WO2019142907A1 - Optical analysis device, and optical analysis method - Google Patents
Optical analysis device, and optical analysis method Download PDFInfo
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- 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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- 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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Abstract
This optical analysis device is provided with: a light source which generates pump light and Stokes light; a first objective lens capable of focusing on a boundary between an object of analysis and a member supporting the object of analysis; a second objective lens which is disposed facing the first objective lens across the object of analysis and the member and which is capable of focusing on the boundary between the object of analysis and the member; and a spectroscope which receives light generated by the object of analysis and the member, through the second objective lens. In the optical analysis method: an object of analysis is supported by a member through which pump light and Stokes light can pass; pump light and Stokes light are radiated onto the object of analysis and the member, through a first objective lens which has a first numerical aperture and which is capable of focusing on a boundary between the object of analysis and the member; and a spectroscope receives scattered light generated by the object of analysis and the member, through a second objective lens which is disposed facing the first objective lens and which is capable of focusing on the boundary between the object of analysis and the member.
Description
本発明は、光学分析装置および光学分析方法に関する。
The present invention relates to an optical analyzer and an optical analysis method.
コヒーレントアンチストークスラマン散乱光(以降の記載においては「コヒーレントアンチストークスラマン散乱」を「CARS」と記載する)のスペクトルにより分析対象に含まれる物質を分析する光学分析方法がある(例えば、特許文献1参照)。
特許文献1 国際公開第2016/009548号 There is an optical analysis method of analyzing a substance included in an analysis target by a spectrum of coherent anti-Stokes Raman scattering light (in the following description, "coherent anti-Stokes Raman scattering" is described as "CARS") (for example, Patent Document 1) reference).
Patent Document 1 International Publication No. 2016/009548
特許文献1 国際公開第2016/009548号 There is an optical analysis method of analyzing a substance included in an analysis target by a spectrum of coherent anti-Stokes Raman scattering light (in the following description, "coherent anti-Stokes Raman scattering" is described as "CARS") (for example, Patent Document 1) reference).
分析対象に励起光を照射した場合、CARS光の発生と同時に、4光波混合による非共鳴バックグラウンドも発生するので、検出されたスペクトルは、自発ラマンスペクトルと異なるプロファイルを有するものとなる。
When the analysis target is irradiated with excitation light, 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.
本発明の第一態様においては、ポンプ光およびストークス光を発生する光源と、分析対象と分析対象を支持する部材との境界に焦点を結ぶことが可能な第1の対物レンズと、分析対象および部材を挟んで第1の対物レンズに対向して配置され、分析対象および部材の境界に焦点を結ぶことが可能である第2の対物レンズと、 分析対象および部材で発生した光を、第2の対物レンズを通じて受光する分光器と を備える光学分析装置が提供される。
In a first aspect of the present invention, there is provided 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.
本発明の第二態様においては、ポンプ光およびストークス光が透過可能な部材に分析対象を支持させ、第1の開口数を有して分析対象および部材の境界に焦点を結ぶことが可能な第1の対物レンズを通じて、ポンプ光およびストークス光を分析対象および部材に照射し、第1の対物レンズに対向して配置され、分析対象および部材の境界に焦点を結ぶことが可能な第2の対物レンズを通じて、分析対象および部材で発生した散乱光を分光器で受光する光学分析方法が提供される。
In the second aspect of the present invention, 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.
上記の発明の概要は、本発明の必要な特徴の全てを列挙したものではない。これらの特徴群のサブコンビネーションもまた発明となり得る。
The above summary of the invention does not enumerate all of the necessary features of the present invention. Subcombinations of these features may also be inventive.
以下、発明の実施の形態を通じて本発明を説明するが、以下の実施形態は請求の範囲にかかる発明を限定するものではない。実施形態の中で説明されている特徴の組み合わせの全てが発明の解決手段に必須であるとは限らない。
Hereinafter, the present invention will be described through the embodiments of the invention, but the following embodiments do not limit the invention according to the claims. Not all combinations of features described in the embodiments are essential to the solution of the invention.
図1は、レーザ顕微分光器100によるスペクトルの分析対象となるサンプル110の構成を示す模式的断面図である。図示のサンプル110は、分析対象112を、保護液116に浸した状態でサンプル容器114に収容して形成される。分析対象112は、例えば細胞シートのような生体標本であり、保護液116として培養液が用いられている。サンプル容器114は、ガラス製も用いることができるが、ガラスに限定されるわけではない。
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. As shown in FIG. 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.
図2は、レーザ顕微分光器100の構造を示す模式図である。レーザ顕微分光器100は、レーザ光源120、励起光発生部130、光学系150、ステージ160、ポリクロメータ170、および制御部180を備える。
FIG. 2 is a schematic view showing the structure of the laser microspectroscope 100. As shown in FIG. 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.
レーザ顕微分光器100において、分析対象となるサンプル110は、ステージ160に置かれる。レーザ光源120が発生したレーザ光は、励起光発生部130においてポンプ光およびストークス光に変換された後、励起光側対物レンズ152等を通じてサンプル110に照射される。励起光を照射されたサンプル110において発生した散乱光は、信号光側対物レンズ154を通じてポリクロメータ170に検出される。ポリクロメータ170による検出結果は、制御部180において処理され、サンプル110の分光スペクトルが出力される。
In the laser microspectroscope 100, 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.
レーザ顕微分光器100において、レーザ光源120は、ピコ秒パルスレーザを発生して、励起光発生部130に入射する。レーザ光源120としては、モードロックピコ秒Nd:YVO4レーザ、モードロックピコ秒イットリビウムレーザー等を用いることができる。また、レーザ光源120に、ピコ秒パルスの第2高調波を励起光とする光パラメトリック発振器を設けて、ピコ秒パルスレーザの波長を変化させ出力してもよい。
In the laser microspectroscope 100, the laser light source 120 generates a picosecond pulse laser and enters the excitation light generator 130. As the laser light source 120, a mode locked picosecond Nd: YVO 4 laser, a mode locked picosecond ytterbium laser, or the like can be used. Alternatively, 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.
励起光発生部130は、光分岐器132、フォトニック結晶ファイバ134、反射鏡136、および光合波器138を有する。光分岐器132は、レーザ光源120から入射されたピコ秒パルスレーザを2つに分岐させる。
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.
光分岐器132の一方の射出端は一対の反射鏡136を含む光路に結合される。よって、こちら側に入射したピコ秒パルスレーザは、フォトニック結晶ファイバ134を迂回して、光合波器138の一方の入射端に結合された光路に誘導される。これにより、分岐されたピコ秒パルスレーザの一方は、光合波器138に到達するまでの時間を調整され、フォトニック結晶ファイバ134を経由した他方のピコ秒パルスレーザとタイミングを揃えて光合波器138に入射される。
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.
光分岐器132の他方の射出端は、フォトニック結晶ファイバ134の入射端に結合される。フォトニック結晶ファイバ134に入射したピコ秒パルスレーザは、フォトニック結晶ファイバ134において生じる自己位相変調により、当初入射したピコ秒パルスレーザよりも波長が長い帯域で広スペクトル化する。フォトニック結晶ファイバ134の射出端は、光合波器138の他方の入射端に結合される。
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.
広スペクトル化ピコ秒パルスレーザと、それにタイミングを合わせたピコ秒パルスレーザとは、光合波器138で合波された後、励起光発生部130から合成されたピコ秒パルスレーザとして射出される。励起光発生部130から射出されたピコ秒パルスレーザは、最終的に励起光としてサンプル110に照射される。ここで、当初の狭帯域のピコ秒パルスレーザはポンプ光として、フォトニック結晶ファイバ134で広帯域化されたピコ秒パルスレーザはストークス光として、それぞれサンプル110に照射される。
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. Here, the original narrow band picosecond pulse laser is irradiated as a pump light, and 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.
なお、レーザ光源120は、単一波長のピコ秒パルスレーザを発生するものに限られず、複数の波長のピコ秒パルスレーザを発生するものを用いてもよい。また、ポンプ光となるピコ秒パルスレーザも、フォトニック結晶ファイバにより波長を変換してから出力してもよい。
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. In addition, a picosecond pulse laser to be pump light may also be output after wavelength conversion by a photonic crystal fiber.
レーザ顕微分光器100の光学系150は、励起光発生部130とステージ160との間に配された励起光側対物レンズ152を含む。励起光側対物レンズ152は、ステージ160に置かれたサンプル110の内部に焦点を結び、励起光発生部130から伝播した励起光をサンプル110内に集光する。これにより、サンプル110内の焦点付近において、励起光は非線形効果を生じる。
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.
ステージ160は、圧電素子によりステージ160を少なくともX-Y方向に移動させる駆動部162を有する。これにより、光学系を移動させることなく、ステージ160上のサンプル110を励起光で走査できる。
The stage 160 has a drive unit 162 that moves the stage 160 at least in the XY direction by a piezoelectric element. Thus, the sample 110 on the stage 160 can be scanned with excitation light without moving the optical system.
また、光学系150は、ステージ160に置かれたサンプル110に対して、励起光側対物レンズ152と反対側に配された信号光側対物レンズ154を有する。信号光側対物レンズ154は、ステージ160に置かれたサンプル110の内部に焦点を結び、サンプル110から射出された散乱光を集光する。なお、レーザ顕微分光器100において、励起光側対物レンズ152と、信号光側対物レンズ154とは、互いに異なる開口数(NA)を有することが好ましい。この点については、図6、7、8を参照して後述する。
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. In the laser microspectroscope 100, 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.
光学系150は、更に、信号光側対物レンズ154から射出された散乱光の光路上に、光学フィルタ156および結像レンズ158を有する。光学フィルタ156は、サンプル110から射出された散乱光から不要な光学的成分を取り除く。
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.
ここで、不要な成分とは、サンプル110を透過して射出された照射光の一部の帯域を含む。このため、光学フィルタ156は、サンプル110の種類、分析対象の組成、検出目的等に応じて変更される。結像レンズ158は、後述するポリクロメータ170の受光面に、サンプル110で発生した散乱光を結像させる。
Here, 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.
なお、レーザ顕微分光器100は、励起光および散乱光の光路上に、反射鏡140、142を有する。これにより、励起光および散乱光の光路が折り曲げられ、レーザ顕微分光器100の寸法の拡大を抑制する。
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.
ポリクロメータ170は、広帯域の照射光をサンプル110に照射した場合に、分析対象112から射出された光を回折格子で分光して複数の受光素子で同時に受光する。これにより、ポリクロメータ170は、照射光が照射された領域におけるサンプル110のスペクトルを検出する検出部として動作する。
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.
なお、ポリクロメータ170は、光学系150の像面のひとつと共役な位置に配された分光器の入射スリットに相当する狭い領域を通して受光した光を分光して検出する。このため、ポリクロメータを用いて射出光を受光する場合は、サンプル110に照射する励起光の光路を変位させることができない。
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.
よって、ポリクロメータ170の1回の検出により得られるスペクトルは、分析対象112のあるひとつの位置における成分に対応する。しかしながら、レーザ顕微分光器100は、駆動部162により、サンプル110を置いたステージ160を変位させることができる。よって、レーザ顕微分光器100では、サンプル110の異なる位置のスペクトルも検出することができる。
Thus, the spectrum obtained by one detection of the polychromator 170 corresponds to the component at one position of the object to be analyzed 112. However, the laser microspectroscope 100 can displace the stage 160 on which the sample 110 is placed by the drive unit 162. Thus, the laser microspectroscope 100 can also detect spectra at different positions of the sample 110.
制御部180は、キーボード182、マウス184、情報処理装置186および表示装置188を有する。キーボード182およびマウス184は、情報処理装置186に接続され、情報処理装置186にユーザの指示を入力する場合に操作される。情報処理装置186は、汎用パーソナルコンピュータに制御手順を実行させるプログラムを実装して形成できる。
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.
表示装置188は、キーボード182およびマウス184による操作に対するフィードバックをユーザに返すと共に、情報処理装置186が生成した画像または文字列をユーザに向かって表示する。制御部180は、レーザ光源120、駆動部162、ポリクロメータ170等の動作を制御すると共に、ユーザによる指示をレーザ顕微分光器100に設定する。また、ポリクロメータ170の検出結果を映像化して、表示装置188に表示する画像を生成する。
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.
なお、レーザ顕微分光器100は、上記の他に、励起光の光路を変位させて固定されたサンプル110を励起光により走査するガルバノスキャナや、散乱光によるサンプル110のイメージングをする場合に使用する光電子増倍管190等を付加的に設けてもよい。光電子増倍管190等を付加的に設けてもよい。光電子増倍管190等の複数の検出部を設けた場合は、例えば、散乱光の光路において最も下流側に挿抜式反射鏡142を設けて、複数の検出部を選択的に使用してもよい。
In addition to the above, 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. When a plurality of detection units such as the photomultiplier tube 190 are provided, for example, 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. .
図3は、サンプル110における分析対象112に、集光された励起光が照射された場合に生じるCARS過程を説明する図である。CARS過程は、互いに異なる角周波数ωp、ωsを有するポンプ光およびストークス光を含む励起光をサンプル110に照射して、ポンプ光の光周波数ωpとストークス光の光周波数ωsとの差[ωp-ωs]が、サンプルに含まれる分子の固有振動の角振動数ω0と共鳴した場合に発生する。
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 ω p and ω s , and the difference between the light frequency ω p of the pump light and the light frequency ω s of the Stokes light This occurs when [ω p −ω s ] resonates with the angular frequency ω 0 of the natural vibration of the molecules contained in the sample.
CARS過程により、サンプルに含まれる特定の分子構造の振動モードが励振されると、分子振動が角周波数ω3を有する第3のレーザ光であるプローブ光と相互作用することにより、三次の非線形分極に由来するCARS光がラマン散乱光として発生する。
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.
更に、ポンプ光はプローブ光としても利用できるので、[ωp=ω3]という条件の下で、CARS光が発生する。サンプルにおいて発生するCARS光は、[ωCARS=ωp-ωs+ω3=2ωp-ωs]を満たす光周波数を有する。よって、サンプルから射出されたCARS光を検出することにより、サンプルに含まれる特定の分子構造、例えば官能基の存在を検出できる。更に、照射光をサンプルに照射する位置を変えながら繰り返しCARS光を検出することにより、分析対象112における特定の分子構造の分布を画像化することができる。
Furthermore, since pump light can also be used as probe light, CARS light is generated under the condition of [ω p = ω 3 ]. The CARS light generated in the sample has an optical frequency that satisfies [ω CARS = ω p −ω s + ω 3 = 2 ω p −ω s ]. Thus, by detecting CARS light emitted from the sample, it is possible to detect the presence of a specific molecular structure, such as a functional group, contained in the sample. Furthermore, the distribution of specific molecular structures in the object of analysis 112 can be imaged by repeatedly detecting the CARS light while changing the position where the irradiation light is irradiated to the sample.
ここで、CARS光は自発ラマン散乱光等に比べると光強度が高いので、光電気変換素子を用いて検出する場合に蓄積時間が短く、高速に検出できる。これにより、ビデオレートでの観察も可能になる。また、特定分子構造の分布だけではなく、分布の変化も検出することができる。更に、サンプルに照射する照射光の帯域を、生細胞に与えるダメージが少ない赤外帯域とすることにより、観察対象の生細胞を生かしたまま観察することができる。
Here, since the light intensity of CARS light is higher than that of spontaneous Raman scattering light and the like, 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.
また更に、サンプルの特定の位置に照射する照射光の光周波数を変化させることにより、当該照射位置から射出されたラマン散乱光の周波数分布(波数分布)を示すスペクトル画像が得られる。更に、照射光の光路に対して交差する方向にサンプルを移動させながら照射光を繰り返し照射することにより、観察平面における特定の分子の分布を画像化することもできる。
Furthermore, by changing the light frequency of the irradiation light irradiated to a specific position of the sample, 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.
図4は、サンプル110における分析対象112に、集光された励起光が照射された場合に生じる、CARS過程とは別の現象である四光波混合を説明する図である。四光波混合は、励起光が照射された場合に、分析対象112の3次非線形感受率χ(3)に起因して、図示のように、CARS光と同じ角周波数2ωp-ωsの散乱光がCARS光と同時に発生する現象である。
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 2ω p -ω s as CARS light, as illustrated, due to the third-order nonlinear susceptibility 感(3) of the analysis object 112 when the excitation light is irradiated. Light is a phenomenon that occurs simultaneously with CARS light.
このような四光波混合過程に起因する散乱光は、試料の分子振動に依存することなく発生するので非共鳴バックグラウンドと呼ばれ、CARS光像のコントラストを低下させる。また、指紋領域と呼ばれるCARS信号レベルが低い領域では、非共鳴バックグラウンドの影響が相対的に強く、イメージングが困難な場合があった。
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. In addition, in an area called 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.
更に、レーザ波長を掃引してスペクトルを検出した場合に、CARS光と非共鳴バックグラウンドとが重畳して検出されるので、自発ラマン散乱により検出したスペクトルとは異なるプロファイルのスペクトルが検出される原因となる。このため、生体内で凝集しやすく分子振動が強い脂質の検出に限られる等、CARS顕微法の適用範囲が制限されている。スペクトル検出後に画像処理等による後処理でスペクトルを補正することも試みられているが、処理負荷および処理時間が増加するので、有効な検出までの積算時間が短いというCARS信号の利点が損なわれる。
Furthermore, when the spectrum is detected by sweeping the laser wavelength, the CARS light and the non-resonant background are detected in an overlapping manner, so a spectrum of a profile different from the spectrum detected by spontaneous Raman scattering is detected It becomes. For this reason, the scope of application of CARS microscopy is limited, for example, it is limited to the detection of lipids that easily aggregate in vivo and have strong molecular vibrations. Although it has been attempted to correct the spectrum by post-processing such as image processing after spectrum detection, the processing load and processing time increase, so the advantage of the CARS signal that the integration time to effective detection is short is lost.
図5は、レーザ顕微分光器100における励起光側対物レンズ152および信号光側対物レンズ154の光学的配置を模式的に示す図である。図示のように、励起光側対物レンズ152および信号光側対物レンズ154は、それぞれ、分析対象112とガラス製のサンプル容器114との境界に焦点を結ぶ。
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.
このため、励起光側対物レンズ152が集光した励起光は、分析対象112とサンプル容器114の両方において四光波混合を生じる。更に、分析対象112においては、励起光の差周波(ωp-ωs)が分子振動共鳴周波数と一致した場合に増強されてCARS光が発生する。
For this reason, 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 (ω p −ω s ) of the excitation light matches with the molecular vibration resonance frequency.
よって、分子振動共鳴に依存しない非共鳴バックグラウンドは、3次非線形感受率χ(3)の実部に起因する。一方、CARS光強度は|χ(3)|2に比例するので、3次非線形感受率χ(3)の実部と虚部の両方を反映する。換言すれば、非線形感受率の虚部Im{χs
(3)}が、非共鳴バックグラウンドの影響が除かれた自発ラマンスペクトル(自然スペクトル)と同じスペクトルに相当する。
Thus, the non-resonant background, which does not depend on molecular vibrational resonance, is due to the real part of the third-order nonlinear susceptibility χ (3) . On the other hand, since the CARS light intensity is proportional to | χ (3) | 2 , it reflects both the real and imaginary parts of the third-order nonlinear susceptibility χ (3) . In other words, the imaginary part Im {χ 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.
図6は、図5に示した領域Aにおける点像振幅分布を示す図である。なお、図示の例は、励起光側対物レンズ152の開口数NAexが、信号光側対物レンズ154の開口数NAcolよりも大きい場合を示す。
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.
また、図内の右に、分析対象112側の寄与による散乱光と、サンプル容器114側の寄与による散乱光のアルガン図を併せて示す。図中上側のアルガン図は、分析対象112において発生した散乱光の初期位相に対応する。また、図中下側のアルガン図は、サンプル容器114において発生する散乱光の初期位相に対応する。
Further, on the right in the figure, 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.
図7は、図6と同様に、領域Aにおける点像振幅分布を示す図である。ただし、図7に示した例では、励起光側対物レンズ152の開口数NAexが、信号光側対物レンズ154の開口数NAcolと等しい。また、図7の右に、分析対象112側の寄与による散乱光のアルガン図を併せて示す。
FIG. 7 is a diagram showing the point spread distribution in the region A, as in FIG. However, in the example shown in FIG. 7, 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. Further, on the right of FIG. 7, an Argan diagram of scattered light due to the contribution on the side of the analysis target 112 is also shown.
図8は、図6、7と同様に、領域Aにおける点像振幅分布を示す図である。図8に示す例では、励起光側対物レンズ152の開口数NAexが、信号光側対物レンズ154の開口数NAcolよりも小さい。図8の右にも、分析対象112側の寄与による散乱光のアルガン図を併せて示す。
FIG. 8 is a diagram showing a point spread distribution in the region A, as in FIGS. In the example shown in FIG. 8, 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. Also on the right of FIG. 8, an Argan diagram of scattered light due to the contribution of the analysis target 112 is shown together.
図6、7、8に示すアルガン図を比較すると判るように、励起光側対物レンズ152の開口数NAexと、信号光側対物レンズ154の開口数NAcolとの関係の変化は、発生する散乱光の点像振幅分布に反映され、散乱光の偏角θFが変化することが判る。よって、励起光側対物レンズ152の開口数NAexと、信号光側対物レンズ154の開口数NAcolとを適切に設定することにより、上記の式(5)を満足できることが判る。
As can be seen by comparing the Argan diagrams shown in FIGS. 6, 7 and 8, a change in the relationship between the numerical aperture NA ex of the excitation light side objective lens 152 and the numerical aperture NA col of the signal light side objective lens 154 occurs. It can be seen that the angle of deviation θ F of the scattered light changes, which is reflected in the point image amplitude distribution of the scattered light. Therefore, it can be understood that the equation (5) can be satisfied by appropriately setting the numerical aperture NA ex of the excitation light side objective lens 152 and the numerical aperture NA col of the signal light side objective lens 154.
ここで、円形の瞳を有する励起光側対物レンズ152の開口数NAexを[n・sinθex(nは屈折率)]とし、信号光側対物レンズ154の開口数NAcolを[n・sinθcol(nは屈折率)]とした場合、下記の一連の式(6)に示す条件が満たされることにより上記式(5)が満足される。
Here, the numerical aperture NA ex of the excitation light side objective lens 152 having a circular pupil is [n · sin θ ex (n is a refractive index)], and the numerical aperture NA col of the signal light side objective lens 154 is [n · sin θ When col (n is a refractive index) is satisfied, the above-mentioned equation (5) is satisfied by satisfying the conditions shown in the following series of equations (6).
また、励起光側対物レンズ152が、外径n・sinθex(out)、内径n・sinθex(in)の環状瞳を有して輪帯照明が形成されている場合は、信号光側対物レンズ154が半径n・sinθcolの円形瞳を有するものとすると、下記の一連の式(7)に示す条件が満たされることにより上記式5が満足される。
When the excitation light side objective lens 152 has an annular pupil with an outer diameter n · sin θ ex (out) and an inner diameter n · sin θ ex (in) to form annular illumination, the signal light side objective is Assuming that the lens 154 has a circular pupil of radius n · sin θ col , the above equation 5 is satisfied by satisfying the conditions shown in the following series of equations (7).
上記のように、励起光側対物レンズ152が環状の瞳を有する場合は、信号光側対物レンズ154の円形瞳の半径を大きくとることができる。よって、光学分析装置としての分解能が高くなり、信号光を高効率に集光できる。
As described above, when 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.
図9は、式(5)の関係を満たす開口数NAの組合せの一例として、散乱光の角周波数とCARS光強度との関係を示すグラフである。図示の例は、励起光側対物レンズ152の開口数NAexを1とし、信号光側対物レンズ154の開口数NAcolを0.4として、励起光側対物レンズ152の開口数NAexの方が大きくなる開口数を選択した場合を示す。このような開口数の関係が成立している場合、角周波数が低い領域でCARS光の強度がより高くなる傾向がある。よって、指紋領域におけるCARSスペクトルの検出に有利である。
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). In the illustrated example, 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, and 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. When such a numerical aperture relationship is established, the intensity of the CARS light tends to be higher in the region where the angular frequency is low. Thus, it is advantageous for detection of CARS spectra in the fingerprint region.
図10は、式(5)の関係を満たす開口数NAの組合せの一例として、散乱光の角周波数とCARS光強度との関係を示すグラフである。図示の例は、励起光側対物レンズ152の開口数NAexを1とし、信号光側対物レンズ154の開口数NAcolを1.3として、信号光側対物レンズ154の開口数NAcolの方が大きくなる開口数を選択した場合を示す。このような開口数の関係が成立している場合は、角周波数が高い領域でCARS光の強度が高くなる傾向がある。
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). In the illustrated example, 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. When such a numerical aperture relationship is established, the intensity of the CARS light tends to be high in the region where the angular frequency is high.
図11は、上記式(5)に示した関係が成立する条件下で検出したスペクトルのプロファイルを示すグラフである。図示のプロファイルは、自発ラマンスペクトルのプロファイルと略一致する。
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.
図12は、比較のために、レーザ顕微分光器100の励起光側対物レンズ152の開口数NAexと、信号光側対物レンズ154の開口数NAcolとを同じにして測定したスペクトルのプロファイルを示すグラフである。図示のように、このプロファイルは、非共鳴バックグラウンドの影響を受けて、プロファイルが全体に山なりに変形している。また、各ピークの直後にディップが付帯しており、本来のスペクトルとは異なる波形となっている。
For comparison, 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. FIG. As shown, this profile is generally crumply deformed under the influence of non-resonant background. In addition, dips are attached immediately after each peak, and the waveform is different from the original spectrum.
このように、励起光側対物レンズ152および信号光側対物レンズ154の開口数NAを適切に設定することにより、デバイスを付加することなく、自発ラマン散乱光のスペクトルと一致する正確なプロファイルのスペクトルを検出できる。また、付加的な画像処理または信号処理も不要なので、分析器の処理負荷および処理時間が減少して、信号レベルが高いというCARSスペクトルの利点を活かすことができ、例えば、正確なスペクトルをビデオレートで検出することも可能になる。
Thus, by appropriately setting the numerical aperture NA of the excitation light side objective lens 152 and the signal light side objective lens 154, 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
以上、本発明を実施の形態を用いて説明したが、本発明の技術的範囲は上記実施の形態に記載の範囲には限定されない。上記実施の形態に、多様な変更または改良を加えることが可能であることが当業者に明らかである。その様な変更または改良を加えた形態も本発明の技術的範囲に含まれ得ることが、請求の範囲の記載から明らかである。
As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It is apparent to those skilled in the art that various changes or modifications can be added to the above embodiment. It is also apparent from the scope of the claims that the embodiments added with such alterations or improvements can be included in the technical scope of the present invention.
請求の範囲、明細書、および図面中において示した装置、システム、プログラム、および方法における動作、手順、ステップ、および段階等の各処理の実行順序は、特段「より前に」、「先立って」等と明示しておらず、また、前の処理の出力を後の処理で用いるのでない限り、任意の順序で実現しうることに留意すべきである。請求の範囲、明細書、および図面中の動作フローに関して、便宜上「まず、」、「次に、」等を用いて説明したとしても、この順で実施することが必須であることを意味するものではない。
The order of execution of each process such as operations, procedures, steps, and steps in the apparatuses, systems, programs, and methods shown in the claims, the specification, and the drawings is particularly "before", "before" It should be noted that it can be realized in any order, unless explicitly stated as etc., and unless the output of the previous process is used in the later process. With regard to the operation flow in the claims, the specification, and the drawings, even if it is described using “first,” “next,” etc. for convenience, it means that it is essential to carry out in this order. is not.
100 レーザ顕微分光器、110 サンプル、112 分析対象、114 サンプル容器、116 保護液、120 レーザ光源、130 励起光発生部、132 光分岐器、134 フォトニック結晶ファイバ、136、140 反射鏡、138 光合波器、142 挿抜式反射鏡、150 光学系、152 励起光側対物レンズ、154 信号光側対物レンズ、156 光学フィルタ、158 結像レンズ、160 ステージ、162 駆動部、170 ポリクロメータ、180 制御部、182 キーボード、184 マウス、186 情報処理装置、188 表示装置、190 光電子増倍管
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
Claims (8)
- ポンプ光およびストークス光を発生する光源と、
分析対象と前記分析対象を支持する部材との境界に焦点を結ぶことが可能な第1の対物レンズと、
前記分析対象および前記部材を挟んで前記第1の対物レンズに対向して配置され、前記分析対象および前記部材の境界に焦点を結ぶことが可能である第2の対物レンズと、
前記分析対象および前記部材で発生した光を、前記第2の対物レンズを通じて受光する分光器と
を備える光学分析装置。 A light source generating pump light and Stokes light;
A first objective lens capable of focusing on a boundary between an analysis target and a member supporting the analysis target;
A second objective lens disposed opposite to the first objective lens across the analysis target and the member, and capable of focusing on the boundary of the analysis target and the member;
An optical analyzer comprising: a spectroscope which receives the light to be analyzed and the light generated by the member through the second objective lens. - 前記第2の対物レンズは、前記第1の対物レンズの開口数と異なる開口数を有する請求項1に記載の光学分析装置。 The optical analyzer according to claim 1, wherein the second objective lens has a numerical aperture different from the numerical aperture of the first objective lens.
- 前記第1の対物レンズの開口数が、前記第2の対物レンズの開口数よりも大きい請求項2に記載の光学分析装置。 The optical analyzer according to claim 2, wherein the numerical aperture of the first objective lens is larger than the numerical aperture of the second objective lens.
- 前記部材が、前記ポンプ光およびストークス光に対してコヒーレントアンチストークスラマン散乱光を生じない材料で形成される請求項1から3のいずれか一項に記載の光学分析装置。 The optical analyzer according to any one of claims 1 to 3, wherein the member is formed of a material that does not generate coherent anti-Stokes Raman scattered light with respect to the pump light and Stokes light.
- 前記部材がガラス板である請求項4に記載の光学分析装置。 The optical analyzer according to claim 4, wherein the member is a glass plate.
- 前記第1の対物レンズの開口数および前記第2の対物レンズの開口数が、前記ポンプ光および前記ストークス光を照射した場合に、前記分析対象において発生した散乱光の位相と前記部材で発生した散乱光の位相との差2θFが、下記の式(1)に示す条件を満たすように選択されている請求項2から5のいずれか一項に記載の光学分析装置。
65°≦2θF≦115° ・・・(1) When the pump light and the Stokes light were irradiated, the numerical aperture of the first objective lens and the numerical aperture of the second objective lens were generated in the phase of the scattered light generated in the analysis target and in the member The optical analyzer according to any one of claims 2 to 5, wherein the difference 2θF with the phase of the scattered light is selected to satisfy the condition shown in the following equation (1).
65 ° ≦ 2θF ≦ 115 ° (1) - ポンプ光およびストークス光が透過可能な部材に分析対象を支持させ、
第1の開口数を有して前記分析対象および前記部材の境界に焦点を結ぶことが可能な第1の対物レンズを通じて、前記ポンプ光および前記ストークス光を前記分析対象および前記部材に照射し、
前記第1の対物レンズに対向して配置され、前記分析対象および前記部材の境界に焦点を結ぶことが可能な第2の対物レンズを通じて、前記分析対象および前記部材で発生した光を分光器で受光する
光学分析方法。 The analysis target is supported by a member capable of transmitting pump light and Stokes light,
Irradiating the pump light and the Stokes light to the analysis object and the member through a first objective lens having a first numerical aperture and capable of focusing on the analysis object and the boundary of the member;
The light generated by the object to be analyzed and the member is disposed with a spectroscope through a second objective lens disposed opposite to the first objective lens and capable of focusing on the analysis object and the boundary of the member Optical analysis method to receive light. - 前記第2の対物レンズは、前記第1の対物レンズの開口数と異なる開口数を有する請求項7に記載の光学分析方法。 The optical analysis method according to claim 7, wherein the second objective lens has a numerical aperture different from that of the first objective lens.
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