JP6684875B2 - Spectroscopic analyzer - Google Patents

Description

The present invention relates to a spectroscopic analyzer capable of extracting, as output light, an arbitrary wavelength component among wavelength components included in input light.

  In recent years, research and development of regenerative medicine technology and drug discovery using somatic stem cells and embryonic stem cells (ES cells (embryonic stem cells)) and induced pluripotent stem cells (iPS cells (induced pluripotent stem cells)) have risen. ing. In this type of research and development, it is extremely important to efficiently mass-produce the required target cells and tissues.

  If defective or unnecessary cells are mixed in the cell aggregates used for the purpose of regenerative medicine to supplement the damaged tissues and organs of the patient, not only the original effect may not be exhibited, but also tumorigenesis and other patients. It can also have an adverse effect on your health. However, discarding the entire culture container in which unnecessary cells are mixed leads to a decrease in the yield (yield) of the target cells or tissues, which raises the cost of regenerative medicine. In order to improve the yield of target cells or tissues, it is desirable to kill and remove unnecessary cells existing in the culture container and use the remaining cells without wasting them.

  Therefore, it has become possible to selectively kill only unnecessary cells among the proliferated cells by using a laser beam having an excellent light-collecting property (see Patent Document 1 below).

  In order to distinguish which cells are the target cells and which are unnecessary cells in the aggregate of living cells existing in the cell culture vessel, conventionally, observation using a microscope and taking images I was doing the analysis. However, it is not easy to reliably and quickly determine the identity of individual cells.

  As a method for discriminating the identity of cells, it is necessary to use coherent anti-Stokes Raman scattering using coherent anti-Stokes Raman scattering to obtain the type of protein synthesized by gene expression. Is regarded as influential. The SC light is a so-called “white” laser light of high intensity and broadband. The SC light is superposed on the pump light as Stokes light to irradiate the target cell, and the CARS light obtained is dispersed by a diffraction grating spectroscope and the spectrum is analyzed to determine the molecular species and molecular structure in the cell. It has become clear that not only can it be identified, but also the vital activity of cells can be selectively monitored (see Non-Patent Document 1 below).

  When performing CARS light spectroscopic analysis, the diffracted light output from the diffraction grating spectroscope is input to an image sensor such as a CCD or CMOS. The position of the photodiode, which is the light receiving element on the image sensor, corresponds to the wavelength of the spectrum included in the CARS light, and the intensity of the light received by the photodiode is the intensity of the wavelength component.

  With the method using such a diffraction grating spectrometer, it is possible to investigate a narrow spot range of about 1 μm width, but it is not possible to observe a two-dimensionally expanded plane area at once. In order to image a planar region, it is necessary to repeatedly perform a number of spectroscopic analyzes while moving the sample relative to the optical axis. Therefore, for example, it takes a long time to acquire the entire image of the cell aggregates grown in the cell culture dish, which is a practical problem.

Japanese Patent No. 6090891

Hideaki Kano, "Coherent Raman spectroscopy using supercontinuum light", [online], MOLECULAR SCIENCE (Journal of the Society of Molecular Science), [Search on September 1, 2018], Internet <URL: http: // j -molsci.jp/article/2007#1/A0005.pdf>

The present invention is intended to provide a spectroscopic analyzer that can obtain output light containing an arbitrary wavelength component without using a diffraction grating spectroscope and can change the wavelength component rapidly. .

According to the present invention, an objective lens which faces a sample and which receives a response light generated in a two-dimensionally expanded plane area as a result of irradiating the sample with light, and a response light which has passed through the objective lens are incident to p-polarized light. A first polarization beam splitter that splits the light into a s-polarized light component and an s-polarized light component, and that transmits only a part of the wavelength component of the incident light. The first multilayer filter capable of controlling the angle at which the light intersects with the optical axis of the light is transmitted, and only a part of the wavelength component of the incident light is transmitted. A second multilayer filter capable of operating the angle intersecting the optical axis of the other light while making the other light emitted from the beam splitter incident, the light transmitted through the first multilayer filter and the A second polarization beam splitter to obtain output light and second multilayer filter the transmitted light obtained by superimposing both made incident respectively, an imaging lens for incident output light emitted from the second polarization beam splitter, A spectroscopic analysis device is provided, which comprises an image sensor that acquires the image of the planar region at a time by receiving the output light that has passed through the imaging lens .

  As the second multilayer filter, when using a filter having the same characteristics as the first multilayer filter, the attitude of the second multilayer filter and the orientation of the rotation axis of the second multilayer filter. Around the optical axis of light passing through the filter, the posture of the first multilayer filter and the first multilayer filter are displaced by 90 °.

Even if a diffraction grating spectroscope does not exist between the objective lens and the image sensor, it is possible to obtain an image of a flat area by the output light including the wavelength component transmitted through each multilayer filter at one time.

The response light is, for example, Raman scattered light generated from a target sample irradiated with light. In particular, the response light may be coherent anti-Stokes Raman scattered light generated as a result of irradiating a target sample with supercontinuum light that is pump light and Stokes light.

  When the sample of interest is a living cell or other biological sample, the first multilayer filter and the second multilayer filter respectively have, for example, an incident angle of incident light of 45 ° or an angle in the vicinity thereof. It shall be designed to transmit near infrared light.

According to the present invention, it is possible to realize a spectroscopic analysis device that can obtain output light containing an arbitrary wavelength component and can change the wavelength component quickly without using a diffraction grating spectroscope. .

The figure which shows the structure of the spectroscopic analysis apparatus of one Embodiment of this invention. The figure which illustrates the relationship between the incident angle of the incident light of the multilayer filter in the spectral analyzer of the embodiment, and the wavelength of the transmitted light.

  An embodiment of the present invention will be described with reference to the drawings. FIG. 1 shows the configuration of the spectroscopic device of this embodiment. The present spectroscopic device is for performing spectroscopic analysis of the wavelength spectrum included in the response light 102 emitted from the sample 0 irradiated with the light 101.

  The response light 102 is Raman scattered light emitted from the sample 0 as a result of irradiating the sample 0 with the light 101. In particular, the present embodiment aims to investigate and discriminate the biological sample 0 using CARS. Therefore, the light 101 irradiating the sample 0 is the pump light on which the Stokes light is superimposed, and the response light 102 is the CARS light.

  Each of the pump light and the Stokes light 101 is an ultrashort pulse laser such as a picosecond laser or a femtosecond laser. As the pump light, near infrared light, for example, a laser having a wavelength of 1064 nm is used. As the Stokes light, SC light containing various wavelength components is used. To generate the SC light, a non-linear optical fiber called a photonic crystal fiber or an ordinary optical fiber called a tapered fiber is extended to deform the core into an extremely fine shape. In the present embodiment, a laser oscillated by a laser light source for pump light is split into a plurality of beams by a beam splitter or the like, one is used as pump light as it is, and the other is incident on a PCF or a tapered fiber to obtain SC light, After overlapping, the sample 0 is irradiated.

  The spectroscopic device according to the present embodiment includes an objective lens 1 facing a sample 0, a first polarization beam splitter 2 that inputs the response light 102 that has passed through the objective lens 1 as input light, and a first polarization beam splitter 2. From the first multi-layer film filter 3 that allows the p-polarized component 103 of the input light 102 to be emitted from the first polarization beam splitter 2, and the second multi-layer film filter that causes the s-polarized component 104 of the input light 102 to be emitted from the first polarization beam splitter 2. 4 and a second polarization beam splitter 7 that receives the light 105 that has passed through the first multilayer filter 3 and the light 106 that has passed through the second multilayer filter 4 to obtain output light 107 that is a superposition of the two. , An image forming lens 8 on which the output light 107 emitted from the second polarization beam splitter 7 is made incident, and the output light 107 having passed through the image forming lens 8 is received and an image is taken. ; And a Mejisensa 9.

  The first multilayer filter 3 and the second multilayer filter 4 respectively transmit only some wavelength components 105 and 106 contained in the light 103 and 104 incident on the filters. Each of the filters 3 and 4 is an interference filter that typically functions as a bandpass filter. However, it does not prevent the adoption of filters other than interference filters, such as dichroic filters, as the multilayer filters 3 and 4. The general multilayer filters 3 and 4 have a structure in which dielectric optical thin films are stacked in multiple layers on the surface of a transparent substrate, and a predetermined amount of interference occurs due to intra-film multiple reflection in the process of incident light passing through the multilayer film. Of the wavelengths are selectively transmitted, other wavelengths are not transmitted, and a very sharp change in spectral transmittance is achieved.

  As a feature of the multilayer filters 3 and 4, the wavelengths of the lights 105 and 106 that are transmitted through the filters 3 and 4 and the wavelengths of the light that are not transmitted are changed by changing the angle intersecting the optical axes of the lights 103 and 104 that are incident thereon. Can change. As a tendency, the larger the incident angle, which is the angle formed by the normal line of the boundary surface of the filters 3 and 4 on which the light 103 and 104 is incident and the optical axis of the incident light 103 and 104, the more the light can pass through the filters 3 and 4. The wavelengths of the lights 105 and 106 are shortened. This is because the optical path length in the optical thin film expands and contracts depending on the incident angle, the interference condition changes, and as a result, the spectral transmittance characteristic changes.

  In the present embodiment, when the incident angles of the incident lights 103 and 104 are intermediate predetermined angles smaller than 90 ° and larger than 0 °, for example, 45 °, the light 105 having a predetermined wavelength belonging to the near infrared light, Bandpass filters 3 and 4 designed to transmit 106 and not transmit light of other wavelengths are used. The definition of near-infrared light is light with a wavelength of about 700 nm to about 2500 nm, but the center wavelength of the transmitted light 105, 106 of the bandpass filters 3, 4 at an incident angle of 45 ° or in the vicinity thereof is about 900 nm. To a value in the range of about 1300 nm. FIG. 2 shows an example of the incident angle of the p-polarized incident light 103 on the bandpass filter 3, the central wavelength and the bandwidth of the light 105 transmitted through the bandpass filter 3, and the transmittance of the p-polarized component. . In the illustrated example, the central wavelength of the transmitted light 105 at an incident angle of 45 ° or in the vicinity thereof is 1064 nm, which is equal to or substantially equal to the wavelength of the pump light. The band width of the transmitted light 105 is several nm or less.

  The multilayer filters 3 and 4 as described above are rotated about the rotation shafts 31 and 41 by a rotary drive device such as a galvano scanner motor, and the incident angles of the lights 103 and 104 incident on the filters 3 and 4 are increased or decreased. As a result, it is possible to change the wavelengths of the lights 105 and 106 that pass through the filters 3 and 4. When CARS spectroscopic analysis is performed on the biological sample 0, it is preferable that the wavelengths of the transmitted lights 105 and 106 can be changed within the range of near infrared light, particularly within the range of about 900 nm to about 1300 nm. On the other hand, the transmitted light 105, 106 having a short wavelength whose wavelength is less than 900 nm is not necessarily required.

  In the present embodiment, the characteristics of the first multilayer filter 3 and the characteristics of the second multilayer filter 4 are equivalent. It is needless to say that the “equivalent characteristic” mentioned here includes the existence of a minute difference such as an individual difference between the characteristics of the filters 3 and 4.

  However, the relationship between the incident angles of the lights 103 and 104 that enter the multilayer filters 3 and 4 and the wavelengths of the lights 105 and 106 that are transmitted through the filters 103 is affected by the polarization directions of the incident lights 103 and 104. When a filter having characteristics equivalent to those of the first multilayer film filter 3 is used as the second multilayer film filter 4, the polarization directions of the light beams 103 and 104 divided by the first polarization beam splitter 2 are different from each other. Must be taken into consideration. In the present embodiment, the attitude of the second multilayer filter 4 and the rotation axis 41 are incident on the second multilayer filter 4 relative to the attitude of the first multilayer filter 3 and the rotation axis 31. It is rotated 90 ° around the optical axis of the transmitted light 104, 106. For example, when the rotation axis 31 of the first multilayer film filter 3 is orthogonal to the virtual plane (paper surface of FIG. 1) including the optical axes of the incident lights 103 and 104, the second multilayer film filter The rotation axis 41 of No. 4 is parallel to the plane. As a result, the first multilayer film filter 3 on which the p-polarized light component 103 of the input light 102 is incident and a control system for rotationally driving the filter 3, the second multilayer film filter 4 on which the s-polarized light component 104 is incident, and the filter concerned. The control system for rotating and driving 4 can be shared.

  The light 106 transmitted through the second multilayer filter 4 is reflected by the plurality of total reflection mirrors 5 and 6, and is orthogonal or substantially orthogonal to the light 105 transmitted through the first multilayer filter 3. The positions and angles of the mirrors 5 and 6 can be adjusted. Further, it does not prevent that a lens for focus adjustment, a beam expander or the like is interposed between the mirrors 5 and 6.

  The second polarization beam splitter 7 is located at a position where the optical axis of the light 105 transmitted through the first multilayer filter 3 and the optical axis of the light 106 transmitted through the second multilayer filter 4 intersect. Then, upon receipt of the incident lights 105 and 106, an output light 107 that is a combination of the two lights is emitted.

  The output light 107 enters the image sensor 9 such as CCD or CMOS through the imaging lens 8. The image sensor 9 receives the output light 107 and takes an image. If necessary, a notch filter or a band stop filter for blocking unnecessary components such as the wavelength component of the light 101 with which the sample 0 is irradiated may be arranged upstream of the image sensor 9.

  A feature of the light receiving device of the present embodiment is that a planar region of the target sample 0 excited by the irradiation of the pump light and the SC light 101 can be imaged at one time. The size of the imaging area depends on the focal lengths of the objective lens 1 and the imaging lens 8, the visual field size of the image sensor 9, the pixel resolution, etc., but it is possible to image an area of several tens μm square to 2 mm square. Is. The limit resolution of the captured image can be 1 μm or less.

  As described above, the wavelength included in the output light 107, that is, the wavelength extracted from the CARS light 102 is determined by the incident angle of the multilayer filters 3 and 4. The incident angles of the multilayer filters 3 and 4 can be changed automatically and at high speed without manual operation. If the process of receiving and imaging the output light 107 by the image sensor 9 is repeated while executing the control for gradually changing the incident angles of the multilayer filters 3 and 4, the planes of various wavelengths that can be included in the CARS light 102 can be obtained. You can get an image of the area. That is, the wavelength spectrum of the response light 102 generated at each position in the plane area can be investigated in a short time. As a result, identification of molecular species and molecular structure existing at each position in the plane area, discrimination of cells existing at each position, monitoring of life activity, etc. can be performed in a practically required time.

  If the size of the target sample 0 exceeds the size of the plane area that can be imaged at one time, repeat the imaging while displacing the sample 0 relative to the optical axes of the irradiation light 101 and the response light 102. Needless to say.

  In the present embodiment, the first polarization beam splitter 2 that splits the incident input light 102 into a p-polarized component 103 and an s-polarized component 104, and transmits only a part of the wavelength component 105 of the incident light 103. One of the light beams 103 emitted from the first polarization beam splitter 2 is made incident, and the light beam 103 is made incident on the first multilayer film filter 3 whose angle intersecting with the optical axis of the light 103 can be manipulated. An angle at which only a part of the wavelength component 106 of the light 104 is transmitted, the other light 104 emitted from the first polarization beam splitter 2 is incident, and an angle intersecting with the optical axis of the light 104. The second multilayer filter 4 which can be operated, the light 105 transmitted through the first multilayer filter 3 and the light 106 transmitted through the second multilayer filter 4 The Configure spectroscopic apparatus comprising a second polarizing beam splitter 7 to obtain an output light 107 obtained by superimposing both made incident.

  If the wavelength component transmitted through the filter is to be selected by changing the incident angle of the multilayer filter, the wavelength of the transmitted light is affected by the polarization direction of the light incident on the filter. However, as in the present embodiment, the input light 102 is once separated into the p-polarized component 103 and the s-polarized component 104, and these are separately incident on the multilayer filters 3 and 4, and the transmitted lights 105 and 106 are re-generated. With the overlapping structure, it is possible to extract the accurate output light 107 without the influence of the polarization direction.

  According to the present embodiment, it is possible to realize a spectroscopic device that can obtain the output light 107 including an arbitrary wavelength component without using a diffraction grating spectroscope and can change the wavelength component rapidly.

  The present invention is not limited to the embodiment described in detail above. In particular, the practical application of the present invention is not limited to CARS spectroscopy. The present invention can naturally be applied to Raman spectroscopic analysis other than CARS. In that case, the input light 102 that is the response light is Raman scattered light generated by irradiating the sample 0 with the excitation light 101, and the output light 107 is an arbitrary wavelength component included in the Raman scattered light 102.

  Furthermore, the present invention can be applied to applications other than Raman spectroscopy, such as infrared spectroscopy. In that case, the input light 102 which is the response light is the light which is not absorbed by the sample 0 as a result of irradiating the sample 0 with the infrared light 101 (the sample 0 is transmitted or the sample 0 is reflected), and the output light 107 is It is an arbitrary wavelength component included in the light 102.

  INDUSTRIAL APPLICABILITY The present invention can realize direct imaging, that is, an optical system that forms an image of a region that expands in a plane at one time, but can also be used for the purpose of performing imaging by scanning a small spot region.

  In addition, the specific configuration of each part can be variously modified without departing from the spirit of the present invention.

0 ... Sample 2 ... 1st polarization beam splitter 3 ... 1st multilayer film filter 4 ... 2nd multilayer film filter 7 ... 2nd polarization beam splitter 9 ... Image sensor 101 ... Light irradiating a sample 102 ... Input light (Raman scattered light, CARS light)
103 ... p-polarized component 104 ... s-polarized component 105 ... transmitted light 106 ... transmitted light 107 ... output light

Claims (6)

An objective lens that faces the sample and irradiates the sample with a response light generated in a two-dimensionally expanded planar region as a result of irradiating the sample with light.
A first polarization beam splitter that receives the response light that has passed through the objective lens and splits it into a p-polarized component and an s-polarized component;
It transmits only a part of the wavelength component of the incident light, and allows one of the light emitted from the first polarization beam splitter to be incident and the angle intersecting with the optical axis of the light can be controlled. A first multilayer filter,
It transmits only part of the wavelength component of the incident light, and allows the other light emitted from the second polarization beam splitter to be incident and the angle at which it intersects the optical axis of the light can be manipulated. Second multilayer filter,
A second polarization beam splitter that obtains output light that is obtained by superimposing both the light transmitted through the first multilayer filter and the light transmitted through the second multilayer filter, respectively .
An image forming lens which makes the output light emitted from the second polarization beam splitter incident,
A spectroscopic analyzer comprising: an image sensor that receives an image of the planar region at a time by receiving output light that has passed through the imaging lens . As the second multilayer filter, a filter having the same characteristics as the first multilayer filter is used,
The orientation of the second multilayer filter and the orientation of the rotation axis of the second multilayer filter are set around the optical axis of the light passing through the filter, and the orientation of the first multilayer filter and the first The spectroscopic analyzer according to claim 1, wherein the spectroscopic analyzer is displaced by 90 ° with respect to the multilayer filter. The spectroscopic analyzer according to claim 1 or 2, wherein the diffraction grating spectrometer is not present between the objective lens to the image sensor. The spectroscopic analyzer according to claim 1, 2 or 3, wherein the response light is Raman scattered light generated from a target sample irradiated with light. The spectroscopic analyzer according to claim 4, wherein the response light is coherent anti-Stokes Raman scattered light generated as a result of irradiating a target sample with supercontinuum light that is pump light and Stokes light. 6. The first multilayer filter and the second multilayer filter each transmit near infrared light when an incident angle of incident light is 45 ° or an angle in the vicinity thereof. Spectroscopic analyzer .

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