JP2006258990A - Optical microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical microscope achieving the shortening of measuring time. <P>SOLUTION: The optical microscope is equipped with: a laser beam source 11; a cylindrical lens 15 converting a laser beam into a linear light beam; a galvano mirror 23 making the linear light beam scan; an objective 27 condensing the light beam made to scan by the galvano mirror 23 to be linear; a 1st beam splitter 22 branching emitted light 62 whose wavelength is different from laser wavelength and which is emitted from a sample 28 to the objective 27 side from the laser beam; a spectroscope 33 having an incident slit 32 arranged on an incident side on which the emitted light 62 branched by the 1st beam splitter 22 is made incident and spatially dispersing the emitted light 62 passing through the incident slit 32 in accordance with the wavelength; and a detector 34 detecting the emitted light 62 dispersed by the spectroscope 33. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an optical microscope, and more particularly to an optical microscope that detects light having a wavelength different from that of laser light applied to a sample.

  The Raman spectroscopic measurement is possible regardless of whether the sample is a gas, liquid, crystal, or amorphous solid, and the temperature can be high or low, and there is an advantage that a special measurement atmosphere such as a vacuum is not required for the measurement. Have. Furthermore, there is an advantage that the sample can be measured as it is without requiring any pre-treatment of the sample, and many measurements are made by taking advantage of these advantages. By using Raman spectroscopy, molecules can be observed unstained and impurities in semiconductors can be observed

In order to perform such Raman spectroscopic measurement, a Raman microscope using a spectroscope has been disclosed (Patent Documents 1 and 2). In this Raman microscope, a sample is irradiated with focused laser light. A Raman spectrum can be measured by spectroscopically analyzing Raman scattered light from the sample. Furthermore, in these Raman microscopes, the spatial distribution of the Raman scattered light intensity at a specific wavelength can be measured by moving the sample and measuring. A Raman microscope is also disclosed in which a sample is illuminated in a line by a cylindrical lens and detected by a CCD camera (Non-Patent Document 1).
JP 2002-14043 A Japanese Patent Laid-Open No. 2003-344776 CHARLENE A. DRUMM and 1 other "Microscopic Raman Line-Imaging With Principle Component Analysis" APPLIED SPECTROCOPY, 1995, 49, No. 9, p. 1331-1337

  However, the conventional Raman microscope has the following problems. For example, since the Raman scattering of a living body is weak, the measurement time (integrated time) of the Raman spectrum per point becomes long. For example, if the integration time per point is 1 second, it takes 1 hour or more (4096 seconds = 64 × 64 seconds) to acquire a 64 × 64 pixel image. Further, when the wavelength resolution is improved by increasing the integration time, there is a problem that the measurement time becomes longer if the number of measurement points is increased in order to increase the resolution or measure a wider area.

  In order to avoid such long-time measurement, it is necessary to narrow the measurement range, that is, the observation range. Also, in order to widen the measurement range, it is necessary to lower the resolution. Alternatively, it is necessary to narrow the range of the spectrum to be measured or to measure only for a specific wavelength. As described above, the conventional Raman microscope has a problem in that the measurement is limited because the measurement time is long.

As described above, the conventional Raman microscope has a problem that the measurement time is long and the measurement is limited.
The present invention has been made in view of the above-described problems, and an object thereof is to provide an optical microscope that can shorten the measurement time.

  An optical microscope according to a first aspect of the present invention includes a laser light source (for example, the laser light source 11 according to an embodiment of the present invention) and light conversion for converting a light beam from the laser light source into a linear light beam. Means (for example, the third lens cylindrical 15 and / or the iris diaphragm 39 according to the embodiment of the present invention) and scanning means for scanning the light beam converted into the line shape (for example, the embodiment of the present invention) The objective lens (for example, the objective lens 27 according to the embodiment of the present invention) for condensing the light beam scanned by the scanning means into a line and entering the sample, and the sample Out of the light beam incident on the sample, the output light that has a different wavelength and exits from the sample to the objective lens side is linearly incident on the sample from the laser light source. A first beam splitter (for example, the first beam splitter 22 according to an embodiment of the present invention) that branches off from the beam, and the line-like shape on the incident side on which the outgoing light branched by the first beam splitter enters. An incident slit (for example, an incident slit 32 according to an embodiment of the present invention) arranged along a direction corresponding to the light beam, and the outgoing light that has passed through the incident slit is spatially dependent on the wavelength. (For example, the spectrometer 33 according to the embodiment of the present invention) and a two-dimensional array photodetector (for example, the embodiment of the present invention) that detects the emitted light dispersed by the spectrometer. And such a detector 34). Thereby, measurement time can be shortened.

  The optical microscope according to the second aspect of the present invention is characterized in that, in the above-described optical microscope, the scanning means is a galvanometer mirror. As a result, the scanning speed can be improved, and the fluctuation of the light beam applied to the sample can be reduced. Therefore, convenience can be improved.

  An optical microscope according to a third aspect of the present invention is the above-described optical microscope in which the scanning unit scans the light beam in a direction perpendicular to the line direction of the light beam. Thereby, measurement time can be shortened.

  An optical microscope according to a fourth aspect of the present invention is characterized in that, in the above-described optical microscope, the light conversion means has a cylindrical lens. Thereby, the light from the laser light source can be efficiently incident on the sample, and the measurement time can be shortened.

  An optical microscope according to a fifth aspect of the present invention is the above-described optical microscope, wherein an iris diaphragm having a slit-like opening at a position where the incident light is condensed by the cylindrical lens (for example, an embodiment of the present invention). The iris diaphragm 39) is provided with an iris diaphragm whose opening size is variable. Thereby, measurement time can be shortened.

  An optical microscope according to a sixth aspect of the present invention is the above-described optical microscope, wherein a filter (for example, a filter that blocks light having a laser wavelength of the laser light source) is interposed between the first splitter and the two-dimensional array photodetector. An edge filter 38) according to the embodiment of the present invention is provided. Thereby, measurement time can be shortened.

    An optical microscope according to a seventh aspect of the present invention is the optical microscope described above, wherein the surface condensed by the light conversion means and the sample surface are conjugated with each other, and the sample surface and the entrance slit are conjugated with each other. It has become a relationship. Thereby, the resolution can be improved.

  An optical microscope according to an eighth aspect of the present invention is characterized in that, in the above-described optical microscope, a relative distance between the objective lens and the sample is variable. Thereby, three-dimensional measurement can be performed with a simple configuration.

  An optical microscope according to a ninth aspect of the present invention is an illumination light source (for example, according to an embodiment of the present invention) that is disposed on the opposite side of the sample from the objective lens and emits illumination light. An illumination light source 41), a ring slit (for example, a ring slit 43 according to an embodiment of the present invention) that converts the illumination light from the illumination light source into a ring illumination light, and the ring illumination light is condensed. A condenser lens (for example, a condenser lens 44 according to an embodiment of the present invention) that is incident on the sample, and second light that is collected by the condenser lens and transmitted through the sample is incident through the objective lens. Beam splitter (for example, the second beam splitter 21 according to an embodiment of the present invention), and the optical path of the transmitted light from the laser light source A phase plate that provides a phase difference in a ring-shaped region corresponding to the ring slit of the transmitted light branched by the second beam splitter branched by the optical path of the incident light (for example, the present invention) A phase plate 46) according to the first embodiment, and an image pickup device (for example, a CCD camera 48 according to the embodiment of the present invention) that images the transmitted light from the phase plate. Thereby, since transmitted light can be detected, the convenience can be improved.

  An optical microscope according to a tenth aspect of the present invention is the optical microscope described above, wherein the first beam splitter is disposed on the sample side of the second beam splitter, and the transmission is performed via the first beam splitter. The light is incident on the second beam splitter. Thereby, since the emitted light from the sample can be efficiently incident on the detector, the measurement time can be shortened.

  An optical microscope according to an eleventh aspect of the present invention is the above-described optical microscope, wherein the imaging element observes the distribution of phase objects having different phases in the sample. Thereby, since the phase object can be observed with a simple configuration, the convenience can be improved.

  ADVANTAGE OF THE INVENTION According to this invention, the optical microscope which can shorten measurement time can be provided.

  Hereinafter, embodiments to which the present invention can be applied will be described. The following description is to describe the embodiment of the present invention, and the present invention is not limited to the following embodiment. For clarity of explanation, the following description is omitted and simplified as appropriate. Further, those skilled in the art will be able to easily change, add, and convert each element of the following embodiments within the scope of the present invention. In addition, what attached | subjected the same code | symbol in each figure has shown the same element, and abbreviate | omits description suitably.

  An optical microscope according to an embodiment of the present invention will be described with reference to FIG. FIG. 1 is a diagram schematically showing the configuration of the optical system of the optical microscope according to the present embodiment. 1 is an optical microscope, 11 is a laser light source, 12 is a mirror, 13 is a first second cylindrical lens 14 is a second third cylindrical lens 15 is a third cylindrical lens, 16 is a lens, and 21 is a second , 22 is a galvano mirror, 24 is a lens, 25 is a mirror, 26 is a lens, 27 is an objective lens, 28 is a sample, 31 is a lens, 32 is an entrance slit, and 33 is a spectroscopic beam. , 34 is a detector, 35 is a processing device, 38 is an edge filter, 39 is an iris diaphragm, 41 is an illumination light source, 42 is a lens, 43 is a ring slit, 44 is a condenser lens, 45a is a lens, 45b is a lens, 46 Is a phase plate, 47 is a lens, and 48 is an image sensor. In addition, 61 is incident light incident on the sample 28 from the laser light source 11, 62 is light emitted from the sample 28 after being Raman scattered by the sample 28, and 71 is incident on the sample 28 from the illumination light source 41. Illumination light 72 is transmitted light transmitted through the sample 28 among the illumination light 71 incident from the illumination light source 41.

  The optical microscope 1 includes a laser light source 11, a spectroscope 33 that splits the outgoing light 62, a detector 34 that detects the outgoing light 62 split by the spectroscope 33, and an optical system between the laser light source 11 and the spectroscope 33. A Raman microscope, an illumination light source 41, a CCD camera 48 that detects the transmitted light 72 incident from the illumination light source 41 and transmitted through the sample 28, and a phase contrast microscope having an optical system from the illumination light source 41 to the CCD camera 48 are provided. . In the phase contrast microscope and the Raman microscope, the optical system has an overlapping portion. In the optical microscope 1 according to the present embodiment, the laser light source 11 or the illumination light source 41 can be turned off to observe either the Raman microscope or the phase contrast microscope. That is, a Raman microscope and a phase contrast microscope are used exclusively. In addition to turning off the light source, a shutter that blocks light from the laser light source 11 or the illumination light source 41 may be provided to block light from either one.

  First, the configuration of the Raman microscope of the optical microscope 1 will be described with reference to FIG. The laser light source 11 emits monochromatic laser light. As the laser light source 11, for example, Millennia manufactured by Spectra Physics Co., Ltd. can be used. The laser light source 11 is an Nd / YVO4 laser having a laser wavelength of 532 nm, a line width of 0.24 nm, and a maximum output of 10 W. The laser light source 11 emits laser light having this laser wavelength. Laser light from the laser light source 11 propagates through an optical system described later to become incident light 61 incident on the sample 28.

  Incident light 61 from the laser light source 11 becomes a parallel light beam by a lens (not shown) or the like and enters the mirror 12. The incident light 61 is reflected by the mirror 12 and is incident on the first first cylindrical lens 13. The first cylindrical lens 13 refracts the incident light 61 only in one direction. In the following description, it is assumed that the light propagation direction, that is, the optical axis direction is the Z direction, and the plane perpendicular to the Z direction is the XY plane. The first cylindrical lens 13 is a lens having a focal length of 50 mm in the Y direction, and refracts incident light 61 in the Y direction. Incident light 61 refracted in the Y direction by the first cylindrical lens 13 is collected in a line. That is, the incident light 61 is once converted into a linear light beam along the X direction by the first cylindrical lens 13. The incident light 61 condensed in a line shape spreads and enters the second cylindrical lens 14. The second cylindrical lens 14 is a lens having a focal length of 100 mm in the Y direction. The second cylindrical lens 14 refracts the incident light 61 in the Y direction and converts it into a parallel light beam. That is, the incident light 61 from the second cylindrical lens 14 has a beam shape extending in the Y direction. In other words, the first cylindrical lens 13 and the second cylindrical lens 14 are beam expanders that expand the beam diameter in the Y direction.

  Incident light 61 from the second cylindrical lens 14 enters the third cylindrical lens 15. The third cylindrical lens 15 is a lens having a focal length of 100 mm in the X direction. The third cylindrical lens 15 refracts the incident light 61 in the X direction. The incident light 61 is condensed in the X direction by the third cylindrical lens 15. Therefore, the third cylindrical lens 15 converts the incident light 61 into a linear light beam along the Y direction. By using a cylindrical lens, it can be efficiently converted into line light. The third cylindrical lens 15 forms the incident light 61 at the position of the iris diaphragm 39. That is, the third cylindrical lens 15 condenses the incident light 61 at a position of the iris diaphragm 39 so as to form a light beam in a line shape. In other words, the iris diaphragm 39 is disposed on the image plane portion behind the third cylindrical lens 15. The iris diaphragm 39 has a slit-shaped opening corresponding to the direction of the line-shaped incident light 61. The iris diaphragm 39 sets the size of the line-shaped incident light 61 condensed on the sample 28. By making the size of the opening of the iris diaphragm 39 variable, the size of the spot of the light beam of the incident light 61 on the sample 28, that is, the illumination area in the Raman microscope can be adjusted.

  Incident light 61 refracted by the third cylindrical lens 15, passing through the iris diaphragm 39 and converted into a line shape, spreads in the X direction and enters the lens 16. The lens 16 is, for example, a lens having a focal length of 160 mm. The lens 16 is once condensed into a line shape by the third cylindrical lens 15 and then refracts the incident light 61 which is spread and enters into a parallel light flux. Part of the incident light 61 from the lens 16 passes through the second beam splitter 21 and the first beam splitter 22 and enters the galvanometer mirror 23. The galvanometer mirror 23 scans the incident light 61 and reflects it in the direction of the lens 24. That is, the galvanometer mirror 23 is a scanning unit that scans the light beam converted into a line shape. Incident light 61 can be scanned by inputting an electrical signal from the processing device 35 and changing the angle of the galvanometer mirror 23.

  Incident light 61 reflected by the galvanometer mirror 23 enters the lens 24. For example, the lens 24 is a lens having a focal length of 200 mm, and refracts the incident light 61. Incident light 61 refracted by the lens 24 enters the mirror 25. Incident light 61 incident on the mirror 25 is reflected and incident on the lens 26. The lens 26 is, for example, a lens having a focal length of 200 mm. The lens 26 refracts the incident light 61 and makes it incident on the objective lens 27. The objective lens 27 collects the incident light 61 and makes it incident on the sample 28. For the objective lens 27, for example, Nikon Apochromat NA 1.2 x60 can be used. The objective lens 27 collects the incident light 61 converted into a line shape by the third cylindrical lens 15 in a line shape on the sample surface. That is, the iris diaphragm 39 and the sample 28 are arranged in a conjugate relationship with each other.

  A part of the incident light 61 incident on the sample 28 is subjected to Raman scattering. Out of the incident light 61 incident on the sample 28, light emitted to the objective lens 27 side by Raman scattering is referred to as outgoing light 62. That is, out of the Raman scattered light, the light incident on the objective lens 27 is set as the outgoing light 62. The Raman scattered outgoing light 62 has a wavelength different from that of the incident light 61. In other words, the outgoing light 62 is scattered by being shifted from the frequency of the incident light 61 by the Raman shift. The spectrum of the emitted light 62 becomes a Raman spectrum. Therefore, by measuring the spectrum of the emitted light 62, the chemical structure and physical state of the substance contained in the sample 28 can be specified. That is, since the Raman spectrum includes information on the frequency of the substance constituting the sample 28, the substance in the sample 28 can be specified by detecting the emission light 62 with the spectrometer 33. . Then, by scanning the incident light 61 in the XYZ directions and measuring the spectrum of the emitted light 62 from the entire surface or a partial region of the sample 28, a three-dimensional measurement of the Raman spectrum can be performed. By paying attention to a specific wavelength in the measured Raman spectrum, it is possible to measure a three-dimensional spatial distribution of a specific substance. Specifically, when the sample 28 is a living cell, the spatial distribution of nucleic acids and lipids or the spatial distribution of sucrose and polystyrene spheres can be measured.

  The outgoing light 62 that has been Raman-scattered and entered the objective lens 27 propagates through the same optical path as the incident light 61. That is, the light is refracted by the objective lens 27, reflected by the mirror 25, refracted by the lens 24, reflected by the galvanometer mirror 23, and incident on the first beam splitter 22. The first beam splitter 22 reflects a part of the incident outgoing light 62 in the direction of the lens 31. For example, the first beam splitter 22 has a reflectance of 80% and a transmittance of 20%. That is, 80% of the outgoing light 62 incident on the first beam splitter 22 enters the lens 31. An edge filter 38 is disposed between the lens 31 and the first beam splitter 22. The edge filter 38 removes scattered light having the same wavelength as the laser wavelength. That is, the edge filter 38 removes the Rayleigh scattered light based on the difference in wavelength between the Rayleigh scattered light and the Raman scattered light. Thereby, the scattered light having the same wavelength as the laser wavelength is separated from the outgoing light 62 by Raman scattering. It should be noted that a dichroic mirror may be used as the first beam splitter 22 instead of the edge filter that blocks the light having the laser wavelength. Thereby, only Raman scattered light can be detected efficiently.

  The outgoing light 62 that has passed through the edge filter 38 enters the lens 31. The light that has passed through the lens 31 enters the spectroscope 33 that the incident slit 32 has on the incident side. That is, the outgoing light 62 passes through the entrance slit 32 and is split by the spectroscope 33. The lens 31 refracts the outgoing light 62 and forms an image on the entrance slit 32. Here, since the incident light 61 is imaged in a line shape on the surface of the sample 28, the emitted light 62 is collected in a line shape on the incident slit 32. The direction of the incident slit 32 and the direction of the line-shaped outgoing light 62 are matched. That is, the line-shaped incident light on the sample 28 and the opening of the incident slit 32 are arranged so as to be conjugated with each other. Therefore, the Raman microscope is configured as a confocal optical system. That is, the iris diaphragm 39 and the surface of the sample 28 are arranged so as to be conjugated with each other, and the surface of the sample 28 and the entrance slit 32 are arranged so as to be conjugated with each other. Accordingly, the incident light 61 is collected in a line along the Y direction on the XY plane on which the iris diaphragm 39 is provided and on the surface on which the sample 28 is provided. Then, the outgoing light 62 scattered and emitted from the sample 28 is collected in a line along the Y direction on the entrance slit 32. The entrance slit 32 has an opening along the Y direction, and only the light incident on the opening is transmitted to the detector 34 side. By using such an imaging optical system for the illumination optical system from the laser light source 11 to the sample 28 and the observation optical system from the sample 28 to the detector 34, a confocal Raman microscope can be obtained. Thereby, measurement with high resolution in the Z direction can be performed.

  The spectroscope 33 includes a spectroscopic element such as a diffraction grating (grating) or a prism, and spatially disperses light incident from the incident slit 32 according to the wavelength. In the case of the spectroscope 33 using a reflective diffraction grating, an optical system such as a spherical mirror that guides the light from the incident slit 32 to the spectroscopic element and a spherical mirror that guides the light dispersed by the spectroscopic element to the detector 34 is provided. It has been. The outgoing light 62 is dispersed by the spectroscope 33 in a direction perpendicular to the direction of the entrance slit 32. The outgoing light 62 split by the spectroscope 33 enters the detector 34. The detector 34 is an area sensor in which light receiving elements are arranged in a matrix. That is, the detector 34 is a two-dimensional array photodetector such as a two-dimensional CCD in which pixels are arranged in an array.

  The detector 34 may be a cooled CCD with an image intensifier. The detector 34 may be, for example, an electronically cooled CCD (−25 ° C.) of 1024 × 256 pixels manufactured by Princeton Instruments. The pixels of the detector 34 are arranged along the direction corresponding to the entrance slit 32. Therefore, one arrangement direction of the pixels of the detector 34 coincides with the direction of the entrance slit 32, and the other arrangement direction coincides with the dispersion direction of the spectrometer 33. Here, the direction corresponding to the direction of the incident slit 32 of the detector 34 is defined as the Y direction, and the direction perpendicular to the incident slit 32, that is, the direction in which the emitted light 62 is dispersed by the spectroscope 33 is defined as the X direction.

  The detector 34 outputs a detection signal corresponding to the light intensity of the emitted light 62 received by each pixel to the processing device 35. The processing device 35 is an information processing device such as a personal computer (PC), and stores a detection signal from the detector 34 in a memory or the like. Then, the detection result is subjected to a predetermined process and displayed on the monitor. Further, the processing device 35 controls scanning of the galvanometer mirror 23 and driving of the stage of the sample 28 (not shown). Here, the X direction of the detector 34 corresponds to the wavelength (frequency) of the outgoing light 62. That is, in the pixel array arranged in the X direction, one pixel detects the outgoing light 62 having a long wavelength (low frequency), and the other pixel detects the outgoing light 62 having a short wavelength (high frequency). To do. Thus, the light intensity distribution in the X direction of the detector 34 indicates the distribution of the Raman spectrum.

  On the other hand, since the linear incident light 61 is used, the Y direction of the detector 34 corresponds to the Y direction of the sample 28. That is, in the pixel array arranged in the Y direction, one pixel detects the emitted light 62 from one end of the region illuminated by the linear incident light 61, and the other pixel detects the linear incident light 61. The emitted light 62 from the other end of the area illuminated by is detected. In other words, the positions of the pixels arranged in the Y direction of the detector 34 correspond to the positions on the sample 28, and the light intensity distribution in the Y direction represents the spatial distribution of the emitted light 62 on the sample 28. Will show. As described above, the position in the X direction on the light receiving surface of the detector 34 corresponds to the wavelength of the Raman spectrum, and the position in the Y direction corresponds to the position on the sample 28. Then, the incident light 61 is scanned by the galvanometer mirror 23, and the relative position between the sample 28 and the incident light 61 is shifted in the X direction. By scanning the illumination area on the sample 28 in the X direction, an area corresponding to the length and scanning distance of the linear incident light 61 can be illuminated. Thereby, the two-dimensional spatial distribution of the Raman spectrum can be measured. Furthermore, when the scanning distance of the galvanometer mirror 23 is not sufficient, the illumination area may be widened using the sample 28 as a drive stage.

  In the present embodiment, the line-shaped incident light 61 is scanned. Thereby, the Raman spectrum of an arbitrary point on the XY plane of the sample 28 can be measured. Furthermore, since the scanning speed can be improved as compared with the case of scanning point illumination or the stage, the spectrum measurement time can be shortened. That is, the measurement of the Raman spectrum in a wide region of the sample 28 can be performed in a short time. Therefore, even when the observation area of the sample 28 is widened, a long measurement time is not required. Thereby, even when the wavelength resolution of the Raman spectrum is improved or when a spectrum in a wider range is measured, the measurement time does not take a long time, and practical observation can be performed. For example, observation of a two-dimensional image paying attention to Raman scattered light of an arbitrary wavelength (frequency) and measurement of a Raman spectrum at a specific point of the sample 28 can be performed with high resolution.

  Furthermore, in this embodiment, the Raman microscope is configured as a confocal optical system that collects light in a line shape. Thereby, since the resolution in the Z direction can be improved, three-dimensional measurement of the Raman spectrum becomes possible. That is, the relative distance between the objective lens 27 and the sample 28 can be changed, and the focal position on the sample 28 can be scanned in the Z direction. As a result, scanning in the XYZ directions becomes possible, and the Raman spectrum can be measured for a three-dimensional spatial distribution. In other words, the Raman spectrum can be measured at an arbitrary point of the three-dimensional sample 28. Even when such a three-dimensional spatial distribution of a Raman spectrum is measured, the measurement time can be shortened by illuminating in a line shape. Therefore, measurement can be performed in a practical time.

  Next, the configuration of the phase contrast microscope will be described. A lamp light source is used as the illumination light source 41 of the phase contrast microscope. The illumination light source 41 is disposed on the side opposite to the objective lens 27 of the Raman microscope. The illumination light 71 emitted from the illumination light source 41 is refracted by the lens 42 and enters the ring slit 43. The ring slit 43 is a ring zone in which a transmission part that transmits light is provided in a ring-shaped region. The ring slit 43 converts the illumination light 71 from the illumination light source 41 into a ring-shaped illumination light 71. The illumination light 71 converted into a ring shape by the ring slit 43 enters the sample 28 collected by the condenser lens 44. The condenser lens 44 collects light so that the ring-shaped illumination light 71 overlaps on the sample 28.

  Of the illumination light 71 collected by the condenser lens 44, the transmitted light 72 that has passed through the sample 28 enters the objective lens 27. The transmitted light 72 propagates through the same optical component as the optical component on which the outgoing light 62 is incident. That is, the transmitted light 72 is refracted by the objective lens 27, refracted by the lens 26, reflected by the mirror 25, refracted by the lens 24, and enters the galvanometer mirror 23. When a phase contrast microscope is used, observation is performed without scanning with the galvanometer mirror 23. For example, the galvanometer mirror 23 is disposed with an inclination of 45 ° with respect to the optical axis of the lens 24. Therefore, the galvanometer mirror 23 reflects the transmitted light at an angle of 90 ° and reflects it in the direction of the first beam splitter 22. Part of the transmitted light that has entered the first beam splitter 22 enters the second beam splitter 21. As described above, since the transmittance of the first beam splitter 22 is 20%, 20% of the transmitted light 72 incident on the first beam splitter 22 is incident on the second beam splitter 21. A part of the transmitted light 72 incident on the second beam splitter 21 is reflected and incident on the lens 45a. The transmitted light 72 is refracted by the lens 45a and enters the lens 45b. The transmitted light 72 refracted by the lens 45a forms an image on the front side of the lens 45a and enters the lens 45b. The lens 45b refracts the incident transmitted light 72. The transmitted light 72 refracted by the lens 45 b enters the phase plate 46.

  The phase plate 46 is provided with a ring-shaped phase film. The light incident on the ring-shaped phase film is given a phase difference of π / 2 from the light incident on other regions. That is, the phase of the transmitted light 72 incident on the region where the ring-shaped phase film is provided is delayed in phase by π / 2 relative to the transmitted light 72 incident on the inner side and the outer side thereof. The transmitted light 72 that has passed through the phase plate 46 is refracted by the lens 47 and enters the CCD camera 48. The lens 47 is arranged to form an image of transmitted light on the light receiving surface of the CCD camera 48.

  By providing the phase contrast microscope in this way, it is possible to observe the distribution of the phase objects having different refractive indexes of the sample 28. Therefore, when cells are injected into a buffer solution that can maintain the state of the cells, even if the substance has almost no difference in transmittance between the buffer solution and the cells, the cells can be observed by the difference in refractive index. it can. That is, the cell becomes a phase object with respect to the buffer solution. The distribution of the phase object, that is, the spatial distribution of the cells can be observed with a phase contrast microscope. Then, the three-dimensional spatial distribution of the Raman spectrum is measured only in the region where the cell density is high. That is, even when transparent cells are injected into a transparent buffer solution, the spatial distribution of the cells can be measured. As described above, since efficient measurement can be performed with the above configuration, a Raman spectrum can be measured for a substantially long time. Therefore, the convenience of the optical microscope can be improved.

  Since the optical microscope 1 is configured to include a phase contrast microscope and a Raman microscope, various observations can be performed. The application range of the optical microscope can be widened, and observation that has been impossible until now can be performed. With this optical system, a Raman spectrum can be measured with high resolution. Moreover, even when the three-dimensional spatial distribution of the Raman spectrum is measured with high resolution, the measurement time can be shortened. For example, observation of cells such as Hela cells can be performed with high resolution.

  Next, the configuration in the vicinity of the sample 28 will be described with reference to FIGS. FIG. 2 is a diagram schematically showing an optical system in the vicinity of the sample 28. FIG. 3 is a plan view showing spots of the incident light 61 on the sample 28. FIG. 4 is a plan view showing the configuration of the ring slit 43.

  First, incident light 61 from the laser light source 11 incident on the sample 28 in the Raman microscope will be described. Incident light 61 is focused on the sample 28 by the objective lens 27. The sample 28 is placed on a transparent stage 29. The stage 29 is preferably an XYZ stage. Here, a state of focusing on the surface of the sample 28 on the objective lens 27 side is illustrated, but when scanning in the Z direction, the objective lens 27 or the stage 29 is moved to move the objective lens 27 and the sample 28. The distance between the two may be changed along the optical axis. For example, the distance between the objective lens 27 and the stage 29 can be changed by the processing device 35. In this way, by making the distance between the objective lens 27 and the sample 28 variable, the three-dimensional spatial distribution of the Raman spectrum can be measured. In place of the galvanometer mirror 23, the stage 29 may scan in the X direction. The entire surface of the sample 28 can also be observed by scanning the stage 29 in this way.

  The incident light 61 is irradiated on the sample 28 as shown in FIG. Since the incident light 61 is converted into a line shape by the cylindrical lens 15, the light beam spot of the incident light 61 is in a line shape on the sample 28. The incident light 61 has a line shape along the direction converted by the cylindrical lens 15, that is, the Y direction. For example, the spot of the line-shaped incident light 61 can have a length of about 60 μm in the Y direction. The line-shaped incident light 61 is scanned by the galvanometer mirror 23 in the X direction, for example. That is, scanning is performed in a direction perpendicular to the direction in which the incident light 61 extends in a line. Thereby, the fixed area | region of the sample 28 can be illuminated. Out of this incident light 61, the outgoing light 62 scattered and emitted by the sample 28 is dispersed by the spectroscope 33 as described above and detected by the detector 34.

  Next, the illumination light 71 from the illumination light source 41 in the phase contrast microscope will be described. The illumination light 71 enters the ring slit 43 via the lens 42. The ring slit 43 converts the incident illumination light 71 into a ring-shaped illumination light 71. The shape of the ring slit 43 is as shown in FIG. FIG. 5 is a plan view showing the configuration of the ring slit 43. The ring slit 43 is constituted by a disc in which a ring-shaped transmission part 43a is formed. A light shielding part 43b is formed on the outer side and the inner side of the transmission part 43a. The light incident on the transmission part 43 a is transmitted through the ring slit 43, and the light incident on the light shielding part 43 b is blocked by the ring slit 43. In addition, the connection part 43c which connects the inner side light-shielding part 43b and the outer side light-shielding part 43b of the transmission part 43a is arrange | positioned so that the transmission part 43a may be crossed. The ring-shaped transmission part 43a is divided into three by this connection part 43c. It is preferable to provide the connecting portions 43c equally, that is, every 120 °. The light incident on the connection portion 43c is shielded in the same manner as the light shielding portion 43b.

  The ring slit 43 can be constituted by a circular plate having such a transmission part 43a. In the above description, the ring slit 43 is formed of a circular plate, but is not limited thereto. For example, the ring slit 43 may be configured by a liquid crystal element or the like. Alternatively, the light shielding portion 43b may be formed by forming a light shielding film on a transparent plate.

  The illumination light 71 spreads inside and outside the ring slit 43 and enters the condenser lens 44. The condenser lens 44 refracts the illumination light 71 incident from the ring slit 43 to the sample 28. The illumination light 71 illuminates the sample 28 through the transparent stage 29. Thereby, a predetermined region of the sample 28 is illuminated by the illumination light 71. Of this illumination light 71, transmitted light 72 that has passed through the sample 28 is detected by the CCD camera 48. In this manner, the illumination light 71 from the illumination light source 41 and the laser light from the laser light source are incident from the front side and the back side of the sample 28. Thereby, the optical microscope 1 which has a phase-contrast microscope and a Raman microscope with a simple optical system can be comprised.

  Next, the beam spot of the emitted light 62 on the entrance slit 32 of the spectrometer 33 will be described with reference to FIG. FIG. 5 is a plan view showing the configuration of the entrance slit 32, and also shows the outgoing light 62. The entrance slit 32 has an opening 32a extending in the Y direction. The light that has passed through the opening 32 a is split by the spectroscope 33 and enters the detector 34. The width of the entrance slit 32, that is, the width of the opening 32a of the entrance slit 32 in the X direction can be set to 10 μm, for example. Outgoing light 62 having a linear beam spot is incident on the entrance slit 32. That is, since the incident light 61 is converted into a line shape by the cylindrical lens 15 and the iris diaphragm 39 and then incident on the sample 28, a linear spot along the Y direction is formed on the incident slit 32.

  The Raman microscope is a slit confocal optical system because the line-shaped incident light 61 on the sample and the incident slit 32 of the spectroscope 33 are in an imaging relationship. That is, the iris diaphragm 39 and the line-shaped incident light 61 on the sample 28 are conjugated with each other, and the opening 32a of the incident slit 32 and the line-shaped incident light 61 on the sample 28 are conjugated with each other. It has become a relationship. Thereby, the outgoing light 62 emitted from a surface other than the surface where the incident light 61 is condensed can be removed by the incident slit 32. For example, as shown in FIG. 2, when the light is condensed on the surface of the sample 28 on the objective lens side, the Raman scattered light from the other surface enters the outside of the opening 32a of the entrance slit 32 and is removed. Is done. That is, the outgoing light 62 from the inside of the sample 28 enters the outside of the opening 32a of the entrance slit 32 as shown in FIG. Therefore, outgoing light 62 from other than a predetermined surface is shielded by the entrance slit 32 and does not contribute to detection. Thus, the resolution in the Z direction can be improved by using a confocal optical system. The magnification of the image on the sample surface and the light receiving surface of the detector 34 can be set to 50 times, for example. In this case, the line-shaped illumination having a length of 60 μm is about 3 mm on the light receiving surface of the detector 34.

  Thus, the outgoing light 62 that has passed through the entrance slit 32 is split by the spectroscope 33 and enters the detector 34. The spectroscope 33 disperses the slit-shaped outgoing light 62 in the X direction according to the wavelength (frequency). That is, the spectroscope 33 disperses the slit-shaped outgoing light 62 in a direction perpendicular to the direction. The emitted light 62 is detected by the detector 34 of a two-dimensional array in the form of slits dispersed by the spectroscope 33. Thereby, the spatial distribution of the Raman spectrum can be measured.

  Specifically, it is assumed that the pixels of the detector 34 are arranged in the XY directions. The X direction indicates the wavelength of the emitted light, and the Y direction indicates the position of the emitted light 62 on the sample 28. That is, the emitted light 62 received by the pixels at the same position in the X direction has the same wavelength. Even the emitted light emitted from the same position on the sample surface is received by different pixels in the X direction when the wavelengths are different. On the other hand, the emitted light 62 received by the pixels at the same position in the Y direction is emitted from the same position in the Y direction on the sample 28. The emitted light 62 having the same wavelength is received by different pixels in the Y direction on the light receiving surface according to the position in the Y direction on the sample surface. In other words, the light intensity distribution in the XY plane on the light receiving surface of the detector 34 indicates the intensity distribution in the ω-Y plane of the emitted light 62. Note that ω is the angular frequency of the outgoing light 62. By illuminating in this way, measurement time can be shortened. Specifically, the measurement time can be shortened by 1 / (number of pixels for one line). Furthermore, even when a wide observation range is measured, the measurement can be performed without reducing the resolution. Further, since the laser light is illuminated as a line, the laser intensity per unit area can be reduced as compared with a conventional Raman microscope. That is, since the laser light from the laser light source, which is a point light source, is spatially dispersed to provide line illumination, the laser intensity per unit area can be reduced. Therefore, even when an attempt is made to irradiate the same dose as in point illumination, the laser dose per unit time and unit area can be reduced. Thereby, the sample can be prevented from being damaged by the laser beam. This is particularly effective when a living body that is easily damaged by laser light is used as a sample.

  Here, the emitted light 62 detected by the detector 34 when the incident light 61 is scanned will be described with reference to FIG. FIG. 6 is an image diagram showing a Raman spectrum measured two-dimensionally in the optical microscope 1 according to the present invention. FIG. 6A is a diagram schematically showing incident light 61 scanned on the sample surface. FIG. 6B is a diagram conceptually showing the light receiving surface of the detector when the incident light 61 is scanned as shown in FIG.

  As shown in FIG. 6A, on the sample 28, the line-shaped incident light 61 along the Y direction is scanned in the X direction. Here, a description will be given assuming that scanning is performed in the order of the line-shaped incident light 61a, incident light 61b, incident light 61c, and incident light 61d on the sample 28. At this time, it is assumed that the Raman spectrum of the incident light 61a, the incident light 61b, the incident light 61c, and the outgoing light from the respective positions of the incident light 61d is measured by the detector 34. Here, the light receiving surface when the outgoing light 62 from the incident light 61a enters the detector is referred to as a light receiving surface 65a. That is, the outgoing light 62, which is Raman scattered light from the position of the incident light 61a, enters the light receiving surface 65a. Similarly, the light receiving surfaces when the incident light 61b, the incident light 61b, and the outgoing light 62 from the incident light 61c enter the detector are a light receiving surface 65b, a light receiving surface 65c, and a light receiving surface 65d, respectively. The light intensity distribution in each of the light receiving surfaces 65a to 65d represents the distribution of the Raman spectrum in the Y direction. Therefore, as shown in FIG. 6B, the distribution of the Raman spectrum in the XY plane can be measured by arranging the measurement results from the light receiving surfaces 65a to 65d in the X direction. Furthermore, XYZ-ω four-dimensional measurement can be performed by scanning in the Z direction. Various spectrum analysis can be performed by performing the four-dimensional measurement.

  Next, the configuration of the phase contrast microscope will be described with reference to FIGS. FIG. 7 is a diagram showing the configuration of the optical system of the phase contrast microscope. FIG. 8 is a plan view showing the configuration of the phase plate. In FIG. 7, the stage 29 and the optical system from the lens 26 to the lens 45b are not shown. In FIG. 7, only the illumination light 71 passing through only one of the light transmission portions of the ring slit 43 and only the 0th-order diffracted light 74 and the + 1st-order diffracted light 73 are shown. The illumination light 71 from the illumination light source 41 is refracted by the lens 42 and enters the ring slit 43. For example, the ring slit 43 having the shape shown in FIG. 4 can be used. Since the illumination light 71 incident on the ring slit 43 can pass only through the transmission part 43a, it is converted into a ring shape. The ring-shaped illumination light 71 is condensed by the condenser lens 44 and illuminates the sample 28. The ring-shaped illumination light 71 is collected on the sample 28 so as to overlap with a point on the optical axis as a center. Here, the ring-shaped illumination light 71 is condensed by the condenser lens 44 so as to illuminate a region including a point on the optical axis. Of the illumination light 71 incident on the sample 28, the transmitted light 72 that has passed through the sample 28 includes zero-order diffracted light (direct light) 74 and first-order diffracted light 73. That is, if there is a difference in refractive index, that is, a phase difference in the sample 28, diffraction occurs. When the amount of phase difference is small, the zero-order diffracted light 74 and the first-order diffracted light 73 are shifted by a quarter wavelength (π / 2).

  The zero-order diffracted light 74 is transmitted through the sample 28 in the same direction as the incident direction of the illumination light 71. On the other hand, the first-order diffracted light 73 irradiates the sample 28 with a predetermined angle (diffraction angle) from the incident direction of the illumination light 71. Here, in order to simplify the description, refraction at the sample 28 and the stage is omitted. The transmitted light 72 including the 0th-order diffracted light 74 and the first-order diffracted light 73 is incident on the objective lens 27. Then, the light is refracted by the objective lens 27 and propagates through the optical system shown in FIG. At this time, the zero-order diffracted light 74 and the first-order diffracted light 73 propagate through different paths and enter the phase plate 46. The phase plate 46 is provided with a ring-shaped phase film 46a as shown in FIG. That is, the phase plate 46 includes a transparent disk and a ring-shaped phase film 46a provided thereon. The zero-order diffracted light 74 is incident on a region of the phase plate 46 where the phase film 46a is provided. On the other hand, the first-order diffracted light 73 is incident on a region of the phase plate 46 where the phase film 46a is not provided. That is, the primary diffracted light 73 is incident on a region inside the ring-shaped phase film 46a. 7 shows only the + 1st order diffracted light 73, the first order diffracted light 73 passes only inside the phase film 46a, but the −1st order diffracted light is outside the phase film 46a. Pass through.

  The phase film 46a emits the incident light with a phase delayed by a quarter wavelength. That is, the 0th-order diffracted light 74 incident on the region where the phase film 46a is provided is emitted with a quarter wavelength delay compared to the first-order diffracted light 73 incident on the region where the phase film 46a is not provided. The ring slit 43 and the phase plate 46 are conjugated with each other. Therefore, the transmission part 43 a of the ring slit 43 and the phase film 46 a are conjugated with each other through the sample 28. Accordingly, only the 0th-order diffracted light 74 enters the region where the phase film 46a is provided, and the first-order diffracted light 73 enters the region where the phase film 46a is not provided. As described above, the phase plate 46 provides a phase difference in a region corresponding to the transmission part 43 a of the ring slit 43.

  The first-order diffracted light 73 and the zero-order diffracted light 74 are collected by the lens 47 and enter the light receiving surface of the CCD camera 48. The first-order diffracted light 73 and the zero-order diffracted light 74 are incident on the same region on the light receiving surface of the CCD camera 48. That is, the lens 47 condenses the transmitted light 72 so that the 1st-order diffracted light 73 and the 0th-order diffracted light 74 overlap on the light receiving surface of the CCD camera 48 around the point on the optical axis of the lens 47.

  Due to the phase film 46a, the zero-order diffracted light 74 and the first-order diffracted light 73 are shifted by ¼ wavelength, and the phase object becomes bright due to interference. Thereby, the distribution in the XY plane of the phase object can be measured.

  If the intensity of the first-order diffracted light 73 is weaker than that of the zero-order diffracted light 74, the transmittance of the region where the phase film 46a is provided may be lowered. Specifically, the absorption film is provided in the region where the phase film 46a is provided. As a result, the contrast can be increased. In the above description, the phase of the region where the 0th-order diffracted light 74 is incident is delayed by π / 2, but it may be advanced by π / 2. For example, the phase can be advanced by reducing the thickness of the phase plate 46 only in the ring-shaped region. The configuration of the phase contrast microscope is not limited to the above-described configuration.

  Here, the first boom splitter 22 that branches the optical path of the emitted light 62 from the optical path of the incident light 61 is arranged closer to the sample 28 than the second beam splitter 21. That is, the transmitted light 72 branched by the second beam splitter 21 is incident on the second beam splitter 21 via the first beam splitter 22. A second beam splitter 22 is disposed between the first beam splitter 22 and the third cylindrical lens 15. With such an arrangement, an optical microscope including a Raman microscope and a phase-contrast microscope can be observed without reducing the intensity of Raman scattered light. That is, incident light 61 passes through the second beam splitter 22, but outgoing light 62 does not pass through the second beam splitter 21. Thereby, the loss of light by the second beam splitter 21 is eliminated, and a decrease in the intensity of Raman scattered light can be suppressed. Therefore, it is possible to prevent the measurement time from becoming long.

  In the above description, the optical microscope 1 including the Raman microscope has been described, but the present invention is not limited to this. Any microscope that detects the outgoing light 62 emitted from the sample at a wavelength different from the laser wavelength of the incident light 61 may be used. For example, a microscope that detects fluorescence excited by excitation light or a microscope that detects infrared absorption may be used. Even with these microscopes, the spatial distribution of the spectrum can be measured in a short time.

  In the above description, the outgoing light 62 reflected from the first beam splitter 22 and the transmitted light 72 reflected from the second beam splitter 21 are detected, but the light transmitted through the beam splitter is detected by changing the arrangement of the optical system. You may make it do.

  As described above, the scanning speed can be increased by scanning the light beam of the incident light 61. Thereby, measurement time can be shortened. In the above description, the scanning unit is described as the galvanometer mirror 23, but the scanning unit may be limited to this. For example, the incident light 61 may be scanned using an acousto-optic polarizer (AOD). The light conversion means for converting the laser light from the laser light source 11 into a line shape can be constituted by a cylindrical lens, an iris diaphragm having a line-shaped opening, and a Powell lens alone or in combination.

It is a figure which shows the structure of the optical microscope concerning this invention. In the optical microscope concerning this invention, it is a figure which shows typically the structure of a sample vicinity. In the optical microscope concerning this invention, it is a figure for demonstrating the incident light which injects into a sample from a laser light source. It is a top view which shows the structure of the ring slit used in the optical microscope concerning this invention. It is a figure for demonstrating the emitted light which injects into the slit used in the optical microscope concerning this invention. It is an image figure which shows the Raman spectrum measured two-dimensionally in the optical microscope concerning this invention. It is a figure which shows an example of the optical system of the phase-contrast microscope which is a part of optical microscope concerning this invention. It is a top view which shows the structure of the phase plate used in the optical microscope concerning this invention.

Explanation of symbols

11 laser light source, 12 mirror, 13 first cylindrical lens,
14 2nd 3rd cylindrical lens 15 3rd cylindrical lens,
16 lens, 21 second beam splitter, 22 first beam splitter,
23 Galvano mirror, 24 lenses, 25 mirrors, 26 lenses,
27 objective lens, 28 sample, 31 lens, 32 slit, 33 spectroscope,
34 detector, 35 processing device, 38 edge filter, 39 iris diaphragm,
41 Illumination light source, 42 lens, 43 ring slit, 43a transmission part,
44 condenser lens, 45 lens,
46 phase plate, 46a retardation film, 47 lens, 48 CCD camera,
61 incident light, 62 outgoing light, 71 illumination light, 73 0th order diffracted light, 74 1st order diffracted light

Claims (11)

A laser light source;
A light converting means for converting a light beam from the laser light source into a linear light beam;
Scanning means for scanning the light beam converted into the line shape;
An objective lens for condensing the light beam scanned by the scanning means into a line and entering the sample; and
Of the light beam incident on the sample, a first beam splitting the light emitted from the sample toward the objective lens with a different wavelength from the line light beam incident on the sample from the laser light source A splitter,
An incident slit disposed along a direction corresponding to the line-shaped light beam on an incident side on which the outgoing light branched by the first beam splitter is incident; and the outgoing light that has passed through the incident slit is A spectroscope that spatially disperses according to the wavelength;
An optical microscope comprising: a two-dimensional array photodetector that detects outgoing light dispersed by the spectrometer.   The optical microscope according to claim 1, wherein the scanning unit is a galvanometer mirror.   The optical microscope according to claim 1, wherein the scanning unit scans the light beam in a direction perpendicular to a line direction of the light beam.   The optical microscope according to claim 1, wherein the light conversion unit is a cylindrical lens.   5. The optical microscope according to claim 4, wherein an iris diaphragm having a slit-like opening at a position where the incident light is collected by the cylindrical lens, the iris diaphragm having a variable opening size is provided. .   The optical according to any one of claims 1 to 5, wherein a filter for blocking light having a laser wavelength of the laser light source is provided between the first splitter and the two-dimensional array photodetector. microscope.   7. The optical microscope according to claim 1, wherein the surface condensed by the light conversion means and the sample surface are conjugated with each other, and the sample surface and the entrance slit are conjugated with each other. .   The optical microscope according to claim 7, wherein a relative distance between the objective lens and the sample is variable. An illumination light source arranged on the opposite side of the sample from the objective lens and emitting illumination light;
A ring slit that turns the illumination light from the illumination light source into a ring-shaped illumination light; and
A condenser lens that collects the ring-shaped illumination light and enters the sample; and
A second beam splitter for allowing the transmitted light collected by the condenser lens and transmitted through the sample to enter through the objective lens; and the optical path of the transmitted light branches off from the optical path of the incident light from the laser light source A second beam splitter that
A phase plate providing a phase difference in a ring-shaped region corresponding to the ring slit of transmitted light branched by the second beam splitter;
The optical microscope according to claim 1, further comprising an image sensor that images transmitted light from the phase plate. The first beam splitter is disposed on the sample side of the second beam splitter;
The optical microscope according to claim 9, wherein the transmitted light is incident on the second beam splitter through the first beam splitter. The optical microscope according to claim 8 or 10, wherein the imaging element observes a distribution of phase objects having different phases in the sample.

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