JP2008309706A - Spectrometer and spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-sensitivity spectrometer with high spatial and time resolution, and also to provide its method. <P>SOLUTION: A group of light beams (also called object beam) such as scattered light and transmitted light radially emitted in various directions from one bright spot on a measuring object S, enter an objective lens 12, pass therethrough, and then arrive at a fixed mirror part 15 and a movable mirror part 16 of a phase shifter 14. The rays are reflected by the mirror parts 15 and 16, respectively, and then form an interference figure on an imaging surface of a detection part 24 through an image-forming lens 22. Moving the mirror part 16 in such a state, gradually changes the intensity of interference light on the imaging surface of the detection part, providing a waveform of an imaging intensity change (interference light intensity change) called an interferogram. By Fourier-transforming this interferogram spectral characteristics are acquired which are the wavelength-by-wavelength relative intensities of the light emitted from the one bright spot on the measuring object S. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a spectroscopic measurement apparatus and a spectroscopic measurement method that are useful as a biocomponent analysis technique in a very small single cell in the field of life science, and as a material evaluation technique of a fine structure device in the field of nanotechnology.

  The Human Genome Project has ended, and we have reached an era called post-genome in the life science field. In the post-genome era, research focused on clarifying the functional role of base sequences composed of approximately 3 billion base pairs, also called human blueprints that were analyzed by the Human Genome Project. Is underway. However, it is said that the human body is composed of an enormous number of cells of approximately 60 trillion. Using such a human body, the biological function due to the difference in the base sequence of a part of 3 billion base pairs. It is not easy to assess the impact. Therefore, in the post-genome era, research for elucidating functions using living cells cultured in petri dishes is being strongly promoted.

  In addition, clinical trials have been undertaken to select treatment methods suitable for individual constitutions, called tailor-made treatments and tailor-made treatments. Specifically, for example, cancer cells (tumor cells) collected surgically are cultured on a petri dish, and the effects of a plurality of anticancer agents are experimentally verified using the cultured cells. The right drug. For this purpose, it is necessary to observe in detail changes in biological components inside a very small single cell having a diameter of about 10 microns.

Against the background of research and development and demand in the life science field, a technique for observing the distribution of biological components inside a single cell in detail with high spatial resolution has been intensively researched and developed. As a typical observation method, there is a method in which a specific component is labeled with a fluorescent substance and the spatial distribution of the biological component is measured by observing the spatial position of the fluorescence.
For example, quantum dots and green fluorescent protein (GFP) are used as fluorescent substances for labeling specific biological components. Quantum dots are ultrafine particles of about several tens of nanometers that emit fluorescence of different colors according to their particle sizes. Therefore, if one or more types of quantum dots having a uniform particle size are made and chemically bonded to a specific biological component, the spatial distribution of the fluorescence emitted by the quantum dot is observed to observe the specific biological component. Spatial distribution can be indirectly measured.

As a technique for measuring the distribution of fluorescence in a minute particle, a technique using a spectroscopic technique called dispersion spectroscopy or Fourier spectroscopy has been proposed (see Non-Patent Document 1).
In wavelength dispersion spectroscopy, when a diffraction grating is irradiated with light transmitted through a measurement sample or light reflected from the measurement sample surface (hereinafter referred to as object light), the diffraction angle varies depending on the wavelength of the object light. It is a spectroscopic method using the principle.

  On the other hand, Fourier spectroscopy is a spectroscopic measurement technique based on phase shift interference using a Michelson-type two-beam interference optical system. The object light is branched into two by a beam splitter such as a half mirror, the respective light beams are reflected by the mirror, reach the half mirror again, and the two light beams are merged to observe the interference phenomenon. A mirror that reflects one of the two branched light beams (reference light) is called a reference mirror. In Fourier spectroscopy, the reference mirror is moved with high accuracy at a resolution shorter than the wavelength of light to change the interference light intensity, so-called interferogram is detected, and this interferogram is mathematically Fourier transformed. Obtain spectral characteristics.

The direction of the light beam of the object light emitted from the measurement sample surface is varied depending on scattering, refraction, reflection, and the like. When light components in various directions are irradiated on the diffraction grating and the reference mirror in this way, the spectral accuracy is lowered. Therefore, to increase the spatial coherency (coherence) of the object light in any spectroscopic method, only a light component in a specific direction of the object light using a pinhole or slit having a minute aperture is used as a diffraction grating or a reference. The mirror is illuminated. Depending on the required spectral performance, a pinhole having a hole diameter of about several tens of microns is used in the dispersion type spectroscopy, and a slit having an opening width of about several millimeters is used in the Fourier spectroscopy.
Jiro Hiraishi, "Fourier Transform Infrared Spectroscopy" Society Publishing Center, November 1985

  However, when pinholes and slits are used, most of the object light does not pass through the pinholes and slits and is not used for measurement, so the light utilization efficiency is low. The fluorescence emitted from the quantum dots and GFP described above is extremely weak light that can be finally observed with a high-sensitivity cooled CCD camera with high quantum efficiency. For this reason, the conventional spectroscopic technique is unsuitable for weak light measurement, and it has been difficult to observe the fluorescence emitted at an arbitrary position inside a single cell or discriminate the fluorescence color.

In any of the spectroscopic methods, since all of light generated from a predetermined measurement region on the measurement sample is subjected to spectroscopy, an average spectral characteristic in the measurement region is acquired. If the area of the measurement region is reduced, the spatial resolution is improved, but the total amount of detected object light is reduced and the spectral sensitivity is lowered. Further, since it is a technique for measuring object light generated from one point on the sample, the measurement region must be scanned in two dimensions in order to perform spectroscopic measurement in two dimensions. Therefore, the spatial resolution of the two-dimensional spectral image greatly depends not only on the area of the measurement region but also on the interval between the measurement points. Therefore, in order to perform two-dimensional spectroscopy with a high spatial resolution, it is necessary to reduce the area of the measurement region and provide measurement points with a high spatial density, resulting in a long measurement time. When the measurement time becomes long, when a sample such as a living cell that moves is spectroscopically measured, the measurement object moves within the measurement time, and the image is blurred.
Further, when the measurement sample is optically transparent, it is not possible to perform spectroscopy by limiting the measurement region in the depth direction. For this reason, for example, a three-dimensional spectral absorptance distribution cannot be measured.

  The above-mentioned issues are not limited to the field of life science, but the development of nanotechnology that has achieved remarkable development of electronic device products with fine structures on the order of nanometers such as semiconductor memory, liquid crystal, plasma, and organic EL flat panel displays. It also exists in the field. That is, as a material evaluation technique based on spectroscopic measurement of a fine structure device, there is a high demand for a spectroscopic optical method having a high two-dimensional or three-dimensional spatial resolution.

  The problem to be solved by the present invention is to provide a highly sensitive spectroscopic measuring apparatus with high spatial and temporal resolution and a method therefor.

The spectroscopic measurement device according to the present invention, which has been made to solve the above problems,
a) a splitting optical system in which light emitted in various directions from each measurement point of the object to be measured is incident;
b) An imaging optical system for guiding the light transmitted through the splitting optical system to substantially the same point to form an interference image;
c) a detection unit for detecting the light intensity of the interference image;
d) optical path length difference expansion / contraction means for expanding / contracting a relative optical optical path length difference between a part of the light traveling from the splitting optical system toward the imaging optical system and the remaining light;
e) Obtaining an interferogram at each measurement point of the object to be measured based on a change in light intensity detected by the detection unit by expanding / contracting the optical optical path length difference by the optical path length difference expansion / contraction means, and the interferogram A processing unit that obtains a spectrum by Fourier transforming
It is characterized by providing.

Moreover, although it is the same principle, the spectroscopic measurement apparatus which concerns on this invention as another structure is
a) a splitting optical system that guides light emitted from each measurement point of the object to be measured in various directions into a first reflecting part and a second reflecting part;
b) an imaging optical system for guiding the light reflected by the first and second reflecting portions to substantially the same point to form an interference image;
c) By moving the first and second reflecting parts relatively, light traveling from the splitting optical system to the imaging optical system via the first reflecting part and the second reflecting part from the splitting optical system. An optical path length difference expansion / contraction means for expanding / contracting the optical optical path length difference of the light passing through the imaging optical system,
d) a detection unit for detecting the light intensity of the interference image;
e) Obtaining an interferogram at each measurement point of the object to be measured based on a change in light intensity detected by the detection unit by expanding / contracting the optical optical path length difference by the optical path length difference expansion / contraction means, and the interferogram A processing unit that obtains a spectrum by Fourier transforming
It is characterized by providing.

  In the above configuration, when the reflecting surfaces of the first and second reflecting parts are arranged at an angle of 45 ° with respect to the optical axis of the parallel light beam transmitted through the split optical system, the first and second reflecting parts reflect the reflecting surfaces. The reflected light can be directly guided to the imaging optical system.

Furthermore, the spectroscopic measurement method of the present invention provides:
a) The light emitted from each measurement point of the object to be measured in various directions is divided into a phase-fixed light beam group and a phase-variable light beam group by a splitting optical system,
b) While expanding or contracting the optical path length difference of the phase variable beam group, the phase variable beam group and the phase fixed beam group are guided to substantially the same point by an imaging optical system to form an interference image,
c) Obtaining an interferogram of each measurement point of the object to be measured based on a change in light intensity of the interference image, and obtaining a spectrum by performing a Fourier transform on the interferogram.

The spectroscopic measurement apparatus and the spectroscopic measurement method according to the present invention use an imaging optical system, and the light generated from each bright spot that optically configures the object to be measured is split by the split optical system. The interferogram of the object to be measured is obtained using the interference phenomenon between the lights. Here, the term “divided optical system” is used to distinguish the light from each luminescent spot simply from the “spectral optical system” that optically divides the light for each wavelength.
In the present invention, since all the light beams that have passed through the split optical system can be used for analysis, the light utilization efficiency is extremely high, and it is suitable for weak light measurement.
In addition, since the present invention uses an imaging optical system, high-sensitivity one-dimensional spectroscopic measurement is possible if a one-dimensional detection device is used as the detection unit, and high-sensitivity 2 is achieved if a two-dimensional detection device is used. Dimensional spectroscopic measurement is possible.

  In general, it is known that the spatial resolution of the imaging optical system is determined in proportion to λ / NA. Λ is the wavelength of light, and NA is the numerical aperture of the objective lens. Therefore, high resolution can be obtained by using a high NA objective lens. Further, if a super-resolution technique such as an immersion lens or modified illumination is used in combination, the resolution can be further improved.

Furthermore, in the present invention, it is possible to measure the spectral characteristics of only the light rays that are emitted from the focus position of the splitting optical system and that affect the image formation. Thereby, the three-dimensional spectral characteristics can be acquired by moving the objective lens or the sample constituting the split optical system in the focal depth direction and moving the in-focus position.
Since the depth of focus is optically determined in proportion to λ / NA ² , the spatial resolution in the depth direction can be easily improved using a high NA optical system using super-resolution technology. .

  When an object is irradiated with light, object light is generated due to various optical phenomena such as reflection, refraction, scattering, and fluorescence. When an object is optically modeled by these generated lights, it can be regarded as an aggregate of bright spots that are ideal point light sources. Although directivity differs depending on the illumination method and the optical phenomenon that generates the object light, light rays are emitted radially from one bright point that is an ideal point light source. The imaging optical system forms the optically conjugate bright spot group as an image by reconstructing the bright spot group that optically constitutes an object in this way on the imaging surface using a lens. In the present invention, this imaging optical system is used.

  In the present invention, the object light generated from each bright spot that optically constitutes the object is divided into two light beam groups, and the interference light intensity (condensation) formed on the imaging surface of the detector by the interference phenomenon between these light beam groups. Image intensity) is detected. When the relative optical path length difference between the two light groups is changed, the interference light intensity of the light beams of various wavelengths constituting both light groups changes periodically according to the lengths of the wavelengths. Light intensity change, that is, an interferogram can be acquired. Spectral characteristics, which are relative intensities for each wavelength, can be acquired by Fourier transforming this interferogram.

  Further, if the in-focus position of the objective lens on which the object light generated from each bright spot constituting the object is incident can be scanned, a three-dimensional image of the object can be acquired. For example, when a specific component that will be contained in an object is labeled with a fluorescent dye, the fluorescent dye becomes a self-luminous substance by irradiating the object with excitation light, and light rays are emitted in various directions. By detecting the interference light intensity of this light beam, the detailed component distribution inside can be observed while the cells are still alive. The same applies to scattered light that is an electric field component generated from an electric dipole excited by illumination light.

In the case of fluorescent emission or scattered light, the initial phase of the light beam between the bright spots does not necessarily match. In other words, it can be considered that an optical model is formed when a large number of bright spots having an initial phase that are not aligned are distributed on the object. However, in the space from the object plane to the imaging plane, if the optical path of each ray is traced, the light rays generated from one bright spot that optically composes the object are aligned in phase on the imaging plane. It can be considered that the image is formed by focusing on one point.
Hereinafter, specific examples in which the present invention is applied to a spectral tomographic image measuring apparatus which is a spectral measuring apparatus will be described.

1 to 8 show a first embodiment of the present invention, and FIG. 1 is a schematic diagram of an overall configuration of a spectral tomographic image measurement apparatus 10 according to the present embodiment. When a light source (not shown) irradiates the object S with light, a group of rays (such as “object light”), such as scattered light and fluorescence emitted radially from one bright spot of the object S to be measured in various directions. Is also incident on the objective lens 12 and converted into a parallel light beam.
The objective lens 12 is configured to be movable in the optical axis direction by a lens driving mechanism 13. The lens driving mechanism 13 is for scanning the in-focus position of the objective lens 12 and can be constituted by, for example, a piezo element.

  The light beam after passing through the objective lens 12 does not have to be a perfect parallel light beam. As will be described later, it suffices if the light ray group generated from one bright spot can be expanded to such a degree that it can be divided into two or more. However, if the light beam is not a parallel light beam, an error is likely to occur in the phase difference amount generated according to the phase shift amount described later. Therefore, in order to obtain higher spectroscopic measurement accuracy, it is desirable to use a parallel beam as much as possible.

  The parallel light beam that has passed through the objective lens 12 reaches the phase shifter 14. For example, as shown in FIG. 2, the phase shifter 14 includes a rectangular plate-like fixed mirror portion 15, a columnar movable mirror portion 16 inserted into a circular hole portion 15 a at the center thereof, and a holding portion that holds the movable mirror portion 16. 17, a drive stage 18 that moves the holding unit 17 is provided. The surfaces of the fixed mirror unit 15 and the movable mirror unit 16 are optically flat and are optical mirror surfaces capable of reflecting the wavelength band of light to be measured by the apparatus 10.

In this embodiment, the phase shifter 14 corresponds to the optical path length difference expansion / contraction means of the present invention, and the fixed mirror portion 15 and the movable mirror portion 16 correspond to the first and second reflecting portions, respectively.
In the following description, among the light beams that have reached the phase shifter 14, the light beam that has reached the reflection surface of the fixed mirror unit 15 and reflected is reached and reflected by the fixed light beam group and the reflection surface of the movable mirror unit 16. The luminous flux is also referred to as a movable ray group.

  The drive stage 18 is composed of, for example, a piezoelectric element having a capacitance sensor, and moves the holding unit in the direction of arrow A in response to a control signal from the control unit 20. Thereby, the movable mirror part 16 moves to the arrow A direction with the accuracy according to the wavelength of light. Depending on the spectroscopic measurement capability, for example, in the visible light region, highly accurate position control of about 10 nm is required.

  The phase shifter 14 is arranged so that the reflecting surfaces of the fixed mirror unit 15 and the movable mirror unit 16 are inclined by 45 degrees with respect to the optical axis of the parallel light flux from the objective lens 12. The drive stage 18 moves the movable mirror unit 16 while maintaining the inclination of the reflecting surface of the movable mirror unit 16 with respect to the optical axis at 45 degrees. With this configuration, the moving amount of the movable mirror unit 16 in the optical axis direction is 1 / √2 of the moving amount of the drive stage 18. Further, the optical path length difference that gives a relative phase change between the two light beams of the fixed light beam group and the movable light beam group is twice the amount of movement of the movable mirror unit 16 in the optical axis direction.

  If the fixed mirror unit 15 and the movable mirror unit 16 are arranged obliquely in this way, a beam splitter for branching the light beam is not necessary, and the utilization efficiency of the object light can be increased. Further, since the movable mirror unit 16 is tilted, the amount of movement of the movable mirror unit 16 in the optical axis direction with respect to the amount of movement of the drive stage 18 is reduced, so that the influence of deterioration of the stage movement error on the spectral measurement accuracy can be reduced. .

  The fixed ray group and the movable ray group that reach the phase shifter 14 and are reflected by the reflecting surfaces of the fixed mirror unit 15 and the movable mirror unit 16 are converged by the imaging lens 22 and enter the imaging plane of the detection unit 24. . The detection unit 24 is composed of, for example, a two-dimensional CCD camera. The reflecting surface of the fixed mirror unit 15 and the reflecting surface of the movable mirror unit 16 are configured in parallel with an accuracy that does not shift the condensing positions of the two light beam groups on the imaging surface of the detecting unit 24.

The optical action of the spectral tomographic image measurement apparatus 10 having the above configuration will be described.
First, a group of light rays whose initial phases are not necessarily aligned, such as fluorescent light and scattered light, are focused on one point as waves whose phases are aligned on the imaging surface of the detection unit 24 via the objective lens 12 and the imaging lens 22, Description will be made based on an optical model for forming a bright spot image (interference image).

  As described above, a group of rays emitted from one bright spot of the measurement object S reaches the surfaces of the fixed mirror portion 15 and the movable mirror portion 16 of the phase shifter 14 via the objective lens 12. At this time, as shown in FIG. 4A, the light beam group reaches the surface of the fixed mirror unit 15 and the surface of the movable mirror unit 16 in two parts. It should be noted that the amount of light on the surface of the movable mirror 16 is fixed so that the amount of light in the group of light that has reached the surface of the fixed mirror 15, that is, the group of fixed rays, and the amount of light on the surface of the movable mirror 16 Although the area is set, it is also possible to equalize the light quantity by adjusting the relative light quantity difference by installing a neutral density filter in one or both of the optical paths of the fixed light beam group and the movable light beam group.

  The light beam groups reflected by the surfaces of the fixed mirror unit 15 and the movable mirror unit 16 enter the imaging lens 22 as a fixed beam group and a movable beam group, respectively, and form an interference image on the imaging surface of the detection unit 24. At this time, the light beams emitted from the object S to be measured include light of various wavelengths (and the initial phases of the light of the respective wavelengths are not necessarily aligned). By changing the optical path length difference between the group and the movable beam group, a waveform of an imaging intensity change (interference light intensity change) called an interferogram as shown in FIG. 6A is obtained. FIG. 6A is an interferogram in one pixel of the detection unit 24. In FIG. 6A, the horizontal axis represents the optical path length difference between the fixed light beam group and the movable light beam group as the movable mirror unit 16 moves, and the vertical axis represents the imaging intensity at one point on the imaging surface.

  By performing a Fourier transform on the interferogram, it is possible to obtain spectral characteristics that are relative intensities for each wavelength of light emitted from one bright spot of the measurement object S (see FIG. 6B). If the spectral characteristics can be obtained in all the pixels of the detection unit 24, the two-dimensional spectroscopic measurement of the object S can be performed.

Here, the principle of generating the interferogram will be described.
First, the relationship between the optical path length difference and the interference light intensity when the measurement wavelength is a single wavelength will be described with reference to FIGS. In FIG. 7, the horizontal axis indicates the relative optical path length difference between the fixed light beam group and the movable light beam group as the movable mirror unit moves, and the vertical axis indicates the imaging intensity in one pixel of the detection unit. .

  7A to 7C show the relationship between the optical path length difference and the interference light intensity of three types of monochromatic light (λa> λb> λc) having different wavelength lengths. The phase shift origin (indicated by the alternate long and short dash line in the figure) shown in the vicinity of the center of FIG. 7 indicates that the reflecting surface of the movable mirror unit 16 shown in FIG. 3B matches the reflecting surface of the fixed mirror unit 15. Say. When the reflecting surfaces of the movable mirror unit 16 and the fixed mirror unit 15 are coincident, there is no relative phase difference between the fixed light beam group and the movable light beam group. That is, the light beams of these two light beam groups reach each other on the imaging plane, and thus strengthen each other. For this reason, bright bright spots are formed on the imaging surface, and the imaging intensity is increased.

On the other hand, when the movable mirror section 16 is moved from the position shown in FIG. 3B to cause a relative optical path length difference between the fixed light beam group and the movable light beam group, the optical path length difference is reduced by half. When the wavelength (λ / 2) becomes an odd multiple, the destructive interference condition is reached, so that the imaging intensity is reduced. When the optical path length difference is an integral multiple of one wavelength, the interference condition between the two light beams is intensified, and the imaging intensity is increased.
Therefore, when the movable mirror unit 16 is moved from FIG. 3A to FIG. 3B to the state of FIG. 3C and the optical path length difference is sequentially changed, the imaging intensity due to the interference phenomenon between the two light beams is It will change periodically. As shown in FIGS. 7A to 7C, the cycle of the imaging intensity change is long for light having a long wavelength and short for light having a short wavelength.

  In a spectroscopic measurement device that measures multi-wavelength light, changes in the intensity of interference light with various lengths of wavelengths are detected as luminance value changes. This is the interferogram shown in FIG. At the phase shift origin where there is no relative optical path length difference between the fixed light beam group and the movable light beam group, the two beams are intensified without depending on the wavelength. It becomes strength. However, when the optical path length difference is increased, the period of intensity change of each wavelength does not match, so that the imaging intensity does not increase even when the intensity changes of multiple wavelengths are added. For this reason, in the interferogram, a change in imaging intensity is observed in which the luminance value gradually decreases as the optical path length difference increases. In this way, since the interferogram is a waveform in which single-wavelength imaging intensity changes of a single wavelength are added, spectral characteristics that are relative intensities for each wavelength can be obtained by Fourier transforming this waveform data. Can do.

Next, an optical model in the case where the light emitted from the measurement object S includes 0th-order diffracted light and 1st-order or higher-order diffracted light will be described.
The 0th-order diffracted light and the higher-order diffracted light emitted from the measurement object S can be separated most spatially on the Fourier transform plane that is the back focal point of the objective lens 12. Therefore, when the object to be measured S generates diffracted light, the reflecting surfaces of the fixed mirror unit 15 and the movable mirror unit 16 are arranged on the Fourier transform plane as shown in FIG. Thereby, each diffracted light component can be easily separated and irradiated onto the imaging lens 22.

In this case, as shown in FIG. 5, the movable mirror unit 16 and the fixed mirror unit 15 are irradiated with 0th-order diffracted light as a movable light beam group and high-order diffracted light such as ± 1st-order diffracted light as a fixed light beam group. The 0th-order diffracted light may be a fixed light beam group, and the higher-order diffracted light may be a movable light beam group. In short, it is only necessary that a relative optical path length difference can be generated between the two light beam groups.
For example, an object to be measured S having a pattern composed of bright and dark stripes having a specific spatial frequency generates ± first-order diffracted light in the orthogonal direction of the bright and dark stripes. FIG. 4B shows an example of the irradiation distribution of the most basic diffracted light on the reflecting surfaces of the movable mirror portion 16 and the fixed mirror portion 15.

  According to the imaging theory, it is possible to acquire an imaging image as an interference image in which only the light rays generated from the in-focus position of the objective lens 12 have the same phase on the imaging surface. That is, light generated from other than the in-focus position does not contribute to the formation of a clear interferogram. Therefore, the spectral tomographic image measurement apparatus 10 can measure only the spectral characteristics at the in-focus position. Therefore, if the lens drive mechanism 13 moves the objective lens 12 in the optical axis direction and scans the in-focus position, three-dimensional spectral characteristics can be measured.

  FIG. 8 shows an example of a spectral tomographic image of a single cell obtained by the spectral tomographic image measuring apparatus 10 of the present invention. Here, glycoproteins on the surface of the cell membrane are labeled with quantum dots, and the fluorescence emission of the quantum dots is observed. The cell tomogram shown in FIG. 8 is a two-dimensional spectroscopic image that is sequentially obtained by moving the objective lens 12 and scanning the focal plane (the plane including the focal position). A three-dimensional spectral image can be acquired from these two-dimensional spectral images.

  The resolution on the focal plane is determined by the numerical aperture of the objective lens 12 and the wavelength of light, and the resolution in the depth direction is determined by the depth of focus. Therefore, high resolution can be obtained by using an objective lens having a high numerical aperture such as an immersion lens. It is also possible to use a super-resolution technique such as modified illumination that achieves higher resolution by imaging using higher-order diffracted light components.

  9 and 10 show a second embodiment of the present invention. As shown in FIG. 9, the spectral tomographic image measurement apparatus 10 of the present embodiment is configured so that the optical axes of the objective lens 12 and the imaging lens 22 coincide. Between the objective lens 12 and the imaging lens 22, the first and second glass plates 30 and 32 constituting the optical path length difference expansion / contraction means are orthogonal to the optical axes of the objective lens 12 and the imaging lens 22. Has been placed. The first and second glass plates 30 and 32 are arranged so that a part of a light beam group from the objective lens 12 toward the imaging lens 22 transmits the first glass plate 30 and the remaining light passes through the second glass plate 32. Has been. In the following description, the light ray groups that pass through the first and second glass plates 30 and 32 are referred to as a fixed light ray group and a movable light ray group, respectively. In the present embodiment, the light quantity of the fixed light beam group and the movable light beam group are configured to be equal.

FIGS. 10A and 10B show the shapes of the first and second glass plates 30 and 32 viewed from the left or right in FIG. The first and second glass plates 30 and 32 are both made of optical glass having low wavelength dependency. The first glass plate 30 is composed of a single plate-like member having a substantially rectangular cross-sectional shape.
The second glass plate 32 is composed of two plate members 32a and 32b having a trapezoidal cross section. The inclined surfaces of the large and small plate-like members 32a and 32b have the same inclination (angle). The large plate-like member 32a can be slid in the direction of arrow B by the slide mechanism 34, and the thickness dimension of the second glass plate 32 changes continuously by sliding.

Since the optical glass which comprises the 1st and 2nd glass plates 30 and 32 has a refractive index larger than air, optical optical path length changes according to the thickness dimension. For this reason, when the thickness dimension of the second glass plate 32 changes, the optical path length difference between the fixed light beam group and the movable light beam group expands and contracts.
Specifically, when the plate-like member 32a is present at the position indicated by the solid line, the thickness dimension of the second glass plate 32 is equal to the thickness dimension of the first glass plate 30, and therefore the optical characteristics of the fixed light beam group P1 and the movable light beam group P2. The optical path lengths are equal. On the other hand, since the thickness dimension of the second glass plate 32 when the plate-like member 32a is at the position indicated by the alternate long and short dash line is smaller than the thickness dimension of the first glass plate 30, the optical optical path length of the movable light beam group P2 is It becomes shorter than the optical optical path length of the fixed light beam group P1. On the other hand, since the thickness dimension of the second glass plate 32 when the plate-like member 32a is located at the position indicated by the two-dot chain line is larger than the thickness dimension of the first glass plate 30, the optical optical path length of the movable light beam group P2 is a fixed beam. It becomes longer than the optical path length of the group P1.

  Therefore, by sliding the plate-like member 32a of the second glass plate 32, the optical optical path length difference between the fixed light beam group and the movable light beam group can be continuously expanded and contracted. Then, based on the light intensity change detected by the detection unit 24 while expanding / contracting the optical path length difference between the fixed light beam group and the movable light beam group, an interferogram of each measurement point of the object to be measured is obtained. A spectrum can be obtained by Fourier transforming this interferogram.

Further, the present invention is not limited to the above-described embodiments, and for example, the following modifications and expansions are possible.
The movable mirror portion 16 constituting the phase shifter 14 does not need to be a cylinder, and may be processed into a shape that is easy to manufacture, such as a prism.
In the above-described embodiment, the movable mirror unit 16 is arranged at the center of the fixed mirror unit 15. However, for example, as shown in FIGS. 11 and 12, the rectangular plate-like fixed mirror unit 15 and the movable mirror unit 16 are moved left and right or up and down. It may be arranged. In order to obtain a clear change in interference intensity, it is desirable that the light amounts of the fixed light beam group and the movable light beam group are equal. According to the said structure, the light quantity of a fixed ray group and a movable ray group can be arrange | equalized easily equally.
The number of divisions of the object beam is not limited to two. When an optical system capable of measuring the interference light intensity change between three or more light beam groups is used, the light beam of the object light can be divided into three or more light beam groups.

  In the above embodiment, the optical path length difference is continuously expanded and contracted. For example, the optical path length difference expanding and contracting glass plate 40 as shown in FIG. (Discretely) may be changed. On one surface of the optical path length difference expansion and contraction glass plate 40, a large number of concave portions 40a whose depth dimension changes stepwise and a large number of convex portions 40b whose protrusion size changes stepwise are provided. The portions 40c located between the concave portions 40a and between the convex portions 40b are all set to the same thickness dimension. By moving the glass plate 40 in the direction of arrow C, the fixed light beam group moves the portion 40c into the movable light beam. The group is configured to pass through the concave portion 40a or the convex portion 40b.

  Therefore, for example, as shown in FIG. 13A, when the fixed light beam group P1 passes through the portion 40c and the movable light beam group P2 passes through the recess 40a, the movable light beam group P2 has an optical optical path length longer than the fixed light beam group P1. Becomes shorter. As shown in FIG. 13B, when both the fixed light beam group P1 and the movable light beam group P2 pass through the portion 40c, the optical light path lengths of the fixed light beam group P1 and the movable light beam group P2 are equal. Further, as shown in FIG. 13C, when the fixed light beam group P1 passes through the portion 40c and the movable light beam group P2 passes through the convex portion 40b, the movable light beam group P2 has an optical path length longer than the fixed light beam group P1. become longer.

  In the above embodiment, the light beam group from the objective lens 12 toward the imaging lens 22 is divided into two, and one optical optical path length is expanded and contracted. However, the relative optical optical path length difference between the two light beam groups is expanded and contracted. If it can be made, the structure which expands / contracts the optical optical path length of both light beam groups may be sufficient.

You may make it scan the focus surface of the objective lens 12 by moving the to-be-measured object S instead of the objective lens 12. FIG.
The angle between the reflection surface of the fixed mirror portion 15 and the movable mirror portion 16 of the phase shifter 14 and the optical axis may not be 45 degrees. However, in this case, there is a high possibility that the spectral characteristics are deteriorated due to an angle error between the optical axis and the moving direction of the movable mirror section 16. This is because the amount of movement of the piezo stage with the capacitance sensor in the optical axis direction has an important meaning on the spectral accuracy. The amount of movement in the optical axis direction is the amount of phase shift given to the movable light beam group, and this amount of phase shift is a parameter necessary for spectroscopic measurement.

If the positional relationship between the objective lens and the object to be measured is set so that the light emitted from the object to be measured and transmitted through the objective lens is focused on one point, the splitting optical system and the imaging optical system are configured by one lens. Is also possible.
In addition, when the light to be measured is ultraviolet light or long wavelength light, the split optical system and the imaging optical system may be configured by a reflective optical system.
Furthermore, in an actual optical system, the installation angle of the movable mirror unit may be shifted due to an adjustment error. If this angle deviation is a problem in spectral characteristics, calibrate the amount of tilt between the phase shifter and the optical axis by measuring the spectral characteristics using a light source with a known emission line spectrum such as a mercury lamp. Can do.

1 is a schematic system configuration diagram of a spectral tomographic image measurement apparatus showing a first embodiment of the present invention. FIG. The whole block diagram of a phase shifter. FIG. 6 is an operation explanatory diagram of a phase shifter. The figure which shows the irradiation distribution (a) of the object light in the reflective surface of a fixed mirror part and a movable mirror part, and the irradiation distribution (b) of 0th-order diffracted light and +/- 1st-order diffracted light. The figure which shows a mode that 0th order diffracted light and +/- 1st order diffracted light are separated spatially. Interferogram (a) and waveform diagram (b) of spectrum obtained by Fourier transform of the interferogram (a). The figure for demonstrating the production | generation principle of an interferogram. The figure which shows a mode that the spectrum tomogram of the single cell which labeled the glycoprotein of the cell membrane surface layer with the quantum dot is measured. FIG. 1 is a view corresponding to FIG. 1 showing a second embodiment of the present invention. The figure which shows the structure of an optical path length difference expansion-contraction means. The equivalent figure of FIG. 1 which shows the modification of a fixed mirror part and a movable mirror part. FIG. The figure which shows another Example of an optical path length difference expansion-contraction means.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Spectral tomographic image measuring device 12 ... Objective lens 13 ... Lens drive mechanism 14 ... Phase shifter 15 ... Fixed mirror part 16 ... Movable mirror part 18 ... Drive stage 20 ... Control part 22 ... Imaging lens 24 ... Detection part 30 ... 1st 1 glass plate 32 ... 2nd glass plate 40 ... glass for optical path length difference expansion / contraction

Claims (6)

a) a splitting optical system in which light emitted in various directions from each measurement point of the object to be measured is incident;
b) An imaging optical system for guiding the light transmitted through the splitting optical system to substantially the same point to form an interference image;
c) a detection unit for detecting the light intensity of the interference image;
d) optical path length difference expansion / contraction means for expanding / contracting a relative optical optical path length difference between a part of the light traveling from the splitting optical system toward the imaging optical system and the remaining light;
e) Obtaining an interferogram at each measurement point of the object to be measured based on a change in light intensity detected by the detection unit by expanding / contracting the optical optical path length difference by the optical path length difference expansion / contraction means, and the interferogram A processing unit that obtains a spectrum by Fourier transforming
A spectroscopic measurement device comprising: a) a splitting optical system that guides light emitted from each measurement point of the object to be measured in various directions into a first reflecting part and a second reflecting part;
b) an imaging optical system for guiding the light reflected by the first and second reflecting portions to substantially the same point to form an interference image;
c) By moving the first and second reflecting parts relatively, light traveling from the splitting optical system to the imaging optical system via the first reflecting part and the second reflecting part from the splitting optical system. An optical path length difference expansion / contraction means for expanding / contracting the optical optical path length difference of the light passing through the imaging optical system,
d) a detection unit for detecting the light intensity of the interference image;
e) Obtaining an interferogram at each measurement point of the object to be measured based on a change in light intensity detected by the detection unit by expanding / contracting the optical optical path length difference by the optical path length difference expansion / contraction means, and the interferogram A processing unit that obtains a spectrum by Fourier transforming
A spectroscopic measurement device comprising:   The spectroscopic measurement apparatus according to claim 2, wherein the reflection surfaces of the first and second reflection portions are arranged in a state inclined by 45 ° with respect to the optical axis of the light beam transmitted through the split optical system. .   4. The spectroscopic measurement apparatus according to claim 1, wherein the processing unit obtains a spectrum of light emitted from a measurement point located at a focusing position of the divided optical system in the object to be measured.   The spectroscopic measurement apparatus according to claim 4, further comprising: a focus position changing unit that relatively changes a focus position of the split optical system with respect to the object to be measured. a) The light emitted from each measurement point of the object to be measured in various directions is divided into a phase-fixed light beam group and a phase-variable light beam group by a splitting optical system,
b) While expanding or contracting the optical path length difference of the phase variable beam group, the phase variable beam group and the phase fixed beam group are guided to substantially the same point by an imaging optical system to form an interference image,
c) A spectroscopic measurement method for obtaining an interferogram at each measurement point of the object to be measured based on a change in light intensity of the interference image, and acquiring a spectrum by Fourier transforming the interferogram.