In addition, unlike the related art, a mechanism for irradiating light onto the sample in the present embodiment is not fixed to a main body of the SPM. Accordingly, the light irradiating mechanism can be easily attached to or detached from an SPM commercially available, as a stage for scanning probe microscopy. In addition, by configuring the stage for scanning probe microscopy in the same shape as stages for scanning probe microscopy of other SPMs commercially available, an exchange can be easily performed. As a result, attaching and detaching can be performed quickly and accordingly, and the stage for scanning probe microscopy can be commonly used in a plurality of SPMs. As the sample substrate12, only a mica substrate which fixes the sample100thereon may be used or that obtained by fixing the mica substrate on an upper surface of a substrate formed of a material having optical transparency, such as slide glass.

Furthermore, a tube (having the hole H11) having an opening, in which a surface at the position where the sample substrate12is disposed is opened, may be used as the container11as shown inFIG. 1or a plate11B having a hole H11B with an opening provided on a surface at the position where the sample substrate12is placed may be used as shown inFIG. 2. The sample substrate12is disposed to overlap the opening of the hole H11of the tube or the opening of the hole H11B of the plate11B in plan view (when viewed in parallel to the depth direction of a hole). In addition, the area of the sample substrate12in plan view is set to be greater than or equal to the opening area of the opening of each of the hole H11and the hole H11B in plan view. The hole H11B is formed in a direction perpendicular to a surface of the plate11B. The openings of the hole H11and hole H11B and the inside light source13form a light irradiating portion20that irradiates light onto the sample100.

Furthermore, in the case shown inFIG. 2, a stage for scanning probe microscopy1is configured to include: a plate11B that has a planar substrate shape with a hole H11B and has an opening in an upper portion (surface on a side where the sample substrate12to be described later is disposed) of the hole H11B; the sample substrate12that is a planar substrate which is provided on an upper portion of a tube, which is formed by an inner peripheral surface of the hole H11B in the plate11B, in the axial direction thereof such that the substrate surface is perpendicular to the axial direction, serves to fix the sample100to be observed on an upper surface thereof, and is formed of mica, for example; a light source13that irradiates light, which has a wavelength set beforehand, from the bottom surface direction of the sample substrate12to the sample through the hole H11B; a power source14of the light source13; and a switch15that switches between ON (irradiation of light) and OFF (no irradiation of light) of the light source13. In addition, glass, acrylic, metal, and the like may be used as a base material for forming the container11and the plate11B.

Since the sample substrate12on which the sample is fixed is disposed on the opening of the hole H11of the container11or the opening of the hole H11B of the plate11B and the light source13is disposed below the sample substrate12within the hole H11or the hole H11B, light is irradiated onto the sample100from a direction opposite the cantilever101. Therefore, as already described above, light irradiated from the light source13is not blocked by the cantilever101and a sufficient amount of light can be irradiated onto the sample100. In addition, if a material having high light reflectance, such as metal, can be used as a base material for forming the container11and the plate11B, it is possible to increase the intensity of light irradiated onto the sample100from a bottom surface of the sample substrate12placed on the opening of the container11or the plate11B.

Since the switch15which performs the ON/OFF control of the light source13is provided at the end of the wiring line16, mechanical vibration generated when touching a switch does not cause any trouble in measurement, and irradiation of light can be easily performed at the necessary timing during the measurement. In addition, since a battery is used, a DC voltage is not generated from an AC power source by a regulator, for example. Accordingly, noise caused by the emission of light of the light source13and the measurement system can be suppressed, causing no problems in observation.

Furthermore, the wavelength of light irradiated to a sample or a solution near the sample can be easily changed by using an LED (light emitting diode) or a laser diode as the light source13. Accordingly, the influence of various lights on other kinds of samples can be observed by changing the LED or the laser diode to that of the required wavelength. For example, as wavelengths of the LED, there are wavelengths corresponding to infrared light to UV (ultraviolet) light, and the wavelength of light emitted is determined by a semiconductor material. The wavelength range of LEDs commercially available is 360 nm to 1550 nm in many cases. Furthermore, in the case of emitted light of the LED, a luminescent color is a single color, that is, the wavelength range is narrow, unlike sunlight or an electric lamp. Accordingly, only light with a required wavelength can be irradiated onto a sample. Moreover, in addition to LEDs that emit light (visible light) having a wavelength in a range of about 380 nm to 780 nm (approximately, 450 nm in the case of a blue color, 520 nm in the case of a green color, 590 nm in the case of a yellow color, and 660 nm in the case of a red color) which is visible to human beings, many LEDs that emit ultraviolet light (ultraviolet ray) having a wavelength shorter than 380 nm and infrared light (infrared ray) having a wavelength exceeding 780 nm also exist.

As described above, by using LEDs having different wavelengths and adopting the configuration where light is not blocked by the cantilever, deterioration of a material of a sample to be observed may also be observed on a nanometer scale. For example, it also becomes possible to observe how harmful UV light acts and affects a biological sample with a nanometer size.

In addition, as shown inFIGS. 3A and 3B, it is also possible to dissociate a caged compound by irradiation of UV light.FIG. 3Aillustrates that ligand is bonded to a photodissociable protecting group to become a caged compound, resulting in a physiologically inactive state with respect to the sample100. Here,FIGS. 3A and 3Bare conceptual views illustrating receptor protein of the sample100which is fixed on the sample substrate12and is active in the solution102. On the other hand,FIG. 3Billustrates a state where ligand, that is, a material that is bonded to receptor protein to become active, or a material that suppresses the activity is separated from a photodissociable protecting group by irradiating UV light, resulting in a bioactive state bonded to the sample100(for example, receptor protein).

Here, the caged compound refers to a compound which confines a material (ligand), which gives the activity, with another molecule, which reacts to light having a specific wavelength, as a method of controlling the activity in a biomolecule. Specifically, the caged compound indicates a compound obtained by bonding a photodissociable protecting group, such as a nitrobenzyl group, to ligand, that is, a bioactive material. Therefore, the ligand is physiologically inactive in a state where the photodissociable protecting group is bonded to the ligand, but the state of the ligand can be changed to a bioactive state by dissociating the bonding between the ligand and the photodissociable protecting group by irradiation of UV light. As a result, drug stimulation can be given to a biomolecule at an arbitrary timing.

As shown inFIG. 3A, the caged compound is contained in the solution102and there is a site (ligand binding site), to which ligand is bonded, in the receptor protein. However, the caged compound on its own is not bonded to the ligand binding site. In addition, by irradiating UV light to the solution102, the caged compound in the solution102is dissociated to thereby discharge the ligand into the solution102, as shown inFIG. 3B. As a result, since the ligand is bonded to the ligand binding site of receptor protein, the shape of the receptor protein is changed due to the drug stimulation that the receptor protein receives.

In the case of dissociating a caged compound by the use of UV light, various kinds of stimuli may be given depending on the kind of ligand that forms a caged compound. Examples of ligand include ATP (Adenosine TriPhosphate), AMP (Adenosine MonoPhosphate), GTP (Guanosine TriPhosphate), GMP (Guanosine MonoPhosphate), glutamic acid, Ca2+, and an inositol phosphoric acid. Moreover, in the case where the UV light is used and the caged compound is not used, the response excited by photoreaction can be observed in a nanometer level. For example, applications to molecular level observation of deterioration caused by UV light, molecular structure observation of protein related to photosynthesis performed by visible light, and a structural change of polymer, such as a light curing resin, caused by light may be made.

In addition, by using cleaved mica for the sample substrate12, it is possible to easily obtain a transparent (light transmissive) substrate, which has a smooth flat surface without contamination in an atomic level, as the sample substrate12each time (whenever a sample is observed). In addition, a change in an electrical property caused by light irradiation may also be monitored by coating a metal, such as ITO (Indium Tin Oxide), on a mica surface. The configuration described can be easily used in the same manner in any kind of SPM. In addition, in configurations where the temperature of the sample substrate12can be changed, the sample can be observed at various temperatures in the stage for scanning probe microscopy. In this case, in order to change the temperature of the sample substrate12, there are methods such as warming, by building a heater into the sample substrate12, cooling by building a peltiert device into the sample substrate12, and circulation of a solvent of glycerol and the like through a tube in close contact with the sample substrate12that can be used.

Second Embodiment

The configuration in which only one LED is used may be adopted as described in the first embodiment, but a configuration in which one of a plurality of LEDs is selected according to the wavelength used and light having an arbitrary wavelength is irradiated onto the sample100may also be adopted like a second embodiment. Moreover, in the second embodiment, it may be possible to use a configuration in which the plurality of LEDs are sequentially selected one by one and a plurality of light beams having different wavelengths are irradiated onto the sample100in a time-sequential manner such that a change corresponding to a wavelength may be observed.

FIGS. 4A to 4Cillustrate that an LED box30where a plurality of LEDs (LED1, LED2, LED3, . . . ), from which light beams having different wavelengths are emitted, are built is provided in a stage for scanning probe microscopy according to the second embodiment.FIG. 4Ais a top view illustrating the stage for scanning probe microscopy according to the present embodiment, andFIGS. 4B and 4Care cross-sectional views taken along the lines A-A ofFIG. 4A. Also in the present embodiment, the cantilever101shown inFIGS. 1 and 2is provided at a position opposite an upper surface (surface of an upper portion of the sample substrate12shown inFIGS. 4B and 4C) of the sample substrate12. Therefore, since light irradiated onto the sample100is irradiated from a bottom surface of the sample substrate12onto the sample100through a hole H11B, the light is not blocked by the cantilever101in the same manner as inFIGS. 1 and 2.

In the configuration shown inFIGS. 4A to 4C, the LED box30is disposed at the position distant from the sample substrate12and light emitted from each LED is guided to the hole H11B through an optical fiber using an optical waveguide40(for example, a slab type waveguide) corresponding to each LED and is then reflected by a mirror51, such that the light emitted from each LED is irradiated onto the sample100. Although not shown in the drawings, in each LED, a power source14and a switch15that controls whether to supply driving power from the power source14are provided at positions distant from the stage for scanning probe microscopy in the same manner as inFIG. 1or2. Light having an arbitrary wavelength can be irradiated onto the sample100by turning on/off the switch15of each LED.

In the configuration shown inFIG. 4B, the LED box30is disposed on an upper surface of the plate11B, and a hole53is provided below the LED box30disposed, and a mirror52is provided in a bottom portion of the hole53for every LED. Light is emitted from each LED to the mirror52through the optical fiber. In addition, for every mirror52, the optical waveguide40is provided between the hole53and the hole11B corresponding to the LED. Light that each LED of the LED box30emits is incident on the mirror52provided for every LED of the LED box30and is then emitted to the optical waveguide40. Then, the light emitted from each optical waveguide40is reflected by the mirror51, which is provided in a bottom portion of the hole H11B for every optical waveguide40, and is then emitted to the sample100. That is, the light emitted from each LED to the sample100is irradiated from the lower portion of the sample substrate12.

Furthermore, in the configuration shown inFIG. 4C, the LED box30is disposed within the hole53, and light emitted from each LED is directly incident on the optical waveguide40corresponding to the LED. Then, the light emitted from each optical waveguide40is reflected by the mirror51, which is provided in the bottom portion of the hole H11B for every optical waveguide40, and is then emitted to the sample100, such that the light emitted from each LED is irradiated onto the sample100. That is, the light emitted from each LED to the sample100is irradiated from the lower portion of the sample substrate12.

InFIGS. 4B and 4C, the opening of the hole H11B described above and the mirror51positioned in the bottom portion of the hole H11B form the light irradiating portion20serving to irradiate light onto the sample100. Then, the light emitted from each optical waveguide40is emitted to the sample100by the mirror51, which is provided in the bottom portion of the hole H11B for every optical waveguide40. In addition, by turning on an LED corresponding to a wavelength required for observation of a sample by making a selective ON/OFF control using the outside switch15, the light from the opening of the hole H11B can be irradiated onto the sample. Since the sample100can be placed on the periphery including a portion where light is irradiated, it is possible to irradiate light from the lower portion of the sample substrate12while making an observation from the upper portion of the sample100with the cantilever101.

Furthermore, as described above, the optical waveguide40may be built within the stage for scanning probe microscopy or be provided on a stage surface as long as a mechanism in which light is irradiated from the lower portion of the sample substrate12onto the sample100is eventually obtained. The optical waveguide40needs to be formed of a material allowing propagation of light with a desired wavelength, which is emitted from an LED corresponding to each optical waveguide40, so as not to attenuate. Although not shown inFIGS. 4A to 4C, the power source14or the switch15may also be placed to be distant from the stage for scanning probe microscopy in the same manner as in the first embodiment. In addition, regarding which LED in the LED box30is to be selected, a program for sequence control may be created beforehand so that the switch15can be automatically controlled by operating a computer using the program.

Since the light source13is distant from the sample substrate12, noise can be avoided more efficiently. In addition, by the size of the opening of the hole H11B, irradiation of light onto the sample substrate12over a wide or narrow range (i.e., an irradiated region) can be controlled. Although a plurality of LEDs are disposed in the LED box30in the above description, it is also possible to dispose only one LED in the LED box30and to use only one wavelength in the same manner as in the first embodiment.

Next,FIGS. 5A and 5Billustrate that the LED box30is disposed at the position distant from a stage body for scanning probe microscopy and light emitted from each LED is incident on an optical fiber60corresponding to each LED. In addition, each optical fiber60is connected to the optical waveguide40, which is provided for every optical fiber60, using an optical connector55.FIG. 5Ais a top view illustrating a stage for scanning probe microscopy having another configuration according to the present embodiment, andFIG. 5Bis a cross-sectional view taken along the line A-A ofFIG. 5A. The optical waveguide40is provided with a light receiving port, that is, a connecting portion for connecting the optical connector55on a side surface of the plate11B.

In the configuration shown inFIGS. 5A and 5B, the opening of the hole H11B described above and the mirror51positioned in the bottom portion of the hole H11B form the light irradiating portion20serving to irradiate light onto the sample100. Also in the present embodiment, the cantilever101shown inFIGS. 1 and 2is provided at the position opposite the upper surface (surface of the upper portion of the sample substrate12shown inFIG. 5B) of the sample substrate12. Therefore, since light irradiated onto the sample100is irradiated from the bottom surface of the sample substrate12onto the sample100through the hole H11B, the light is not blocked by the cantilever101in the same manner as inFIGS. 1 and 2.

Light emitted from each LED of the LED box30is irradiated onto the sample100from the lower direction of the sample substrate12through the optical fiber60, the optical connector55, and the minor51. The mirror51is provided for each optical waveguide40in the same manner as the configuration shown inFIGS. 4A to 4C. The light emitted from each LED of the LED box30is incident on the optical waveguide40through the optical fiber60and the optical connector55. Light emitted from the optical waveguide40corresponding to each LED is reflected by the mirror51and is then irradiated from the bottom surface of the sample substrate12to the sample100through the hole H11B. Although not shown in the drawings, in each LED, the switch15that controls whether to supply driving power from the power source14is provided in the same manner as inFIG. 1or2. Light having an arbitrary wavelength can be irradiated onto the sample100by turning on/off the switch15of each LED. The power source14and the switch15are disposed at positions distant from the LED box30and are connected to each LED of the LED box by wiring lines.

Next,FIGS. 6A and 6Billustrate that a light source box35, which emits light irradiated onto a sample, is disposed at the position distant from the plate11B in the same manner as inFIGS. 5A and 5B.FIG. 6Ais a top view illustrating a stage for scanning probe microscopy having still another configuration according to the present embodiment, andFIG. 6Bis a cross-sectional view taken along the line A-A ofFIG. 6A. The light source box35is different from the LED box30shown inFIGS. 4A to 4Cand5A and5B. In the light source box35, a light source which emits natural light having electromagnetic waves (light) with a number of wavelengths is provided instead of an LED. The light with a number of wavelengths that the light source emits is incident on an AWG (Arrayed Waveguide Grating) wavelength demultiplexer70as multiple wavelength light by the optical fiber61. The AWG wavelength demultiplexer70is an example of a wavelength divider and divides the incident multiple wavelength light into light having each wavelength (wavelength range region) set beforehand. A switch71is provided corresponding to each divided light. Each divided light is incident on the optical waveguide40from each optical switch71through the corresponding optical fiber60. The light incident on the optical waveguide40is reflected by the mirror51and is then emitted from the bottom surface of the sample substrate12to the sample100through the hole H11B, in the same manner as the configuration shown inFIGS. 4A to 4C.

Although not shown in the drawings, in the light source box35, the power source14and the switch15that controls whether or not to supply driving power from the power source14are provided at positions distant from the stage for scanning probe microscopy in the same manner as inFIG. 1or2. In addition, light having an arbitrary wavelength can be irradiated onto the sample100by turning on/off the switch71provided for every divided light. In addition, the optical waveguide40provided in the plate11B may be configured inside the plate11B as shown inFIGS. 6A and 6Bor may be provided on a surface of the plate11B. The AWG wavelength demultiplexer70may be provided on the upper surface of the stage in the same manner as inFIGS. 4A to 4C. In this case, light emitted from the light source box35is incident on the optical waveguide40from a side surface of the plate11B through the optical connector55by the optical fiber60. A silicon substrate is generally used as the plate11B. However, a conductor, a semiconductor, and an insulator may also be used as long as they do not allow light to be transmitted therethrough (the same is true for the container11shown inFIGS. 1 and 2).

Next, an example of the application using the stages for scanning probe microscopy according to the first and second embodiments will be described with reference toFIGS. 7A to 7Cand8A to8C.FIGS. 7A to 7Cand8A to8C illustrate AFM images (surface images of receptor protein on the sample substrate12observed by the AFM) indicating results, which are obtained by observing receptor protein reconstructed in an artificial lipid bimolecular membrane, and conceptual views of the receptor protein analyzed by the AFM images. In this application, the observation was performed by using the AFM as an SPM.

FIGS. 7A and 8Aillustrate AFM images (that is, images observed by the cantilever101from the upper surface) of receptor protein,FIGS. 7B and 8Billustrate conceptual views obtained when the receptor protein is viewed from the side surface, andFIGS. 7C and 8Cillustrate conceptual views obtained when the receptor protein is viewed from the upper surface. In addition,FIGS. 7A to 7Care views illustrating a result of observation before irradiation of UV light, andFIGS. 8A to 8Care views illustrating a result of observation after irradiation of UV light.

The sample100is generated by reconstructing receptor protein in the artificial lipid bimolecular membrane, and the reconstructed sample100is incubated on mica, which is the sample substrate12, in temperature and time set beforehand for every sample and is then washed, thereby removing a sample that is not fixed on the sample substrate12. Then, the sample100was observed in the solution102using the cantilever101of the AFM. In the solution102, a caged glutamic acid of 2 mM (mol) is dissolved.

As described above,FIGS. 7A to 7Cshow the AFM image before receiving irradiation of UV light, andFIGS. 8A to 8Cshow the AFM image after receiving irradiation of UV light. In each of the drawings, a lump that a protruding portion constitutes indicates one molecule of receptor protein. That is, in each ofFIGS. 7A and 8A, a portion surrounded by a dotted line is the protruding portion and one molecule of the receptor protein is formed by each protruding portion. InFIG. 7A, one large protruding object and two small protruding objects are observed, and a planar shape including these three protruding objects is a shape similar to a square (approximately square shape).

That is, the structure of the receptor protein shown inFIG. 7Ais theoretically a tetramer structure as shown inFIGS. 7B and 7C. Accordingly, subunits of the receptor protein are disposed at the positions symmetrical four times. In the AFM image shown inFIG. 7A, however, four protruding objects are not observed but one large protruding object and two small protruding objects, which are indicated by portions surrounded by dotted lines, are observed. Presumably, this is because the large protruding object surrounded by the dotted line is actually configured to include two small protruding objects (subunits) and accordingly, the distance between the protruding objects is so short as not to be observable with the resolution of the AFM, resulting in being observed as one protruding object (subunit).

Alternatively, it may also be considered that two protruding objects were observed as one protruding object since the protruding object shakes due to heat, for example, while observing the receptor protein of the sample100. In any case, it is thought that the square shape was observed as shown inFIG. 7Asince UV light was not irradiated, the caged glutamic acid which was a caged compound was not dissolved, and the receptor protein was placed under the environment of a non-bioactive state and accordingly, the distance between subunits of the receptor protein was narrow.

Next, inFIG. 8A, two large protruding objects surrounded by dotted lines are observed and a shape including the two protruding objects is a shape similar to a long and narrow elliptical shape. Presumably, this is because the caged glutamic acid in the solution102was dissolved, that is, the glutamic acid and a photodissociable protecting group were dissociated from each other by irradiation of UV light and accordingly, the receptor protein was activated, that is, stimulated by the glutamic acid and as a result, the three-dimensional shape between subunits changed and the structure of the receptor protein changed.

Furthermore, regarding observation of the two protruding objects in the AFM image shown inFIG. 8A, it is thought that as shown inFIGS. 8B and 8C, two subunits which form a tetramer were met and accordingly, the distance between the subunits became so small as to be less than the resolution and as a result, two protruding portions were observed as shown in an image view ofFIG. 8C. Furthermore, inFIG. 8Ait can be observed that a distance D between the two met protruding portions is long compared with that shown inFIG. 7A.

A graph shown inFIG. 9shows a change in the distance D between the two met protruding objects (structures). A vertical axis indicates the distance D and a horizontal axis indicates the time-series number (that is, equivalent to time) on a screen. Referring toFIG. 9, irradiation of UV light is performed in timing between a thirty-ninth frame and a fortieth frame of an AFM image. As can be seen from the graph shown inFIG. 9, it can be seen that before irradiation of UV light, the distance D between the structures fluctuates, that is, the distance D becomes small or large.

However, after irradiation of UV light, it could be checked that the distance D between the two structures, each of which was viewed as a circular shape, became large apparently and the fluctuation in the distance D was rarely observed. It is thought that this observation result was obtained since the caged glutamic acid in the solution102was dissociated by irradiation of UV light and the receptor protein was stimulated by the dissociated glutamic acid and as a result, the three-dimensional structure of the subunit changed as described above.