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WO2016009548A1 - Dispositif d'analyse optique - Google Patents

Dispositif d'analyse optique Download PDF

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Publication number
WO2016009548A1
WO2016009548A1 PCT/JP2014/069132 JP2014069132W WO2016009548A1 WO 2016009548 A1 WO2016009548 A1 WO 2016009548A1 JP 2014069132 W JP2014069132 W JP 2014069132W WO 2016009548 A1 WO2016009548 A1 WO 2016009548A1
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Prior art keywords
light
cars
sample
detector
cells
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PCT/JP2014/069132
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English (en)
Japanese (ja)
Inventor
秀治 三上
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株式会社日立製作所
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Priority to PCT/JP2014/069132 priority Critical patent/WO2016009548A1/fr
Priority to JP2016534064A priority patent/JP6279081B2/ja
Publication of WO2016009548A1 publication Critical patent/WO2016009548A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the present invention relates to high performance optical analyzers.
  • the optical microscope is an indispensable observation tool in the natural science, engineering, and industrial fields. Particularly in recent years, more advanced microscopes using a laser as an illumination light source have become essential in advanced technology development.
  • a typical example is a coherent anti-Stokes Raman scattering (CARS) microscope (Patent Documents 1, 2 and Non-Patent Document 1).
  • the CARS microscope irradiates a sample with two types of laser light, excitation light and Stokes light, It is a microscope for observing anti-Stokes light (hereinafter referred to as CARS light) generated as a result of the difference frequency of these lights resonating with the natural vibration of the sample molecule.
  • the substance in the sample can be quantitatively analyzed and observed by the spectrum of the CARS light, and has attracted attention as a non-invasive quantitative analysis means.
  • CARS is light emission by third-order polarization, and excitation light, Stokes light, and probe light are required to generate CARS.
  • the probe light is substituted with excitation light.
  • ⁇ r (3) ( ⁇ ) is a resonance term of molecular vibration of third-order electrical susceptibility
  • ⁇ nr (3) is a non-resonance term.
  • the electric field of excitation light and probe light is represented by E P
  • the electric field of Stokes light is represented by E S.
  • the non-resonant term has no frequency dependence.
  • An asterisk attached to the shoulder of the E S of the formula (1) denotes a complex conjugate.
  • the intensity of CARS light is the square of the absolute value of P ( ⁇ ). The mechanism of CARS light generation will be described using the energy level diagram of the molecule shown in FIG. FIG. 22 illustrates the resonance term process. 1401 represents the vibrational ground state of the molecule, and 1402 represents the vibrational excited state. Simultaneously irradiate excitation light with frequency ⁇ P and Stokes light with frequency ⁇ S.
  • the molecule is excited to a vibration excitation level having 1402 through the virtual level 1403.
  • this excited molecule is irradiated with probe light having the frequency ⁇ P
  • the molecule returns to the vibrational ground state while generating CARS light having the frequency ⁇ AS via the virtual level 1404.
  • this resonance CARS light is generated only when the difference in frequency ⁇ P ⁇ S between the excitation light and the Stokes light coincides with a certain vibration excitation state of the observation sample.
  • the Planck unit system is adopted here, and the Planck constant is 1. Therefore, when a broadband light source is used as Stokes light, the generated CARS light is also broadband light, but has a spectrum having a sharp peak at a wavelength corresponding to the vibrationally excited state. This spectrum reflects the distribution of vibrationally excited states of the molecules in the sample and can be used to identify the molecular species.
  • FIG. 23 is a diagram illustrating one process related to the non-resonant term of Equation (1).
  • the frequency of the Stokes light is not a vibrationally excited state, but a process through the virtual level 1405.
  • 'Participating virtual level 1405 such as electrons are excited by the simultaneous irradiation of the P of the probe light, yet the frequency omega' exciting light frequency omega P and the frequency omega by Stokes light of S, the frequency omega via a virtual level 1406
  • Non-resonant CARS light is generated. Since this non-resonant CARS light is generated regardless of the vibration excitation state, when a wide-band Stokes light is used, a wide-band non-resonant CARS light having no wavelength dependency of intensity is generated.
  • Non-Patent Document 2 a Raman spectrum is extracted using the maximum entropy method, and the magnitude of each peak value is used as quantitative information.
  • the magnitude of each resonance component is specified by fitting data using the assumed resonance component.
  • Non-Patent Documents 4 and 5 differences and ratios are used as quantitative values of the resonance component for the maximum value and the minimum value of data corresponding to a specific resonance frequency.
  • Non-Patent Document 6 a quantitative value is calculated by calculation from a total of three data values of an assumed resonance frequency and a frequency equally spaced from the frequency.
  • Excitation light having a predetermined frequency and Stokes light in a lower frequency region are incident on the sample, and CARS light is generated in a frequency region larger than the excitation light.
  • the CARS microscope performs a plurality of measurements on the Raman spectrum obtained as described above by changing the position where the excitation light and the Stokes light are collected, and as a result, acquires an image of the spatial distribution for each molecular species.
  • Non-Patent Documents 2 and 3 are complicated in data processing, and particularly take a long time to process a large number of spectra, so a large amount of data is processed at high speed. There is a problem that it is not suitable for.
  • Non-Patent Documents 4, 5, and 6 are suitable for high-speed data processing because of simple data processing.
  • the quantitative value in Non-Patent Document 4 is based on the premise that the resonance component is sufficiently smaller than the non-resonant component. If this condition is not satisfied (for example, when the concentration of the measurement target substance is high), the quantitative value is obtained. There is a problem that an error occurs and the applicable samples are limited.
  • Non-Patent Document 6 since the obtained quantitative value also depends on the size of the non-resonant component, it is serious when evaluating a sample with a variation in the size of the non-resonant component. Cause an error.
  • an accurate quantitative value is acquired regardless of the magnitude of the resonance component and non-resonance component by measuring the relationship between the concentration of the substance to be measured and the quantitative value (calibration data) in advance. Since the measurement is required in advance, the analysis process becomes complicated, and there is a problem that the net analysis time becomes long.
  • Non-Patent Document 6 needs to know the resonance frequency of the measurement target accurately in advance, but it is known that the resonance frequency changes depending on the concentration of the measurement target substance and the surrounding environment (for example, Non-Patent Document 1). There is a problem that accurate quantitative values can be obtained only under very limited conditions.
  • CARS spectrum an analysis that acquires a spectrum of CARS light at one or more points
  • an object of the present invention is to provide an optical analyzer that enables analysis of a sample at high speed and accurately.
  • the basis of the present invention is to calculate the following quantitative value from the measurement data. That is, the square root of the maximum value and the minimum value of the spectrum before and after the resonance frequency is calculated for a specific resonance component to be measured, and the difference between them is used as a quantitative value. Thereby, it is possible to perform quantification at high speed and accurately (regardless of the size of the resonance component and the non-resonance component) with respect to an isolated resonance component in which other resonance signals do not overlap on the spectrum. Specifically, the following means were used.
  • At least two light sources such as a short pulse laser, a photonic crystal fiber, and an optical parametric oscillator, an irradiation optical system such as an objective lens that irradiates a light beam generated from at least two light sources to the same portion of the sample, and a sample
  • an irradiation optical system such as an objective lens that irradiates a light beam generated from at least two light sources to the same portion of the sample
  • a sample A spectroscope that detects coherent anti-Stokes Raman scattering light generated from the detector, a detector such as a CCD camera or a photomultiplier tube, and a signal processing unit such as a computer that takes in the output of the detector and performs signal processing,
  • the signal processing unit calculates and outputs a value proportional to the difference between the absolute values of the electric field of the coherent anti-Stokes Raman scattering at the wavelength where the coherent anti-Stokes Raman scattering becomes a maximum and the wavelength at which the detector becomes a minimum.
  • the detector acquires the spectrum of the coherent anti-Stokes Raman scattering light, and the difference in the absolute value of the electric field is the difference between the square roots of the maximum and minimum values of the spectrum. It was supposed to be.
  • the detector detects coherent anti-Stokes Raman scattered light at at least two wavelengths, and the difference between the absolute values of the electric field is determined at two predetermined wavelengths specified by the user. The difference between the square roots of the detector output was determined.
  • At least one wavelength of the light sources is alternately modulated between two types of set wavelengths.
  • a light source a sample holding unit that holds a plurality of cells as a sample, an observation unit that observes the cells held in the sample holding unit, and a light beam from the light source is condensed on the cells held in the sample holding unit
  • the irradiation optical system for irradiating the light the spectroscopic unit for dispersing the light generated from the cells by the light irradiation, the detection unit for detecting the light dispersed by the spectroscopic unit, and the light irradiation position on the cell by the irradiation optical system
  • a signal processing unit comprising: an irradiation control unit for controlling; a cell destruction means for destroying cells held in the sample holding unit; and a biomolecule capturing device for capturing biomolecules in the cells released from the cells by the destruction. Calculates a value proportional to the difference between the absolute values of the electric field of the coherent anti-Stokes Raman scattering at the wavelength at which the coherent anti-Stokes Raman scattering becomes maximum and the wavelength at
  • the cell destruction means destroyed the cells by laser light irradiation. Thereby, the apparatus can be reduced in size.
  • the schematic diagram which shows the structural example of an optical analyzer.
  • the sequence diagram of data acquisition operation A diagram representing a typical CARS spectrum
  • the figure showing the principle of quantitative value calculation of the present invention Measured adipocyte CARS spectrum and restored Raman spectrum
  • Configuration diagram when using a scan mirror The block diagram of the optical analyzer which detects the backscattering of CARS light.
  • the schematic diagram which shows the structural example of embodiment which switches the wavelength of Stokes light into two types, and acquires data Schematic diagram showing a configuration example of an embodiment that switches the set values of two types of wavelengths at high speed
  • the schematic diagram which shows the structural example of embodiment which applies heterodyne detection The figure showing the irradiation state of the light on the CCD camera in embodiment which applies heterodyne detection
  • the schematic diagram which shows another structural example of embodiment which applies heterodyne detection The schematic diagram which shows the structural example of an optical analyzer.
  • the flowchart which shows the procedure of gene expression data analysis.
  • the figure which shows the result of a main factor analysis.
  • An energy diagram representing a resonant CARS process An energy diagram representing a non-resonant CARS process.
  • the figure which shows the relationship of the frequency of excitation light, Stokes light, and CARS light.
  • FIG. 1 is a schematic diagram showing a basic configuration example of the optical analyzer of the present invention. The operation of this embodiment will be described below with reference to FIG.
  • the light is divided into two, that is, transmitted light and reflected light.
  • the reflected light is coupled to the photonic crystal fiber 104 by the condensing lens 103, and broadband supercontinuum light is generated inside the fiber.
  • the generated supercontinuum light is collimated by the collimating lens 105 and then enters the long pass filter 106 to block the wavelength of the short pulse laser light source and the shorter wavelength component.
  • the dichroic mirror 108 has a property of reflecting light having a wavelength of excitation light and a shorter wavelength region and transmitting light having a wavelength region longer than the excitation light. Therefore, the excitation light is reflected, and the Stokes light is transmitted and combined as a result.
  • This combined light beam is condensed at one point of the sample 110 by an objective lens 109 (NA 0.9, magnification 40 times) that constitutes an irradiation optical system that collects and irradiates the light beam from the light source onto the sample.
  • CARS light reflecting the resonance vibration of molecules present at the location is generated.
  • the CARS light is converted into parallel light by the condenser lens 111 (NA 0.65), passes through the short-pass filter 112, and the excitation light and the Stokes light, which are coaxial components, are blocked. And is detected separately for each wavelength by the detection unit 115, and the spectrum is output as a detection signal.
  • the spectroscope 113 includes a spectroscopic unit 114 that diffracts incident light in a different direction for each wavelength by a diffraction grating, and a one-dimensional or two-dimensional detector array (CCD camera, CMOS camera, etc.) of the light diffracted by the spectroscopic unit 114. It consists of the detection part 115 detected by.
  • a CCD camera is used as the detection unit 115, and the light receiving unit 201 has two-dimensionally arranged pixels 202 as shown in FIG.
  • the light split by the spectroscopic unit 114 enters the light receiving unit as a horizontally long beam 203, and the wavelength varies depending on the position in the horizontal direction.
  • the CCD camera of the detection unit 115 is in an exposure state for a predetermined time by external control, that is, in a state in which each pixel is exposed to incident light and converts incident light into electric charge to accumulate electric charge.
  • the total charge accumulated in the vertically aligned pixel columns is transferred to the buffer 204 (full vertical binning), and the charge in the buffer 204 is output to the outside as a serial signal. Therefore, the output signal is a signal proportional to the intensity of each wavelength of incident light, that is, a spectrum signal of incident light.
  • the XYZ stage 12 holding the sample 110 is driven, and the sample is scanned three-dimensionally or two-dimensionally at the condensing position of the excitation light and Stokes light onto the sample. A spectral signal is acquired each time the position changes. Accordingly, spectral data from each position of the sample is finally obtained.
  • the data acquisition sequence of this embodiment is as shown in FIG. 3, and the operations of exposure, data transfer, and position movement are repeated for the number of data points. Note that the order of data transfer and position movement may be reversed or may be performed simultaneously.
  • the maximum value and the minimum value of each acquired spectrum are used as Imax and Imin.
  • the setting method is not limited to this.
  • a representative one of the acquired spectra is selected and the maximum value is selected.
  • the values at ⁇ 1 and ⁇ 2 in all spectra may be set as Imax and ⁇ ⁇ ⁇ ⁇ Imin, respectively, using values on the horizontal axis that generate values and local minimum values (respectively ⁇ 1 and ⁇ 2; the unit is wavelength or wave number). If the values on the horizontal axis that generate the maximum value and the minimum value are substantially constant over all points of the sample, this is substantially equivalent to reading the maximum value and the minimum value, and such a setting method is appropriate.
  • ⁇ 2 ′ spectral values at ⁇ 1 ′ and ⁇ 2 ′ may be Imax and Imin.
  • ⁇ 1 ′ and ⁇ 2 ′ may be determined from spectra acquired by prior measurement, and the spectrum values at ⁇ 1 ′ and ⁇ 2 ′ may be Imax and Imin.
  • Equation 1 the principle of obtaining a quantitative value by Equation 1 and its features are described.
  • the resonance frequency of the target substance is only one in the observed spectrum range
  • the electric field of light generated from the sample by CARS is
  • ANR is the electric field of the non-resonant component
  • AR is a coefficient representing the magnitude of the resonant component
  • the frequency observed by the ⁇ spectrometer ⁇ 0 is the resonant frequency
  • ⁇ is a relaxation constant (a value unique to the resonant component). Since AR is proportional to the concentration of the substance to be measured and the number of molecules, this is the value to be quantified.
  • the observed spectrum (CARS spectrum) is the intensity of the electric field of this light, that is, the square of the absolute value
  • FIG. 1 A typical CARS spectrum is shown in FIG.
  • the CARS spectrum takes a minimum value at a frequency lower than the resonance frequency ⁇ 0 and a maximum value at a higher frequency.
  • the locus when the ⁇ of the electric field E ( ⁇ ) of CARS is changed is illustrated on the complex plane as shown in FIG. That is, it is a circle with a radius AR / ⁇ that contacts the real axis at ANR, and when it increases ⁇ , it makes a round clockwise starting from ANR, and returns to ANR again at ⁇ ⁇ + ⁇ .
  • the diameter AR / ⁇ of this circle is a quantitative value proportional to the amount of the substance.
  • the relationship between the maximum and minimum of the spectrum and the diameter of the circle is as follows.
  • the observed spectrum value is the square of the absolute value of one point having the locus in FIG. 5, that is, the square of the distance from the origin, according to Equation 3. Therefore, the local maximum and minimum values of the spectrum are the closest point and the farthest point from the origin in the circle's trajectory, that is, the two intersections of the straight line drawn from the origin toward the center of the circle as shown in FIG.
  • the diameter of the circle is the distance between these two intersections, and is therefore expressed by the following equation (1).
  • the quantitative value AR / ⁇ is expressed by Equation 1.
  • number 1 and AR are in a proportional relationship means more than simply that number 1 is a quantitative value. Because the amount to be truly quantified (in this case, the concentration of the substance or the number of molecules) and the quantitative value are in a proportional relationship, the sum of the quantitative values obtained from multiple spatial points is taken, and the quantitative value of the entire sample Because it becomes. If equation 1 is not proportional to AR, the sum of the values of equation 1 for each measurement point is not the entire quantitative value, and additional calculation or calibration is required to obtain the entire quantitative value. (This is obvious if the number 1 is proportional to the square of AR, for example.) Therefore, the quantification method of the present invention is particularly effective in quantifying the amount of a substance in a predetermined region. .
  • Quantitative value expressed by Equation 1 has the following features. (1) The magnitude of the non-resonant component does not depend on the ANR. (2) The value does not change even if the resonance frequency changes. (3) It can be treated as a quantitative value regardless of the magnitude relationship between the resonant component and the non-resonant component. Applicable even when the resonance frequency is unknown. (5) No complicated signal processing is required. None of the conventional quantification methods satisfy these features at the same time, and this quantification value enables faster and more accurate quantification for isolated resonance signals.
  • FIG. 7 shows a plot of the correlation between the three types of quantitative values and the reference value. Whereas the conventional quantitative value is not proportional to the reference value, the quantitative value of the present invention clearly has a linear correlation. Therefore, even in a situation where a plurality of non-measurement molecules are mixed and the resonance frequency is not isolated, Equation 1 can be used as the quantitative value for the total amount.
  • the XYZ stage 12 is used as an irradiation control unit for controlling the light irradiation position on the sample by the irradiation optical system, and the sample position is scanned for scanning the measurement point.
  • the light irradiation position is controlled by the irradiation control unit.
  • the method is not limited to this.
  • a scanning mirror such as a galvanometer mirror or a MEMS mirror that scans the incident angle of the excitation light / Stokes light to the sample by external control may be used as the irradiation control unit, or the position of the objective lens 109 may be scanned. Absent.
  • a combination of the methods described above may be used.
  • a galvanometer mirror 1601 is inserted between the dichroic mirror 108 and the objective lens 109 so that the excitation light / Stokes light is reflected before entering the objective lens 109.
  • the installation angle of the galvanometer mirror is controlled by the external control from the computer 13, and thereby the angle of the luminous flux of the excitation light / Stokes light can be controlled.
  • the excitation light and Stokes light whose angle has been changed by the galvanometer mirror is condensed at a position different from that before the angle change in the sample, and the generated CARS light also enters the light receiving surface of the CCD camera at a different position.
  • the angular scanning direction of the galvanometer mirror is set so that the position of the CARS light on the light receiving surface of the CCD camera changes in the vertical direction in FIG.
  • the beam 203 of the CARS light moves in the vertical direction.
  • the output signal is not affected even if the beam position changes. Absent.
  • the other axes are scanned using the XYZ stage 12. This operation is the same when another scan mirror such as a MEMS mirror is used. Since these scan mirrors usually operate at a higher speed than an XYZ stage, it is possible to perform a higher-speed measurement by applying them.
  • the spectroscope is arranged on the side opposite to the excitation light / Stokes light incident side of the sample.
  • the spectroscope is arranged on the same side, and the backscattered light from the sample is converted into parallel light by the objective lens 109. You may detect with a spectrometer.
  • the excitation light / Stokes light and the CARS light are coaxial, it is necessary to separate the CARS light from the excitation light / Stokes light using a beam splitter 301 or the like.
  • a CCD camera is assumed as a detector.
  • the detector is not limited to this, and a similar effect can be obtained when a line sensor that is a COMS camera or a one-dimensional detector array is used.
  • imaging is possible by outputting different spectra for each position of the sample, but it goes without saying that the same method can be applied to analysis means for analyzing the spectrum of one or more points of the sample. Yes.
  • a short pulse laser is used as the excitation light source and a photonic crystal fiber is used as the Stokes light source.
  • the configuration of the light source is not limited to this.
  • a mode-locked laser having a pulse width of about 10 fs may be used instead of the photonic crystal, or excitation light and probe light may be used from separate short pulse laser light sources.
  • This example is an embodiment in which data is acquired by switching the wavelength of Stokes light to two types.
  • a configuration diagram of this embodiment is shown in FIG. The difference from the first embodiment is that an optical parametric oscillator 1001 is used as a Stokes light generation source, and a photomultiplier tube 1002 is used to detect a CARS signal.
  • the optical parametric oscillator 1001 is input with pumping light that has passed through the nonlinear crystal 1003 and having a half wavelength, and outputs signal light and idler light of different wavelengths by a parametric process, which is one of the nonlinear optical processes. Yes, if the frequencies of excitation light, signal light, and idler light are ⁇ p, ⁇ sig, and ⁇ idler,
  • the wavelength of signal light and idler light can be adjusted within a predetermined range while maintaining this relationship.
  • signal light is used as Stokes light.
  • two wavelengths of Stokes light are set by adjusting the optical parametric oscillator, and CARS light is detected by a photomultiplier tube at each wavelength.
  • the set wavelength of Stokes light is determined in advance as follows by preliminary measurement.
  • the wavelength of the optical parametric oscillator is continuously scanned before and after the wavelength at which the CARS light is just equal to the resonance frequency to be measured, and the wavelength at which the CARS signal takes the maximum value and the minimum value is set as the set wavelength. Or you may set arbitrarily the wavelength estimated that a CARS signal will become the maximum value and the minimum value from the relaxation constant assumed, the magnitude
  • the detector output of the CARS signal from each point of the sample at these two set wavelengths is set to Imax and Imin, and the quantitative value is calculated by Equation 1 as in the first embodiment. In this example, it can be considered that only two points used for calculation of the quantitative value in the CARS spectrum in Example 1 are measured, and it is obvious that the same result as in Example 1 can be obtained.
  • the Stokes light source in this embodiment is not limited to the optical parametric oscillator, and may be another short pulse laser light source prepared separately. In this case, it is necessary to synchronize the pulse oscillation timings of the two short pulse light sources, but this can be realized as reported in Non-Patent Document 7 and the like.
  • the CARS light detector is not limited to a photomultiplier tube, and an avalanche photodiode or PIN photodiode may be used.
  • FIG. 11 shows a configuration diagram of this embodiment.
  • another short pulse laser light source 1101 in this embodiment, a titanium sapphire laser having an oscillation wavelength of about 800 nm
  • the light used as the pump light in the first and second embodiments is used as the Stokes light
  • the idler light of the optical parametric oscillator is used as the first pump light
  • the light emitted from the short pulse laser light source is used as the second pump light. Use.
  • the first excitation light and the second excitation light are combined by the polarization beam splitter 1102 in a state where the polarizations are orthogonal to each other, and the combined light flux is converted into a predetermined polarization state by the pocket lens 1103 and then passes through the polarizer 1104. Thus, only the same polarization component as the excitation light is output.
  • the Pockels cell 1103 is an element that converts the polarization state of light input according to the drive voltage.
  • the output of the polarizer 1104 is the first excitation light and the second excitation light according to the two types of drive voltages. It is set to be one of the light. That is, the excitation light is switched according to the driving voltage of the Pockels cell.
  • the drive voltage to the Pockels cell is generated by the function generator 1105 and is a rectangular wave signal having a frequency of 500 kHz and a duty ratio of 1.
  • the reference frequency signal of the rectangular wave signal is sent to the lock-in amplifier 1106, and the reference frequency component of the output from the photomultiplier tube input to the lock-in amplifier is amplified and output.
  • a method for measuring the CARS signal by switching the wavelength of the excitation light at this high speed is called Frequency Modulation CARS (FM-CARS), and is described in detail in Non-Patent Document 8.
  • the wavelengths are set in advance so that the first excitation light and the second excitation light have the maximum value and the minimum value of the CARS spectrum, respectively.
  • the wavelength can be switched at a higher speed than in the second embodiment, so that a high-speed measurement is possible, and the S / N ratio can be improved by using a lock-in amplifier.
  • the output of the photomultiplier tube 1002 may be directly captured by the computer 11 and the quantitative value may be calculated by Equation 1 with the maximum value and the minimum value as Imax and Imin, respectively. .
  • the output of the photomultiplier tube 1002 may be input to an analog square root arithmetic circuit and the output may be detected by a lock-in amplifier.
  • the configuration of the light source it is sufficient that light beams of three types of wavelengths can be prepared, and three separate lasers may be used.
  • This example is an embodiment to which heterodyne detection is applied.
  • a configuration diagram of this embodiment is shown in FIG.
  • the excitation light and Stokes light are generated and combined in the same manner as in the first embodiment, and then the combined light beam is split into two by the beam splitter 1201. , 1203 is condensed.
  • the sample 110 is a measurement sample, and CARS light is generated and converted into parallel light by the objective lens 111, and the excitation light and Stokes light are blocked through the filter 112 as in the first embodiment.
  • a sample 1203 (a glass plate in this embodiment) generates CARS light that does not include a resonance component and includes only a non-resonance component.
  • This CARS light is converted into parallel light by an objective lens 1204 and passes through a filter 1205 to be excitation light. , Stokes light is blocked. These two CARS lights are combined by a beam splitter 1206 to generate two interference light beams. Both of these interference light beams enter the spectroscope 114 and are detected by the CCD camera 115. Here, the two interference light beams are condensed at different positions in the slit direction in the entrance slit of the spectrometer. For this reason, as shown by 203 and 1301 in FIG. 13, they are separated and irradiated and output as separate spectral signals (in order to detect these lights separately, full vertical binning described in Embodiment 1). Do not do).
  • I1 ( ⁇ ) and I2 ( ⁇ ), respectively are captured by the computer 11.
  • Equation 7 is the product of
  • the locus of E1 ( ⁇ ) on the complex plane is a circle of diameter AR / ⁇ as described above, and therefore the projection of this locus has a length AR regardless of the size of ⁇ . / ⁇ line segment.
  • the orbit of E1 ( ⁇ ) is circular, and the quantitative value AR / ⁇ can be calculated by the calculation of Equation 7 regardless of the value of ⁇ . If the value of ⁇ can take the entire range of 0-2 ⁇ , the acquired spectrum range needs to be sufficiently wide before and after the resonance frequency (otherwise, the portion close to the ANR of the circular orbit is missing. Because it will be.)
  • the other light that interferes with the CARS light from the sample 110 need not be the CARS light from another sample.
  • the same wavelength as the CARS light is used. Ingredients may be used.
  • heterodyne detection can be performed by preparing light having the same wavelength as the CARS light, and the same quantitative value as in the present embodiment can be calculated. It is.
  • the idler light of the optical parametric oscillator has the same wavelength as the CARS light. Therefore, the interference light may be detected by causing it to interfere with the CARS light with the configuration shown in FIG.
  • the two interference lights are detected by separate photomultiplier tubes 1002 and 1501, the difference between these outputs is calculated by the differential circuit 1502, and the output is input to the computer 11.
  • the calculation of the difference may be performed on a computer.
  • the setting wavelength for the maximum value and the minimum value can be determined in the same manner by the method described in the second embodiment.
  • This example is an example of a biomolecule analysis device in which the optical analysis device of the present invention is applied to single cell analysis, and is an example of acquiring a CARS spectrum as one form of cell analysis.
  • FIG. 16 and FIG. 17 are schematic views showing a configuration example of the biomolecule analyzing apparatus according to the present embodiment.
  • FIG. 16 is a schematic view showing an optical system portion of the present apparatus
  • FIG. 17 is a detailed view of the periphery of a sample showing a configuration example of a biomolecule collection system.
  • FIG. 17 includes a biomolecule collection system 2 that captures the mRNA of a sample cell for gene expression analysis. The optical system part and the biomolecule collection system are controlled by the computer 11 and data acquisition is performed.
  • the optical system portion of the apparatus shown in FIG. 16 includes, in addition to the configuration, a differential observation system, a cell destruction laser 5 (pulse laser with a wavelength of 355 nm, an average output of 2 W, and a repetition frequency of 5 kHz), a driver 602, and an output from the laser 5.
  • a dichroic mirror 603 for making the incident light coaxial with the excitation light is provided.
  • the optical system portion includes three functions: (1) acquisition of differential interference microscope images, (2) acquisition of CARS spectra, and (3) destruction of cells. Each will be described below.
  • the illumination light from the illumination 401 passes through the Wollaston prism 402, is reflected by the dichroic mirror 403, is condensed on the sample 110 by the condenser lens 111, and is subjected to differential interference of the sample 110.
  • the image is imaged on an imaging device such as a CCD camera 408 using the objective lens 109, the dichroic mirror 404, the Wollaston prism 405, the polarizer 406, and the imaging lens 407 to obtain an image of the sample.
  • This configuration is identical to that of the well-known differential interference microscope.
  • the dichroic mirrors 403 and 404 reflect the wavelength (400 to 700 nm) of the visible light region of the illumination 401, and transmit the excitation light, Stokes light, and CARS light (both have a near infrared wavelength of 700 nm or more). It does not affect the generation and detection of CARS signals.
  • the function (2) is as described in the first embodiment.
  • the function (3) is a function of condensing the emitted light from the cell destruction laser 5 on the cell to be observed by the objective lens 109, destroying the cell, and releasing biomolecules such as mRNA inside the cell to the outside. is there. The released mRNA is captured and analyzed by the biomolecule collection system 2 as described later.
  • the biomolecule collection system 2 shown in FIG. 17 includes an array device in which regions for capturing biomolecules such as mRNA released from cells are arranged.
  • a cDNA library can be constructed by capturing mRNA in a plurality of regions of the array device for each single cell and performing a reverse transcription reaction in the array device.
  • the array device is constructed of a transparent porous membrane in which a large number of through holes are formed perpendicular to the surface, and this will be referred to as a pore array sheet 30 hereinafter.
  • a structure in which a cDNA library is formed on the pore array sheet 30 is referred to as a cDNA library pore array sheet.
  • a separation wall 31 can be formed on the pore array sheet 30 to separate regions that capture biomolecules.
  • the separation wall 31 can be formed by a semiconductor process using polydimethylsiloxane (PDMS), for example, and can be brought into close contact with the pore array sheet 30 with a thickness of about 80 ⁇ m.
  • PDMS polydimethylsiloxane
  • FIG. 18 is a top view of the pore array sheet 30.
  • a region 300 for capturing a large number of biomolecules such as mRNA is formed in the pore array sheet 30 (size 2 mm ⁇ 2 mm, thickness 80 ⁇ m).
  • the size of the region 300 is set such that one side is 100 ⁇ m and the interval is 80 ⁇ m (arranged at a cycle of 180 ⁇ m).
  • the size of the region 300 can be freely designed from about 1 ⁇ m to about 10 mm in accordance with the amount of biomolecules to be captured and the ease of diffusion in the plane (molecule size).
  • an array device in addition to the pore array sheet 30 made of a porous membrane formed by anodizing aluminum, a device in which a large number of through holes are formed by anodizing a material such as silicon may be used. . Furthermore, an array device may be constructed by providing a large number of through holes in a silicon oxide or silicon nitride thin film using a semiconductor process.
  • a loop-shaped platinum electrode 32 is joined to the tip of a shield wire 33 as a means for guiding biomolecules released from cells to a specific region in the pore array sheet 30 by electrophoresis. .
  • the diameter of the wire of the platinum electrode 32 is 30 ⁇ m.
  • the loop side is processed into a circle with a diameter of 100 ⁇ m.
  • Two such electrodes are prepared, arranged so as to sandwich the pore array sheet 30, and a direct current of 1.5 V is applied by the power source 35. Since the released mRNA 36 has a negative charge, the upper platinum electrode 32 is used as a positive electrode.
  • a silver-silver chloride reference electrode 39 is provided, and 0.2 V is applied to the lower platinum electrode 32.
  • mRNA 36 can be induced by electrophoresis inside the region 300 for capturing biomolecules.
  • the diameter of the loop of the upper platinum electrode 32 may be set to 50 ⁇ m in order to realize concentration of mRNA by lateral electrophoresis.
  • the wire has a diameter of 10 ⁇ m.
  • FIG. 19 shows an example of a flowchart.
  • a sample composed of adherent cultured cells 21, 22, and 23 is placed on the petri dish 20.
  • the measurement target is a cultured cell
  • the cell is cultured in advance using the petri dish 20 so that the measurement target cell adheres to the bottom surface.
  • the sample is a frozen section, it is placed on the petri dish 20.
  • a sample in which a plurality of cells are three-dimensionally arranged in a gel may be used.
  • a differential interference image of a target cell group is acquired using a microscope system, and a user determines a target cell from which a biomolecule is collected and measured.
  • the computer 11 receives input of information regarding a cell or a cell portion to be measured from a user.
  • a user often uses a plurality of cells as a measurement target.
  • the computer 11 determines the order of cells that capture biomolecules, and first drives the XYZ stage 12 so that the first target cell is placed at the center of the field of view.
  • the CARS spectrum of the cell arranged at the center of the visual field is acquired by the method described in the first embodiment, and the quantitative value data obtained by the method described in the first embodiment is stored in the computer 11 from the acquired spectrum.
  • the computer 11 uses the XYZ stage 34 to locate a specific region (for example, (1, 1)) of the pore array sheet 30 in the vicinity of the cell from which the CARS spectrum was acquired (in the example of FIG. 17, directly above the cell).
  • the address area 300) is approached.
  • the distance between the lower surface of the pore array sheet 30 and the petri dish 20 is set to 300 ⁇ m, but this distance can be changed depending on the type of biomolecule to be collected and the electrode structure. For example, about 1 ⁇ m to 10 mm is preferable.
  • the movement of the pore array sheet 30 by the XYZ stage 34 is automatically performed by the computer 11 according to a prior program.
  • a voltage is applied to the platinum electrode 32 for electrophoresis, and at the same time, in order to destroy the cell membrane of the cell to be measured, the laser light from the cell destruction laser light source 5 is applied to the cell. Irradiate.
  • the irradiation time can be, for example, 10 seconds, and the electrophoresis driving time can be 60 seconds.
  • the computer 11 drives the XYZ stage 12 to position the registered second target cell at the center of the visual field. Thereafter, the CARS spectrum of the second cell is acquired, and the data is stored in the computer 11. Next, the computer 11 drives the XYZ stage 34 to locate a specific region (for example, (1, 2) of the pore array sheet 30 in the vicinity of the second target cell (immediately above the cell in the configuration example of FIG. 17). The address area 300) is approached. Then, the second cell registered in the computer 11 is irradiated with the laser beam from the cell destruction laser 5. At this time, a voltage is simultaneously applied to the platinum electrode 32 as described above.
  • the CARS spectrum is acquired for the sequentially designated cells, the cells are destroyed, the biomolecules in the cells are captured in the specific region 300 of the pore array sheet 30, and then the captured biomolecules are captured.
  • the process for measuring is executed.
  • the portion corresponding to the destroyed cell in the differential interference image, the region 300 in which the biomolecule is captured in the pore array sheet 30, the acquired CARS spectrum and the quantitative value acquired therefrom are associated with each other and presented to the user To do.
  • the cell to be destroyed is one cell.
  • one cell 300 on the array device is released and electrophoresed when a plurality of cells are destroyed.
  • mRNA may be captured.
  • a plurality of cells may be destroyed at the same time, or the cells may be destroyed one by one without moving the array device.
  • the CARS spectrum is acquired and the biomolecule is captured sequentially for different cells. For example, after acquiring the differential interference image of the sample, all the CARS spectra of the target cells are measured. Alternatively, the flow may be such that each cell is sequentially destroyed to supplement biomolecules.
  • This example makes it possible to acquire CARS spectra and gene expression data for individual cells. Using this function, it is possible to confirm the dynamic characteristics of cells with high accuracy. A flowchart for performing such an analysis is shown in FIG.
  • the CARS spectrum is acquired.
  • the cell is destroyed for the cell selected by the user, the biomolecule in the cell is captured on the array device, and the amount of the cell is selected. Measure.
  • the CARS spectrum and the cell state and type can be associated with each other with high accuracy.
  • the CARS spectrum can obtain more information on the chemical species to be measured in that a Raman spectrum can be obtained compared to a fluorescent confocal microscope that is usually used for single cell analysis. Such highly accurate analysis is possible.
  • FIG. PC in the figure is an abbreviation for principal-component, where PC1 indicates the first main factor and PC2 indicates the second main factor.
  • Each point corresponds to gene expression data for one cell. In many cases, it is divided into a plurality of clusters (in this example, 6 clusters) corresponding to the state and type of cells.
  • each point corresponds to a cell, even if it is not possible to determine which cell is what kind of cell only by CARS spectrum, it is possible to correspond based on gene expression analysis data. it can.
  • the computer system is caused to perform machine learning that determines what type of CARS spectrum and quantitative value the cell state and type of when obtained. After learning is completed, it is possible to classify cell states and types only by acquiring CARS spectra and quantitative values.
  • principal factor analysis is used for clustering based on gene expression of cells, but various methods such as hierarchical clustering and k-means method can be applied. Also, as a method of machine learning, various methods used for data mining such as a support vector machine are known, and any of them may be used.
  • this invention is not limited to the above-mentioned Example, Various modifications are included.
  • the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.
  • the present invention makes it possible to provide an analyzer capable of acquiring information from a large amount of sample at a high speed, and accelerate research and development in the medical and pharmaceutical fields.

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Abstract

Le problème décrit par la présente invention est que, dans des procédés classiques d'obtention de valeurs quantitatives de données de mesure de microscope CARS, il est difficile d'obtenir les données rapidement et précisément. La solution selon l'invention consiste en un dispositif d'analyse optique qui acquiert des informations quantitatives d'un spectre CARS comprenant une composante non résonante en utilisant la différence dans les valeurs absolues d'un champ électrique correspondant à des valeurs spectrales maximale et minimale en tant que valeur quantitative.
PCT/JP2014/069132 2014-07-18 2014-07-18 Dispositif d'analyse optique WO2016009548A1 (fr)

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