JP2021181909A - Difference detection conjugate compensation cars measurement apparatus - Google Patents
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Abstract
Description
本発明は、差分検出共役補償CARS(コヒーレント反ストークスラマン散乱、以下、単に「CARS」と言う。)計測装置に関する。 The present invention relates to a differential detection conjugated compensation CARS (coherent anti-Stoke Raman scattering, hereinafter simply referred to as "CARS") measuring device.
分子同定の技術には、赤外振動吸収分光がある。これは各分子の指紋領域の分子振動数の赤外光の吸収を測定するものである。しかし生体内分子の測定には、生体組織の主成分が水であり赤外光は吸収が大きいため、適用が困難である。これに対してラマン散乱測定は入射光に対し分子振動周波数分だけ周波数シフトしたラマン散乱光を測定するため、入射光を水に対する吸収が少なく生体内を透過する近赤外光を選択することで、適用が可能である。しかしラマン散乱光は極めて弱く、高感度化が課題である。 Infrared vibration absorption spectroscopy is a technique for molecular identification. This measures the absorption of infrared light at the molecular frequency of the fingerprint region of each molecule. However, it is difficult to apply the measurement of molecules in the living body because the main component of the living tissue is water and infrared light absorbs a large amount of infrared light. On the other hand, Raman scattering measurement measures Raman scattered light whose frequency is shifted by the molecular vibration frequency with respect to the incident light. , Applicable. However, Raman scattered light is extremely weak, and increasing the sensitivity is an issue.
この感度の問題を解決したのがCARS(コヒーレント反ストークスラマン散乱)技術である。CARSは、図1(a)に示すように分子の振動吸収バンドに対応した周波数差Δωのあるポンプ光パルスとストークス光パルスを時空間的に同時に照射すると、分子はポンプ光パルスにより基底準位V=0から上準位へ上がるとともに、ストークス光パルスにより励起準位V=1に誘導される。さらに、ポンプ光パルスにより上準位へ上がった後、基底準位V=0に緩和する過程でアンチストークス光を生じる。エネルギー準位の関係から、ストークス光とアンチストークス光のそれぞれの周波数はポンプ光ωpを中心にΔω折り返された周波数であり、ωs=ωp−Δω、ωas=2ωp−ωsとなる。ωpとωsはあらかじ設定可能なので、近赤外域でこれを設定すれば近赤外域の2波長のポンプ光、ストークス光により、赤外振動吸収のΔωの情報をアンチストークス光により計測できる。 CARS (coherent anti-Stoke Raman scattering) technology solves this sensitivity problem. As shown in FIG. 1 (a), when CARS is simultaneously irradiated with a pump light pulse and a Stokes light pulse having a frequency difference Δω corresponding to the vibration absorption band of the molecule, the molecule is subjected to the ground level by the pump light pulse. As it rises from V = 0 to the upper level, it is guided to the excitation level V = 1 by the Stokes optical pulse. Further, after rising to the upper level by the pump light pulse, anti-Stokes light is generated in the process of relaxing to the base level V = 0. Due to the energy level, the frequencies of Stokes light and anti-Stokes light are the frequencies folded back by Δω around the pump light ω p , and ω s = ω p − Δω, ω as = 2 ω p −ω s . Become. Since ω p and ω s can be roughly set, if this is set in the near infrared region, the information of Δω of infrared vibration absorption can be measured by anti-Stokes light by pump light and Stokes light of two wavelengths in the near infrared region. ..
CARS信号強度ICARS(Ω)は、励起過程を反映してポンプ光強度Ppumpの2乗とストークス光強度Pstokesの積に比例し、[数1]の特性を持つ。この特性により、ラマン散乱光測定に
(数1)
ICARS(Ω) ∝ Ppump 2・Pstokes
対し大きな信号強度が確保できる。通常パルスレーザーのパルスエネルギー安定度は±3%程度である。するとCARS信号安定度は(0.97)3=0.91から、略略±10%となる。このCARS信号の安定度の悪さが、CARSは物質同定には適しているが、物質定量には不向きとされる点である。CARSを定量計測に応用する場合の大きな課題である。
CARS signal intensity I CARS (Ω) is proportional to the product of the square of the pump light intensity P pump and the Stokes light intensity P stokes , reflecting the excitation process, and has the characteristic of [Equation 1]. Due to this characteristic, Raman scattered light measurement (Equation 1)
I CARS (Ω) ∝ P pump 2・ P stokes
On the other hand, a large signal strength can be secured. The pulse energy stability of a normal pulse laser is about ± 3%. Then, the CARS signal stability is (0.97) 3 = 0.91, which is approximately ± 10%. This poor stability of the CARS signal is that CARS is suitable for substance identification, but unsuitable for substance quantification. This is a major issue when applying CARS to quantitative measurement.
CARS信号の安定化には上述したパルスエネルギーの安定化以外にも課題が残る。CARSではポンプ光パルスとストークス光パルスが、時間(t)、偏光(p)、周波数差(ω)、空間3軸(x,y,z)の6要素で一致しなければならない。ポンプ光パルスとストークスパルスの時間軸での一致に対しては、OPOの発振2波長をCARS励起に用いることで解決できることが開示されている。また分子振動レベルに対応したポンプ光とストークス光の周波数差については、分子振動スペクトル幅範囲内の変動は許容される。一方ポンプ光とストークス光の偏光軸の変動はCARS励起効率に大きく影響する。また通常レーザー光源はレーザー光のポインティング変動があり、ポンプ光およびストークス光のそれぞれの集束位置変動につながり、CARS信号の変動要因となる。これらCARS信号の変動要因を抑えたCARS信号の安定化はCARSを定量計測に応用する場合の大きな課題である。 Stabilization of the CARS signal has problems other than the above-mentioned stabilization of pulse energy. In CARS, the pump optical pulse and the Stokes optical pulse must match in six elements: time (t), polarized light (p), frequency difference (ω), and three spatial axes (x, y, z). It is disclosed that the coincidence between the pump optical pulse and the Stokes pulse on the time axis can be solved by using the two wavelengths of OPO oscillation for CARS excitation. Further, the frequency difference between the pump light and the Stokes light corresponding to the molecular vibration level is allowed to fluctuate within the molecular vibration spectrum width range. On the other hand, fluctuations in the polarization axes of pump light and Stokes light greatly affect the CARS excitation efficiency. In addition, the laser light source usually has a pointing fluctuation of the laser light, which leads to a fluctuation of the focusing position of the pump light and the Stokes light, which becomes a fluctuation factor of the CARS signal. Stabilization of the CARS signal, which suppresses the fluctuation factors of the CARS signal, is a major issue when applying CARS to quantitative measurement.
さらにCARSの課題として非共鳴信号がある。CARS信号には、分子振動のV=1準位を介したアンチストークス光生成によるV=0準位への緩和である共鳴過程(図1(a))とV=1準位を介さない(仮想準位を介した)アンチストークス光生成によるV=0準位への緩和である非共鳴過程(図1(b))に基づく2種類がある。CARS信号のS/N比は、実質共鳴信号/非共鳴信号比が律速している。このため共鳴信号/非共鳴信号比確保する各種手法が検討されている。 Another issue of CARS is the non-resonant signal. The CARS signal does not go through the resonance process (Fig. 1 (a)), which is relaxation to the V = 0 level by anti-Stokes light generation via the V = 1 level of molecular vibration, and not through the V = 1 level (Fig. 1 (a)). There are two types based on the non-resonant process (FIG. 1 (b)), which is relaxation to the V = 0 level by anti-Stokes light generation (via virtual level). The S / N ratio of the CARS signal is determined by the real resonance signal / non-resonance signal ratio. Therefore, various methods for securing the resonance signal / non-resonance signal ratio are being studied.
この共鳴信号/非共鳴信号比の向上を実現するものとして、周波数変調CARS技術が知られている。CARSは3次の非線形光学効果の一種で、CARS光の光電界は3次非線形感受率χ(3)に比例し、CARS信号強度の差周波数特性ICARS(Ω)は、下記の数2によって定まることが既に知られている。
(数2)
ICARS(Ω) ∝ ∣χ(3) R(Ω)+χ(3) NR ∣2
= ∣χ(3) R(Ω)∣2+(χ(3) NR)2+2Re{χ(3) R(Ω)} χ(3) NR
となる。ここで、χ(3) R(Ω)は共鳴信号の3次非線形感受率、χ(3) NRは非共鳴信号の3次非線形感受率である。図2(a)は[数1]の3つの項の特性を共鳴周波数近傍について示したものである。図2(b)は[数1]の3つの項の和としてICARS(Ω) と(χ(3) NR)2を示したものである。いまポンプ光とストークス光の差周波数δ=ωp−ωsを時間的に変化させる(周波数変調)と、ICARS(Ω)は直流成分の非共鳴信号に交流成分の共鳴信号が重畳した信号となる。すなわちICARS(Ω)の直流成分は非共鳴信号、交流成分は共鳴信号のため、交流成分の振幅レベルを測定することで、共鳴信号のみの選択的検出が可能となる。しかし直流成分となるはずの非共鳴信号の安定度が悪いと、共鳴信号の交流成分に非共鳴信号が混在することとなり共鳴信号/非共鳴信号比の向上につながらない。周波数変調CARSの本来の特徴を活かすためには、CARS信号の安定化は必須である。
The frequency modulation CARS technique is known to realize the improvement of the resonance signal / non-resonance signal ratio. CARS is a kind of third-order nonlinear optical effect. The optical electric field of CARS light is proportional to the third-order nonlinear susceptibility χ (3), and the difference frequency characteristic I CARS (Ω) of CARS signal intensity is calculated by the following
(Number 2)
I CARS (Ω) ∝ ∣ χ (3) R (Ω) + χ (3) NR ∣ 2
= ∣ χ (3) R (Ω) ∣ 2 + (χ (3) NR ) 2 + 2Re {χ (3) R (Ω)} χ (3) NR
Will be. Here, χ (3) R (Ω) is the third-order nonlinear susceptibility of the resonance signal, and χ (3) NR is the third-order nonlinear susceptibility of the non-resonant signal. FIG. 2A shows the characteristics of the three terms of [Equation 1] in the vicinity of the resonance frequency. FIG. 2 (b) shows I CARS (Ω) and (χ (3) NR ) 2 as the sum of the three terms of [Equation 1]. Now, when the difference frequency δ = ωp−ωs between pump light and Stokes light is changed over time (frequency modulation), I CARS (Ω) becomes a signal in which the resonance signal of the AC component is superimposed on the non-resonance signal of the DC component. .. That is, since the DC component of I CARS (Ω) is a non-resonant signal and the AC component is a resonance signal, it is possible to selectively detect only the resonance signal by measuring the amplitude level of the AC component. However, if the stability of the non-resonant signal, which should be a DC component, is poor, the non-resonant signal is mixed with the AC component of the resonance signal, which does not lead to an improvement in the resonance signal / non-resonance signal ratio. In order to utilize the original characteristics of frequency modulation CARS, it is essential to stabilize the CARS signal.
共鳴信号/非共鳴信号比の向上を実現するもう一つのものとして、偏光差分CARSがある。共鳴信号は実成分と虚数成分を有し、虚数成分が自発ラマンスペクトルに直接関係していることと、非共鳴信号は実成分のみを有していることに着目し、直交偏光において2つのCARS信号を同時生成し、2つの信号を減算することで実成分は打ち消される一方、虚数成分は増強され共鳴信号のみを選別するものである。ここでも2つの信号の減算処理が定常に機能するにはCARS信号の安定化が必要である。 Another thing that realizes the improvement of the resonance signal / non-resonance signal ratio is the polarization difference CARS. Focusing on the fact that the resonance signal has a real component and an imaginary component, the imaginary component is directly related to the spontaneous Raman spectrum, and the non-resonant signal has only the real component, two CARS in orthogonal polarization. By simultaneously generating signals and subtracting the two signals, the real component is canceled, while the imaginary component is enhanced and only the resonance signal is selected. Again, the CARS signal needs to be stabilized for the subtraction process of the two signals to function steadily.
さらに共鳴信号/非共鳴信号比の向上を実現するものに、干渉差分CARSがある。パルス幅を伸長したポンプ光とパルス幅は元のままのポンプ光を励起光とした光パラメトリック増幅器(OPA)からのアイドラー光をストークス光としてCARS励起し、ポンプ光パルス単独領域に発生する共鳴CARS光を、OPAのシグナル光と干渉させて検出し共鳴CARS光を選別検出するものである。これも干渉成分(交流成分)を検出するのでCARS信号の安定度が求められる。 Further, there is an interference difference CARS that realizes an improvement in the resonance signal / non-resonance signal ratio. The pump light with the extended pulse width and the idler light from the optical parametric amplifier (OPA) with the original pump light as the excitation light are CARS excited as Stokes light, and the resonance CARS generated in the pump light pulse single region. Light is detected by interfering with OPA signal light, and resonance CARS light is selected and detected. Since this also detects the interference component (AC component), the stability of the CARS signal is required.
このようにCARSを定量計測に応用する場合に、CARS信号の安定化と共鳴信号/非共鳴信号比の確保が大きな課題としてある。 When applying CARS to quantitative measurement in this way, stabilizing the CARS signal and securing the resonance signal / non-resonance signal ratio are major issues.
CARS信号を安定化し、共鳴CARS信号の選別計測を実現する。 It stabilizes the CARS signal and realizes selective measurement of the resonant CARS signal.
同一光学系を有する被測定試料のCARS励起検出系と参照試料の非共鳴CARS励起検出系を備え、{(被測定CARS信号/非共鳴CARS信号)−1}の信号処理系を有することを特徴とするCARS計測装置 It is characterized by having a CARS excitation detection system for the sample to be measured having the same optical system and a non-resonant CARS excitation detection system for the reference sample, and having a signal processing system of {(CARS signal to be measured / non-resonant CARS signal) -1}. CARS measuring device
同一光学系を有する被測定試料のCARS励起検出系と参照試料の非共鳴CARS励起検出系を備え、参照試料の非共鳴CARS励起検出系において、非共鳴CARS光を二分し、一方の非共鳴CARS光を検出する補償信号検出系を有し、被測定試料のCARS光ともう一方の非共鳴CARS光からの光電流の差分を出力する差分検出器を備え、(差分信号/非共鳴CARS信号)の信号処理系を有することを特徴とするCARS計測装置。 A CARS excitation detection system for the sample under test and a non-resonant CARS excitation detection system for the reference sample, which have the same optical system, are provided. It has a compensation signal detection system that detects light, and is equipped with a difference detector that outputs the difference between the CARS light of the sample to be measured and the optical current from the other non-resonant CARS light (difference signal / non-resonant CARS signal). CARS measuring device characterized by having a signal processing system of.
CARS励起のポンプ光パルスとストークス光パルスにおいて、ストークス光パルスに対しポンプ光パルスが遅延していることを特徴とする上記記載のCARS計測装置。 The CARS measuring device according to the above description, wherein the pump optical pulse is delayed with respect to the Stokes optical pulse in the CARS-excited pump optical pulse and the Stokes optical pulse.
CARS励起のポンプ光パルスとストークス光パルスにおいて、ストークス光パルスに対するポンプ光パルスの遅延時間Dtが、両光パルスのパルス幅が半値全幅(FWHM)でTとしたとき、0.83T≦Dt≦1.03Tであることを特徴とする上記記載のCARS計測装置。
In the CARS-excited pump light pulse and Stokes light pulse, the delay time Dt of the pump light pulse with respect to the Stokes light pulse is 0.83T ≤ Dt ≤ 1 when the pulse width of both light pulses is T at half-value full width (FWHM). The CARS measuring device according to the above description, which is characterized by being .03T.
同一光学系を有する被測定試料のCARS励起検出系と参照試料の非共鳴CARS励起検出系を備え、{(被測定CARS信号/非共鳴CARS信号)−1}の信号処理を行うことで、安定した共鳴信号成分の計測を実現する。 It is equipped with a CARS excitation detection system for the sample under test and a non-resonant CARS excitation detection system for the reference sample, which have the same optical system, and is stable by performing signal processing of {(CARS signal under test / non-resonant CARS signal) -1}. Realizes the measurement of the resonance signal component.
表1はOPO2波長をグルコースのCO伸縮振動バンドである1130cm-1に対応するポンプ光波長1003nm、ストークス光波長1133nmに調整し、グルコース1mol水溶液と蒸留水をそれぞれCARS励起した時のCARS信号の安定度と(グルコースCARS信号/蒸留水CARS信号)の安定度を比較した結果である。OPOの繰り返し周波数が45Hzなので、45パルスの平均、標準偏差(SD)、標準偏差/平均を比較している。45パルスの変動を標準偏差/平均で比較すると、グルコースCARS信号と蒸留水CARS信号はCARS励起の各種変動要因の影響から各々約20%と大きな変動となっている。一方、(グルコースCARS信号/蒸留水CARS信号)では同一光学系で得られた信号による補償処理(共役補償処理)により3.8%までに抑えられている。 Table 1 adjusts the OPO2 wavelength to the pump light wavelength of 1003 nm and the Stokes light wavelength of 1133 nm corresponding to the CO expansion and contraction vibration band of glucose, 1130 cm -1 , and stabilizes the CARS signal when 1 mol glucose aqueous solution and distilled water are CARS excited respectively. It is the result of comparing the stability of degree and (glucose CARS signal / distilled water CARS signal). Since the repetition frequency of OPO is 45Hz, the mean, standard deviation (SD), and standard deviation / mean of 45 pulses are compared. Comparing the fluctuations of 45 pulses with the standard deviation / average, the glucose CARS signal and the distilled water CARS signal each have a large fluctuation of about 20% due to the influence of various fluctuation factors of CARS excitation. On the other hand, (glucose CARS signal / distilled water CARS signal) is suppressed to 3.8% by compensation processing (conjugate compensation processing) using signals obtained in the same optical system.
次にグルコース濃度に対する
(グルコースCARS信号―蒸留水CARS信号)/蒸留水CARS信号
=(グルコースCARS信号/蒸留水CARS信号)−1
を測定した結果を図3に示す。グルコースCARS信号にはグルコースCARS共鳴信号と非共鳴信号が含まれ、蒸留水CARS信号は非共鳴信号のみであるので、図3の縦軸はグルコースCRAS共鳴信号である。図3よりグルコース濃度1mol〜0.1molの範囲で直線性が得られ、グルコースの定量計測が可能であることが判る。低濃度側が0.1molで抑制されているのは、グルコースCARS信号の内の非共鳴成分に光検出器のダイナミックレンジを取られているためである。
Next, for glucose concentration (glucose CARS signal-distilled water CARS signal) / distilled water CARS signal = (glucose CARS signal / distilled water CARS signal) -1
The result of the measurement is shown in FIG. Since the glucose CARS signal includes a glucose CARS resonance signal and a non-resonance signal, and the distilled water CARS signal is only a non-resonance signal, the vertical axis in FIG. 3 is a glucose CRAS resonance signal. From FIG. 3, it can be seen that the linearity is obtained in the range of glucose concentration of 1 mol to 0.1 mol, and the quantitative measurement of glucose is possible. The low concentration side is suppressed at 0.1 mol because the dynamic range of the photodetector is taken by the non-resonant component in the glucose CARS signal.
そこで、同一光学系を有する被測定試料のCARS励起検出系と参照試料の非共鳴CARS励起検出系を備え、参照試料の非共鳴CARS励起検出系において、非共鳴CARS光を二分し、一方の非共鳴CARS光を検出する補償信号検出系を有し、被測定試料のCARS光ともう一方の非共鳴CARS光からの光電流の差分を出力する差分検出器を備え、(差分信号/非共鳴CARS信号)の信号処理系を有することで、測定ダイナミックレンジの拡大を実現する。 Therefore, the CARS excitation detection system of the sample to be measured and the non-resonant CARS excitation detection system of the reference sample having the same optical system are provided. It has a compensation signal detection system that detects resonant CARS light, and is equipped with a differential detector that outputs the difference between the CARS light of the sample under test and the optical current from the other non-resonant CARS light (difference signal / non-resonant CARS). By having a signal processing system (signal), the measurement dynamic range can be expanded.
図4は差分検出器からの差分信号を共役補償処理した信号をグルコース濃度に対しプロットしたものである。グルコース濃度の下限が0.1molから0.01molへ拡大している。 FIG. 4 is a plot of the signal obtained by performing conjugate compensation processing on the difference signal from the difference detector with respect to the glucose concentration. The lower limit of glucose concentration has expanded from 0.1 mol to 0.01 mol.
さらにダイナミックレンジを拡大するために、CARS励起のポンプ光パルスとストークス光パルスにおいて、ストークス光パルスに対しポンプ光パルスを遅延させることで、共鳴信号成分の増強を実現する。 In order to further expand the dynamic range, the resonance signal component is enhanced by delaying the pump optical pulse with respect to the Stokes optical pulse in the CARS-excited pump optical pulse and the Stokes optical pulse.
図1に示した共鳴過程では被測定分子が励起準位のV=1に存在する。このため励起準位寿命内にプローブ用ポンプ光(後段のポンプ光)が照射されれば、必ずしも共鳴CARS光の発生にはプローブ用ポンプ光とストークス光の同時性は必要ない。一方非共鳴過程では仮想準位を介して非共鳴CARS光が発生するため、ポンプ光とストークス光の同時性は必須である。この特性に違いに着目すると、ストークス光パルスに対しポンプ光パルスを遅延させてCARS励起を行うことで、共鳴信号の増強を計れる。図5にその様子を示した。図中(1)のポンプ光パルスとストークス光パルスが同時に被測定試料に照射されると、両パルスの重なり合う時間領域で共鳴過程と非共鳴過程のCARS励起が生じる。グルコース水溶液では共鳴CARS光と非共鳴CARS光が発生し、非共鳴CARS光の比率の方が大きい。蒸留水からは非共鳴CARS光のみが発生する。一方ストークス光パルスに対しポンプ光パルスがパルス幅(FWHM)の半幅遅延した(2)の場合では、グルコース水溶液では両パルスの重なりあう時間領域(グレーハッチ部)では共鳴CARS光と非共鳴CARS光が発生し、ストークス光パルスと重ならないポンプ光パルスの時間領域(斜め線ハッチ部)では共鳴CARS光のみが発生する。蒸留水では両パルス光の重なり合う時間領域で非共鳴CARS光が発生する。したがって(グルコースCARS信号―蒸留水CARS信号)の信号処理により残存するCARS共鳴信号は(1)より(2)の方が増強される。 In the resonance process shown in FIG. 1, the molecule under test exists at V = 1 at the excited level. Therefore, if the pump light for the probe (pump light in the subsequent stage) is irradiated within the excitation level lifetime, the simultaneous generation of the pump light for the probe and the Stokes light is not necessarily required to generate the resonance CARS light. On the other hand, in the non-resonant process, non-resonant CARS light is generated via the virtual level, so the simultaneity of the pump light and the Stokes light is indispensable. Focusing on this difference in characteristics, it is possible to enhance the resonance signal by delaying the pump optical pulse with respect to the Stokes optical pulse and performing CARS excitation. The situation is shown in FIG. When the pump light pulse and the Stokes light pulse in (1) in the figure are simultaneously irradiated to the sample to be measured, CARS excitation of the resonance process and the non-resonance process occurs in the time domain where both pulses overlap. Resonant CARS light and non-resonant CARS light are generated in an aqueous glucose solution, and the ratio of non-resonant CARS light is larger. Only non-resonant CARS light is generated from distilled water. On the other hand, in the case of (2) where the pump light pulse is delayed by half the pulse width (FWHM) with respect to the Stokes light pulse, the resonant CARS light and the non-resonant CARS light are in the time region (gray hatch part) where both pulses overlap in the glucose aqueous solution. Is generated, and only resonance CARS light is generated in the time region (diagonal line hatch portion) of the pump light pulse that does not overlap with the Stokes light pulse. In distilled water, non-resonant CARS light is generated in the time domain where both pulsed lights overlap. Therefore, the CARS resonance signal remaining by the signal processing of (glucose CARS signal-distilled water CARS signal) is enhanced in (2) rather than (1).
図6はストークス光パルスに対するポンプ光パルスの時間遅延量と共鳴信号/非共鳴信号の比の関係を測定した結果である。両パルス光のパルス幅6nsの時に遅延量Dtが5ns<Dt<6.2nsの範囲で共鳴信号/非共鳴信号≧1となり、共鳴信号の増強が見られた。 FIG. 6 shows the result of measuring the relationship between the time delay amount of the pump light pulse and the ratio of the resonance signal / non-resonance signal with respect to the Stokes light pulse. When the pulse width of both pulsed lights was 6 ns, the delay amount Dt became resonance signal / non-resonance signal ≧ 1 in the range of 5 ns <Dt <6.2 ns, and the resonance signal was enhanced.
図7はポンプ光パルスを光遅延路を通し、ストークス光パルスに対し5.6ns遅延させた差分信号共役補償により、差分共役補償信号をグルコース濃度に対しプロットした結果である。ポンプ光パルス遅延による共鳴信号増強により、差分共役補償信号はグルコース濃度1mol〜0.002molの範囲で直線性が確保できている。これは0.002mol=36mg/dlのグルコース濃度まで測定できることを示すものである。 FIG. 7 shows the result of plotting the differential conjugate compensation signal against the glucose concentration by the differential signal conjugate compensation in which the pump optical pulse is passed through the optical delay path and delayed by 5.6 ns with respect to the Stokes optical pulse. By enhancing the resonance signal by the pump optical pulse delay, the linearity of the differential conjugate compensation signal can be ensured in the range of glucose concentration of 1 mol to 0.002 mol. This indicates that the glucose concentration of 0.002 mol = 36 mg / dl can be measured.
以上、ポンプ光パルス遅延励起の同一光学系からなる差分共役補償により36mg/dlまでの定量計測が実現できる。 As described above, quantitative measurement up to 36 mg / dl can be realized by differential conjugated compensation consisting of the same optical system of pump optical pulse delayed excitation.
実施例1を図8、実施例2を図9、実施例3を図10、実施例4を図11、実施例5を図12のそれぞれ示す。実施例1と実施例2の違いは、偏光直交のOPO2波長光を光学軸が45度の偏光子を介してCARS励起に用いるか、偏光直交のOPO2波長光を一旦偏光ビームスプリッターで分離し、片方をλ/2板で偏光方向を90度回転させた後、再度ダイクロイックミラーで同軸に合波しCARS励起に用いるかの相違である。実施例3と実施例4の違いも同様である。また実施例1および実施例2と実施例3および実施例4との違いは、光検出において光電変換し増幅した信号同士の差分処理をし補償処理の信号処理を行うか、光検出において差分検出器により光電流の状態で差分し増幅した信号を取り出し補償処理の信号処理を行うかの相違である。実施例5は実施例4にポンプ光パルスの時間遅延を付与する光遅延路を設けたものである。 Example 1 is shown in FIG. 8, Example 2 is shown in FIG. 9, Example 3 is shown in FIG. 10, Example 4 is shown in FIG. 11, and Example 5 is shown in FIG. The difference between Example 1 and Example 2 is that OPO 2-wavelength light with polarized light is used for CARS excitation via a polarizing element with an optical axis of 45 degrees, or OPO 2-wavelength light with polarized light is once separated by a polarizing beam splitter. The difference is whether one is rotated 90 degrees in the polarization direction with a λ / 2 plate, and then the dichroic mirror is used to re-wavelength coaxially and use it for CARS excitation. The same applies to the difference between Example 3 and Example 4. Further, the difference between Example 1 and Example 2 and Example 3 and Example 4 is that the difference processing between the signals obtained by photoelectric conversion and amplification in the light detection is performed and the signal processing of the compensation processing is performed, or the difference detection in the light detection. It is the difference between taking out the signal that is differentiated and amplified in the state of the optical current by the device and performing the signal processing of the compensation processing. In the fifth embodiment, an optical delay path for imparting a time delay of the pump optical pulse is provided in the fourth embodiment.
図8に実施例1を示す。101光パラメトリック発振器(OPO)は励起レーザー(図中には記載なし)により励起されシグナル光とアイドラー光は同軸に発振する。励起レーザー光がP偏光の場合、シグナル光はS偏光、アイドラー光はP偏光となる。101OPOの発振2波長光でCARS励起を行う場合、シグナル光がポンプ光、アイドラー光がストークス光となる。 FIG. 8 shows Example 1. The 101 optical parametric oscillator (OPO) is excited by an excitation laser (not shown in the figure), and the signal light and idler light oscillate coaxially. When the excitation laser light is P-polarized, the signal light is S-polarized and the idler light is P-polarized. When CARS excitation is performed with the oscillation two-wavelength light of 101OPO, the signal light becomes the pump light and the idler light becomes the Stokes light.
101OPOからの同軸上の1ポンプ光(S偏光)と2ストークス光(P偏光)は3反射鏡を介して4ハーフビームスプリッターで2分される。一方が測定用、他方は参照用となる。 Coaxial 1-pump light (S-polarized light) and 2-Stokes light (P-polarized light) from 101OPO are split into two by a 4-half beam splitter via a 3-reflector. One is for measurement and the other is for reference.
測定用の同軸の1ポンプ光と2ストークス光は、光学軸が45度の5偏光子により偏光軸が45度の揃った光として、6a集光レンズにより測定用の7a試料セルに集光される。集光域において発生したCARS光は、8aコリメートレンズにより集められ平行光となり、9可変NDフィルターとポンプ光とストークス光をカットする10aフィルターを介して、11a光検出器により測定される。102光路長調整部は4ハーフビームスプリッターから6a集光レンズまでの測定側光路長と4ハーフビームスプリッターから6b集光レンズまでの光路長を等しくするものである。これにより測定側と参照側の共役光学系が成立する。なお9可変NDフィルターは、測定前に測定側と参照側に蒸留水セルを配置し、測定側CARS信号強度が後述の参照用CARS信号強度と同じレベルとなるよう校正するためのものである。 Coaxial 1-pump light and 2-Stokes light for measurement are focused on a 7a sample cell for measurement by a 6a condenser lens as light with a 45-degree polarization axis aligned by a 5-polarizer with an optical axis of 45 degrees. NS. The CARS light generated in the condensing region is collected by an 8a collimated lens to become parallel light, and is measured by an 11a photodetector via a 9-variable ND filter and a 10a filter that cuts pump light and Stokes light. The 102 optical path length adjusting unit equalizes the measurement side optical path length from the 4 half beam splitter to the 6a condenser lens and the optical path length from the 4 half beam splitter to the 6b condenser lens. As a result, a conjugated optical system on the measurement side and the reference side is established. The 9-variable ND filter is for arranging distilled water cells on the measurement side and the reference side before measurement and calibrating the measurement side CARS signal strength to the same level as the reference CARS signal strength described later.
参照用の同軸の1ポンプ光と2ストークス光は、測定用と同様に5b偏光子、6b集光レンズを介して参照用の7b試料セルに集光される。以下測定用と同様にCARS光は11b光検出器で測定される。 The coaxial 1-pump light and 2 Stokes light for reference are focused on the 7b sample cell for reference via a 5b polarizing element and a 6b condenser lens as in the case of measurement. Hereinafter, the CARS light is measured by the 11b photodetector in the same manner as for the measurement.
測定側の試料をグルコース水溶液、参照側の試料を蒸留水とすると、11a光検出器からの12G信号はグルコースのCARS共鳴信号と水のCARS非共鳴信号から成っている。一方13W信号は水のCARS非共鳴信号のみである。103信号処理部では12G信号と13W信号の減算、さらに13W信号で除算の処理を行うことで、14差分共役補償信号{(グルコースCARS共鳴信号/CARS非共鳴信号)−1}を出力している。この差分共役補償処理を行うことで、CARS励起にともなう種々の変動要因を一括補償して、安定した被測定分子のCARS共鳴信号が測定できる。 Assuming that the sample on the measurement side is an aqueous glucose solution and the sample on the reference side is distilled water, the 12G signal from the 11a photodetector consists of a CARS resonance signal of glucose and a CARS non-resonance signal of water. On the other hand, the 13W signal is only the CARS non-resonant signal of water. The 103 signal processing unit outputs a 14-difference conjugate compensation signal {(glucose CARS resonance signal / CARS non-resonance signal) -1} by subtracting the 12G signal and the 13W signal, and further performing division processing with the 13W signal. .. By performing this difference-conjugated compensation processing, various variable factors associated with CARS excitation can be collectively compensated, and a stable CARS resonance signal of the molecule to be measured can be measured.
図9に実施例2を示す。OPOからの同軸の1ポンプ光(S偏光)と2ストークス光(P偏光)を15偏光ビームスプリッターで分離し、1ポンプ光(S偏光)を16λ/2板により1ポンプ光(P偏光)にし、17ダイクロイックミラー上で1ポンプ光(P偏光)と2ストークス光(P偏光)を同軸に合波するものである。それ以降の動作を実施例1と同じである。ただし実施例では1ポンプ光と2ストークス光は同一偏光なので、実施例1で配置した5偏光子は測定側、参照側ともに不要である。偏光子は直交偏光の偏光を45度方向に揃えられるが、光パワーが1/√2に減じる。一方実施例2では偏光子が不要なため、光パワーを有効に利用できる効果がある。 FIG. 9 shows Example 2. Coaxial 1-pump light (S-polarized light) and 2-Stokes light (P-polarized light) from OPO are separated by a 15-polarized beam splitter, and 1-pump light (S-polarized light) is converted to 1-pump light (P-polarized light) by a 16λ / 2 plate. , 1 pump light (P polarized light) and 2 Stokes light (P polarized light) are coaxially combined on a 17-dycroic mirror. Subsequent operations are the same as in the first embodiment. However, since the 1-pump light and the 2-Stokes light have the same polarization in the embodiment, the 5-polarizer arranged in the first embodiment is unnecessary on both the measurement side and the reference side. The transducer aligns the polarization of orthogonally polarized light in the 45 degree direction, but the optical power is reduced to 1 / √2. On the other hand, in the second embodiment, since the polarizing element is not required, there is an effect that the optical power can be effectively used.
実施例1および実施例2では、測定側と参照側のCARS信号の差分処理を、それぞれの光検出器からの出力信号同士での差分を取っている。測定側の信号は被測定分子のCARS共鳴信号と非共鳴信号の合算であり、共鳴信号/非共鳴信号=1/5程度であり出力信号のほとんどが非共鳴信号成分である。このため光検出器のダイナミックレンジが非共鳴信号で占められ、共鳴信号のダイナミックレンジが十分に確保できない。この点を改善したものが実施例3である。図9に実施例3を示す。測定側と参照側のCARS光による光電変換素子の光電流自体での差分をおこなえる21差分検出器を用いている。これにより光電流時点でCARS非共鳴成分が打ち消され、CARS共鳴成分のみが光電流として残る。したがって検出器のダイナミックレンジ全てをCARS共鳴信号に振り分けることができる。なお測定側および参照側の19集光レンズと20光ファイバーは21差分検出器への光導光のためのものである。実施例1との他の相違点は、差分検出器からの22直接差分信号の共役補償用の23CARS非共鳴信号を取り出すため、参照側の8bコリメートレンズ後に18ハーフミラーを介して10bフィルターと11b光検出器を設けている。21差分検出器からの22直接差分信号と23CARS非共鳴信号は103信号処理部において(22直接差分信号/23CARS非共鳴信号)の除算処理により24直接差分共役補償信号に成形される。これにより差分信号に光検出器のダイナミックレンジがすべて割り振られる。 In the first and second embodiments, the difference processing of the CARS signals on the measurement side and the reference side is performed by taking the difference between the output signals from the respective photodetectors. The signal on the measurement side is the sum of the CARS resonance signal and the non-resonance signal of the molecule to be measured, and the resonance signal / non-resonance signal = about 1/5, and most of the output signals are non-resonance signal components. Therefore, the dynamic range of the photodetector is occupied by the non-resonant signal, and the dynamic range of the resonance signal cannot be sufficiently secured. Example 3 is an improvement in this respect. FIG. 9 shows Example 3. A 21 difference detector is used that can make a difference in the photocurrent itself of the photoelectric conversion element by CARS light on the measurement side and the reference side. As a result, the CARS non-resonant component is canceled at the time of the photocurrent, and only the CARS resonance component remains as the photocurrent. Therefore, the entire dynamic range of the detector can be allocated to the CARS resonance signal. The 19 condenser lenses and 20 optical fibers on the measurement side and the reference side are for optical guidance to the 21 difference detector. Another difference from Example 1 is that the 23 CARS non-resonant signal for conjugate compensation of the 22 direct difference signals from the difference detector is taken out, so that the 10b filter and 11b are passed through an 18 half mirror after the 8b collimating lens on the reference side. A photodetector is provided. The 22 direct difference signal and the 23 CARS non-resonant signal from the 21 difference detector are formed into a 24 direct difference conjugate compensation signal by the division process of (22 direct difference signal / 23 CARS non-resonant signal) in the 103 signal processing unit. This allocates the entire dynamic range of the photodetector to the difference signal.
図11に実施例4を示す。実施例4は実施例2に直接差分共役補償信号が取得できるように実施例3に示した構成を付加したものである。 FIG. 11 shows Example 4. In the fourth embodiment, the configuration shown in the third embodiment is added so that the differential conjugated compensation signal can be directly acquired in the second embodiment.
図12に実施例5を示す。実施例5は実施例4のダイナミックレンジをさらに広げる機能を付加したものである。CARS励起のポンプ光パルスとストークス光パルスにおいて、ストークス光パルスに対しポンプ光パルスを遅延させることで、共鳴信号成分の増強を実現ものである。101OPOからの直交偏光の1ポンプ光(S偏光)と2ストークス光(P偏光)を15偏光ビームスプリッターで分離し、1ポンプ光(S偏光)を16λ/2板で1ポンプ光(P偏光)とし、2ストークス光に対し時間遅延を発生させる104光遅延部にて、15偏光ビームスプリッターと17ダイクロイックミラー間のポンプ光とストークス光の光路長差を調整した後、再び17ダイクロイックミラーで同軸上に合波する。1ポンプ光(P偏光)光路内の25ビーム縮小器は、1ポンプ光(P偏光)の長光路長伝搬により生じる17ダイクロイックミラー上での2ストークス光のビーム径との不整合を補正するものである。17ダイクロイックミラー以降の機能は実施例4と同様である。これにより、遅延ポンプ光パルスの効果により測定側の被測定分子のCARS共鳴信号が増強され、実施例4よりさらにCARS共鳴信号検出のダイナミックレンジが拡大し、最小検出感度が向上する。 FIG. 12 shows Example 5. The fifth embodiment has a function of further expanding the dynamic range of the fourth embodiment. In the CARS-excited pump light pulse and Stokes light pulse, the resonance signal component is enhanced by delaying the pump light pulse with respect to the Stokes light pulse. 1 pump light (S polarized light) and 2 Stokes light (P polarized light) of orthogonal polarization from 101OPO are separated by a 15 polarized beam splitter, and 1 pump light (S polarized light) is separated by 1 pump light (P polarized light) with a 16λ / 2 plate. Then, after adjusting the optical path length difference between the pump light and the Stokes light between the 15 polarization beam splitter and the 17 dichroic mirror at the 104 light delay part that generates a time delay for the 2 Stokes light, it is coaxially on the 17 dichroic mirror again. To meet the waves. The 25-beam reducer in the 1-pump light (P-polarized) optical path corrects the inconsistency with the beam diameter of the 2-Stokes light on the 17-dichroic mirror caused by the long-optical path length propagation of the 1-pump light (P-polarized). Is. The functions after the 17 dichroic mirror are the same as those in the fourth embodiment. As a result, the CARS resonance signal of the molecule to be measured on the measurement side is enhanced by the effect of the delay pump optical pulse, the dynamic range of CARS resonance signal detection is further expanded as compared with Example 4, and the minimum detection sensitivity is improved.
これらは従来実用性が極めて乏しかったCARSに、大きな実用性をもたらし、本来CARS技術が有する生体内分子計測の特徴を汎用化させる効果がある。 These bring great practicality to CARS, which has been extremely poor in practicality, and have the effect of generalizing the characteristics of in-vivo molecular measurement originally possessed by CARS technology.
1 ポンプ光
2 ストークス光
3 反射鏡
4 ハーフビームスプリッター
5 偏光子
6 集光レンズ
7 試料セル
8 コリメートレンズ
9 可変NDフィルター
10 フィルター
11 光検出器
12 G信号
13 W信号
14 差分共役補償信号
15 偏光ビームスプリッター
16 λ/2板
17 ダイクロイックミラー
18 ハーフミラー
19 集光レンズ
20 光ファイバー
21 差分検出器
22 直接差分信号
23 CARS非共鳴信号
24 直接差分共役補償信号
25 ビーム縮小器
101 光パラメトリック発振器(OPO)
102 光路長調整部
103 信号処理部
104 光遅延部
同一番号におけるaは測定側、bは参照側を示す
1 Pump light 2 Stokes light 3
102 Optical path
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WO2022224999A1 (en) * | 2021-04-22 | 2022-10-27 | Atonarp Inc. | Method and system for acquiring cars spectrum |
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WO2022224999A1 (en) * | 2021-04-22 | 2022-10-27 | Atonarp Inc. | Method and system for acquiring cars spectrum |
JP7477933B2 (en) | 2021-04-22 | 2024-05-02 | アトナープ株式会社 | Method and system for acquiring CARS spectra |
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