JP2005309415A - Optical microscope and optical observation method - Google Patents

Optical microscope and optical observation method Download PDF

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JP2005309415A
JP2005309415A JP2005086550A JP2005086550A JP2005309415A JP 2005309415 A JP2005309415 A JP 2005309415A JP 2005086550 A JP2005086550 A JP 2005086550A JP 2005086550 A JP2005086550 A JP 2005086550A JP 2005309415 A JP2005309415 A JP 2005309415A
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light
light beam
specimen
optical microscope
light source
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JP2005309415A5 (en
JP4819383B2 (en
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Shigeru Kobayashi
Ryoji Saito
Hajime Sasaki
元 佐崎
茂 小林
良治 斎藤
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Olympus Corp
オリンパス株式会社
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical microscope suitable to observe a transparent sample in real time in a non-destructive state. <P>SOLUTION: The optical microscope 100 is equipped with: an objective 107; a light source 101 for emitting a beam; a polarizer 102 generating the beam of linearly polarized light; a beam dividing and composing element 106 dividing the beam into two beams according to a polarizing component and also composing two beams returned from the sample 116; a two-dimensional scanning part 104 scanning with a light spot formed on the sample 116; a beam separating element 103 separating the beam returned from the sample 116; a pinhole 108 through which only the beam from the vicinity of an observation surface out of the beam returned from the sample 116 is selectively made to pass; an analyzer 109 taking out the beam of the prescribed linearly polarized light from the beam passing through the pinhole 108; and a detector 110 detecting the beam passing through the analyzer 109. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microscope (observation method) for optically observing the surface shape (step) of a transparent substance with high resolution.

  Conventionally, surface shapes of crystals, minerals, biological cells, and the like have been observed using an optical microscope, an electron microscope, an atomic force microscope, or the like.

  In particular, since the surface shape needs to capture minute changes in shape, the observation method needs to be devised, and an observation method suitable for the observation conditions is used.

  In general, an electron microscope has a high observation resolution and is excellent when a fine shape is viewed. However, since an electron microscope observes a specimen in a vacuum, it is used for observing a static specimen such as a material or a cell whose shape has already been fixed.

  In addition, the atomic force microscope (AFM) makes a cantilever equipped with a probe at the free end close to the sample, and scans the cantilever with respect to the sample to detect the deflection of the cantilever, thereby changing the shape of the sample surface. taking measurement. For example, if the probe is scanned while feedback controlling the height of the probe so that the atomic force acting on the atom at the tip of the probe and the atom on the sample surface is constant, the tip of the probe is at a certain distance from the sample surface. Trace the surface while moving up and down according to the unevenness of the surface. The vertical position of the tip of the probe reflects the unevenness of the sample surface, and is obtained by detecting the deflection of the cantilever. Therefore, the unevenness of the sample surface can be obtained from the scanning position and the vertical position of the probe. Examples of means for measuring the deflection of the cantilever include an optical lever method, an optical interferometer, and a piezoresistor. Of these, the optical lever method, which is the simplest means, is often used.

  AFM can capture a minute step of a few nanometers on a specimen. Some researchers have reported examples of observing fine shapes of protein crystals using AFM in the following literature.

1) SD Durbin, WE Carlson, J. Crystal Growth 122 (1992) 71.
2) SD Durbin, WE Carlson, MT, Saros, J. Phys. D 26 (1993) B128.
3) A. McPherson, AJ MaIkin, Yu.G. Kuznetsov, Annu. Rev. Biophys. Biomol. Struct. 29 (2000) 361.
4) JJ De Yoreo, CA Orme, TA Land, in: K. Sato, Y. Furukawa, K. Nakajima (Eds.), Advances in Crystal Growth Research Elsevier, Tokyo, 2001, p. 361.
5) AJ Malkin, A. McPherson, in: XY Lin, JJ De Yoreo (Eds.), Nanoscale Structure and Assembly at Solid-fluid Interface, Plenum Press / Kluwer Academic Publisher, New York, Dordrecht, in press.
In addition, there is a proposal of an example of measuring a fine step using light.

  Japanese Patent Application Laid-Open No. 9-42938 detects the amount of phase change of interference fringes by irradiating a sample with laser light that has passed through a Nomarski prism using an incident differential optical system and causing the reflected light to interfere with the Nomarski prism. Discloses a method for measuring the surface shape by measuring with a vessel. As a result, submicron order measurement is possible.

  Optical observation has advantages such as not causing damage due to impact from the outside, and not disturbing the crystal surface. It is a non-destructive and real-time technique that can be observed in-situ and can image dynamic changes. However, although the planar resolution of the optical microscope is limited to submicrons because it is limited by the wavelength of light, the vertical resolution can be made comparable to the vertical resolution of AFM by selecting an observation method. .

  There are several observation methods for optical microscopes, but there are many points that are difficult to visualize if the specimen to be observed is colorless and transparent. For example, when the sample absorbs little light and there is only a change of several tens of nanometers or less in the optical axis direction, the change in brightness and darkness of the optical image is only small, so that observation is difficult with a normal transmission observation method. Also in the reflective optical system, if the specimen is colorless and transparent and has a low reflectance, the return light reflected from the middle of the specimen or the optical system is much more than the return light that depends on the level difference of the specimen. It is difficult to observe a specimen having a step of several tens of nm or less in the axial direction.

  For this reason, a phase contrast microscope and a differential interference microscope are widely used as observation methods for observing the fine structure of a colorless and transparent specimen as a difference in brightness.

  The phase contrast microscope method is an observation method in which a difference in brightness of an observation image is generated by changing the phase of diffracted light from a sample reflecting information on a fine structure. Actually, the phase of the light that is directly transmitted (or reflected) without change in the sample is different from the phase of the diffracted light that reflects the fine structure of the sample, so the phase of the component of the directly transmitted (or reflected) light is the phase of the diffracted light. The light and the diffracted light are made to interfere with each other so that there is a difference between light and dark. This method is widely used for observing unstained culture specimens because a change in specimen structure in the optical axis direction of about 10 nm can be detected by the difference in brightness of the image. However, the contour portion of a specimen having a large shape change may be too bright to observe clearly because not only diffracted light but also scattered light and refracted light are mixed. Also, the phase contrast microscope has a low resolution due to the system structure that illuminates with a NA that is significantly smaller than the NA of the objective lens. For this reason, the differential interference microscopy described below is widely used to perform high-resolution observation in the direction perpendicular to the optical axis direction.

  Differential interference (shearing interference) microscopy is an observation method in which a sample is irradiated with two light beams separated by several tens of nanometers to several μm, and after passing through the sample, these two light beams are combined and interfered with each other. Similarly, it is an observation method for visualizing the fine structure of a transparent specimen as light and dark. Normally, in a microscope, polarized light is incident on a birefringent prism (Nomarski prism), combined with an illumination condenser lens, illuminated with two light beams that are slightly separated, and placed in the objective lens and the observation optical path. The birefringent prism (Nomarski prism) makes the light beam one again, and finally the polarizer makes the two superposed light beams interfere with each other to visualize the fine structure of the specimen. In this method, the phase difference (for example, step difference or refractive index difference) of the sample in the optical path through which the two light beams pass can be observed as bright and dark, and a minute step of about 10 nm in the optical axis direction can be detected as in the case of the phase contrast microscope. . Further, unlike the phase contrast microscope, there is no restriction on the illumination NA, so that the resolving power in the direction perpendicular to the optical axis can be exhibited to the limit of the microscope objective lens. Furthermore, the contour does not shine compared to the phase difference method, and an image with high resolution can be obtained.

An epi-illumination example in which fine irregularities on the specimen surface are observed using such an observation system is also introduced in Applied Physics Vol. 60, No. 9, 1991.
JP-A-9-42938 SD Durbin, WE Carlson, J. Crystal Growth 122 (1992) 71. SD Durbin, WE Carlson, MT, Saros, J. Phys. D 26 (1993) B128. A. McPherson, AJ MaIkin, Yu.G. Kuznetsov, Annu. Rev. Biophys. Biomol. Struct. 29 (2000) 361. JJ De Yoreo, CA Orme, TA Land, in: K. Sato, Y. Furukawa, K. Nakajima (Eds.), Advances in Crystal Growth Research Elsevier, Tokyo, 2001, p. 361. AJ Malkin, A. McPherson, in: XY Lin, JJ De Yoreo (Eds.), Nanoscale Structure and Assembly at Solid-fluid Interface, Plenum Press / Kluwer Academic Publisher, New York, Dordrecht, in press. Applied Physics Vol.60 No.9 1991

  In order to perform in-situ observation of a specimen, it is necessary to observe the specimen in a state where a predetermined storage environment is maintained, and preferably to be able to freely control the environment during observation. For example, in the case of biological specimens or protein crystal specimens, the specimens should be observed while stored in a predetermined medium, and the conditions (temperature, solution concentration, etc.) of the medium should be controlled during observation using a reflux device. Is done.

  The electron microscope has a low degree of freedom, such as the condition settings cannot be easily changed during observation. There are also restrictions on the specimens that can be observed. Electron microscopes are effective for observing materials with fixed shapes, but cannot maintain a vacuum on specimens that are filled with water or solutions, such as cultured cells and protein crystal growth processes. Moreover, since the target specimen cannot be directly irradiated with electrons, it cannot be observed in the first place.

  An atomic force microscope (AFM) cannot sufficiently increase the scanning speed due to poor followability of cantilever scanning with respect to a surface step. For example, when a specimen stored in a medium is to be observed, the cantilever must be approached from a position far from the specimen on the specimen storage container (such as a petri dish). Therefore, since the scanning portion is enlarged, the scanning speed cannot be increased, and it takes several minutes to acquire an image of one frame. That is, even if in-situ observation is possible, the observation speed is insufficient. If the scanning speed is increased forcibly, the risk that the probe supported by the cantilever contacts the stepped portion of the sample surface increases. When the probe comes into contact with the sample, the contact portion may be destroyed, making non-destructive observation difficult.

  In recent years, a light beam AFM has been developed that can acquire images of 10 frames or more per second. In this type of AFM, a cantilever scanning device is placed close to the observation surface (surface) of the specimen in order to ensure high speed. It is difficult to observe the specimen placed in the medium. It is also difficult to incorporate a control device for setting and changing the sample conditions.

  In Japanese Patent Laid-Open No. 9-42938, a stage is used as a scanning method for obtaining an entire image of a specimen. For this reason, the mass of the drive part is large, and it is not suitable for fast scanning, that is, high-speed image acquisition. Therefore, the various methods described above are not suitable for dynamic specimen observation because they lack real-time properties.

  It is an optical microscope that can observe a dynamic specimen without contact. The optical microscope can be observed from a distance from the sample to some extent, and since there is no movable probe such as AFM, the sample has a degree of freedom in setting the sample and is suitable for in-situ observation. Real time observation is also satisfied. However, the conventional optical microscope has a surface with insufficient observation capability.

  In the optical microscope, the observation methods that can detect the fine structure of a transparent specimen are the phase contrast microscopy and the differential interference microscopy as described above. To observe the fine structure of a transparent specimen, in transmitted illumination observation, if the step in the optical axis direction is about several nanometers (that is, one molecule of protein), phase difference microscopy or differential interference microscopy is used. However, observation is difficult because the detection signal is weak and buried in various noises. In addition, in epi-illumination observation, when phase contrast or differential interference microscopy is used, light passes through the specimen twice, so the amount of change in the optical path is doubled and the signal from the specimen structure becomes stronger. Reflected / scattered light from the surface and reflected light from the middle of the illumination optical system (especially the objective lens) is 1 digit or more stronger than transmitted illumination observation. Fine structure observation is difficult.

  The present invention has been made in consideration of such actual situations, and its purpose is to provide a new technique suitable for observing a transparent specimen in a non-destructive and real-time manner while ensuring the degree of freedom of installation. Is to provide.

  One aspect of the present invention is directed to an optical microscope suitable for observing a transparent specimen. An optical microscope according to the present invention includes an objective lens arranged near a specimen, a light source for emitting a light beam for observation, a polarizer for generating a linearly polarized light beam from the light beam emitted from the light source, and a polarizer A beam splitting / synthesizing element that divides the light beam into two light beams that are spatially separated according to the polarization component and combines the two light beams returning from the sample, and a light spot formed on the sample are two-dimensionally scanned. A two-dimensional scanning unit for directing the light beam from the polarizer to the objective lens, and separating the light beam returning from the sample through the objective lens from the light beam directed to the objective lens, and the light beam separation element Pinhole for selectively passing only the light beam from the vicinity of the desired observation surface in the sample out of the light beam returning from the sample separated by And an analyzer for extracting a light beam of a specific linear polarization from the light beam that has passed through the pinhole, a detector for detecting the light beam that has passed through the analyzer, and information detected by the detector and obtained from the two-dimensional scanning unit. And an image processing unit that forms an image of the observation surface in the specimen based on the position information of the light spot.

  One aspect of the present invention is directed to an optical observation method suitable for observing a transparent specimen. The optical observation method of the present invention is an optical observation method suitable for observing a transparent specimen, and irradiates the specimen by dividing the linearly polarized light flux into two light fluxes that are spatially separated according to the polarization component. The light spot formed on the sample is scanned two-dimensionally, the two light beams reflected by the sample are combined, the combined light beam is separated from the linearly polarized light beam irradiated on the sample, and the combined light beam Only a light beam from the vicinity of a desired observation surface in the sample is selectively extracted, a light beam of a specific linear polarization is detected from the light beam from the vicinity of the observation surface, and based on the detected information and the position information of the light spot To form an image of the observation surface of the specimen.

  ADVANTAGE OF THE INVENTION According to this invention, the new technique suitable for ensuring the freedom degree of an installation state and observing a transparent sample with non-destructive and real-time property is provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment This embodiment is directed to an optical microscope suitable for observing a transparent specimen. FIG. 1 schematically shows an optical microscope according to the first embodiment of the present invention.

  As shown in FIG. 1, an optical microscope 100 according to this embodiment includes a stage 115 for placing a specimen 116, an objective lens 107 disposed near the specimen 116, and a light source for emitting a light beam for observation. 101, a polarizer 102 disposed on the optical path between the light source 101 and the objective lens 107, a beam splitting / combining element 106 disposed on the optical path between the polarizer 102 and the objective lens 107, and the polarizer 102 and the beam splitting / combining. A two-dimensional scanning unit 104 disposed on the optical path between the elements 106, a deflection mirror 105 disposed on the optical path between the two-dimensional scanning unit 104 and the light beam splitting / combining element 106, a polarizer 102, and a two-dimensional scanning unit. And a light beam separation element 103 disposed on the optical path between the two.

  The optical microscope 100 of this embodiment is an inverted type. For this reason, the objective lens 107 is disposed below the sample 116. However, the optical microscope 100 may be an upright type, in which case the objective lens 107 is disposed above the specimen 116.

  The light source 101 is preferably a laser light source. The laser light source is not particularly limited in wavelength range or oscillation form. The laser light source may be a gas laser or a semiconductor laser. For example, a gas laser includes an Ar laser that emits light having a wavelength of 488 nm and a HeNe laser that emits light having a wavelength of 543 nm and 633 nm. In addition to laser light, light with high brightness and good light collecting properties can be used.

  The polarizer 102 is a polarizing plate and selectively transmits only a light beam having a specific polarization component among incident light beams. For this reason, the polarizer 102 generates a linearly polarized light beam from the light beam emitted from the light source 101.

  The light beam splitting / combining element 106 splits the light beam from the polarizer 102 into two light beams according to the polarization component. The two light beams are spatially separated, and their polarization components are orthogonal to each other. The two light beams split by the light beam splitting / combining element 106 pass through the objective lens 107 and irradiate the specimen 116 to form a light spot. Further, the beam splitting / combining element 106 combines the two beams returning from the sample 116 through the objective lens 107. Although not limited to this, the beam splitting / combining element 106 is generally composed of a Nomarski prism.

  The two-dimensional scanning unit 104 scans the light spot formed on the specimen 116 two-dimensionally. The two-dimensional scanning unit 104 may use a galvanometer mirror, for example. The two-dimensional scanning unit 104 may also use an acousto-optic element. FIG. 2 schematically shows a two-dimensional scanning unit 104 using a galvanometer mirror. As shown in FIG. 2, the two-dimensional scanning unit 104 includes, for example, a galvano mirror 121 having a reflecting surface that can swing around the X axis, and a galvano mirror having a reflecting surface that can swing around the Y axis. 122.

  The light beam sequentially reflected by the galvanometer mirror 121 and the galvanometer mirror 122 forms a light spot irradiated on the specimen 116. The light spot formed on the specimen 116 moves along the X axis corresponding to the swing of the galvano mirror 121 around the X axis, and along the Y axis corresponding to the swing of the galvano mirror 122 around the Y axis. Move. Accordingly, the light spot formed on the specimen 116 is formed into a rectangular area as shown in FIG. 2 by appropriately combining the swing of the galvano mirror 121 around the X axis and the swing of the galvano mirror 122 around the Y axis. It is also possible to perform raster scanning within the observation range.

  The deflection mirror 105 directs the light beam that has passed through the two-dimensional scanning unit 104 to the light beam dividing / combining element 106 and directs the light beam returned from the sample 116 to the two-dimensional scanning unit 104.

  The light beam separation element 103 directs the light beam from the polarizer 102 to the objective lens 107 and separates the light beam returning from the sample 116 through the objective lens 107 from the light beam directed to the objective lens 107. The light beam separating element 103 is constituted by, for example, a mirror that partially reflects and partially transmits light. The light beam separating element 103 is constituted by, for example, a mirror that reflects 20% of the light beam from the light source 101 and transmits 80% of the light beam returned from the sample 116. A mirror having an appropriate transmittance and reflectance may be selected according to the reflectance of the specimen.

  Further, the optical microscope 100 includes a pinhole 108 for selectively allowing only a light beam from the vicinity of a desired observation surface in the sample 116 out of the light beam returned from the sample 116 separated by the light beam separation element 103, and a pin An analyzer 109 for extracting a light beam having a specific linear polarization from a light beam that has passed through the hole 108, a detector 110 for detecting the light beam that has passed through the analyzer 109, information detected by the detector 110, and a two-dimensional scanning unit And an image processing unit 111 that forms an image of the observation surface of the specimen 116 based on the position information (scanning information) of the light spot obtained from 104.

  The pinhole 108 is disposed at a position optically conjugate with the observation surface in the specimen 116. As a result, the pinhole 108 allows the light beam from the vicinity of the observation surface in the sample 116 among the light beams returning from the sample 116 separated by the light beam separation element 103 to pass, but in the vicinity of the observation surface in the sample 116. It does not allow the light flux from the surface that is off the surface to pass. Here, “near the observation surface” means a range of about the focal depth including the observation surface.

  The analyzer 109 is a polarizing plate like the polarizer 102, and selectively transmits only a light beam having a specific polarization component among incident light beams. The analyzer 109 is arranged in a crossed Nicols relationship with respect to the polarizer 102. Therefore, the analyzer 109 selectively transmits linearly polarized light that is orthogonal to the linearly polarized light that passes through the polarizer 102. In other words, the analyzer 109 extracts a linearly polarized light beam orthogonal to the linearly polarized light transmitted through the polarizer 102 from the light beam that has passed through the pinhole 108.

  The detector 110 is composed of, for example, a photomultiplier tube. However, the detector 110 is not limited to this, as long as it has a photoelectric conversion function, and may be composed of a photodiode, a CMD, a CCD, or the like.

  As can be seen from the above description, the optical microscope 100 includes a scanning confocal epi-differential differential interference optical system.

  The light beam emitted from the light source 101 passes through the polarizer 102 and becomes a linearly polarized light beam. The light beam that has passed through the polarizer 102 is partially reflected by the light beam separation element 103 and travels toward the two-dimensional scanning unit 104. The light beam incident on the two-dimensional scanning unit 104 is deflected two-dimensionally by the two-dimensional scanning unit 104. The light beam that has passed through the two-dimensional scanning unit 104 is reflected by the deflecting mirror 105 and travels toward the light beam splitting / combining element 106. The light beam incident on the light beam splitting / combining element 106 is split into two linearly polarized light beams. The two divided light beams are spatially separated from each other in parallel to the optical axis, and the polarization components are orthogonal to each other. The two light beams are converged by the objective lens 107 and irradiated on the specimen 116 to form a light spot. The light spot formed on the specimen 116 is scanned two-dimensionally (for example, raster scanning) corresponding to the deflection of the light beam by the two-dimensional scanning unit 104.

  The two light beams reflected by the specimen 116 return to the light beam splitting / combining element 106 through the objective lens 107 and are combined into one light beam by the light beam splitting / combining element 106. The light beam from the light beam splitting / combining element 106 is reflected by the deflection mirror 105 and travels toward the two-dimensional scanning unit 104. The light beam that has passed through the two-dimensional scanning unit 104 is partially transmitted through the light beam separation element 103. The light beam that has passed through the light beam separation element 103 enters the pinhole 108. The pinhole 108 selectively allows only the light beam from the vicinity of the observation surface in the sample 116 out of the light beam returning from the sample 116, and blocks the light beam from other parts. The light beam that has passed through the pinhole 108 enters the analyzer 109. The analyzer 109 selectively transmits linearly polarized light that is orthogonal to the linearly polarized light that is transmitted through the polarizer 102. The light beam that has passed through the analyzer 109 enters the detector 110 and is converted into an electrical signal reflecting the intensity.

  The detector 110 outputs an electric signal reflecting the intensity of the incident light beam to the image processing unit 111. The two-dimensional scanning unit 104 outputs an electric signal reflecting the position of the light spot formed on the specimen 116, for example, a signal indicating the swing angle of the galvano mirror to the image processing unit 111. The image processing unit 111 forms an image of the observation surface of the specimen 116 by processing the electrical signal from the detector 110 and the electrical signal from the two-dimensional scanning unit 104 in synchronization.

  In other words, in this embodiment, the linearly polarized light beam is split into two linearly polarized light beams that are spatially separated according to the polarization component and irradiated onto the sample, and the light of the two light beams formed on the sample. The spot is scanned two-dimensionally, the two beams reflected by the sample are combined, the combined beam is separated from the linearly polarized beam irradiated on the sample, and the desired beam within the sample is synthesized. Only a light beam from the vicinity of the observation surface is selectively extracted, a light beam of a specific linearly polarized light is extracted from the light beam from the vicinity of the observation surface, detected, and based on the detected information and the position information (scanning information) of the light spot An image of the observation surface of the specimen is formed.

  In the optical microscope 100, the light beam divided into two by the light beam splitting / combining element 106 is spatially separated and is irradiated to two different points in the specimen. A phase difference (retardation) corresponding to the difference between the optical path lengths of the portions where the two light beams pass through the sample is generated between the two light beams. That is, a phase difference occurs between the two light beams due to the difference in the optical thickness of the portion through which the two light beams pass. The phase difference between the two light beams generates a component of linearly polarized light that is orthogonal to the linearly polarized light that is transmitted through the polarizer 102. Therefore, the phase difference between the two light beams is detected as the intensity of the light beam that has passed through the analyzer 109.

  That is, when the optical thickness of the portion in the sample 116 through which the two light beams pass is equal, no signal is output from the detector 110, but the optical thickness of the portion in the sample 116 through which the two light beams pass. If the distances are different, a signal corresponding to the difference in optical thickness is output. Thereby, even if the observation object in the specimen 116 is optically transparent, information on the level difference and inclination of the surface can be acquired.

  Since the optical microscope 100 observes the specimen in a non-contact manner using light, the specimen does not receive mechanical damage due to contact, which is a concern in AFM. There is also a degree of freedom in the state of specimen installation.

  Since the optical system of the optical microscope 100 is an epi-illumination type, the optical path length in the specimen is twice that of the transmission type. For this reason, the phase change of the observation surface is detected with twice the sensitivity compared to the transmission type.

  Further, since the optical system of the optical microscope 100 is a confocal optical system having a pinhole arranged at a position conjugate with the sample surface, reflected light and scattered light from a part off the vicinity of the observation surface, Undesired light from other than the observation surface such as reflected light from the optical element is blocked by the pinhole 108. As a result, it is possible to acquire information on the fine structure of the specimen that has been buried in noise in the conventional optical microscope. Further, laser light as a point light source is irradiated onto the aperture sample by the objective lens, and the generated light is imaged by the same objective lens, and only the light from the focal plane is detected through the pinhole. Therefore, since the image is obtained by converging the light beam twice, the plane spatial resolution is about 1.4 times better than the normal microscope observation. Further, in the optical axis direction, it has a high resolution of submicron that is not comparable to a conventional optical microscope.

  Furthermore, since the optical system of the optical microscope 100 is a scanning type, the irradiation range of the light beam can be limited to a minute region. As a result, the light beam is not irradiated to an area in the specimen 116 that is not an observation target. As a result, damage to the specimen 116 is minimized.

  In addition, the optical microscope 100 can acquire an image of the observation surface of the specimen 116 in real time. Here, “can be acquired with real-time characteristics” means that approximately two or more images can be acquired per second.

  As a result, the optical microscope 100 can observe the fine structure of a transparent specimen in a non-destructive manner and with a good quality image in real time. The specimen can be observed non-destructively with the same resolution as AFM. A step of several nm in the transparent material can also be observed.

Second Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen. More specifically, the present invention is directed to an optical microscope capable of in-situ observation of the fine structure of a protein crystal surface and its growth process under various conditions.

  FIG. 3 schematically shows an optical microscope according to the second embodiment of the present invention. In FIG. 3, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted.

  As shown in FIG. 3, the optical microscope 200 according to the present embodiment includes a heat stage 207 for placing the protein crystal specimen 208 instead of the stage 115 of the first embodiment, and a temperature for controlling the heat stage 207. And a control unit 206. The heat stage 207 and the temperature control unit 206 constitute a condition setting changing unit for setting and changing various conditions of the protein crystal specimen 208. Specifically, the heat stage 207 and the temperature control unit 206 control the temperature of the protein crystal specimen 208 and its peripheral part.

  The optical microscope 200 is roughly divided into an inverted microscope main body 201 and a scanning / detecting unit 202. The inverted microscope body 201 accommodates the objective lens 107, the beam splitting / combining element 106, and the deflection mirror 105. The scanning / detecting unit 202 accommodates a polarizer 102, a light beam separation element 103, a two-dimensional scanning unit 104, a pinhole 108, an analyzer 109, and a detector 110.

The optical microscope 200 further includes a vibration isolation table 203 for supporting the inverted microscope main body 201. The vibration isolation table 203 interrupts transmission of vibration from the outside to the inverted microscope main body 201, and the image between the objective lens 107 and the protein crystal specimen 208 is changed by vibration during image acquisition, and the observation image is disturbed. The image processing unit 111 that effectively prevents the control includes a control / storage unit 204 and a display unit 205 for displaying an image. The control / storage unit 204 controls the two-dimensional scanning unit 104 and stores image information. The control / storage unit 204 also controls the temperature control unit 206.

  FIG. 4 schematically shows a protein crystal specimen 208. As shown in FIG. 4, the protein crystal 215 is hermetically sealed between two cover glasses 211 and 212 having a thickness of 0.17 mm and spaced apart by about 1 mm. The space between the two cover glasses 211 and 212 is filled with the protein solution 214, and molecules in the solution are bonded to the surface of the protein crystal 215 and grow. The crystal growth rate can be changed by directly changing the concentration of the solution 214 or changing the saturation concentration by changing the temperature. In order to change the concentration of the solution 214, a reflux device (not shown) may be combined with the sample sealed container of FIG. The size of the crystal is about 50 to 100 μm square. If the working distance of the objective lens is within an allowable range, the crystal size is not limited and can be imaged.

  As the objective lens 107 to be used, a type having a large NA is suitable for high resolution. In addition, the type in which the space between the specimen is filled with oil generally has a high refractive index and high resolution, and sufficient reflected light can be obtained. Further, not only oil but also a type using water or a dry (air) type, sufficient reflected light can be obtained.

  FIG. 5 shows a reflection image (reflection differential interference image) obtained by the optical microscope of the present embodiment. FIG. 6 shows a reflection image obtained by a conventional laser scanning microscope as a comparative example. The observation image of FIG. 6 is an image in which the position of the step portion is a line added on the surface, and the height relationship of each step is difficult to recognize. On the other hand, the reflection differential interference image of FIG. 5 is an image in which the level difference of one molecular layer of the crystal is clearly displayed, and the height relation of each level difference can be identified. This step is 5.6 nm. Note that this image is an XYZ observation used for observation of a three-dimensional structure with a confocal microscope (a three-dimensional stereoscopic image is constructed by acquiring a plurality of XY images while moving the focal position of the objective lens with respect to the specimen in the optical axis direction). Unlike the above, it is obtained with the focus position fixed.

  In the images captured for each passage of time by this observation, a single molecular layer on the crystal surface further grows and bonds to neighboring layers. In addition, the next growth layer will be newly generated. The following paper has been published that captures the growth of protein crystals using the same technique.

G. Sazaki, A. Moreno, K. Nakajima, "Novel coupling effects of the magnetic and electric fields on protein crystallization", J. Crystal Growth, 262 (2004) 499-502.
In such image observation of the protein crystal growth process, the optical microscope 200 of the present embodiment can realize optimal observation conditions. Factors controlling the crystal growth rate are the concentration of the solution filled in the surroundings and the temperature change in the surroundings, which can be changed by the temperature control unit 206. A necessary image acquisition time is set in accordance with the growth rate, and an image is recorded every time (time lapse). For example, a desired image acquisition interval (several seconds interval to several hours interval) can be set so that a useless image is not acquired in accordance with the growth rate so that the state of crystal growth can be understood by frame advance. These time intervals are determined and set by the control / storage unit 204 reading the signal conditions of the temperature control unit 206.

  In addition, even when the growth conditions are changed in the middle, the image acquisition time interval can be changed by the control / storage unit 204 based on the signal from the temperature control unit 206, and an image that follows the crystal growth rate of the specimen can be obtained. it can.

  A conventional AFM can observe a minute change of several nanometers, but has a low degree of freedom in a sample setting environment and lacks real-time property, and therefore cannot perform in-situ observation in various flow fields of protein crystals. In addition, protein crystals are soft and may be destroyed. On the other hand, with a normal optical microscope, the protein crystals are transparent, so that flare appears on the image and sufficient observation is impossible.

  According to the present embodiment, the state of the surface step can be easily observed by optical observation with an order exceeding the resolution of normal light, and in-situ observation in various flow fields is possible. For example, it is possible to easily observe a specimen enclosed in a sealed container together with a medium and use a medium reflux device. This makes it possible to observe in situ the growth state and growth rate, and the crystal defect generation process during the growth process.

  Further, in the optical microscope 200 of the present embodiment, since the inverted microscope body 201 including the objective lens 107 and the like can be separated from other configurations such as the scanning / detecting unit 202, it is not necessary to control the temperature of the entire apparatus, and the protein crystal specimen Observation is possible by controlling the temperature of only 208 and its surroundings. For this reason, it is possible to observe while changing the various conditions by arranging the specimen and the control equipment on the microscope. The state of crystal growth over time can be easily seen on the spot. This eliminates the need for special environmental equipment and expensive observation equipment. Furthermore, the image quality of the observed image is less affected by the overall temperature change.

  In addition, since the inverted microscope body 201 is mounted on the vibration isolation table 203, the image quality of the observed image is less affected by the overall vibration.

  The above method is not limited to the specimen described in the embodiment, but can be widely used when it is desired to obtain surface step information with a transparent specimen.

Third Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 7 schematically shows an optical microscope according to the third embodiment of the present invention. In FIG. 7, members indicated by the same reference numerals as those shown in FIG. 3 are similar members, and detailed description thereof is omitted.

  As shown in FIG. 7, the optical microscope 300 of the present embodiment has a configuration in which the light source 101 in the optical microscope 200 of the second embodiment is changed to an SLD (super luminescent diode) light source 301. Other configurations are the same as those of the optical microscope 200 of the second embodiment.

  The surface reflection of a transparent substance such as a protein crystal includes reflection from the opposite surface in addition to reflection from the surface of interest. The reflectivity of the surface of the transparent material depends on the refractive index difference of the medium defining the surface. For example, in FIG. 4, in addition to the reflected light from the lower surface 215 b of the protein crystal 215, that is, the boundary surface between the protein crystal 215 and the cover glass 211, from the upper surface 215 a of the protein crystal 215, that is, from the boundary surface between the protein crystal 215 and the solution 214. There is also reflected light. The reflectance of the upper surface 215a of the protein crystal 215 depends on the difference in refractive index between the protein crystal 215 and the solution 214, and the amount of reflected light is determined according to the reflectance.

  When there is a difference between the refractive indices of the protein crystal 215 and the solution 214, the laser light incident on the protein crystal 215 from below is reflected by the lower crystal surface 215b and also by the upper crystal surface 215a. The The reflected light reflected by the upper crystal surface 215a interferes with the laser light reflected by the lower crystal surface 215b to form interference fringes on the image.

  8 and 10 show images of protein crystals observed using an optical microscope in which a HeNe laser emitting light having a wavelength of 633 nm is applied to the light source 101 of the second embodiment. 8 and 10 clearly show interference fringes due to interference between the reflected light from the upper surface 215a of the protein crystal and the reflected light from the lower surface 215b.

  The pitch of the interference fringes is an integral multiple of half the wavelength of the laser light, and reflects the distance between the substances that create the interference fringes. Therefore, information on the thickness of the transparent material can be acquired by measuring the pitch of the interference fringes. The interference fringes are also useful for acquiring information such as uniformity and non-uniformity (distortion) of the entire surface.

  Interference fringes are useful for acquiring this information, but for surface observation of transparent materials in the order of a few nanometers, interference fringes overlap the surface image and often prevent accurate acquisition of surface information. . For example, the surface information of a protein crystal is obtained by imaging a slight difference in light intensity caused by fine unevenness on a molecular scale existing on the surface of the protein crystal. In this case, since a highly coherent laser is used, the interference fringes generated according to the refractive index conditions may appear on the observation image, and the fine surface information to be actually observed may be buried in the interference fringes. . In this case, although the height information can be obtained, the original surface information cannot be obtained.

  In order to avoid this problem, the optical microscope 300 of this embodiment includes an SLD (super luminescent diode) light source 301. The SLD light source 301 constitutes a light source that emits low-coherent light that is light with reduced coherence.

  The SLD light source 301 is relatively similar in structure to a semiconductor laser, but employs a structure that suppresses laser oscillation by reducing the reflectance of the optical waveguide end face. For this reason, the SLD light source 301 outputs light having a broad spectrum spread compared to a normal semiconductor laser. Since light having a broad spectrum spread has poor monochromaticity, the coherence distance is shortened.

  The SLD light source is a high-intensity light source that emits light with sufficient brightness, although it does not have excellent coherence characteristics like a laser light source. For example, a HeNe laser, which is one of laser light sources, has good monochromaticity, has a very narrow spectral spread, is 0.1 nm or less, and a coherence distance is said to be approximately 10 km or more. On the other hand, the SLD light source generally has a spectral spread width of 0.1 to 10 nm, and a coherence distance is about 1 to 100 μm.

  For example, when an approximate coherence distance is estimated, the coherence distance L can be approximately expressed as L = λ0 × λ0 / Δλ. Here, λ0 is the center wavelength, and Δλ is the full width at half maximum of the oscillation spectrum. If the SLD light source has λ0 = 680 nm and Δλ = 10 nm, the coherence distance is about 46 μm.

  Thus, the coherence distance of the SLD light source 301 is very short, and is usually smaller than the thickness D of the protein crystal 215. For this reason, there is a difference between the refractive indexes of the protein crystal 215 and the solution 214, and even when there is reflected light from the upper surface 215a of the protein crystal 215, it is avoided that interference fringes appear in the observed image. Can do. Since the thickness D of the protein crystal is usually about 50 to 100 μm as described above, the occurrence of interference fringes can be prevented by using an SLD light source (coherence distance is about 46 μm) as in this embodiment. In the case where the thickness D of the crystal is further thinner, light having a coherence distance shorter than the thickness D may be used as illumination light.

  9 and 11 show protein crystal images observed using the optical microscope 300 of the present embodiment. 9 is an observation image of the same protein crystal as in FIG. 8, and FIG. 11 is an observation image of the same protein crystal as in FIG. As can be seen by comparing FIG. 9 and FIG. 11 with FIG. 8 and FIG. 10, respectively, the image of the protein crystal obtained by the optical microscope 300 of the present embodiment has no interference fringes, and the surface (FIG. 4). It can be seen that the fine unevenness information of 215a) is captured. For this reason, according to the optical microscope 300 of this embodiment, the information of the protein crystal which was buried in the interference fringes and could not be observed with the optical microscope 200 of the second embodiment can be acquired.

  In the present embodiment, an example in which the light source that emits low-coherent light is configured by the SLD light source 301 has been described. However, the SLD light source 301 may be replaced with an ASE (Amplified Spontaneous Emission) light source. The ASE light source is a light source using spontaneous emission light obtained by optically exciting and amplifying a fiber doped with a rare earth element or a rare earth compound such as Er (erbium), Nd (neodymium), or Yb (yttrium). It emits light with low coherence at high output.

Fourth Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 12 schematically shows an optical microscope according to the fourth embodiment of the present invention. 12, members indicated by the same reference numerals as those shown in FIG. 3 are the same members, and detailed description thereof is omitted.

  As shown in FIG. 12, the optical microscope 400 of this embodiment has a configuration in which an SLD light source 301 and an optical path synthesis element 401 are added to the optical microscope 200 of the second embodiment. The details of the SLD light source 301 are as described in the third embodiment. The optical path combining element 401 is disposed between the light source 101 and the polarizer 102, transmits the light beam emitted from the light source 101, and reflects the light beam emitted from the SLD light source 301 to direct it to the polarizer 102. In other words, the optical path combining element 401 combines the optical path of the light beam emitted from the SLD light source 301 with the optical path of the light beam that travels from the light source 101 to the polarizer 102. Other configurations are the same as those of the optical microscope 200 of the second embodiment.

  In the optical microscope 400 of the present embodiment, when observing the protein crystal specimen 208, either the light source 101 or the SLD light source 301 is selectively driven. When a normal laser light source, which is the light source 101, is driven, thickness information of the protein crystal 215, surface shape distortion information, and the like can be acquired. In addition, when the SLD light source 301 is driven, as described in the third embodiment, the information on the protein crystal 215 (fine irregularities on the molecular surface of the crystal surface) can be obtained well without being disturbed by the interference fringes. can do.

  Thus, according to the optical microscope 400 of this embodiment, more information can be acquired about the protein crystal 215 by observing while switching between the light source 101 and the SLD light source 301 to be used.

  In this embodiment, the optical path synthesis element 401 is an optical element that transmits the light beam emitted from the light source 101 and reflects the light beam emitted from the SLD light source 301. However, the optical path synthesis element 401 is an ordinary mirror. The light flux incident on the polarizer 102 may be switched by being appropriately arranged on the optical path between the light source 101 and the polarizer 102.

Fifth Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 13 schematically shows an optical microscope according to the fourth embodiment of the present invention. In FIG. 13, members indicated by the same reference numerals as those shown in FIG. 3 are similar members, and detailed description thereof is omitted.

  As shown in FIG. 12, the optical microscope 500 of this embodiment has a configuration in which a photonic crystal fiber 501 is added to the optical microscope 200 of the second embodiment. The light source 101 is a normal laser light source. The photonic crystal fiber 501 is connected to the light source 101, guides the light emitted from the light source 101, and emits a light beam toward the polarizer 102. Furthermore, the photonic crystal fiber 501 is composed of a nonlinear fiber, and functions as an optical functional device that converts light emitted from the light source 101 into low-coherent light with reduced coherence. That is, the light source 101 and the photonic crystal fiber 501 cooperate to constitute a light source that emits low-coherent light. More specifically, the photonic crystal fiber 501 converts the light emitted from the light source 101 into high-luminance supercontinuum light having a broad spectrum very close to white. Other configurations are the same as those of the optical microscope 200 of the second embodiment.

  In the optical microscope 500 of the present embodiment, the light emitted from the light source 101 is converted into low-coherent light by the photonic crystal fiber 501 and enters the polarizer 102. Therefore, as in the third embodiment, an image without interference fringes can be acquired. In addition, light emitted from the light source 101 is converted into supercontinuum light by the photonic crystal fiber 501. Supercontinuum light has a shorter coherence distance than light emitted from the SLD light source 301. For this reason, an image without interference fringes can be acquired more stably than in the third embodiment.

Sixth Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 14 schematically shows an optical microscope according to the fourth embodiment of the present invention. 14, members indicated by the same reference numerals as those shown in FIG. 12 are the same members, and detailed description thereof is omitted.

  As shown in FIG. 14, the optical microscope 600 of the present embodiment has a configuration in which the SLD light source 301 in the optical microscope 400 of the fourth embodiment is replaced with a laser light source 601 and a photonic crystal fiber 501. The laser light source 601 and the photonic crystal fiber 501 constitute a light source that emits low-coherent light, like the light source 101 and the photonic crystal fiber 501 of the fifth embodiment. Other configurations are the same as those of the optical microscope 400 of the fourth embodiment.

  In the optical microscope 600 of the present embodiment, as in the fourth embodiment, either the light source 101 or the laser light source 601 is selectively driven when the protein crystal specimen 208 is observed. When a normal laser light source, which is the light source 101, is driven, thickness information of the protein crystal 215, surface distortion information, and the like can be acquired. Further, when the laser light source 601 is driven, light emitted from the laser light source 601 is converted into low coherent light by the photonic crystal fiber 501 and enters the polarizer 102, so that an image without interference fringes can be acquired. In addition, since the light emitted from the laser light source 601 is converted into supercontinuum light by the photonic crystal fiber 501, an image without interference fringes can be acquired more stably than in the fourth embodiment.

  According to the optical microscope 600 of the present embodiment, more information can be acquired about the protein crystal 215 as in the fourth embodiment. Moreover, an image without interference fringes can be acquired more stably than in the fourth embodiment.

Seventh Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 15 schematically shows an optical microscope according to the fourth embodiment of the present invention. In FIG. 15, members indicated by the same reference numerals as those shown in FIG. 13 are the same members, and detailed description thereof is omitted.

  As shown in FIG. 15, the optical microscope 700 of this embodiment has a configuration in which a wavelength selection filter 701 is added to the optical microscope 500 of the fifth embodiment. In the present embodiment, the photonic crystal fiber 501 functions as an optical functional device that simply converts light emitted from the light source 101 into low-coherent light. The wavelength selection filter 701 is appropriately disposed on the optical path between the photonic crystal fiber 501 and the polarizer 102 by a moving mechanism such as a slider (not shown). The wavelength selection filter 701 controls the wavelength range of the light beam incident on the polarizer 102, thereby adjusting the coherence distance of the light beam. That is, the wavelength selection filter 701 functions as a coherence distance adjusting element that adjusts the coherence distance of the light beam. Other configurations are the same as those of the optical microscope 500 of the fifth embodiment.

  As the wavelength selection filter 701, a filter having a wavelength transmissive characteristic in which a desired center wavelength and a full width at half maximum of a spectrum are desired values may be selected and used by using the above-described formula for estimating the coherence distance L. . Thereby, observation can be performed with light having a coherent distance that matches the thickness of the protein crystal 215 to be observed. Furthermore, the wavelength selection filter 701 may be exchanged according to the protein crystal specimen 208 to be observed in order to optimize the observation. Instead of exchanging the wavelength selection filter 701, the wavelength selection filter 701 may be switched by a turret switching mechanism.

  In the optical microscope 700 of the present embodiment, the light emitted from the light source 101 is converted into low-coherent light by the photonic crystal fiber 501 and enters the polarizer 102. Thereby, an image without interference fringes can be acquired. In addition, when the wavelength selection filter 701 is disposed on the optical path, the coherence distance of the light beam is appropriately adjusted by the wavelength selection filter 701. For this reason, an image without interference fringes can be acquired more stably.

Eighth Embodiment This embodiment is directed to another optical microscope suitable for observing a transparent specimen.

  FIG. 16 schematically shows an optical microscope according to the fourth embodiment of the present invention. In FIG. 16, members indicated by the same reference numerals as those shown in FIG. 14 are similar members, and detailed description thereof is omitted.

  As shown in FIG. 16, the optical microscope 800 of this embodiment has a configuration in which a wavelength selection filter 701 is added to the optical microscope 600 of the sixth embodiment. In the present embodiment, the photonic crystal fiber 501 functions as an optical functional device that simply converts light emitted from the light source 101 into low-coherent light. The details of the wavelength selection filter 701 are as described in the seventh embodiment. Other configurations are the same as those of the optical microscope 600 of the sixth embodiment.

  In the optical microscope 800 of this embodiment, either the light source 101 or the laser light source 601 is selectively driven when the protein crystal specimen 208 is observed. When a normal laser light source, which is the light source 101, is driven, thickness information of the protein crystal 215, surface distortion information, and the like can be acquired. When the laser light source 601 is driven, light emitted from the laser light source 601 is converted into low coherent light by the photonic crystal fiber 501 and enters the polarizer 102, so that an image without interference fringes can be acquired. In addition, when the wavelength selection filter 701 is disposed on the optical path, the coherence distance of the light flux is appropriately adjusted by the wavelength selection filter 701, so that an image without interference fringes can be acquired more stably.

  According to the optical microscope 800 of the present embodiment, more information about the protein crystal 215 can be acquired as in the sixth embodiment. In addition, an image without interference fringes can be acquired stably. The combination of the wavelength selection filter 701 and the light source is not limited, and may be combined with the SLD light source 301 or the ASE light source.

  The above method is not limited to the specimen described in the embodiment, but can be widely used when it is desired to obtain surface step information with a transparent specimen.

  The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. Also good.

  In the embodiment described above, an example in which the present invention is applied to an inverted microscope has been described. However, the present invention may be applied to an upright microscope. The scanning method is not limited to raster scanning, and arbitrary scanning can be applied. There are no restrictions on the scanning speed or the scanning size.

1 schematically shows an optical microscope according to a first embodiment of the present invention. 2 schematically shows a two-dimensional scanning unit using a galvanometer mirror. The optical microscope of 2nd embodiment of this invention is shown roughly. 4 schematically shows a protein crystal specimen shown in FIG. 3. The reflection image obtained by the optical microscope of 2nd embodiment of this invention is shown. The reflection image obtained by the conventional laser scanning microscope is shown. The optical microscope of 3rd embodiment of this invention is shown roughly. The image of the protein crystal | crystallization observed using the optical microscope of 2nd embodiment is shown. The image of the same protein crystal as FIG. 8 observed using the optical microscope of 3rd embodiment is shown. The image of the protein crystal | crystallization observed using the optical microscope of 2nd embodiment is shown. The image of the same protein crystal as FIG. 10 observed using the optical microscope of 3rd embodiment is shown. 10 schematically shows an optical microscope according to a fourth embodiment of the present invention. 6 schematically shows an optical microscope according to a fifth embodiment of the present invention. 10 schematically shows an optical microscope according to a sixth embodiment of the present invention. 9 schematically shows an optical microscope according to a seventh embodiment of the present invention. 9 schematically shows an optical microscope according to an eighth embodiment of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Optical microscope, 101 ... Light source, 102 ... Polarizer, 103 ... Light beam splitting element, 104 ... Two-dimensional scanning part, 105 ... Deflection mirror, 106 ... Light beam splitting / combining element, 107 ... Objective lens, 108 ... Pinhole, 109 ... Analyzer 110 ... Detector 111 ... Image processing unit 115 ... Stage 116 ... Sample 121 ... Galvano mirror 122 ... Galvano mirror 200 ... Optical microscope 201 ... Inverted microscope main body 202 ... Scanning / detection unit 203 DESCRIPTION OF SYMBOLS ... Vibration isolator, 204 ... Control / storage unit, 205 ... Display unit, 206 ... Temperature control unit, 207 ... Heat stage, 208 ... Protein crystal specimen, 211 ... Cover glass, 212 ... Cover glass, 214 ... Solution, 215 ... Protein crystal, 215a ... upper surface, 215b ... lower surface, 300 ... optical microscope, 301 ... SLD light , 400 ... optical microscope, 401 ... optical path combining element, 500 ... optical microscope, 501 ... photonic crystal fiber, 600 ... optical microscope, 601 ... laser light source, 700 ... optical microscope, 701 ... wavelength selective filter, 800 ... optical microscope.

Claims (13)

  1. An optical microscope suitable for observing transparent specimens,
    An objective lens placed near the specimen;
    A light source for emitting a light beam for observation;
    A polarizer to create linearly polarized light from the light emitted from the light source;
    A beam splitting and synthesizing element that divides the light beam from the polarizer into two light beams that are spatially separated according to the polarization component and combines the two light beams returning from the sample;
    A two-dimensional scanning unit for two-dimensionally scanning a light spot formed on the specimen;
    A light beam separating element for directing the light beam from the polarizer to the objective lens and separating the light beam returning from the sample through the objective lens from the light beam directed to the objective lens;
    A pinhole for selectively passing only the light beam from the vicinity of a desired observation surface in the sample among the light beams returned from the sample separated by the light beam separation element;
    An analyzer for extracting a light beam of a specific linear polarization from the light beam that has passed through the pinhole;
    A detector for detecting the light beam that has passed through the analyzer;
    An optical microscope comprising: an image processing unit that forms an image of an observation surface in a specimen based on information detected by a detector and position information of a light spot obtained from a two-dimensional scanning unit.
  2.   The optical microscope according to claim 1, wherein the light source is a laser light source.
  3.   2. The optical microscope according to claim 1, further comprising a condition setting changing unit for setting or changing various conditions of the specimen.
  4.   4. The optical microscope according to claim 3, wherein the condition setting changing unit controls the temperature of the specimen and its peripheral part.
  5.   5. The optical microscope according to claim 1, further comprising a vibration isolation table for removing vibration.
  6.   2. The optical microscope according to claim 1, wherein the light source is a light source that emits low-coherent light.
  7.   7. The optical microscope according to claim 6, wherein the light source that emits low-coherent light is an SLD light source.
  8.   7. The optical microscope according to claim 6, wherein the light source that emits low coherent light includes a laser light source and a photonic crystal fiber.
  9.   7. The optical microscope according to claim 6, further comprising a coherence distance adjusting unit that adjusts a coherence distance of the light beam applied to the specimen.
  10.   12. The optical microscope according to claim 11, wherein the coherent distance adjusting element includes a wavelength selection filter.
  11.   3. The second light source for emitting low-coherent light and an optical path synthesis element for synthesizing the optical path of the light beam emitted from the second light source with the optical path of the light beam from the laser light source toward the polarizer. Optical microscope equipped.
  12. An optical observation method suitable for observing a transparent specimen,
    The linearly polarized light beam is split into two light beams that are spatially separated according to the polarization component, and the sample is irradiated.
    Two-dimensional scanning of the light spot formed on the specimen,
    Combining the two luminous fluxes reflected by the specimen,
    Separating the combined luminous flux from the linearly polarized luminous flux irradiated to the specimen,
    Of the combined luminous flux, selectively extract only the luminous flux from the vicinity of the desired observation surface in the sample,
    Detecting a linearly polarized light beam from the light beam near the observation surface,
    An optical observation method for forming an image of an observation surface of a specimen based on detected information and light spot position information.
  13.   The optical observation method according to claim 12, wherein the light beam applied to the specimen is low coherent light.
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