WO2019142907A1 - Dispositif d'analyse optique et procédé d'analyse optique - Google Patents

Dispositif d'analyse optique et procédé d'analyse optique Download PDF

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Publication number
WO2019142907A1
WO2019142907A1 PCT/JP2019/001454 JP2019001454W WO2019142907A1 WO 2019142907 A1 WO2019142907 A1 WO 2019142907A1 JP 2019001454 W JP2019001454 W JP 2019001454W WO 2019142907 A1 WO2019142907 A1 WO 2019142907A1
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light
objective lens
analysis
numerical aperture
optical
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PCT/JP2019/001454
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English (en)
Japanese (ja)
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福武　直樹
武志 川野
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株式会社ニコン
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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 an optical analyzer and an optical analysis method.
  • Patent Document 1 International Publication No. 2016/009548
  • a non-resonant background is also generated by four-wave mixing simultaneously with the generation of the CARS light, so that the detected spectrum has a profile different from that of the spontaneous Raman spectrum.
  • a light source generating pump light and Stokes light
  • a first objective lens capable of focusing on the boundary between an analysis object and a member supporting the analysis object, an analysis object and A second objective lens disposed opposite to the first objective lens across the member and capable of focusing on the analysis object and the boundary of the member; light generated by the analysis object and the member; And a spectroscope for receiving light through the objective lens.
  • a member capable of transmitting pump light and Stokes light supports an object to be analyzed, and has a first numerical aperture so as to focus on the boundary between the object and member.
  • a second objective which irradiates the pump light and the Stokes light to the object to be analyzed and the member through the one objective lens, is disposed opposite to the first objective lens, and can focus on the boundary of the object to be analyzed and the member.
  • An optical analysis method is provided in which scattered light generated by an object to be analyzed and a member is received by a spectroscope through a lens.
  • FIG. 2 is a schematic cross-sectional view of a sample 110.
  • 1 is a schematic view of a laser microspectroscope 100.
  • FIG. It is a schematic diagram explaining a CARS process. It is a schematic diagram explaining a four-wave mixing process. It is a schematic diagram which shows the focus vicinity of an objective lens. It is a figure which shows point-image amplitude distribution of the scattered light by the optical linear optical effect. It is a figure which shows point-image amplitude distribution of the scattered light by the optical linear optical effect. It is a figure which shows point-image amplitude distribution of the scattered light by the optical linear optical effect. It is a figure which shows point-image amplitude distribution of the scattered light by the optical linear optical effect. It is a graph which shows the relationship between the angular frequency of scattered light, and CARS light intensity. It is a graph which shows the relationship between the angular frequency of scattered light, and CARS light intensity. It is a figure which shows the detected spectrum. It is a figure which shows the
  • FIG. 1 is a schematic cross-sectional view showing a configuration of a sample 110 which is an analysis target of a spectrum by the laser microspectroscope 100.
  • the illustrated sample 110 is formed by accommodating the analyte 112 in the sample container 114 in a state of being immersed in the protective liquid 116.
  • the analysis target 112 is a biological sample such as a cell sheet, for example, and a culture solution is used as the protective solution 116.
  • the sample container 114 may also be made of glass, but is not limited to glass.
  • FIG. 2 is a schematic view showing the structure of the laser microspectroscope 100.
  • the laser microspectroscope 100 includes a laser light source 120, an excitation light generator 130, an optical system 150, a stage 160, a polychromator 170, and a controller 180.
  • a sample 110 to be analyzed is placed on a stage 160.
  • the laser light generated by the laser light source 120 is converted into pump light and Stokes light in the excitation light generator 130, and then irradiated to the sample 110 through the excitation light side objective lens 152 or the like.
  • the scattered light generated in the sample 110 irradiated with the excitation light is detected by the polychromator 170 through the signal light side objective lens 154.
  • the detection result by the polychromator 170 is processed by the control unit 180, and the spectrum of the sample 110 is output.
  • the laser light source 120 generates a picosecond pulse laser and enters the excitation light generator 130.
  • a mode locked picosecond Nd: YVO 4 laser, a mode locked picosecond ytterbium laser, or the like can be used.
  • the laser light source 120 may be provided with an optical parametric oscillator that uses the second harmonic of the picosecond pulse as excitation light, and the wavelength of the picosecond pulse laser may be changed and output.
  • the excitation light generator 130 has an optical splitter 132, a photonic crystal fiber 134, a reflecting mirror 136, and an optical coupler 138.
  • the optical splitter 132 splits the picosecond pulse laser incident from the laser light source 120 into two.
  • One exit end of the light splitter 132 is coupled to a light path including a pair of reflecting mirrors 136. Therefore, the picosecond pulse laser incident on this side bypasses the photonic crystal fiber 134 and is guided to the optical path coupled to one incident end of the optical coupler 138. Thereby, one of the branched picosecond pulse lasers is adjusted in time until it reaches the optical multiplexer 138, and the optical multiplexer is aligned in timing with the other picosecond pulse laser via the photonic crystal fiber 134. It is incident on 138.
  • the other exit end of the light splitter 132 is coupled to the incident end of the photonic crystal fiber 134.
  • the picosecond pulse laser incident on the photonic crystal fiber 134 broadens the spectrum in a wavelength band longer than that of the initially incident picosecond pulse laser due to the self phase modulation occurring in the photonic crystal fiber 134.
  • the exit end of the photonic crystal fiber 134 is coupled to the other entrance end of the optical coupler 138.
  • the broad spectrum picosecond pulse laser and the picosecond pulse laser whose timing is matched are multiplexed by the optical multiplexer 138 and then emitted from the excitation light generator 130 as a picosecond pulse laser synthesized.
  • the picosecond pulse laser emitted from the excitation light generator 130 is finally irradiated to the sample 110 as excitation light.
  • the original narrow band picosecond pulse laser is irradiated as a pump light
  • the picosecond pulse laser whose band is broadened by the photonic crystal fiber 134 is irradiated as a Stokes light to the sample 110 respectively.
  • the laser light source 120 is not limited to one generating a picosecond pulse laser of a single wavelength, but may be one generating a picosecond pulse laser of a plurality of wavelengths.
  • a picosecond pulse laser to be pump light may also be output after wavelength conversion by a photonic crystal fiber.
  • the optical system 150 of the laser microspectroscope 100 includes an excitation light side objective lens 152 disposed between the excitation light generation unit 130 and the stage 160.
  • the excitation light side objective lens 152 focuses on the inside of the sample 110 placed on the stage 160, and condenses the excitation light propagated from the excitation light generation unit 130 in the sample 110. As a result, near the focal point in the sample 110, the excitation light produces a non-linear effect.
  • the stage 160 has a drive unit 162 that moves the stage 160 at least in the XY direction by a piezoelectric element.
  • the sample 110 on the stage 160 can be scanned with excitation light without moving the optical system.
  • the optical system 150 also has a signal light side objective lens 154 disposed on the opposite side of the excitation light side objective lens 152 with respect to the sample 110 placed on the stage 160.
  • the signal light side objective lens 154 focuses on the inside of the sample 110 placed on the stage 160 and collects the scattered light emitted from the sample 110.
  • the excitation light side objective lens 152 and the signal light side objective lens 154 preferably have mutually different numerical apertures (NA). This point will be described later with reference to FIGS.
  • the optical system 150 further includes an optical filter 156 and an imaging lens 158 on the optical path of the scattered light emitted from the signal light side objective lens 154.
  • the optical filter 156 removes unwanted optical components from the scattered light emitted from the sample 110.
  • the unnecessary component includes a partial band of the irradiation light emitted through the sample 110. Therefore, the optical filter 156 is changed according to the type of the sample 110, the composition of the analysis target, the purpose of detection, and the like.
  • the imaging lens 158 focuses the scattered light generated by the sample 110 on the light receiving surface of the polychromator 170 described later.
  • the laser microspectroscope 100 has reflecting mirrors 140 and 142 on the optical path of the excitation light and the scattered light. Thereby, the optical paths of the excitation light and the scattered light are bent, and the enlargement of the size of the laser microspectroscope 100 is suppressed.
  • the polychromator 170 When the irradiation light of a wide band is irradiated to the sample 110, the polychromator 170 splits the light emitted from the object of analysis 112 with a diffraction grating and simultaneously receives the light by a plurality of light receiving elements. Thus, the polychromator 170 operates as a detection unit that detects the spectrum of the sample 110 in the area irradiated with the irradiation light.
  • the polychromator 170 splits and detects light received through a narrow area corresponding to the entrance slit of a spectroscope disposed at a position conjugate to one of the image planes of the optical system 150. For this reason, when the emitted light is received using a polychromator, it is not possible to displace the optical path of the excitation light irradiated to the sample 110.
  • the spectrum obtained by one detection of the polychromator 170 corresponds to the component at one position of the object to be analyzed 112.
  • the laser microspectroscope 100 can displace the stage 160 on which the sample 110 is placed by the drive unit 162.
  • the laser microspectroscope 100 can also detect spectra at different positions of the sample 110.
  • the control unit 180 includes a keyboard 182, a mouse 184, an information processing device 186, and a display device 188.
  • the keyboard 182 and the mouse 184 are connected to the information processing apparatus 186, and are operated when the user inputs an instruction to the information processing apparatus 186.
  • the information processing device 186 can be formed by mounting a program that causes a general purpose personal computer to execute a control procedure.
  • the display device 188 returns feedback to the user on the operation by the keyboard 182 and the mouse 184, and displays the image or character string generated by the information processing device 186 to the user.
  • the control unit 180 controls the operations of the laser light source 120, the drive unit 162, the polychromator 170 and the like, and sets an instruction from the user in the laser microspectroscope 100. Further, the detection result of the polychromator 170 is visualized, and an image to be displayed on the display device 188 is generated.
  • the laser microspectroscope 100 is used when imaging a sample 110 by scattered light or a galvano scanner that scans the sample 110 fixed by displacing the optical path of the excitation light with the excitation light.
  • a photomultiplier tube 190 or the like may be additionally provided.
  • a photomultiplier tube 190 or the like may be additionally provided.
  • the insertion / extraction reflection mirror 142 may be provided on the most downstream side in the optical path of the scattered light to selectively use the plurality of detection units. .
  • FIG. 3 is a view for explaining the CARS process that occurs when the analysis target 112 in the sample 110 is irradiated with the collected excitation light.
  • the CARS process irradiates the sample 110 with excitation light including pump light and Stokes light having different angular frequencies ⁇ p and ⁇ s , and the difference between the light frequency ⁇ p of the pump light and the light frequency ⁇ s of the Stokes light This occurs when [ ⁇ p ⁇ s ] resonates with the angular frequency ⁇ 0 of the natural vibration of the molecules contained in the sample.
  • the CARS process the vibration mode of a specific molecular structure contained in the sample is excited by molecular vibration interacts with the probe beam is a third laser beam having an angular frequency omega 3, third order nonlinear polarization
  • the CARS light derived from is generated as Raman scattered light.
  • a specific molecular structure such as a functional group
  • the accumulation time is short and detection can be performed at high speed when detecting using a photoelectric conversion element. This also enables observation at the video rate. Not only the distribution of the specific molecular structure but also changes in the distribution can be detected. Furthermore, by setting the band of the irradiation light to be irradiated to the sample to be an infrared band with less damage to the living cells, it is possible to observe the living cells to be observed as it is.
  • a spectral image showing the frequency distribution (wave number distribution) of the Raman scattered light emitted from the irradiation position can be obtained. Furthermore, the distribution of specific molecules in the observation plane can be imaged by repeatedly irradiating the irradiation light while moving the sample in a direction intersecting the optical path of the irradiation light.
  • FIG. 4 is a diagram for explaining four-wave mixing which is a phenomenon different from the CARS process which occurs when the analysis target 112 in the sample 110 is irradiated with the condensed excitation light.
  • Four-wave mixing causes scattering at the same angular frequency 2 ⁇ p - ⁇ s as CARS light, as illustrated, due to the third-order nonlinear susceptibility ⁇ (3) of the analysis object 112 when the excitation light is irradiated.
  • Light is a phenomenon that occurs simultaneously with CARS light.
  • non-resonant background Scattered light resulting from such a four-wave mixing process is called “non-resonant background” because it is generated independently of the molecular vibration of the sample and reduces the contrast of the CARS light image.
  • CARB signal area where the CARS signal level is low, the influence of the non-resonant background is relatively strong, which may make imaging difficult.
  • FIG. 5 is a view schematically showing an optical arrangement of the excitation light side objective lens 152 and the signal light side objective lens 154 in the laser microspectroscope 100. As shown in FIG. As shown, the excitation light side objective lens 152 and the signal light side objective lens 154 respectively focus on the boundary between the analysis object 112 and the glass sample container 114.
  • the excitation light collected by the excitation light side objective lens 152 causes four-wave mixing in both the analysis object 112 and the sample container 114. Furthermore, in the analysis object 112, CARS light is generated when the difference frequency ( ⁇ p ⁇ s ) of the excitation light matches with the molecular vibration resonance frequency.
  • the non-resonant background which does not depend on molecular vibrational resonance, is due to the real part of the third-order nonlinear susceptibility ⁇ (3) .
  • the CARS light intensity is proportional to
  • the imaginary part Im ⁇ s (3) ⁇ of the nonlinear susceptibility corresponds to the same spectrum as the spontaneous Raman spectrum (natural spectrum) from which the influence of the non-resonant background is removed.
  • FIG. 6 is a view showing a point spread distribution in the region A shown in FIG. The illustrated example shows the case where the numerical aperture NA ex of the excitation light side objective lens 152 is larger than the numerical aperture NA col of the signal light side objective lens 154.
  • an argan diagram of scattered light due to the contribution on the side of the analysis target 112 and scattered light due to the contribution on the side of the sample container 114 is shown together.
  • the upper Argan diagram in the figure corresponds to the initial phase of the scattered light generated at the object of analysis 112. Further, the Argan diagram on the lower side in the figure corresponds to the initial phase of the scattered light generated in the sample container 114.
  • ASF (x) is a point spread function of the whole system including the excitation side and the signal collection side.
  • FIG. 7 is a diagram showing the point spread distribution in the region A, as in FIG.
  • the numerical aperture NA ex of the excitation light side objective lens 152 is equal to the numerical aperture NA col of the signal light side objective lens 154.
  • an Argan diagram of scattered light due to the contribution on the side of the analysis target 112 is also shown.
  • FIG. 8 is a diagram showing a point spread distribution in the region A, as in FIGS.
  • the numerical aperture NA ex of the excitation light side objective lens 152 is smaller than the numerical aperture NA col of the signal light side objective lens 154.
  • an Argan diagram of scattered light due to the contribution of the analysis target 112 is shown together.
  • the numerical aperture NA ex of the excitation light side objective lens 152 having a circular pupil is [n ⁇ sin ⁇ ex (n is a refractive index)]
  • the numerical aperture NA col of the signal light side objective lens 154 is [n ⁇ sin ⁇
  • the above-mentioned equation (5) is satisfied by satisfying the conditions shown in the following series of equations (6).
  • the signal light side objective is Assuming that the lens 154 has a circular pupil of radius n ⁇ sin ⁇ col , the above equation 5 is satisfied by satisfying the conditions shown in the following series of equations (7).
  • the excitation light side objective lens 152 has an annular pupil
  • the radius of the circular pupil of the signal light side objective lens 154 can be made large. Therefore, the resolution as an optical analyzer becomes high, and the signal light can be condensed with high efficiency.
  • FIG. 9 is a graph showing the relationship between the angular frequency of scattered light and the CARS light intensity as an example of the combination of the numerical aperture NA satisfying the relationship of the equation (5).
  • the numerical aperture NA ex of the excitation light side objective lens 152 is 1
  • the numerical aperture NA col of the signal light side objective lens 154 is 0.4
  • the numerical aperture NA ex of the excitation light side objective lens 152 is The case where the numerical aperture which becomes large is selected is shown.
  • the intensity of the CARS light tends to be higher in the region where the angular frequency is low.
  • FIG. 10 is a graph showing the relationship between the angular frequency of scattered light and the CARS light intensity as an example of the combination of the numerical aperture NA satisfying the relationship of the equation (5).
  • the numerical aperture NA ex of the excitation light side objective lens 152 is 1, the numerical aperture NA col of the signal light side objective lens 154 as 1.3, towards the aperture NA col of the signal light side objective lens 154 The case where the numerical aperture which becomes large is selected is shown.
  • the intensity of the CARS light tends to be high in the region where the angular frequency is high.
  • FIG. 11 is a graph showing a profile of a spectrum detected under the condition that the relationship shown in the above equation (5) holds.
  • the illustrated profile substantially matches the profile of the spontaneous Raman spectrum.
  • FIG. 12 shows a profile of a spectrum measured with the numerical aperture NA ex of the excitation light side objective lens 152 of the laser microspectroscope 100 and the numerical aperture NA col of the signal light side objective lens 154 being the same.
  • this profile is generally crumply deformed under the influence of non-resonant background.
  • dips are attached immediately after each peak, and the waveform is different from the original spectrum.
  • the spectrum of the accurate profile that matches the spectrum of the spontaneous Raman scattered light without adding any device. can be detected. Also, since no additional image processing or signal processing is required, the processing load and processing time of the analyzer can be reduced to take advantage of the CARS spectrum that the signal level is high, for example the correct spectrum at the video rate It is also possible to detect
  • DESCRIPTION OF SYMBOLS 100 laser microspectrometer, 110 samples, 112 analysis object, 114 sample container, 116 protective liquid, 120 laser light source, 130 excitation light generation part, 132 light branching device, 134 photonic crystal fiber, 136, 140 reflecting mirror, 138 light combining Waver, 142 insertion / retraction mirror, 150 optical system, 152 excitation light side objective lens, 154 signal light side objective lens, 156 optical filter, 158 imaging lens, 160 stage, 162 driving unit, 170 polychromator, 180 control unit , 182 keyboard, 184 mouse, 186 information processor, 188 display device, 190 photomultiplier tube

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un dispositif d'analyse optique comprenant : une source de lumière qui génère une lumière de pompage et une lumière de Stokes ; une première lentille d'objectif apte à se focaliser sur une limite entre un objet d'analyse et un élément maintenant l'objet d'analyse ; une seconde lentille d'objectif disposée face à la première lentille d'objectif à travers l'objet d'analyse et l'élément et capable de se focaliser sur la limite entre l'objet d'analyse et l'élément ; et un spectroscope recevant la lumière générée par l'objet d'analyse et l'élément, à travers la seconde lentille d'objectif. Selon le procédé d'analyse optique : un objet d'analyse est maintenu par un élément à travers lequel une lumière de pompage et une lumière de Stokes peuvent passer ; une lumière de pompage et une lumière de Stokes sont rayonnées sur l'objet d'analyse et l'élément, par l'intermédiaire d'une première lentille d'objectif qui possède une première ouverture numérique et qui est capable de se focaliser sur une limite entre l'objet d'analyse et l'élément ; et un spectroscope reçoit la lumière diffusée générée par l'objet d'analyse et l'élément, à travers une seconde lentille d'objectif qui est disposée en face de la première lentille d'objectif et qui est capable de se focaliser sur la limite entre l'objet d'analyse et l'élément.
PCT/JP2019/001454 2018-01-19 2019-01-18 Dispositif d'analyse optique et procédé d'analyse optique WO2019142907A1 (fr)

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JP2018007660A JP2021056000A (ja) 2018-01-19 2018-01-19 光学分析装置および光学分析方法
JP2018-007660 2018-01-19

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009044834A1 (fr) * 2007-10-01 2009-04-09 Nikon Corporation Système optique à compensation de polarisation et élément optique de compensation de polarisation utilisé dans ledit système

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009044834A1 (fr) * 2007-10-01 2009-04-09 Nikon Corporation Système optique à compensation de polarisation et élément optique de compensation de polarisation utilisé dans ledit système

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
C HENG, JI-XIN: "Laser -Scanning Coherent Anti- Stokes Raman Scattering Microscopy and Applications to Cell Biology", BIOPHYSICAL JOURNAL, vol. 83, no. 1, July 2002 (2002-07-01), pages 502 - 509, XP055313076 *
CHENG, JI-XIN: "Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy and Study of Lipid Vesicles", THE JOURNAL OF PHYSICAL CHEMISTRY B, vol. 106, no. 34, 7 August 2002 (2002-08-07), pages 8493 - 8498, XP055627618 *
KANO, HIDEAKI: "Coherent Raman Spectroscopic Imaging by a Nanosecond White-Light Laser Source", JAPANESE JOURNAL OF OPTICS, vol. 40, no. 8, 2011, pages 421 - 429 *

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