JP2013145199A - Optical interference observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical interference observation device capable of observing and measuring a three-dimentional object such as a biological sample or a fine uneven surface shape.SOLUTION: The optical interference observation device for observing a structure of an observation object, comprises: a light source that emits light having a plurality of wavelengths; an illumination optical system that splits the light from the light source into reference light and illumination light to irradiate the observation object with the illumination light from at least two directions; a detection unit that allows measurement light including structure information of the observation object, the measurement light being generated by irradiating the observation object with the illumination light to interfere with the reference light, and detects first and second interference light generated by interference between the measurement light and the reference light generated by the irradiation from the two directions, respectively; and an image data processing unit that generates image data on the basis of the first interference light and the second interference light detected by the detection unit.

Description

  The present invention relates to an optical interference observation apparatus used for observation of a three-dimensional object such as a biological sample, measurement of a fine uneven surface shape, and the like.

  Conventionally, a confocal microscope, OCT (Optical Coherence Tomography), or the like is used to observe a three-dimensional structure of a biological sample or the like. A confocal microscope can obtain high-resolution three-dimensional information, but requires three-dimensional probe scanning.

  OCT is a promising technique for observing a three-dimensional structure of a cell, having features of a resolution in the depth direction on the order of μm and an observation range in the depth direction on the order of mm (for example, see Non-Patent Document 1). . OCT uses the principle of interference and can obtain high resolution in the optical axis direction (Z direction). This is determined by the width of the light source spectrum. In Fourier domain OCT, information decomposed in the Z direction can be obtained simply by scanning the XY plane with a probe. However, if an attempt is made to obtain a high resolution in the XY plane, the NA of the objective lens must be increased, and the width in the Z direction with little focus shift obtained by one XY in-plane scan is limited. Therefore, to observe a desired region in the Z direction, precise scanning in the XY plane and a step in the Z direction are required.

  The Fourier domain method mainly includes a type in which a white light source is combined with a spectroscope and a type in which a wavelength scanning light source is used. In time domain OCT, in addition to probe scanning in the XY plane, it is necessary to mechanically scan an object or reference plane in the Z direction. Full-field OCT is illuminated with a spatially coherent plane wave, and three-dimensional information can be obtained without two-dimensional scanning in the XY plane. This type of OCT also includes a time domain system that mechanically scans an object or a reference surface and a Fourier domain system that uses a wavelength scanning light source.

D. huang, EA Swanson, cP Lin, JS Schuman, WG Stinson, W Chang, MR Hee, t Flotte, K Gragory, CA Puliafito and et.al, “Optical coherence tomography” Science Vol.254, No.5035, 1178 -1181, 1991

  By the way, even in the above-described full-field OCT, if the focus matching range is narrow, a step in the Z direction is necessary. Further, since full-field OCT irradiates a plane wave, the three-dimensional detection band of the scattering potential of the sample becomes narrower than that of normal OCT. Further, as OCT in general, back-scattered light from a sample is detected using epi-illumination, it is not suitable for observation of an object with weak back-scattering, detection of a surface structure or line structure substantially parallel to the optical axis. There was a problem.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide an optical interference observation apparatus that has a wide detection band and can perform observation in a short time.

  The optical interference observation apparatus of the present invention refers to a light source that emits light of a plurality of wavelengths, an illumination optical system that irradiates an object to be observed from at least two directions, and light from the illumination optical system. A light branching member that branches into light and illumination light, and interference between measurement light including structural information of the observed object generated by irradiating the observed light with the illumination light and the reference light, A detection unit for detecting first and second interference light generated by interference between the measurement light and the reference light generated by irradiation from a direction; the first interference light detected by the detection unit; An image data processing unit for generating image data based on the second interference light, and observing the structure of the object to be observed.

In the optical interference observation apparatus, the image data processing unit generates first image data based on the first interference light, and generates second image data based on the second interference light.
The first and second image data is subjected to a predetermined conversion process for each wave number of light of the light source, thereby generating object scattering potential information as structure information of the object to be observed, and image data of the object to be observed. Preferably it is produced.

  In the optical interference observation apparatus, it is preferable that the light source is a wavelength scanning light source that scans a wavelength of light applied to the object to be observed.

  In the optical interference observation apparatus, the illumination optical system includes a laser light source, a beam splitter that divides the light of the laser light source into a plurality of pieces, and a plurality of optical fibers that guide the divided light to the observation portion. It is preferable.

  In the optical interference observation apparatus, the image data processing unit performs a Fourier transform process on the first and second image data as a function of the wave number, and calculates a complex amplitude distribution for each wave number. A step of obtaining a spectrum of the complex amplitude distribution by performing a two-dimensional Fourier transform process on the complex amplitude distribution; a step of obtaining and synthesizing a three-dimensional spectrum of the object scattering potential based on the obtained spectrum of the complex amplitude distribution; And a step of obtaining a scattering potential of the object to be observed by performing a three-dimensional inverse Fourier transform on the synthesized three-dimensional spectrum of the scattering potential.

  In the optical interference observation apparatus, the illumination optical system includes a first illumination unit and a second illumination unit for illuminating the object to be observed from at least two directions, and the first illumination unit and the second illumination unit The second illumination unit preferably illuminates the object to be observed from at least two directions.

  In the optical interference observation apparatus, the first illumination unit and the second illumination unit of the illumination optical system can emit the illumination light having a predetermined angle with respect to the optical axis of the illumination optical system. It is preferable to have.

  In the optical interference observation apparatus, it is preferable that the illumination optical system includes at least one of a falling-down illumination optical system or a transmission illumination optical system.

  In the optical interference observation apparatus, it is preferable that the illumination optical system is configured by combining a falling-down illumination optical system and a transmission illumination optical system.

  In the optical interference observation apparatus, it is preferable that the optical unit includes a plurality of light emitting units that emit the illumination light at the predetermined angle.

  In the optical interference observation apparatus, it is preferable that the illumination optical system simultaneously emits the illumination light from different directions to the object to be observed from the plurality of light emitting units.

  In the optical interference observation apparatus, it is preferable that the illumination optical system emits the illumination light from the plurality of light emission units to the object to be observed in time order from different directions.

  Moreover, in the said optical interference observation apparatus, it is preferable that the said some light emission part has a predetermined optical path length difference, and is equipped with the optical path length calibration part which calibrates the optical path length difference in these several light emission parts.

  In the optical interference observation apparatus, it is preferable that the optical path length calibration unit also calibrates the coordinates of the position of the object to be observed and the coordinate relationship on the image detector using interference light from the characteristic points of the calibration member.

  In the optical interference observation apparatus, the optical unit includes a scanning mirror that reflects illumination light in different directions, and the scanning mirror transmits the illumination light so that a spot of the illumination light moves on a ring. It is preferable that the light source performs a predetermined wavelength scan for each spot position that is reflected and moves on the ring.

  According to the present invention, observation can be performed in a short time while having a wide detection band without mechanical scanning.

The figure which shows schematic structure of the observation apparatus which concerns on 1st Embodiment. The figure which showed the structure of the light source. The figure which shows the zone | band obtained with the illumination light irradiated from the optical fiber. The figure which shows the zone | band obtained with the illumination light irradiated from the optical fiber 103. FIG. The figure which shows the zone | band obtained with the illumination light irradiated from the optical fiber 104. FIG. The figure which shows the zone | band obtained with the illumination light irradiated from the optical fiber 105. FIG. The figure which shows the zone | band obtained with the illumination light irradiated from the optical fiber. The figure which shows the zone | band obtained by composition. The figure which shows the structure which concerns on the observation apparatus provided with the beam splitter instead of the polarizing beam splitter, and was replaced with the polarizing plate instead of the quarter wavelength plate. The figure which shows schematic structure of the wavelength scanning laser with which the observation apparatus which concerns on 2nd Embodiment is provided. The figure which shows the interference intensity | strength of measurement light and reference light in 2nd Embodiment. The figure which shows the spectrum which carried out the Fourier-transform of the signal shown in FIG. The figure which shows the structure which concerns on the modification of the observation apparatus of 1st and 2nd embodiment. The figure which shows the structure of the transmission illumination system added by 3rd Embodiment. The figure which shows the three-dimensional detection zone | band by transmitted illumination. The figure which shows the three-dimensional detection zone | band which combined epi-illumination and transmitted illumination. The figure for demonstrating the effect in this embodiment. The figure which shows the structure of the observation apparatus which concerns on 4th Embodiment. The figure for demonstrating the calibration method of an optical path length difference. The figure which shows another structure which forms a some point light source.

  Hereinafter, a configuration applied to a three-dimensional observation apparatus as an embodiment of the optical interference observation apparatus of the present invention will be described with reference to the drawings.

(First embodiment)
The observation apparatus according to this embodiment divides the light emitted from the wavelength scanning light source to generate a plurality of illumination light and reference light, illuminates the sample with the illumination light, and backscattered light (measurement light from the sample) ) Is detected by interference with the reference light, and is composed of a full-field type OCT. In this embodiment, Fourier domain OCT using a wavelength scanning light source will be described.

FIG. 1 is a diagram showing a schematic configuration of an observation apparatus according to the first embodiment. FIG. 2A is a diagram showing the configuration of the light source, and FIG. 2B is a diagram showing the positional relationship of the optical fibers that irradiate each illumination light.
In FIG. 1, the optical axis direction is the Z direction, the direction perpendicular to the optical axis and perpendicular to the paper surface is the X direction, and the direction in the paper surface is the Y direction.

  As shown in FIGS. 1 and 2, the observation apparatus 100 according to the present embodiment divides the light from the laser light source unit 51 of the light source (illumination optical system) 50 by a plurality of beam splitters (branching means) 52a to 52f. Reference light and illumination light are generated using optical fibers (light emitting units) 101 to 106.

  As an example of the wavelength scanning condition of the laser light source 51, for example, when a biological sample having a high light scattering property is used as the sample 13, the center wavelength is set to 800 nm and the scanning width is set to 100 nm. Even in the case of a biological sample, if the scattering is weak, the center wavelength does not need to be in the near infrared region, and the wavelength scanning condition is not limited.

  Specifically, the beam splitter 52a is disposed at a position closest to the laser light source unit 51 and transmits a part of the light emitted from the laser light source unit 51 to guide it to the optical fibers 101 to 103, and at the same time, the laser light source unit. A part of the light emitted from 51 is reflected so as to be guided to the optical fibers 104 to 106.

  The beam splitter 52b reflects part of the light transmitted through the beam splitter 52a and guides it to the optical fiber 101 through the lens 53, and transmits other light. Based on such a configuration, the optical fiber 101 constitutes a point light source that emits reference light.

  The beam splitter 52c reflects a part of the light transmitted through the beam splitter 52b, thereby guiding the light to the optical fiber 102 through the lens 53, and transmits the part of the light transmitted through the beam splitter 52b through the lens 53. Through the optical fiber 103.

  The beam splitter 52d transmits or reflects part of the irradiation light from the laser light source 51 reflected by the beam splitter 52a, and divides the irradiation light toward the beam splitters 52f and 52e.

  The beam splitter 52e transmits and reflects the light reflected by the beam splitter 52d, so that the transmitted light is guided to the optical fiber 104 via the lens 53, and the reflected light is guided to the optical fiber 105 via the lens 53. .

  The beam splitter 52f guides the light transmitted through the beam splitter 52d to the optical fiber 106 by reflecting the light. Each of the optical fibers 102 to 106 constitutes a point light source that emits illumination light.

  In the light source 50 according to the present embodiment, the optical path length between the laser light source unit 51 and the optical fiber 101 is different from the optical path length between the laser light source unit 51 and the optical fibers 102 to 106 as shown in FIG. The optical path length between the laser light source 51 and the optical fiber 101 is set shorter, and the optical path length between the laser light source 51 and the optical fibers 102 to 106 is set to the same value. Here, optical fibers 101 to 106 having the same length are used. Further, as shown in FIG. 2B, the optical fibers 102 to 106 are provided at positions where each illumination light is irradiated to the sample 13 from a predetermined different direction at a predetermined angle as will be described later. .

  As shown in FIG. 1, the reference light beam emitted from the optical fiber 101 is converted into a plane wave parallel to the optical axis by the collimator lens 1. Further, each of the illumination light beams emitted from the optical fibers 102 to 106 is converted by the collimator lens 2 into a plane wave having a predetermined angle and direction with respect to the optical axis.

  The light source 50 is configured to irradiate reference light composed of P-polarized linearly polarized light via the optical fiber 101 and irradiate illumination light composed of S-polarized linearly polarized light via the optical fibers 102 to 106. Thereby, the reference light (P-polarized light) irradiated from the optical fiber 101 is transmitted through the polarization beam splitter 3, and each of the illumination light (S-polarized light) irradiated from the optical fibers 102 to 106 is reflected by the polarization beam splitter 3. It has become.

  The reference light emitted from the optical fiber 101 passes through the polarization beam splitter 4 and enters the reference mirror 7 through the quarter-wave plate 5 and the objective optical system 6. The objective optical system 6 includes a first objective lens 6a and a second objective lens 6b.

  The light reflected by the reference mirror 7 enters the polarization beam splitter 4 through the objective optical system 6 and the quarter wavelength plate 5 as reference light. The reference light is reflected by the polarization beam splitter 4 because it passes through the quarter-wave plate 5 twice and its polarization plane is rotated by 90 °.

  The reference light reflected by the polarizing beam splitter 4 enters a two-dimensional image detector (light detection unit) 10 through a relay optical system 8 and a polarizing plate 9. The two-dimensional image detector 10 is composed of a CCD, for example. The relay optical system 8 includes a first relay lens 8a and a second relay lens 8b.

  On the other hand, each illumination light reflected by the polarization beam splitter 4 enters the sample 13 through the quarter-wave plate 11 and the objective optical system 12. The objective optical system (observation optical system) 12 includes a first objective lens 12a and a second objective lens 12b.

  Backscattered light from the sample 13 returns to the optical path as measurement light, passes through the polarization beam splitter 4, and enters the two-dimensional image detector 10. Here, the measurement light and the reference light interfere with each other through the polarizing plate 9. That is, the polarizing plate 9 constitutes a combining unit that combines the reference light and the measurement light. Thereby, an interference image is detected by the two-dimensional image detector 10. Although the optical path length difference of all illumination light is known, the observation apparatus 100 detects the optical path length difference and calibrates the optical path length difference as will be described later.

In the observation apparatus 100 according to the present embodiment, the light source 50 can switch the five illumination lights irradiated via the optical fibers 102 to 106 by a shutter mechanism (not shown), and each can irradiate the sample 13 separately.
Based on such a configuration, in the observation apparatus 100, the two-dimensional image detector 10 detects the interference image in each measurement light and reference light, and the image processing unit 40 connected to the two-dimensional image detector 10 has the sample 13. Data (interference intensity data) can be acquired. The image processing unit 40 calculates the scattering potential of the sample 13 based on the acquired data as described later, generates an image of the sample 13 based on the scattering potential data, and displays the image on a display unit (not shown). Yes.

Subsequently, a data acquisition procedure by the image processing unit 40 in the present embodiment will be specifically described.
First, the observation apparatus 100 controls a shutter (not shown) of the light source 50, emits illumination light from the optical fiber 102, and blocks illumination light from the other optical fibers 103 to 106. And the observation apparatus 100 acquires the set of wavelength scanning data by the two-dimensional image detector 10 in this state.

  Subsequently, the observation apparatus 100 acquires a set of wavelength scanning data by the two-dimensional image detector 10 while switching the fiber from the optical fiber 103 to the optical fiber 106 by sequentially controlling the opening and closing of the shutter of the light source 50.

Here, a set of image data obtained by wavelength scanning of the laser light source unit 51 with each illumination light is defined as D _m (x, y, k). Note that x and y represent the abscissa on the object (sample 13) converted from the coordinates on the two-dimensional image detector 10 in a magnification relationship. Further, k represents the wave number of light that changes in wavelength scanning. m represents each illumination light irradiated from the optical fibers 102 to 106, and is represented by m = 0 to 4 in this embodiment.

Next, a data analysis method and operation by the image processing unit 40 will be described.
First, for each coordinate (x, y), the Fourier transform of D _m in the wave number k, it extracts a signal component by performing a predetermined band-pass filter, DC component, the conjugate components removed. Further, the complex amplitude distribution U _m (x, y, k) is calculated by performing inverse Fourier transform. Thereby, the complex amplitude distribution decomposed | disassembled for every light wave number can be obtained. The complex amplitude distribution U _m (x, y, k) is related to the scattering potential of the sample, the complex amplitude distribution of the illumination light, the pupil function, and Equation (1). Here, W (x, y, z) in Equation (1) is the sample scattering potential (V (x, y, z)) and the complex amplitude distribution of illumination light (Q (x, y, z)). Is the product of The complex amplitude distribution Q of the illumination light is represented by Q (x, y, z) = exp [ik (μ _x x + μ _y y + μ _z z)], and illumination light (m = 0 to 4). Μ _x , μ _y , and μ _z corresponding to are applied.

Subsequently, the influence of the optical path length difference between illumination lights, which is known by calibration or the like, is corrected. When the optical path length difference with respect to the reference illumination light (m = 0) is δ _m (m = 1 to 4), exp (−ik δ _m ) is multiplied by the complex amplitude distribution U _m .

Subsequently, the corrected U _m (x, y, k) is subjected to a two-dimensional Fourier transform with respect to x and y to obtain a spectrum of a complex amplitude distribution. The spectrum of the complex amplitude distribution has a relationship represented by the following equation (2) with the three-dimensional spectrum of the object scattering potential. Moreover, the relational expression of ν _x , ν _y , ν _{z in} the following expression (3) is satisfied. The scattering potential detected by this apparatus has a wave vector corresponding to the difference between the wave vector of the detection light passing through the pupil of the optical system and the illumination light.

    Subsequently, a three-dimensional spectrum of the object scattering potential is synthesized with reference to the wave number of light and the wave vector of illumination light. In the synthesis of the three-dimensional spectrum, spectral data on equidistant grid points is obtained by interpolation from spectral data obtained under each illumination condition. When combining them, if there is only one spectrum data on a lattice point, that data is adopted, and if there are a plurality of them, an average value is adopted. Further, the image processing unit 40 obtains the scattering potential of the object by performing a three-dimensional inverse Fourier transform on the synthesized three-dimensional spectrum of the scattering potential. The synthesized three-dimensional spectrum is expressed by the following formula (4). Then, the following equation (5) is calculated.

By the way, the observation apparatus 100 according to the present embodiment can measure information in the three-dimensional detection band corresponding to the azimuth and angle of illumination light emitted from the optical fibers 102 to 106. FIG. 3 shows a three-dimensional detection band (a component in the spatial frequency region surrounded by this boundary is detected in the object scattering potential) obtained by the illumination light irradiated from the optical fiber 102. For convenience of explanation, FIG. 3 is a two-dimensional representation divided into a left diagram and a right diagram. Ρ _x , ρ _y , and ρ _{z in} FIG. 3 indicate the x, y, and z components of the wave vector. The left diagram of FIG. 3 is a ρ _x -ρ _z cross-sectional view (ρ _y = 0) of the detected spatial frequency region, and the right diagram is an arrow view of this spatial frequency region viewed from the negative direction of ρ _z to the positive direction. FIG. Thus, three-dimensional detection zone in FIG. 3 is a piece-like region by rotating the left as the center of rotation of the [rho _z-axis.
Similarly, FIG. 4 shows a three-dimensional detection band obtained with illumination light emitted from the optical fiber 103, and FIG. 5 shows a three-dimensional detection band obtained with illumination light emitted from the optical fiber 104. 6 shows a three-dimensional detection band obtained with illumination light irradiated from the optical fiber 105, and FIG. 7 shows a three-dimensional detection band obtained with illumination light irradiated from the optical fiber 106. FIG.
Similar to FIG. 3, ρ _x , ρ _y , and ρ _{z in} FIGS. 4 to 7 indicate the x, y, and z components of the wave vector. 4 and 6 show the ρ _x -ρ _z cross section (ρ _y = 0), and the left diagrams of FIGS. 5 and 7 show the ρ _y -ρ _z cross section (ρ _x = 0). Right figure is an arrow view as viewed the spatial frequency domain from a negative [rho _z in the positive direction.

In this embodiment, the averaging operation is performed in the portion where the three-dimensional bands overlap in the synthesis of the three-dimensional spectrum, but the spectrum obtained with the illumination light parallel to the optical axis (m = 0) is used with priority. It is also possible to do. The three-dimensional detection band obtained by such synthesis is shown in FIG. The left diagram of FIG. 8 shows a ρ _x -ρ _z cross-sectional view (ρ _y = 0) of the synthesized detection band, and the right diagram is an arrow view of the synthesized region viewed from the negative to the positive direction of ρ _z . It is.

  Incidentally, the incident angle of the illumination light with respect to the sample 13 can be selected depending on the frequency component of interest of the scattering potential of the object (sample 13). That is, if the incident angle of the illumination light is increased, a high frequency component can be obtained although a gap is generated in the detection band.

  In general, the high-frequency component of the object scattering potential is smaller and the corresponding signal light tends to be weaker. In this embodiment, illumination light having a large incident angle detects a high-frequency component. Therefore, the SN ratio can be improved by increasing the amplitude of the illumination light having a large incident angle or adjusting the amplitude of the illumination light based on the signal intensity.

  As described above, according to the observation apparatus 100 according to the present embodiment, the illumination light is sequentially irradiated from the optical fibers 102 to 106 to the sample 13, and the detection band as described above can be obtained by each of the illumination lights. The scattering potential information of the object (sample 13) in a wider detection band than in the case of single point light source illumination (see FIG. 3) can be acquired.

  In the above embodiment, the illumination light is formed by dividing the light emitted from the laser light source unit 51 into a plurality (five), but a plurality of illumination lights may be obtained by switching the laser light path. In this case, the laser may be divided into reference light and illumination light, and the optical path of the illumination light may be switched. In addition, although the illumination light is divided into five, the illumination optical fibers (102 to 106) may illuminate the sample from at least two directions, and at least two or more optical fibers may be provided. .

  In the above embodiment, the polarization beam splitter 4 and the quarter wavelength plates 11 and 5 are used to control to separate and combine the reference light and the illumination light. However, the polarization beam splitter 4 and the quarter wavelength are used. A configuration in which the plates 11 and 5 are not used is also applicable.

FIG. 9 is a diagram illustrating a configuration of an observation apparatus 100 that includes a beam splitter 20 instead of the polarizing beam splitter 4 and includes polarizing plates 21 and 22 instead of the quarter-wave plates 11 and 5. The configuration shown in FIG. 9 is the same as that of the above-described embodiment except for the beam splitter 20 and the polarizing plates 21 and 22, and therefore detailed description thereof will be omitted.
According to the configuration shown in FIG. 9, although the amount of light incident on the two-dimensional image detector 10 is reduced as compared with the above embodiment, the scattering potential information in a wide detection band can be obtained similarly.

  Further, the wavelength scanning of the light source 50 is desirably performed so that the wave number k of light is at equal intervals. For example, when the wavenumber k interval deviates greatly from the equal interval, the interpolation operation may be performed on the interference intensity signal so that the pixels of the two-dimensional image data have equal intervals at the wavenumber k.

(Second Embodiment)
Subsequently, a configuration according to the second embodiment of the observation apparatus will be described. The difference between the present embodiment and the first embodiment is part of the configuration of the wavelength scanning laser and the data analysis method, and the other configurations are the same. Therefore, in the following description, the same members and configurations as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted or simplified.

  FIG. 10 is a diagram illustrating a schematic configuration of the light source unit 150 included in the observation apparatus according to the second embodiment. Unlike the first embodiment, the light source unit 150 according to the present embodiment is set such that the optical path length of each illumination light has a predetermined different optical path length. In this embodiment, the illumination light is not switched by the shutter, and the wavelength scanning is performed while simultaneously illuminating the sample with each illumination light having different optical path length differences.

  In this embodiment, the optical path difference between the reference light irradiated from the optical fiber 101 and each illumination light irradiated from the optical fibers 102 to 106 is set to 9 mm, 10.5 mm, 12 mm, 13.5 mm, and 15 mm. The lengths of the optical fibers 101 to 106 are made equal, and the optical path length difference is adjusted according to the difference in the arrangement position of the lens 53 as shown in FIG. In this case, high wavelength scanning resolution is required. 10 differs from the diagram shown in FIG. 2 in that the distances d1 to d5 between the beam splitters 52c to 52f and the lens 53 are set so as to increase sequentially.

FIG. 11 is a diagram showing the interference intensity between the illumination light (measurement light) reflected by the sample 13 and the reference light in the present embodiment. The horizontal axis in FIG. 11 represents the wave number (unit: mm ⁻¹ ) of light, and the center wave number was 2000 mm ⁻¹ (wavelength: 500 nm). The vertical axis indicates the interference intensity.
FIG. 11 shows multi-beam interference of light having different optical path length differences, and the spectrum shown in FIG. 12 is obtained by Fourier transforming this signal with a wave number k. As described above, in this embodiment, the reflected and scattered light component from the sample 13 included in the interference intensity is extracted as the spectrum by performing Fourier transform on the interference intensity of the multi-beam interference.

  FIG. 12 shows a spectrum obtained by Fourier-transforming this signal at a wave number k. In FIG. 12, the horizontal axis represents the modulation frequency, and the vertical axis represents the signal spectrum. In FIG. 12, a signal near zero frequency indicates a DC component, and a low-frequency signal indicates an interference component (due to different illumination light) between measurement lights.

The interference component between the reference light and the measurement light necessary for the analysis is a signal having five peaks having a relatively high frequency. Therefore, the information obtained by individually cutting out these five signal components with the band-pass filter is the same information as obtained by the analysis of the first embodiment. However, the information of the optical path length difference of the illumination light given in advance is known as described above, and it is assumed that correction processing corresponding to the optical path length difference is performed.
Specifically, for the complex amplitude distribution U _m (x, y, k) of (Expression 1) described above, the influence of the optical path length difference between illumination lights that is known by calibration or the like is corrected. That is, if the optical path length difference with respect to the reference illumination light (m = 0) is δ _m (m = 1 to 4), exp (−ik δ _m ) is multiplied. Since the subsequent data processing is the same as in the first embodiment, a description thereof will be omitted.

  For example, this unnecessary component (interference component between measurement beams) may be reduced relative to the signal component by adjusting the intensity of the reference beam. If unnecessary components can be ignored in this way, the optical path difference described above can be set to 0.75 mm, 2.25 mm, 3.75 mm, 5.25 mm, and 6.75 mm. According to this, a lower wavelength scanning resolution is allowed as compared with the case described above (when optical path differences are 9 mm, 10.5 mm, 12 mm, 13.5 mm, and 15 mm).

  As described above, according to the present embodiment, unlike the first embodiment, the sample 13 is irradiated simultaneously without performing the switching operation of the five illumination lights, so that data can be acquired at a higher speed. it can. Furthermore, since the optical path length difference detection of illumination light described later can be simultaneously detected in real time for a plurality of illumination lights, high accuracy can be obtained.

  Moreover, in the said 2nd Embodiment, although the case where the five illumination lights were irradiated simultaneously was demonstrated to the example, you may make it switch the group which irradiates illumination light to a plurality of groups by dividing a plurality of illumination lights sequentially. . For example, the measurement may be performed by switching the light corresponding to the center of five illumination lights and the group of light corresponding to one of the surroundings four times. Further, the measurement may be performed by switching the light corresponding to the center of the five illumination lights and the group of light corresponding to the two peripheral lights twice. Each group always includes illumination light corresponding to the central part of the five illumination lights, and the phase relationship of the illumination light is corrected.

Subsequently, a modification of the observation apparatus according to Embodiments 1 and 2 will be described. Note that this modification is different from the above-described embodiment in that observation is performed with dark field illumination, and other configurations are the same. FIG. 13A is a diagram illustrating a configuration of an observation apparatus according to this modification, and FIG. 13B is a diagram illustrating a positional relationship of optical fibers that irradiate each illumination light. FIG. 13 shows a configuration in which the present modification is applied to the configuration according to FIG.
Also in FIG. 13A, the optical axis direction is the Z direction, the direction perpendicular to the optical axis and perpendicular to the paper surface is the X direction, and the direction in the paper surface is the Y direction.

  In this modification, as shown in FIGS. 13A and 13B, illumination light is irradiated through optical fibers 103 to 106 excluding illumination light (optical fiber 102) parallel to the optical axis. The object side NA (numerical aperture) of the relay optical system 8 is made smaller than the image side NA of the objective optical systems 6 and 12. In this state, the incident angle of the illumination light to the objective optical systems 6 and 12 is set to be smaller than the image side NA equivalent of the objective optical system and larger than the object side NA equivalent of the relay optical system 8. (Dark field illumination). In such a configuration, as shown in FIG. 13, when the sample 13 is held in a sample chamber such as a glass substrate (perpendicular to the optical axis) G, the specularly reflected light from the plane of the glass substrate G in the sample chamber is generated. Can be removed.

(Third embodiment)
Subsequently, a configuration according to the third embodiment of the observation apparatus will be described. This embodiment has a configuration in which transmitted illumination is added to the configuration according to the first embodiment, and the other configurations are the same. Therefore, in the following description, the same members and configurations as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted or simplified.

  In the present embodiment, the number of divisions of the light source 50 is increased by four, and the illumination from four directions is also performed for the transmitted illumination at a predetermined incident angle. FIG. 14 is a diagram showing a configuration of a transmission illumination system added according to the present embodiment, FIG. 14 (a) is a diagram showing a configuration of the transmission illumination system, and FIG. 14 (b) is an irradiation of each transmission illumination light. It is a figure which shows the positional relationship of the optical fiber to perform. In addition, in order to suppress the influence of the refractive index dispersion of the optical system, the same configuration as the epi-illumination system is adopted for the transmission illumination system. Also in FIG. 14A, the optical axis direction is the Z direction, the direction perpendicular to the optical axis and perpendicular to the paper surface is the X direction, and the direction in the paper surface is the Y direction.

  In the transmission illumination system 90, as shown in FIG. 14A, each of the light beams of illumination light irradiated through the optical fibers 202 to 205 is a collimator lens 2 and has a predetermined angle and direction with respect to the optical axis. It is converted to a plane wave. In the present embodiment, irradiation is performed via the optical fibers 202 to 205 from four directions excluding illumination light parallel to the optical axis.

The transmitted illumination light is reflected by the polarization beam splitter 108, reflected by the polarization beam splitter 109, and then transmitted through the sample 13 through the quarter wavelength plate 110 and the objective optical system 111. The objective optical system 111 includes a first objective lens 111a and a second objective lens 111b. Transmitted scattered light that has passed through the sample 13 passes through the objective optical system 12, the quarter wavelength plate 11, the polarizing beam splitter 4, the relay optical system 8, and the polarizing plate 9 shown in FIG. Incident to.
Here, a three-dimensional detection band (for example, transmitted light transmitted from the optical fiber 203) by transmitted illumination according to this embodiment is shown in FIG. FIG. 16 shows a three-dimensional detection band obtained by combining epi-illumination and transmission illumination obtained in this embodiment. 15 and 16 are ρ _x -ρ _z cross-sectional views (ρ _y = 0) of the detected spatial frequency domain, and the right diagram shows the spatial frequency domain viewed from the negative of ρ _z to the positive direction. It is a figure.

  As shown in FIG. 16, it can be seen that the band can be further expanded as compared with the band of only the epi-illumination (see FIG. 8 of the first embodiment). According to this embodiment, for example, when a three-dimensional cell as schematically shown in FIG. 17 is observed as the sample 13, the side surface portion 13b can be detected in addition to the upper surface portion 13a of the cell membrane. In the first embodiment, although it cannot be detected when the backscattering property of the sample 13 is weak, good observation can be performed according to the present embodiment to which transmitted illumination is added.

(Fourth embodiment)
Subsequently, a configuration according to the fourth embodiment of the observation apparatus will be described. The present embodiment has a configuration in which the illumination optical system is changed with respect to the first embodiment, and the other configurations are the same. Therefore, in the following description, the same members and configurations as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted or simplified.

  In the first embodiment, a plurality of point light sources fixed as illumination light are used. However, in this embodiment, illumination light is emitted from a scanning light source that rotates on a ring. FIG. 18 is a diagram illustrating a configuration of the observation apparatus 300 according to the present embodiment.

  In the observation apparatus 300 according to the present embodiment, the light from the laser light source unit 51 is divided by the light source 350 into reference light and illumination light. The light source 350 includes a laser light source unit 51, a beam splitter (not shown) that separates light emitted from the laser light source unit 51 into reference light and illumination light, and an optical fiber 301 that emits the separated illumination light. And an optical fiber 302 that emits the reference light after separation.

In the first embodiment, five point light sources separated by a plurality of beam splitters 52 a to 52 f are used for illumination light. In this embodiment, illumination light emitted from the optical fiber 301 is scanned by a scanning mirror via a lens 303. Reflected by MR. Here, the scanning mirror MR reflects illumination light so as to draw a locus of a ring-shaped spot on the focal plane of the condenser lens 304. The light passing through the condenser lens 304 is converted into a plane wave having a predetermined angle and direction with respect to the optical axis by the collimator lens 305. That is, in this embodiment, the illumination light is reflected by the scanning mirror MR, and a plurality of types of illumination light incident on the sample 13 from different directions are generated from different directions as in the above embodiment without using a plurality of optical fibers. can do.
On the other hand, the illumination light irradiated from the optical fiber 302 is converted into a plane wave parallel to the optical axis by the collimator lens 306.

  In the illumination light and the reference light generated in this manner, the interference image is detected by the two-dimensional image detector 10 as in the above embodiment, and the observation apparatus 300 acquires data (interference intensity data) in the sample 13. Can do.

  Here, a data set of wavelength scanning is acquired for each point on the ring, and data analysis is performed. The band obtained in the present embodiment is obtained by increasing the azimuth according to the number of steps except for the central band shown in FIG. 8, and thus a more isotropic detection band can be obtained. A scanning light source that rotates on the ring may be applied to the transmission illumination system described in the third embodiment.

  According to this embodiment, since the locus of the spot moves on the ring by reflecting the illumination light with the scanning mirror MR, a plurality of illumination lights can be irradiated only through the optical fiber 301 as in the above embodiment. it can. Therefore, it has a wider detection band than the case of single point light source illumination.

  As described above, in the present invention, as in the above-described embodiment, the scattered light obtained by illumination light having different angles and directions is detected, and the spectrum of the object scattering potential is synthesized. Is important. Therefore, in the above embodiment, calibration of the optical path length differences of the plurality of illumination lights is performed. Hereinafter, the calibration method will be described.

  FIG. 19 is a diagram for explaining a calibration method of the optical path length difference. As a method for calibrating the optical path length difference, the following three methods can be exemplified. FIG. 19 illustrates the configuration of the observation apparatus 100 according to the first embodiment as an example.

  First, the image processing unit 40 installs the calibration plane mirror 30 shown in FIG. 19 and acquires the wavelength scanning data when the magnification relationship of the optical system is known in advance, for example, and the illumination angle and direction. It becomes possible to extract the optical path length difference between illumination lights. Thereby, the image processing unit 40 can perform high-precision and high-definition observation by calibrating the optical path length difference.

  Second, for example, when the magnification relationship of the optical system (the objective optical systems 6 and 12 and the relay optical system 8 in FIG. 1) is not known, the calibration reference 31 shown in FIG. The optical path length difference can be extracted by acquiring the data. The calibration reference 31 has a plurality of minute scattering points arranged at known coordinates. By irradiating the calibration reference 31 with illumination light from the light source 50, reference is made to scattered light from the scattering points. Based on the interference light, it is possible to extract the magnification of the optical system, the illumination angle, the direction, and the optical path length difference between the illumination lights. Thereby, the image processing unit 40 can perform high-precision and high-definition observation by calibrating the optical path length difference.

  Thirdly, for example, when the sample holder 33 that holds the sample 13 between the pair of glass substrates G has a flat portion, the flat portion is used as a calibration mirror. Specifically, a sample holder 33 in which a plurality of minute scattering points are arranged at known coordinates on the plane portion 33a is used as a holder with a reference point, and illumination light is emitted from the light source 50 to the plane portion 33a. Based on the scattered light from the scattering point and the interference light of the reference light, the magnification of the optical system, the illumination angle, the direction, and the optical path length difference between the illumination light can be extracted. In this case, since the optical path length difference can be extracted while the sample 13 is held in the sample holder 33, real-time calibration is possible, and the influence of drift due to a temperature change of the sample 13 can be reduced.

  As shown in the second embodiment, if illumination light having a plurality of angles and directions is simultaneously incident on the sample holder 33 (plane portion 33a), the optical path length difference between the illumination lights can be detected in real time. Since calibration can be performed, observation with higher precision and higher definition can be performed. In this description, the case where the image processing unit 40 also serves as the optical path length calibration unit that calibrates the optical path length difference has been described. However, the optical path length calibration unit may be provided separately from the image processing unit 40 or may be observed. A part of the control unit that controls the entire apparatus may constitute the optical path length calibration unit.

  In the above-described embodiment, the case where three-dimensional observation is performed on a biological sample or the like has been described as an example. However, the present invention can also be applied to two-dimensional surface shape measurement including fine unevenness. Such a measurement can be realized by the observation apparatus according to the second embodiment in which five illumination lights are simultaneously incident on the test surface and the reflected light is detected. The focus of the optical system is adjusted to the surface to be measured.

  As in the second embodiment, the data analysis when measuring the surface shape is performed by Fourier transforming the image data set obtained by wavelength scanning with the wave number k, and the interference signal component for each illumination angle is obtained with a bandpass filter. Separately extract and inverse Fourier transform each. Thereby, it can be decomposed into a complex amplitude distribution for each illumination angle and for each wave number of light.

  Subsequently, the obtained complex amplitude distribution is corrected for an optical path length difference between illumination lights, and XY two-dimensional Fourier transform is performed on the complex amplitude distribution. Further, the spectrum is shifted and synthesized by the wave number vector given for each illumination angle. Further, inverse Fourier transform is performed to obtain a complex amplitude distribution. The phase is calculated from the obtained complex amplitude distribution, and the shape of the test surface is calculated. Through these series of analysis processes, measured values of the test surface shape are calculated for each wave number of light (test surface shape data corresponding to the number of wavelength scanning points is obtained). In addition, a more accurate measurement result can be obtained by performing an averaging process as necessary at the time of spectrum synthesis.

  As described above, when the observation apparatus according to the second embodiment is applied to two-dimensional surface shape measurement, it is not necessary to switch illumination light, so that measurement can be performed at high speed. At this time, since the detection band of the test surface shape is expanded compared to the case of single point light source illumination, quantitative measurement of a finer structure is possible. Further, if the optical path length difference of the illumination light is calibrated by the above-described method, more accurate measurement can be performed.

  When the configuration according to the present invention is applied to the three-dimensional observation of a biological sample or the like like the observation device according to the above embodiment, the effect of expanding the three-dimensional detection band is obtained by wavelength scanning (wavelength width) and illumination at a plurality of angles. be able to. On the other hand, when the present invention is applied to two-dimensional surface shape measurement, the effects of DC, signal component, conjugate component separation, and phase distribution extraction can be obtained by wavelength scanning. The magnification effect can be obtained by illumination at multiple angles.

  In addition, this invention is not limited to the structure which concerns on the said embodiment or modification, In the range which does not deviate from the meaning of invention, it can change suitably. For example, in the embodiment described above, it is not necessary to use laser light as long as the light source 50 is capable of wavelength scanning.

  Moreover, in the said embodiment, although the optical fiber 101 (reference light) of the light source and the optical fibers 102-106 (illumination light) were arrange | positioned in another place and branched via the polarization beam splitter 3, it is restricted to this. There is nothing and can be arranged in the same place. Specifically, for example, in FIG. 1, the illumination light sources of the optical fibers 102 to 106 are arranged in place of the optical fiber 101 at the place of the optical fiber 101, and the polarization beam splitter 3 is removed. Then, the position on the optical axis of the reference mirror 7 is moved to provide a predetermined optical path length difference from the sample 13. Thus, by changing the configuration of FIG. 1, the light source (optical fiber) can be arranged in one place.

  At this time, in order to branch the emitted light from the optical fiber 102 into illumination light directed to the sample 13 and reference light directed to the reference mirror, two polarization components of S-polarized light and P-polarized light for the PBS 4 are emitted simultaneously. . For example, the emitted light is linearly polarized light, and the polarization plane is set to a state having a predetermined angle that is not parallel to the S-polarized light and P-polarized light of PBS.

  When the optical fiber 102 is a polarization plane preserving fiber, for example, a mechanism for rotating and adjusting the orientation of the polarization plane may be provided so that the intensity ratio of the illumination light and the reference light can be adjusted.

  Further, when the position on the optical axis of the reference mirror 7 of the above modification is not changed, a polarization plane preserving fiber is used for the optical fiber 102, and an optical path length difference is given to two propagating polarization components (orthogonal). What is necessary is just composition.

  Further, in the above two modified examples, the reference light and the illumination light are branched by the polarization control by the PBS 4 or the like. However, it is possible to branch without using the polarized light. In this case, PBS 4 is replaced with BS, and QWPs 5 and 11 (both arms) are removed.

  Further, a light shielding member for cutting light coming from these fibers is arranged at a position corresponding to the exit point of the optical fibers 103 to 106 on the pupil plane of the objective lens 6 (reference arm side), and only the light from the optical fiber 102 passes. Like that.

  Moreover, in the said embodiment, although the optical fibers 101-106 were used for the division | segmentation and routing of the light beam from the light source 50, you may make it the structure which forms a some point light source using a lens array etc. FIG. FIG. 20 is a diagram showing another configuration for forming a plurality of point light sources. As shown in FIG. 20, a plurality of point light sources are formed by combining a multi-lens array 60, a beam splitter 61, and a reflecting mirror 62, and illumination light having a predetermined angle and direction with respect to the optical axis is generated by a collimator lens 63. Can be formed. In this case, the reflection mirror 62 is disposed so that the optical path lengths of the plurality of illumination lights are different from each other. Instead of the beam splitter 61, a combination of a polarizing beam splitter and a quarter wavelength plate may be used.

In the above embodiment, the wavelength scanning type light source is used. However, the present invention is not limited to this. For example, an optical interference observation apparatus that scans the reference surface may be used.
Specifically, it is as follows.
A reference surface scanning type optical interference observation apparatus requires a spatially coherent light source having a wide wavelength width as a light source. For example, an SLD with a wide wavelength width is desirable. Alternatively, a light source in which a white light source is once condensed by a lens and spatial coherence is imparted through a PH (pinhole) can be employed. The detector is a two-dimensional image sensor (CCD) or the like. The reference surface is scanned in the optical axis direction at a constant speed, and the interference intensity between the measurement light and the reference light is detected. As a region used for this detection, a constant velocity portion may be used in acceleration-constant velocity-deceleration control. It is also possible to use a portion that can be regarded as constant speed by scanning in a sine wave shape. Data of “reference plane scanning distance L” vs. “interference intensity I” is obtained for each pixel of the two-dimensional image sensor. The data is subjected to Fourier transform for each pixel of the two-dimensional image sensor, and a signal component is extracted by a bandpass filter (DC component and conjugate component are unnecessary). This extracted component corresponds to the above formula (1). If the frequency is f (f is the number of vibrations when scanning by L), the light wave number k is the following equation:
k = 2πf / 2L
There is a relationship.
The reason that the denominator is 2L is that the optical path length changes twice as long as it goes back and forth. Thus, the information of the above formula (1) is obtained for each wave number of light by the well-known principle of Fourier spectroscopy. Other than the above, the operation is the same as in the wavelength scanning.

DESCRIPTION OF SYMBOLS 9 ... Polarizing plate (synthesis | combination means), 12 ... Objective optical system (observation optical system), 30 ... Calibration flat mirror, 31 ... Calibration reference, 33 ... Sample holder, 40 ... Image processing part, 50, 350 ... Light source ( Illumination optical system), 52a to 52f ... beam splitter (branching means), 90 ... transmission illumination system, 101-106 ... optical fiber (light emitting part), 202-205 ... optical fiber (light emitting part), 100, 300 ... observation device , 301, 302: optical fiber

Claims (15)

A light source that emits light of multiple wavelengths;
An illumination optical system that divides light from the light source into reference light and illumination light and irradiates the observation light from at least two directions;
The measurement light including the structural information of the object to be observed generated by irradiating the object with the illumination light interferes with the reference light, and each of the measurement light generated by irradiation from the two directions A detector for detecting first and second interference light generated by interference with the reference light;
An image data processing unit that generates image data based on the first interference light and the second interference light detected by the detection unit;
An optical interference observation apparatus for observing the structure of the object to be observed. The optical interference observation apparatus according to claim 1,
The image data processing unit generates first image data based on the first interference light, and generates second image data based on the second interference light,
The first and second image data is subjected to a predetermined conversion process for each wave number of light of the light source, thereby generating object scattering potential information as structure information of the object to be observed, and image data of the object to be observed. The optical interference observation apparatus characterized by producing | generating. The optical interference observation apparatus according to claim 1,
The optical interference observation apparatus, wherein the light source is a wavelength scanning light source that scans a wavelength of light applied to the object to be observed. The optical interference observation apparatus according to claim 1,
The illumination optical system is composed of a laser light source, a beam splitter that divides the light of the laser light source into a plurality, and a plurality of optical fibers that guide the divided light to the observed portion. Observation device. The optical interference observation apparatus according to claim 2,
The image data processing unit
Fourier transforming the first and second image data as a function of the wave number;
Calculating a complex amplitude distribution for each wave number;
Obtaining a spectrum of the complex amplitude distribution by performing a two-dimensional Fourier transform on the complex amplitude distribution;
Obtaining and synthesizing a three-dimensional spectrum of the object scattering potential based on the obtained spectrum of the complex amplitude distribution;
An optical interference observation apparatus comprising: a step of obtaining a scattering potential of the object to be observed by performing a three-dimensional inverse Fourier transform process on a three-dimensional spectrum of the synthesized scattering potential. In the optical interference observation apparatus according to any one of claims 1 to 4,
The illumination optical system includes a first illumination unit and a second illumination unit for illuminating the object to be observed from at least two directions,
The first illumination unit and the second illumination unit illuminate the object to be observed from at least two directions. In the optical interference observation apparatus according to claim 6,
The first illumination unit and the second illumination unit of the illumination optical system have an optical unit capable of emitting the illumination light having a predetermined angle with respect to the optical axis of the illumination optical system. apparatus. The optical interference observation apparatus according to claim 1,
The illumination optical system includes at least one of a falling-down illumination optical system or a transmission illumination optical system. The optical interference observation apparatus according to claim 1,
The said illumination optical system is comprised by combining a falling-down illumination optical system and a transmission illumination optical system. The optical interference observation apparatus characterized by the above-mentioned. In the optical interference observation apparatus according to claim 7,
The optical unit includes a plurality of light emitting units that emit the illumination light at the predetermined angle. In the optical interference observation apparatus according to claim 10,
The illumination optical system causes the illumination light to be simultaneously emitted from different directions from the plurality of light emission units to the object to be observed. In the optical interference observation apparatus according to claim 10,
The illumination optical system emits the illumination light from the plurality of light emitting units to the object to be observed in time order from different directions. In the optical interference observation apparatus according to claim 10,
The plurality of light emitting portions have a predetermined optical path length difference,
An optical interference observation apparatus comprising: an optical path length calibration unit that calibrates optical path length differences in the plurality of light emitting units. In the optical interference observation apparatus according to claim 13,
The optical path length calibration unit calibrates the coordinates of the position of the object to be observed and the coordinates on the image detector using the interference light from the feature point of the calibration member. The optical interference observation apparatus according to any one of claims 3 to 5,
The optical unit includes a scanning mirror that reflects illumination light in different directions;
The scanning mirror reflects the illumination light so that the spot of the illumination light moves on a ring,
The light source performs predetermined wavelength scanning for each spot position moving on the ring.

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