WO2017082218A1 - Photon detection device, photon detection method, fluorescence correlation spectroscopy device, fluorescence cross-correlation spectroscopy device, dynamic light scattering device, and fluorescence microscope - Google Patents

Photon detection device, photon detection method, fluorescence correlation spectroscopy device, fluorescence cross-correlation spectroscopy device, dynamic light scattering device, and fluorescence microscope Download PDF

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WO2017082218A1
WO2017082218A1 PCT/JP2016/083010 JP2016083010W WO2017082218A1 WO 2017082218 A1 WO2017082218 A1 WO 2017082218A1 JP 2016083010 W JP2016083010 W JP 2016083010W WO 2017082218 A1 WO2017082218 A1 WO 2017082218A1
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intensity
signal
fluorescence
photon detection
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石井　邦彦
太平 田原
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国立研究開発法人理化学研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • 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
    • G01N21/64—Fluorescence; Phosphorescence

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  • the present invention relates to a photon detection device, a photon detection method, a fluorescence correlation spectroscopy measurement device, a fluorescence cross correlation spectroscopy measurement device, a dynamic light scattering measurement device, and a fluorescence microscope.
  • the photon correlation measurement method is widely used for measuring the size of particles in a sample solution and detecting intermolecular interactions.
  • photon correlation measurement methods include fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS).
  • photon signals derived from fluorescence (FCS) and light scattering (DLS) from a sample solution are measured in time series, and various physical quantities are obtained based on the time fluctuation.
  • a time correlation function is used to quantify the above time fluctuation. Two photon pairs detected with a certain time interval ⁇ T are counted by dedicated hardware or software, and normalized by the average light intensity to obtain a correlation signal at ⁇ T (Equation 1).
  • I (T) represents the light intensity at time T
  • ⁇ . . . > Represents a time average
  • SPAD photon avalanche diode
  • SPAD detects a photon, it outputs one voltage pulse per photon.
  • SPAD outputs a second voltage pulse (hereinafter referred to as an after pulse) with a certain probability within a few microseconds after detecting a photon and outputting a voltage pulse.
  • the after pulse greatly affects the correlation signal in the time domain of several microseconds. Therefore, in photon correlation measurement using SPAD, a false correlation signal due to SPAD after-pulse phenomenon becomes a problem.
  • Non-patent Document 1 In the conventional photon correlation measurement, when it is necessary to avoid the influence of the after pulse, a method of calculating the cross-correlation of these signals using two detectors is generally used (Non-patent Document 1). .
  • the conventional method of Non-Patent Document 1 has a problem in that it requires two detectors and is expensive, and the optical system becomes complicated. Furthermore, since only photon pairs detected by different detectors are used as described above, information on photon pairs detected by the same detector (50% of all photon pairs) is discarded, and the signal-to-noise ratio is reduced. There is a problem that decreases.
  • Non-Patent Document 2 a time reversal symmetry of a correlation signal by using a pulse laser and a time correlated photon coefficient method (Time Correlated Single Photon Counting: TCSPC). Reported (Non-Patent Document 2). However, since the method of Non-Patent Document 2 requires an expensive pulse laser device and a TCSPC device, there is a problem that the cost of the entire measuring device increases, and application to normal photon correlation measurement is not realistic. It was.
  • Non-Patent Document 3 the influence of the after pulse is evaluated from the difference in the temporal behavior of the after pulse signal and the fluorescence signal using a pulse laser and TCSPC. Similarly to the method of Non-Patent Document 2, the method of Non-Patent Document 3 has a problem that the cost of the entire measuring apparatus increases.
  • the present invention has been made in view of such a situation, and provides a technique capable of removing the influence of a false signal due to the after-pulse phenomenon with a simpler configuration.
  • the present application includes a plurality of means for solving the above-described problems.
  • a semiconductor laser that outputs laser light while intensity-modulating according to a modulation signal, and a single photon detector using an avalanche diode as a light receiving element
  • An optical system configured to irradiate the sample with the laser light and guide the light from the sample to the single photon detector; and from the single photon detector in synchronization with the modulation signal.
  • a photon detection device comprising: a signal distributor that distributes a pulse signal to a plurality of outputs; and a correlator that calculates a correlation function based on the plurality of outputs from the signal distributor.
  • FIG. 1 is a configuration diagram of an embodiment of the photon detection device of the present invention.
  • FIG. 1 shows an example in which the photon detection device of the present invention is applied to a fluorescence correlation spectroscopy (FCS) measurement device.
  • the photon detection device 1 includes a semiconductor laser (laser light source) 2, an external modulator 3, an optical system 4, a SPAD 5, a signal distributor 6, and a correlator 7.
  • the semiconductor laser 2 is a CW laser (Continuous wave laser) that emits laser light continuously.
  • the external modulator 3 outputs an external modulation signal to the semiconductor laser 2 and the signal distributor 6.
  • the external modulator 3 is used to modulate the intensity of the laser light from the semiconductor laser 2, and may be any one that can modulate at a frequency of about several tens of MHz, for example.
  • the semiconductor laser 2 outputs laser light while performing intensity modulation according to the external modulation signal from the external modulator 3.
  • the optical system 4 is configured to irradiate the sample 9 placed on the sample stage 8 with the laser light from the semiconductor laser 2 and to guide the light from the sample 9 to the SPAD 5.
  • the optical system 4 includes a dichroic mirror 41, an objective lens 42, and a pinhole 43. Note that only the main components are shown with respect to the optical system 4 in FIG. 1, and other components (mirror, lens, etc.) may be included in addition to this.
  • the dichroic mirror 41 has an optical characteristic of reflecting the laser light (excitation light) from the semiconductor laser 2 and transmitting fluorescence from the sample 9 (for example, fluorescence from a fluorescent dye excited by excitation light).
  • Fluorescence from the sample 9 for example, fluorescence from a fluorescent dye excited by excitation light.
  • Laser light emitted from the semiconductor laser 2 is reflected by the dichroic mirror 41 and then enters the objective lens 42.
  • a sample 9 is disposed at the focal position of the objective lens 42.
  • the fluorescence emitted from the sample 9 reaches the dichroic mirror 41 through the objective lens 42 again.
  • the fluorescence passes through the dichroic mirror 41, passes through the pinhole 43, and is guided to SPAD 5.
  • SPAD5 is a single photon detector using an avalanche diode as a light receiving element. As described above, SPAD 5 outputs one voltage pulse for each photon when it detects a photon. The SPAD 5 outputs a pulse signal to the signal distributor 6 according to the light received through the pinhole 43.
  • the signal distributor 6 distributes the pulse signal from the SPAD 5 to two outputs. More specifically, the signal distributor 6 distributes the pulse signal from the SPAD 5 to two outputs in synchronization with the external modulation signal from the external modulator 3, and distributes these two outputs to the two outputs of the correlator 7. Input to each of the channels (ch1, ch2).
  • Correlator 7 calculates a correlation function based on the signals input to the two channels (ch1, ch2).
  • the processing in the correlator 7 may be realized by hardware by designing with an integrated circuit, for example.
  • the processing in the correlator 7 may be realized by software by the processor interpreting and executing a program corresponding to the processing.
  • FIG. 2A shows excitation by laser light and the signal photon train obtained thereby.
  • the change in light intensity (time fluctuation) is expressed as a function I (T; t) of time (macro time, T) and delay time (micro time, t) after excitation.
  • FIG. 2B is a diagram simply explaining these time-reversal symmetries.
  • the true fluorescence correlation function that does not include the influence of afterpulse is symmetric with respect to the time reversal ⁇ T ⁇ ⁇ T when the system is in an equilibrium state.
  • the correlation function derived from the after pulse shows asymmetric t dependence with respect to the time reversal ⁇ T ⁇ ⁇ T.
  • the magnitude of the influence of the after-pulse phenomenon on the correlation signal is determined using the above-described time symmetry. As a result, it is possible to perform accurate correlation measurement by removing the false signal due to the after-pulse phenomenon.
  • FIG. 3 is a flowchart showing the flow of processing in the photon detection device 1.
  • the semiconductor laser 2 outputs laser light while performing intensity modulation (step 301).
  • the upper diagram in FIG. 4 is a diagram in which the horizontal axis represents time T and the vertical axis represents laser intensity, and is a diagram for explaining intensity modulation in the semiconductor laser 2.
  • the semiconductor laser 2 outputs a laser while performing intensity modulation between a first intensity (High) and a second intensity (Low) in accordance with an external modulation signal from the external modulator 3.
  • the lower diagram in FIG. 4 is a diagram in which the horizontal axis represents time T and the vertical axis represents light intensity I (T), and is a diagram for explaining measurement of signal light intensity temporal fluctuation.
  • the solid line in the lower diagram of FIG. 4 indicates the pulse signal output from SPAD5.
  • the portion surrounded by the dotted line 401 corresponds to the light intensity when the output of the semiconductor laser 2 is the first intensity (High), and the portion surrounded by the dotted line 402 is the output of the semiconductor laser 2. This corresponds to the light intensity at the second intensity (Low).
  • the time fluctuation of the light intensity when the output of the semiconductor laser 2 is the first intensity (High) is defined as I H (T), and the output when the output of the semiconductor laser 2 is the second intensity (Low).
  • the time fluctuation of the light intensity is defined as I L (T).
  • first intensity is larger than the second intensity
  • second strength these simply indicate that the strengths are different, and the magnitudes of the strengths may be reversed.
  • the signal distributor 6 receives an external modulation signal from the external modulator 3 and a pulse signal from the SPAD 5.
  • the signal distributor 6 synchronizes with the external modulation signal from the external modulator 3 and transmits the time fluctuation I H (T) of the light intensity corresponding to the first intensity (High) to the channel (ch 1) of the correlator 7.
  • the time fluctuation I L (T) of the light intensity corresponding to the second intensity (Low) is output to the channel (ch 2) of the correlator 7.
  • the correlator 7 calculates a correlation function G ( ⁇ T) between the fluorescence signal intensity I (T) at a certain time T and the fluorescence intensity I (T + ⁇ T) at the time when ⁇ T has elapsed therefrom (step 303).
  • the correlator 7 includes ⁇ I H (T) I H (T + ⁇ T)>, ⁇ I H (T) I L (T + ⁇ T)>, ⁇ I L (T) I H (T + ⁇ T)>, ⁇ I L (T)
  • the correlation function G ( ⁇ T) is calculated by obtaining I L (T + ⁇ T)>, ⁇ I H (T)>, ⁇ I L (T)>.
  • ⁇ . . . > Represents a time average as described above.
  • the correlator 7 calculates the correlation function G ( ⁇ T) according to the following equation (2).
  • the first term is a term corresponding to a normal correlation signal
  • the second term is a contribution term of the after pulse. Therefore, according to the present embodiment, the magnitude of the influence of the after pulse is obtained by evaluating the magnitude of the temporal asymmetry of the pseudo signal of the after pulse, and the magnitude of the influence of the after pulse is subtracted from the normal correlation signal. Thus, accurate correlation measurement can be performed.
  • FIG. 5 shows a first example of a signal distributor.
  • the signal distributor 6 is configured by a logic operation circuit, and includes a first AND circuit 61, a second AND circuit 62, and a NOT circuit 63.
  • the output of the SPAD 5 is input to one input terminal of the first AND circuit 61, and the external modulation signal from the external modulator 3 is input to the other input terminal of the first AND circuit 61. Therefore, the output of the first AND circuit 61 corresponds to the temporal fluctuation I H (T) of the light intensity at the first intensity (High).
  • the output of SPAD5 is input to one input terminal of the second AND circuit 62, the external modulation signal from the external modulator 3 is input to the input terminal of the NOT circuit 63, and the output of the NOT circuit 63 is This is input to the other input terminal of the second AND circuit 62. Therefore, the output of the second AND circuit 62 corresponds to the time fluctuation I L (T) of the light intensity at the second intensity (Low).
  • the calculation function of the correlation function in the correlator 7 is the same as the expression (2).
  • FIG. 6 shows a second example of the signal distributor 6.
  • the signal distributor 6 includes an AND circuit 64.
  • the output of SPAD5 is directly input to the channel (ch1) of the correlator 7. Therefore, the time fluctuation I H + L (T) of the light intensity in both the first intensity (High) and the second intensity (Low) is input to the channel ch1.
  • the output of SPAD 5 is input to one input terminal of AND circuit 64, and the external modulation signal from external modulator 3 is input to the other input terminal of AND circuit 64. Accordingly, the output of the AND circuit 64 corresponds to the temporal fluctuation I H (T) of the light intensity at the first intensity (High). According to the configuration of FIG. 6, the signal distributor 6 can be configured with a simpler circuit.
  • the calculation function of the correlation function in the correlator 7 is as shown in the following expression (3).
  • I 1 (T) represents the light intensity input to the channel (ch1) of the correlator 7
  • I 2 (T) represents the light intensity input to the channel (ch2) of the correlator 7. .
  • FIG. 7 shows a third example of the signal distributor 6.
  • the signal distributor 6 includes a NOT circuit 65 and an AND circuit 66.
  • the output of SPAD5 is directly input to the channel (ch1) of the correlator 7. Therefore, the time fluctuation I H + L (T) of the light intensity in both the first intensity (High) and the second intensity (Low) is input to the channel ch1.
  • the output of SPAD 5 is input to one input terminal of AND circuit 66, the external modulation signal from external modulator 3 is input to the input terminal of NOT circuit 65, and the output of NOT circuit 65 is the AND circuit 66. To the other input terminal. Accordingly, the output of the AND circuit 66 corresponds to the light intensity temporal fluctuation I L (T) at the second intensity (Low).
  • the calculation function of the correlation function in the correlator 7 is the same as the expression (3).
  • the configuration of the signal distributor 6 is not limited thereto. It suffices if the signal distributor 6 can distribute the pulse signal from the SPAD 5 to each channel of the correlator 7 in synchronization with the external modulation signal, and other configurations may be used as long as the pulse signal distribution function can be realized. Further, the calculation formula of the correlation function is not limited to that described above, and may be changed as appropriate.
  • OBIS 488 LX manufactured by Coherent was used as the semiconductor laser 2.
  • a single photon detector (id100-20) manufactured by IDQ (ID Quantique) was used as SPAD5.
  • a signal for laser modulation (corresponding to the external modulator 3) and a signal from a single photon detector were recorded by PCI-6602 manufactured by National Instruments.
  • the functions of the signal distributor 6 and the correlator 7 were implemented by a PC (Personal Computer) connected to the PCI-6602, and the distribution of the signals recorded in the PCI-6602 and the calculation of the correlation function were performed.
  • the calculation in the correlator 7 was performed according to the above-described equation (2).
  • FIG. 8 shows the result of observing scattered light from the surface of the aluminum foil using an aluminum foil as a reference sample.
  • the laser intensity was reduced until the peak power reached 5 ⁇ W
  • the graph of FIG. 8 shows an average of results obtained by repeating the measurement for about 10 seconds 10 times.
  • Correlation 1
  • FIG. 9 shows the result of measuring the scattered light from an aqueous solution of BSA using a 0.4 mg / mL aqueous solution of BSA (bovine serum albumin) as sample 9.
  • BSA bovine serum albumin
  • the peak power of the laser intensity is 25 mW
  • the graph of FIG. 9 shows the average of the results of repeating the measurement for about 12 seconds 9 times.
  • the originally expected correlation signal was observed after applying afterpulse removal. From the experimental results, it was confirmed that the influence of the false signal due to the after-pulse phenomenon can be removed using only one detector.
  • Non-patent Document 1 the configuration uses two detectors (Non-patent Document 1), there is a problem that the cost is increased and the optical system is complicated. Further, conventionally, there is a problem that the use efficiency of the information of the photon pair detected by the detector is low (50%). On the other hand, in the present embodiment, the influence of the false signal due to the after-pulse phenomenon can be removed with one detector (SPAD). Moreover, the utilization efficiency of the information of the photon pair detected by the detector is improved, and the problem that the signal-to-noise ratio is reduced can be solved.
  • Non-Patent Document 2 since the conventional configuration uses an expensive pulse laser device and a TCSPC device (Non-Patent Document 2), there is a problem that the cost of the entire measuring device increases.
  • the photon detection device 1 is composed of an inexpensive CW semiconductor laser and a normal correlator, the influence of the false signal due to the after-pulse phenomenon can be achieved with a low-cost and simple configuration. Can be removed.
  • a detector other than SPAD photomultiplier tube (PMT), hybrid detector (HPD)
  • PMT photomultiplier tube
  • HPD hybrid detector
  • PMT has a low probability of afterpulses and HPD can almost ignore afterpulses, but their maximum sensitivity is less than SPAD.
  • the influence of the false signal due to the after-pulse phenomenon can be removed, so that SPAD having excellent sensitivity can be used.
  • this invention is not limited to the Example mentioned above, Various modifications are included.
  • the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described.
  • the photon detection device of the present invention is applied to the FCS.
  • the photon detection device of the present invention may be applied to a fluorescence cross correlation spectroscopy (FCCS) measurement device.
  • the photon detection device of the present invention may be applied to a dynamic light scattering (DLS) measurement device.
  • the photon detection device of the present invention may be applied to a fluorescence microscope.
  • the photon detection device of the present invention may be applied to devices other than those described above.
  • the configuration of the optical system 4 may be changed as appropriate according to the device to which it is applied.
  • FCCS since each molecule that interacts is labeled with a different fluorescent dye and observed, usually, as many photon detectors as the number of fluorescent dyes are required.
  • the photon detection device of the present invention can remove the influence of a false signal due to the after-pulse phenomenon with one photon detector (SPAD).
  • PAD photon detector
  • the output of the semiconductor laser 2 is modulated in two stages of High and Low, but more stages may be used.
  • the signal distributor 6 distributes the pulse signal from the SPAD 5 to a plurality of outputs (three or more outputs) according to the intensity modulation, and the number of channels of the correlator 7 is set in accordance with the number of distribution of the signal distributor 6. Increase it.
  • the function of the signal distributor described above can be combined with the correlator. In this case, it is possible to omit the signal distributor 6 shown in FIG.
  • Photon detector 2 Semiconductor laser (laser light source) 3 External modulator 4 Optical system 6 Signal distributor 7 Correlator 8 Sample stage 9 Sample 41 Dichroic mirror 42 Objective lens 43 Pinhole

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Abstract

A photon detection device is provided with: a semiconductor laser for outputting laser light while the intensity thereof is moderated in accordance with a modulation signal; a single photon detector in which an avalanche diode is used as a light-receiving element; an optical system for irradiating a specimen with the laser light, the optical system being configured such that light from the specimen is guided to the single photon detector; a signal splitter for splitting the pulse signal from the single photon detector into a plurality of outputs in synchronization with the modulation signal; and a correlator for calculating a correlation function on the basis of the plurality of outputs from the signal splitter.

Description

光子検出装置、光子検出方法、蛍光相関分光測定装置、蛍光相互相関分光測定装置、動的光散乱測定装置、及び、蛍光顕微鏡Photon detection device, photon detection method, fluorescence correlation spectroscopy measurement device, fluorescence cross correlation spectroscopy measurement device, dynamic light scattering measurement device, and fluorescence microscope
　本発明は、光子検出装置、光子検出方法、蛍光相関分光測定装置、蛍光相互相関分光測定装置、動的光散乱測定装置、及び、蛍光顕微鏡に関する。 The present invention relates to a photon detection device, a photon detection method, a fluorescence correlation spectroscopy measurement device, a fluorescence cross correlation spectroscopy measurement device, a dynamic light scattering measurement device, and a fluorescence microscope.
　光子相関計測法は、試料溶液中の粒子のサイズの測定や分子間相互作用の検出に広く用いられている。例えば、光子相関計測法としては、蛍光相関分光法（Fluorescence correlation spectroscopy：ＦＣＳ）や動的光散乱法（Dynamic Light Scattering：ＤＬＳ）などがある。 The photon correlation measurement method is widely used for measuring the size of particles in a sample solution and detecting intermolecular interactions. For example, photon correlation measurement methods include fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS).
　光子相関計測法においては、試料溶液からの蛍光（ＦＣＳ）や光散乱（ＤＬＳ）に由来する光子信号を時系列に計測し、その時間揺らぎに基づいて様々な物理量を求める。光子相関計測法においては、上記の時間揺らぎを定量化するために、時間相関関数を用いる。ある時間間隔ΔＴを伴って検出される２つの光子の対を専用ハードウェアあるいは専用ソフトウェアで計数し、平均光強度で規格化することでΔＴにおける相関信号を求める（式１）。 In the photon correlation measurement method, photon signals derived from fluorescence (FCS) and light scattering (DLS) from a sample solution are measured in time series, and various physical quantities are obtained based on the time fluctuation. In the photon correlation measurement method, a time correlation function is used to quantify the above time fluctuation. Two photon pairs detected with a certain time interval ΔT are counted by dedicated hardware or software, and normalized by the average light intensity to obtain a correlation signal at ΔT (Equation 1).
Figure JPOXMLDOC01-appb-M000001
 　ここで、Ｉ（Ｔ）は、時刻Ｔでの光強度を表し、＜．．．＞は、時間平均を表す。
Figure JPOXMLDOC01-appb-M000001
 Here, I (T) represents the light intensity at time T, and <. . . > Represents a time average.
　光子相関計測法では、光子検出器として、単一光子アバランシェダイオード（以下、ＳＰＡＤ）が広く採用されている。ＳＰＡＤは、光子を検知すると、光子１個につき１つの電圧パルスを出力する。しかしながら、ＳＰＡＤは、光子を検知して電圧パルスを出力した後、数マイクロ秒以内に、ある確率で２つ目の電圧パルス（以下、アフターパルス）を出力する。アフターパルスは、数マイクロ秒の時間領域の相関信号に大きな影響を与える。したがって、ＳＰＡＤを用いた光子相関計測においては、ＳＰＡＤのアフターパルス現象による偽の相関信号が問題となる。 In the photon correlation measurement method, a single photon avalanche diode (hereinafter referred to as SPAD) is widely adopted as a photon detector. When SPAD detects a photon, it outputs one voltage pulse per photon. However, SPAD outputs a second voltage pulse (hereinafter referred to as an after pulse) with a certain probability within a few microseconds after detecting a photon and outputting a voltage pulse. The after pulse greatly affects the correlation signal in the time domain of several microseconds. Therefore, in photon correlation measurement using SPAD, a false correlation signal due to SPAD after-pulse phenomenon becomes a problem.
　従来の光子相関計測において、特にアフターパルスの影響を回避する必要があるときは、検出器を２台用い、それらの信号の相互相関を計算する方法が一般的であった（非特許文献１）。しかしながら、非特許文献１の従来法では、検出器が２台必要であることからコストがかかり、また、光学系が複雑になるという課題があった。さらに、上記のように異なる検出器で検出された光子対のみを使用することから、同じ検出器で検出された光子対の情報（全光子対の５０％）を捨てることになり、信号雑音比が低下するという課題がある。 In the conventional photon correlation measurement, when it is necessary to avoid the influence of the after pulse, a method of calculating the cross-correlation of these signals using two detectors is generally used (Non-patent Document 1). . However, the conventional method of Non-Patent Document 1 has a problem in that it requires two detectors and is expensive, and the optical system becomes complicated. Furthermore, since only photon pairs detected by different detectors are used as described above, information on photon pairs detected by the same detector (50% of all photon pairs) is discarded, and the signal-to-noise ratio is reduced. There is a problem that decreases.
　上記のアフターパルスの偽信号の除去に関し、本願の発明者らは、パルスレーザーと時間相関光子係数法（Time Correlated Single Photon Counting：ＴＣＳＰＣ）を用いて相関信号の時間反転対称性を解析する方法を報告した（非特許文献２）。しかしながら、非特許文献２の方法では、高価なパルスレーザー装置とＴＣＳＰＣ装置が必要となるため、測定装置全体のコストが増大するという課題があり、通常の光子相関計測への応用は現実的ではなかった。 Regarding the removal of the false signal of the above after pulse, the inventors of the present application analyzed a time reversal symmetry of a correlation signal by using a pulse laser and a time correlated photon coefficient method (Time Correlated Single Photon Counting: TCSPC). Reported (Non-Patent Document 2). However, since the method of Non-Patent Document 2 requires an expensive pulse laser device and a TCSPC device, there is a problem that the cost of the entire measuring device increases, and application to normal photon correlation measurement is not realistic. It was.
　また、非特許文献３では、パルスレーザーとＴＣＳＰＣを用いて、アフターパルス信号と蛍光信号の時間挙動の違いからアフターパルスの影響を評価している。非特許文献３の方法も、非特許文献２の方法と同様に、測定装置全体のコストが増大するという課題がある。 In Non-Patent Document 3, the influence of the after pulse is evaluated from the difference in the temporal behavior of the after pulse signal and the fluorescence signal using a pulse laser and TCSPC. Similarly to the method of Non-Patent Document 2, the method of Non-Patent Document 3 has a problem that the cost of the entire measuring apparatus increases.
　本発明は、このような状況に鑑みてなされたものであり、より簡易な構成でアフターパルス現象による偽信号の影響を除去することが可能な技術を提供する。 The present invention has been made in view of such a situation, and provides a technique capable of removing the influence of a false signal due to the after-pulse phenomenon with a simpler configuration.
　上記課題を解決するために、例えば請求の範囲に記載の構成を採用する。本願は上記課題を解決する手段を複数含んでいるが、その一例を挙げるならば、変調信号に従って強度変調しながらレーザ光を出力する半導体レーザと、アバランシェダイオードを受光素子とする単一光子検出器と、前記レーザ光を試料に照射し、前記試料からの光を前記単一光子検出器に導くように構成された光学系と、前記変調信号に同期して、前記単一光子検出器からのパルス信号を複数の出力に分配する信号分配器と、前記信号分配器からの前記複数の出力に基づいて相関関数を計算する相関器と、を備える光子検出装置が提供される。 In order to solve the above problems, for example, the configuration described in the claims is adopted. The present application includes a plurality of means for solving the above-described problems. For example, a semiconductor laser that outputs laser light while intensity-modulating according to a modulation signal, and a single photon detector using an avalanche diode as a light receiving element An optical system configured to irradiate the sample with the laser light and guide the light from the sample to the single photon detector; and from the single photon detector in synchronization with the modulation signal. There is provided a photon detection device comprising: a signal distributor that distributes a pulse signal to a plurality of outputs; and a correlator that calculates a correlation function based on the plurality of outputs from the signal distributor.
　本発明によれば、より簡易な構成でアフターパルス現象による偽信号の影響を除去することができる。本発明に関連する更なる特徴は、本明細書の記述、添付図面から明らかになるものである。また、上記した以外の、課題、構成および効果は、以下の実施例の説明により明らかにされる。 According to the present invention, the influence of the false signal due to the after-pulse phenomenon can be removed with a simpler configuration. Further features related to the present invention will become apparent from the description of the present specification and the accompanying drawings. Further, problems, configurations and effects other than those described above will be clarified by the description of the following examples.
本発明の光子検出装置の一実施例の構成図である。It is a block diagram of one Example of the photon detection apparatus of this invention. レーザ光による励起と、それによって得られる信号光子列を示す図である。It is a figure which shows the excitation by a laser beam, and the signal photon row | line | column obtained by it. アフターパルスの影響を含まない真の蛍光相関信号と、アフターパルスの由来の相関信号の時間反転対称性を説明する図である。It is a figure explaining the time reversal symmetry of the true fluorescence correlation signal which does not include the influence of an after pulse, and the correlation signal derived from an after pulse. 光子検出装置での処理の流れを示すフローチャートである。It is a flowchart which shows the flow of a process in a photon detection apparatus. 半導体レーザにおける強度変調と、その強度変調に応じた信号光強度の時間揺らぎの計測とを説明する図である。It is a figure explaining intensity modulation in a semiconductor laser and measurement of time fluctuation of signal light intensity according to the intensity modulation. 信号分配器の第１の例を示す図である。It is a figure which shows the 1st example of a signal distributor. 信号分配器の第２の例を示す図である。It is a figure which shows the 2nd example of a signal distributor. 信号分配器の第３の例を示す図である。It is a figure which shows the 3rd example of a signal distributor. アルミホイルの表面からの散乱光を観測した実験例を示すグラフである。It is a graph which shows the experimental example which observed the scattered light from the surface of an aluminum foil. ＢＳＡ（ウシ血清アルブミン）の水溶液からの散乱光を測定した実験例を示すグラフである。It is a graph which shows the experimental example which measured the scattered light from the aqueous solution of BSA (bovine serum albumin).
　以下、添付図面を参照して本発明の実施例について説明する。なお、添付図面は本発明の原理に則った具体的な実施例を示しているが、これらは本発明の理解のためのものであり、決して本発明を限定的に解釈するために用いられるものではない。 Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. The accompanying drawings show specific embodiments in accordance with the principle of the present invention, but these are for the understanding of the present invention, and are never used to interpret the present invention in a limited manner. is not.
　図１は、本発明の光子検出装置の一実施例の構成図である。図１は、本発明の光子検出装置を蛍光相関分光（ＦＣＳ）測定装置に適用した例を示す。光子検出装置１は、半導体レーザ（レーザ光源）２と、外部変調器３と、光学系４と、ＳＰＡＤ５と、信号分配器６と、相関器７とを備える。 FIG. 1 is a configuration diagram of an embodiment of the photon detection device of the present invention. FIG. 1 shows an example in which the photon detection device of the present invention is applied to a fluorescence correlation spectroscopy (FCS) measurement device. The photon detection device 1 includes a semiconductor laser (laser light source) 2, an external modulator 3, an optical system 4, a SPAD 5, a signal distributor 6, and a correlator 7.
　半導体レーザ２は、連続的にレーザ光を出すＣＷレーザ（Continuous wave laser）である。外部変調器３は、外部変調信号を半導体レーザ２及び信号分配器６へ出力する。外部変調器３は、半導体レーザ２のレーザ光を強度変調するために使用されており、例えば、数十ＭＨｚ程度の周波数で変調が可能なものであればよい。半導体レーザ２は、外部変調器３からの外部変調信号に従って強度変調しながらレーザ光を出力する。 The semiconductor laser 2 is a CW laser (Continuous wave laser) that emits laser light continuously. The external modulator 3 outputs an external modulation signal to the semiconductor laser 2 and the signal distributor 6. The external modulator 3 is used to modulate the intensity of the laser light from the semiconductor laser 2, and may be any one that can modulate at a frequency of about several tens of MHz, for example. The semiconductor laser 2 outputs laser light while performing intensity modulation according to the external modulation signal from the external modulator 3.
　光学系４は、半導体レーザ２からのレーザ光を試料台８に載置された試料９に照射し、試料９からの光をＳＰＡＤ５に導くように構成されている。光学系４は、ダイクロイックミラー４１と、対物レンズ４２と、ピンホール４３とを備える。なお、図１の光学系４に関しては主要な構成要素のみを示しており、これ以外に他の構成要素（ミラー、レンズなど）を含んでもよい。 The optical system 4 is configured to irradiate the sample 9 placed on the sample stage 8 with the laser light from the semiconductor laser 2 and to guide the light from the sample 9 to the SPAD 5. The optical system 4 includes a dichroic mirror 41, an objective lens 42, and a pinhole 43. Note that only the main components are shown with respect to the optical system 4 in FIG. 1, and other components (mirror, lens, etc.) may be included in addition to this.
　ダイクロイックミラー４１は、半導体レーザ２からのレーザ光（励起光）を反射して、試料９からの蛍光（例えば、励起光で励起される蛍光色素からの蛍光）を透過する光学特性を有する。半導体レーザ２から出たレーザ光は、ダイクロイックミラー４１で反射し、その後、対物レンズ４２に入射する。対物レンズ４２の焦点位置には、試料９が配置されている。試料９から発した蛍光は、再び対物レンズ４２を介してダイクロイックミラー４１に達する。当該蛍光は、ダイクロイックミラー４１を通過し、ピンホール４３を経たのち、ＳＰＡＤ５に導かれる。 The dichroic mirror 41 has an optical characteristic of reflecting the laser light (excitation light) from the semiconductor laser 2 and transmitting fluorescence from the sample 9 (for example, fluorescence from a fluorescent dye excited by excitation light). Laser light emitted from the semiconductor laser 2 is reflected by the dichroic mirror 41 and then enters the objective lens 42. A sample 9 is disposed at the focal position of the objective lens 42. The fluorescence emitted from the sample 9 reaches the dichroic mirror 41 through the objective lens 42 again. The fluorescence passes through the dichroic mirror 41, passes through the pinhole 43, and is guided to SPAD 5.
　ＳＰＡＤ５は、アバランシェダイオードを受光素子とする単一光子検出器である。ＳＰＡＤ５は、上述の通り、光子を検知すると光子１個につき１つの電圧パルスを出力する。ＳＰＡＤ５は、ピンホール４３を経て受光した光に応じて、パルス信号を信号分配器６に出力する。 SPAD5 is a single photon detector using an avalanche diode as a light receiving element. As described above, SPAD 5 outputs one voltage pulse for each photon when it detects a photon. The SPAD 5 outputs a pulse signal to the signal distributor 6 according to the light received through the pinhole 43.
　信号分配器６は、ＳＰＡＤ５からのパルス信号を２つの出力に分配する。より具体的には、信号分配器６は、外部変調器３からの外部変調信号に同期して、ＳＰＡＤ５からのパルス信号を２つの出力に分配し、それら２つの出力を相関器７の２つのチャネル（ｃｈ１、ｃｈ２）の各々に入力する。 The signal distributor 6 distributes the pulse signal from the SPAD 5 to two outputs. More specifically, the signal distributor 6 distributes the pulse signal from the SPAD 5 to two outputs in synchronization with the external modulation signal from the external modulator 3, and distributes these two outputs to the two outputs of the correlator 7. Input to each of the channels (ch1, ch2).
　相関器７は、２つのチャネル（ｃｈ１、ｃｈ２）に入力された信号に基づいて相関関数を計算する。相関器７における処理は、例えば集積回路で設計する等によりハードウェアで実現されてもよい。また、別の例として、相関器７における処理は、プロセッサがその処理に対応するプログラムを解釈し、実行することによりソフトウェアで実現されてもよい。 Correlator 7 calculates a correlation function based on the signals input to the two channels (ch1, ch2). The processing in the correlator 7 may be realized by hardware by designing with an integrated circuit, for example. As another example, the processing in the correlator 7 may be realized by software by the processor interpreting and executing a program corresponding to the processing.
　次に、アフターパルス現象による偽信号を除去する原理について説明する。図２Ａは、レーザ光による励起と、それによって得られる信号光子列を示す。光強度の変化（時間揺らぎ）を、時刻（マクロタイム、Ｔ）と励起後からの遅延時間（マイクロタイム、ｔ）の関数Ｉ（Ｔ；ｔ）として表す。ある時刻Ｔの蛍光信号強度Ｉ（Ｔ）とそこからΔＴ経過した時点でのｔに依存する蛍光強度Ｉ（Ｔ＋ΔＴ；ｔ）の間の相関関数を考える。 Next, the principle of removing false signals due to the after-pulse phenomenon will be described. FIG. 2A shows excitation by laser light and the signal photon train obtained thereby. The change in light intensity (time fluctuation) is expressed as a function I (T; t) of time (macro time, T) and delay time (micro time, t) after excitation. Consider a correlation function between the fluorescence signal intensity I (T) at a certain time T and the fluorescence intensity I (T + ΔT; t) depending on t at the time when ΔT has passed.
　ここで、アフターパルスの影響を含まない真の蛍光相関信号と、アフターパルスの由来の相関信号の時間反転対称性について説明する。図２Ｂは、これらの時間反転対称性を簡単に説明する図である。本願発明者らが非特許文献２で報告している通り、アフターパルスの影響を含まない真の蛍光相関関数は、系が平衡状態にある場合、時間反転ΔＴ→－ΔＴに対して対称になる。一方、アフターパルスの由来の相関関数は、時間反転ΔＴ→－ΔＴに対して非対称なｔ依存性を示す。 Here, the time-reversal symmetry of the true fluorescence correlation signal not including the influence of the after pulse and the correlation signal derived from the after pulse will be described. FIG. 2B is a diagram simply explaining these time-reversal symmetries. As reported by the present inventors in Non-Patent Document 2, the true fluorescence correlation function that does not include the influence of afterpulse is symmetric with respect to the time reversal ΔT → −ΔT when the system is in an equilibrium state. . On the other hand, the correlation function derived from the after pulse shows asymmetric t dependence with respect to the time reversal ΔT → −ΔT.
　本発明は、上述の時間対称性を利用して相関信号に対するアフターパルス現象の影響の大きさを判定するものである。これにより、アフターパルス現象による偽信号を除去した正確な相関計測を行うことが可能になる。 In the present invention, the magnitude of the influence of the after-pulse phenomenon on the correlation signal is determined using the above-described time symmetry. As a result, it is possible to perform accurate correlation measurement by removing the false signal due to the after-pulse phenomenon.
　次に、図１の光子検出装置１の処理の流れについて説明する。図３は、光子検出装置１での処理の流れを示すフローチャートである。 Next, the process flow of the photon detection device 1 in FIG. 1 will be described. FIG. 3 is a flowchart showing the flow of processing in the photon detection device 1.
　半導体レーザ２は、強度変調しながらレーザ光を出力する（ステップ３０１）。図４の上側の図は、横軸を時間Ｔ、縦軸をレーザの強度とした図であり、半導体レーザ２における強度変調を説明する図である。半導体レーザ２は、外部変調器３からの外部変調信号に従って、第１の強度（Ｈｉｇｈ）と第２の強度（Ｌｏｗ）との間で強度変調をしながらレーザを出力する。 The semiconductor laser 2 outputs laser light while performing intensity modulation (step 301). The upper diagram in FIG. 4 is a diagram in which the horizontal axis represents time T and the vertical axis represents laser intensity, and is a diagram for explaining intensity modulation in the semiconductor laser 2. The semiconductor laser 2 outputs a laser while performing intensity modulation between a first intensity (High) and a second intensity (Low) in accordance with an external modulation signal from the external modulator 3.
　次に、ＳＰＡＤ５及び信号分配器６を用いて、強度変調に同期して、光強度の時間揺らぎＩＨ（Ｔ）、ＩＬ（Ｔ）を別々に計測する（ステップ３０２）。図４の下側の図は、横軸を時間Ｔ、縦軸を光強度Ｉ（Ｔ）とした図であり、信号光強度の時間揺らぎの計測を説明する図である。図４の下側の図の実線は、ＳＰＡＤ５から出力されるパルス信号を示す。符号４０１の点線で囲まれた部分は、半導体レーザ２の出力が第１の強度（Ｈｉｇｈ）のときの光強度に対応し、符号４０２の点線で囲まれた部分は、半導体レーザ２の出力が第２の強度（Ｌｏｗ）のときの光強度に対応する。ここでは、半導体レーザ２の出力が第１の強度（Ｈｉｇｈ）のときの光強度の時間揺らぎをＩＨ（Ｔ）と定義し、半導体レーザ２の出力が第２の強度（Ｌｏｗ）のときの光強度の時間揺らぎをＩＬ（Ｔ）と定義する。なお、ここでは、第１の強度が第２の強度より大きい例を示すが、これに限定されない。本発明において「第１の強度」、「第２の強度」と表現する場合、これらは単に強度が異なることを表しており、強度の大小が逆であってもよい。 Next, the time fluctuations I H (T) and I L (T) of the light intensity are separately measured in synchronization with the intensity modulation using the SPAD 5 and the signal distributor 6 (step 302). The lower diagram in FIG. 4 is a diagram in which the horizontal axis represents time T and the vertical axis represents light intensity I (T), and is a diagram for explaining measurement of signal light intensity temporal fluctuation. The solid line in the lower diagram of FIG. 4 indicates the pulse signal output from SPAD5. The portion surrounded by the dotted line 401 corresponds to the light intensity when the output of the semiconductor laser 2 is the first intensity (High), and the portion surrounded by the dotted line 402 is the output of the semiconductor laser 2. This corresponds to the light intensity at the second intensity (Low). Here, the time fluctuation of the light intensity when the output of the semiconductor laser 2 is the first intensity (High) is defined as I H (T), and the output when the output of the semiconductor laser 2 is the second intensity (Low). The time fluctuation of the light intensity is defined as I L (T). Here, an example in which the first intensity is larger than the second intensity is shown, but the present invention is not limited to this. In the present invention, when expressed as “first strength” and “second strength”, these simply indicate that the strengths are different, and the magnitudes of the strengths may be reversed.
　信号分配器６には、外部変調器３からの外部変調信号と、ＳＰＡＤ５からのパルス信号が入力される。信号分配器６は、外部変調器３からの外部変調信号に同期して、第１の強度（Ｈｉｇｈ）に対応する光強度の時間揺らぎＩＨ（Ｔ）を相関器７のチャネル（ｃｈ１）に出力し、第２の強度（Ｌｏｗ）に対応する光強度の時間揺らぎＩＬ（Ｔ）を相関器７のチャネル（ｃｈ２）に出力する。 The signal distributor 6 receives an external modulation signal from the external modulator 3 and a pulse signal from the SPAD 5. The signal distributor 6 synchronizes with the external modulation signal from the external modulator 3 and transmits the time fluctuation I H (T) of the light intensity corresponding to the first intensity (High) to the channel (ch 1) of the correlator 7. The time fluctuation I L (T) of the light intensity corresponding to the second intensity (Low) is output to the channel (ch 2) of the correlator 7.
　相関器７は、ある時刻Ｔの蛍光信号強度Ｉ（Ｔ）とそこからΔＴ経過した時点での蛍光強度Ｉ（Ｔ＋ΔＴ）の間の相関関数Ｇ（ΔＴ）を計算する（ステップ３０３）。相関器７は、＜ＩＨ（Ｔ）ＩＨ（Ｔ＋ΔＴ）＞、＜ＩＨ（Ｔ）ＩＬ（Ｔ＋ΔＴ）＞、＜ＩＬ（Ｔ）ＩＨ（Ｔ＋ΔＴ）＞、＜ＩＬ（Ｔ）ＩＬ（Ｔ＋ΔＴ）＞、＜ＩＨ（Ｔ）＞、＜ＩＬ（Ｔ）＞を求めることによって、相関関数Ｇ（ΔＴ）を計算する。ここで、＜．．．＞は、上述の通り、時間平均を表す。 The correlator 7 calculates a correlation function G (ΔT) between the fluorescence signal intensity I (T) at a certain time T and the fluorescence intensity I (T + ΔT) at the time when ΔT has elapsed therefrom (step 303). The correlator 7 includes <I H (T) I H (T + ΔT)>, <I H (T) I L (T + ΔT)>, <I L (T) I H (T + ΔT)>, <I L (T) The correlation function G (ΔT) is calculated by obtaining I L (T + ΔT)>, <I H (T)>, <I L (T)>. Where <. . . > Represents a time average as described above.
　より詳細には、相関器７は、以下の式（２）に従って、相関関数Ｇ（ΔＴ）を計算する。
Figure JPOXMLDOC01-appb-M000002
 More specifically, the correlator 7 calculates the correlation function G (ΔT) according to the following equation (2).
Figure JPOXMLDOC01-appb-M000002
　式（２）の右辺において、第１項は通常の相関信号に対応する項であり、第２項はアフターパルスの寄与項である。したがって、本実施例によれば、アフターパルスの偽信号の時間非対称性の大きさを評価することでアフターパルスの影響の大きさを求め、通常の相関信号からアフターパルスの影響の大きさを差し引くことで、正確な相関計測を行うことができる。 On the right side of Equation (2), the first term is a term corresponding to a normal correlation signal, and the second term is a contribution term of the after pulse. Therefore, according to the present embodiment, the magnitude of the influence of the after pulse is obtained by evaluating the magnitude of the temporal asymmetry of the pseudo signal of the after pulse, and the magnitude of the influence of the after pulse is subtracted from the normal correlation signal. Thus, accurate correlation measurement can be performed.
　次に、信号分配器６の構成について説明する。図５は、信号分配器の第１の例を示す。信号分配器６は、論理演算回路によって構成されており、第１のＡＮＤ回路６１と、第２のＡＮＤ回路６２と、ＮＯＴ回路６３とを備える。 Next, the configuration of the signal distributor 6 will be described. FIG. 5 shows a first example of a signal distributor. The signal distributor 6 is configured by a logic operation circuit, and includes a first AND circuit 61, a second AND circuit 62, and a NOT circuit 63.
　ＳＰＡＤ５の出力が、第１のＡＮＤ回路６１の一方の入力端子に入力され、外部変調器３からの外部変調信号が、第１のＡＮＤ回路６１の他方の入力端子に入力される。したがって、第１のＡＮＤ回路６１の出力は、第１の強度（Ｈｉｇｈ）のときの光強度の時間揺らぎＩＨ（Ｔ）に対応する。 The output of the SPAD 5 is input to one input terminal of the first AND circuit 61, and the external modulation signal from the external modulator 3 is input to the other input terminal of the first AND circuit 61. Therefore, the output of the first AND circuit 61 corresponds to the temporal fluctuation I H (T) of the light intensity at the first intensity (High).
　また、ＳＰＡＤ５の出力が、第２のＡＮＤ回路６２の一方の入力端子に入力され、外部変調器３からの外部変調信号が、ＮＯＴ回路６３の入力端子に入力され、ＮＯＴ回路６３の出力が、第２のＡＮＤ回路６２の他方の入力端子に入力される。したがって、第２のＡＮＤ回路６２の出力は、第２の強度（Ｌｏｗ）のときの光強度の時間揺らぎＩＬ（Ｔ）に対応する。図５の構成の場合、相関器７における相関関数の計算式は、式（２）と同様である。 The output of SPAD5 is input to one input terminal of the second AND circuit 62, the external modulation signal from the external modulator 3 is input to the input terminal of the NOT circuit 63, and the output of the NOT circuit 63 is This is input to the other input terminal of the second AND circuit 62. Therefore, the output of the second AND circuit 62 corresponds to the time fluctuation I L (T) of the light intensity at the second intensity (Low). In the case of the configuration of FIG. 5, the calculation function of the correlation function in the correlator 7 is the same as the expression (2).
　図６は、信号分配器６の第２の例を示す。信号分配器６は、ＡＮＤ回路６４を備える。ＳＰＡＤ５の出力が、相関器７のチャネル（ｃｈ１）に直接入力される。したがって、チャネルｃｈ１には、第１の強度（Ｈｉｇｈ）と第２の強度（Ｌｏｗ）の両方の場合の光強度の時間揺らぎＩＨ＋Ｌ（Ｔ）が入力されることになる。 FIG. 6 shows a second example of the signal distributor 6. The signal distributor 6 includes an AND circuit 64. The output of SPAD5 is directly input to the channel (ch1) of the correlator 7. Therefore, the time fluctuation I H + L (T) of the light intensity in both the first intensity (High) and the second intensity (Low) is input to the channel ch1.
　また、ＳＰＡＤ５の出力が、ＡＮＤ回路６４の一方の入力端子に入力され、外部変調器３からの外部変調信号が、ＡＮＤ回路６４の他方の入力端子に入力される。したがって、ＡＮＤ回路６４の出力は、第１の強度（Ｈｉｇｈ）のときの光強度の時間揺らぎＩＨ（Ｔ）に対応する。図６の構成によれば、信号分配器６を、より簡易な回路で構成することができる。 The output of SPAD 5 is input to one input terminal of AND circuit 64, and the external modulation signal from external modulator 3 is input to the other input terminal of AND circuit 64. Accordingly, the output of the AND circuit 64 corresponds to the temporal fluctuation I H (T) of the light intensity at the first intensity (High). According to the configuration of FIG. 6, the signal distributor 6 can be configured with a simpler circuit.
　図６の構成の場合、相関器７における相関関数の計算式は、以下の式（３）のようになる。
Figure JPOXMLDOC01-appb-M000003
 　ここで、Ｉ１（Ｔ）は、相関器７のチャネル（ｃｈ１）に入力された光強度を表し、Ｉ２（Ｔ）は、相関器７のチャネル（ｃｈ２）に入力された光強度を表す。 In the case of the configuration of FIG. 6, the calculation function of the correlation function in the correlator 7 is as shown in the following expression (3).
Figure JPOXMLDOC01-appb-M000003
 Here, I 1 (T) represents the light intensity input to the channel (ch1) of the correlator 7, and I 2 (T) represents the light intensity input to the channel (ch2) of the correlator 7. .
　図７は、信号分配器６の第３の例を示す。信号分配器６は、ＮＯＴ回路６５と、ＡＮＤ回路６６とを備える。ＳＰＡＤ５の出力が、相関器７のチャネル（ｃｈ１）に直接入力される。したがって、チャネルｃｈ１には、第１の強度（Ｈｉｇｈ）と第２の強度（Ｌｏｗ）の両方の場合の光強度の時間揺らぎＩＨ＋Ｌ（Ｔ）が入力されることになる。 FIG. 7 shows a third example of the signal distributor 6. The signal distributor 6 includes a NOT circuit 65 and an AND circuit 66. The output of SPAD5 is directly input to the channel (ch1) of the correlator 7. Therefore, the time fluctuation I H + L (T) of the light intensity in both the first intensity (High) and the second intensity (Low) is input to the channel ch1.
　また、ＳＰＡＤ５の出力が、ＡＮＤ回路６６の一方の入力端子に入力され、外部変調器３からの外部変調信号が、ＮＯＴ回路６５の入力端子に入力され、ＮＯＴ回路６５の出力が、ＡＮＤ回路６６の他方の入力端子に入力される。したがって、ＡＮＤ回路６６の出力は、第２の強度（Ｌｏｗ）のときの光強度の時間揺らぎＩＬ（Ｔ）に対応する。図７の構成の場合、相関器７における相関関数の計算式は、式（３）と同様である。 The output of SPAD 5 is input to one input terminal of AND circuit 66, the external modulation signal from external modulator 3 is input to the input terminal of NOT circuit 65, and the output of NOT circuit 65 is the AND circuit 66. To the other input terminal. Accordingly, the output of the AND circuit 66 corresponds to the light intensity temporal fluctuation I L (T) at the second intensity (Low). In the case of the configuration of FIG. 7, the calculation function of the correlation function in the correlator 7 is the same as the expression (3).
　上述では、信号分配器６の３つの例を説明したが、信号分配器６の構成はこれらに限定されない。信号分配器６が外部変調信号に同期してＳＰＡＤ５からのパルス信号を相関器７の各チャネルに分配できればよく、パルス信号の分配機能が実現できるならば他の構成でもかまわない。また、相関関数の計算式も上述したものに限定されず、適宜変更されてよい。 In the above description, three examples of the signal distributor 6 have been described, but the configuration of the signal distributor 6 is not limited thereto. It suffices if the signal distributor 6 can distribute the pulse signal from the SPAD 5 to each channel of the correlator 7 in synchronization with the external modulation signal, and other configurations may be used as long as the pulse signal distribution function can be realized. Further, the calculation formula of the correlation function is not limited to that described above, and may be changed as appropriate.
　図８及び図９は、実験結果を示すグラフである。当該実験において、半導体レーザ２としてＣｏｈｅｒｅｎｔ社製のＯＢＩＳ　４８８　ＬＸを使用した。また、ＳＰＡＤ５としてＩＤＱ（ID Quantique）社製のシングルフォトンディテクタ（id100-20）を使用した。また、Ｎａｔｉｏｎａｌ　Ｉｎｓｔｒｕｍｅｎｔｓ社製のＰＣＩ－６６０２によって、レーザ変調のための信号の出力（外部変調器３に相当）、及び、シングルフォトンディテクタからの信号の記録を行った。ＰＣＩ－６６０２に接続したＰＣ（Personal Computer）によって信号分配器６及び相関器７の機能を実装し、ＰＣＩ－６６０２に記録された信号の分配及び相関関数の計算を行った。なお、相関器７での計算は、上述の式（２）に従って行った。 8 and 9 are graphs showing the experimental results. In this experiment, OBIS 488 LX manufactured by Coherent was used as the semiconductor laser 2. Further, a single photon detector (id100-20) manufactured by IDQ (ID Quantique) was used as SPAD5. Further, a signal for laser modulation (corresponding to the external modulator 3) and a signal from a single photon detector were recorded by PCI-6602 manufactured by National Instruments. The functions of the signal distributor 6 and the correlator 7 were implemented by a PC (Personal Computer) connected to the PCI-6602, and the distribution of the signals recorded in the PCI-6602 and the calculation of the correlation function were performed. The calculation in the correlator 7 was performed according to the above-described equation (2).
　図８は、参照試料としてアルミホイルを使用し、アルミホイルの表面からの散乱光を観測した結果を示す。なお、当該実験において、レーザ強度はピークパワーで５μＷになるまで減光し、図８のグラフは、約１０秒の測定を１０回繰り返した結果の平均を示す。参照試料での実験に関して、アフターパルスの影響がない場合、相関がない（Correlation = 1）ことが期待されるが、ほぼ期待通りの結果となった。 FIG. 8 shows the result of observing scattered light from the surface of the aluminum foil using an aluminum foil as a reference sample. In this experiment, the laser intensity was reduced until the peak power reached 5 μW, and the graph of FIG. 8 shows an average of results obtained by repeating the measurement for about 10 seconds 10 times. Regarding the experiment with the reference sample, when there was no afterpulse effect, it was expected that there was no correlation (Correlation = 1), but the result was almost as expected.
　図９は、試料９としてＢＳＡ（ウシ血清アルブミン）の０．４ｍｇ／ｍＬ水溶液を使用し、ＢＳＡの水溶液からの散乱光を測定した結果を示す。当該実験において、レーザ強度のピークパワーは２５ｍＷであり、図９のグラフは、約１２秒の測定を９回繰り返した結果の平均を示す。図９に示すように、アフターパルス除去を適用した後に本来期待される相関信号が観測できた。当該実験結果から、１台の検出器のみを用いてアフターパルス現象による偽信号の影響を除去できることが確認できた。 FIG. 9 shows the result of measuring the scattered light from an aqueous solution of BSA using a 0.4 mg / mL aqueous solution of BSA (bovine serum albumin) as sample 9. In the experiment, the peak power of the laser intensity is 25 mW, and the graph of FIG. 9 shows the average of the results of repeating the measurement for about 12 seconds 9 times. As shown in FIG. 9, the originally expected correlation signal was observed after applying afterpulse removal. From the experimental results, it was confirmed that the influence of the false signal due to the after-pulse phenomenon can be removed using only one detector.
　上述した実施例の効果について説明する。従来では、２台の検出器を用いる構成（非特許文献１）であったため、コストがかかり、光学系が複雑になるという課題があった。また、従来では、検出器で検出された光子対の情報の利用効率が低い（５０％）という課題もあった。これに対して、本実施例では、１台の検出器（ＳＰＡＤ）でアフターパルス現象による偽信号の影響を除去することができる。また、検出器で検出された光子対の情報の利用効率も向上し、信号雑音比が低下するという課題を解決することができる。 The effect of the above-described embodiment will be described. Conventionally, since the configuration uses two detectors (Non-patent Document 1), there is a problem that the cost is increased and the optical system is complicated. Further, conventionally, there is a problem that the use efficiency of the information of the photon pair detected by the detector is low (50%). On the other hand, in the present embodiment, the influence of the false signal due to the after-pulse phenomenon can be removed with one detector (SPAD). Moreover, the utilization efficiency of the information of the photon pair detected by the detector is improved, and the problem that the signal-to-noise ratio is reduced can be solved.
　また、従来では、高価なパルスレーザー装置とＴＣＳＰＣ装置を用いる構成（非特許文献２）であったため、測定装置全体のコストが増大するという課題があった。これに対して、上述の実施例によれば、光子検出装置１が安価なＣＷ半導体レーザと通常の相関器から構成されるので、低コストかつ簡易な構成によって、アフターパルス現象による偽信号の影響を除去することができる。 In addition, since the conventional configuration uses an expensive pulse laser device and a TCSPC device (Non-Patent Document 2), there is a problem that the cost of the entire measuring device increases. On the other hand, according to the above-described embodiment, since the photon detection device 1 is composed of an inexpensive CW semiconductor laser and a normal correlator, the influence of the false signal due to the after-pulse phenomenon can be achieved with a low-cost and simple configuration. Can be removed.
　また、光子相関計測では、ＳＰＡＤ以外の検出器（光電子増倍管（ＰＭＴ）、ハイブリッド検出器（ＨＰＤ））を使う場合もあり得る。ＰＭＴはアフターパルスの確率が低く、また、ＨＰＤはアフターパルスをほぼ無視できるが、これらの最高感度はＳＰＡＤに及ばない。本実施例では、アフターパルス現象による偽信号の影響を除去することができるため、感度に優れたＳＰＡＤを利用することが可能となる。 In photon correlation measurement, a detector other than SPAD (photomultiplier tube (PMT), hybrid detector (HPD)) may be used. PMT has a low probability of afterpulses and HPD can almost ignore afterpulses, but their maximum sensitivity is less than SPAD. In this embodiment, the influence of the false signal due to the after-pulse phenomenon can be removed, so that SPAD having excellent sensitivity can be used.
　なお、本発明は上述した実施例に限定されるものではなく、様々な変形例が含まれる。例えば、上述した実施例は本発明を分かりやすく説明するために詳細に説明したものであり、必ずしも説明した全ての構成を備えるものに限定されるものではない。また、実施例の構成の一部について、他の構成の追加・削除・置換をすることが可能である。 In addition, this invention is not limited to the Example mentioned above, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. In addition, it is possible to add, delete, and replace other configurations for a part of the configuration of the embodiment.
　上述では、本発明の光子検出装置をＦＣＳに適用した例を示したが、本発明の光子検出装置は、蛍光相互相関分光（ＦＣＣＳ）測定装置に適用されてもよい。また、本発明の光子検出装置は、動的光散乱（ＤＬＳ）測定装置に適用されてもよい。また、本発明の光子検出装置は、蛍光顕微鏡に適用されてもよい。さらに、本発明の光子検出装置は、上記以外の他の装置に適用されてもよい。なお、光学系４の構成は、適用する装置に応じて適宜変更されてよい。 In the above description, the photon detection device of the present invention is applied to the FCS. However, the photon detection device of the present invention may be applied to a fluorescence cross correlation spectroscopy (FCCS) measurement device. Further, the photon detection device of the present invention may be applied to a dynamic light scattering (DLS) measurement device. The photon detection device of the present invention may be applied to a fluorescence microscope. Furthermore, the photon detection device of the present invention may be applied to devices other than those described above. The configuration of the optical system 4 may be changed as appropriate according to the device to which it is applied.
　なお、本発明をＦＣＣＳに適用した場合、以下の利点がある。ＦＣＣＳでは、相互作用する分子ごとに異なる蛍光色素で標識して観察するため、通常、蛍光色素の数だけ光子検出装置を要する。上述のように、本発明の光子検出装置は、１台の光子検出器（ＳＰＡＤ）でアフターパルス現象による偽信号の影響を除去できることから、ＦＣＣＳにおいては、非特許文献１の従来法に対して、本発明の光子検出装置を備えていることの優位性は一層大きい。 When the present invention is applied to FCCS, there are the following advantages. In FCCS, since each molecule that interacts is labeled with a different fluorescent dye and observed, usually, as many photon detectors as the number of fluorescent dyes are required. As described above, the photon detection device of the present invention can remove the influence of a false signal due to the after-pulse phenomenon with one photon detector (SPAD). The advantage of having the photon detection device of the present invention is even greater.
　上述では、半導体レーザ２の出力をＨｉｇｈとＬｏｗの２段に変調したが、これより多い多段変調にしてもよい。この場合、信号分配器６が強度変調に従ってＳＰＡＤ５からのパルス信号を複数の出力（３つ以上の出力）に分配し、かつ、信号分配器６の分配数に合わせて相関器７のチャネル数を増やせばよい。 In the above description, the output of the semiconductor laser 2 is modulated in two stages of High and Low, but more stages may be used. In this case, the signal distributor 6 distributes the pulse signal from the SPAD 5 to a plurality of outputs (three or more outputs) according to the intensity modulation, and the number of channels of the correlator 7 is set in accordance with the number of distribution of the signal distributor 6. Increase it.
　例えば、入力部分にゲートを備えた相関器が提供されるならば、上述した信号分配器の機能を相関器で兼ねることも可能である。この場合、図１等に記載した信号分配器６を省略することも可能である。 For example, if a correlator having a gate at the input portion is provided, the function of the signal distributor described above can be combined with the correlator. In this case, it is possible to omit the signal distributor 6 shown in FIG.
１　光子検出装置
２　半導体レーザ（レーザ光源）
３　外部変調器
４　光学系
６　信号分配器
７　相関器
８　試料台
９　試料
４１　ダイクロイックミラー
４２　対物レンズ
４３　ピンホール 1 Photon detector 2 Semiconductor laser (laser light source)
3 External modulator 4 Optical system 6 Signal distributor 7 Correlator 8 Sample stage 9 Sample 41 Dichroic mirror 42 Objective lens 43 Pinhole

Claims (8)

  1. 　変調信号に従って強度変調しながらレーザ光を出力する半導体レーザと、
    　アバランシェダイオードを受光素子とする単一光子検出器と、
    　前記レーザ光を試料に照射し、前記試料からの光を前記単一光子検出器に導くように構成された光学系と、
    　前記変調信号に同期して、前記単一光子検出器からのパルス信号を複数の出力に分配する信号分配器と、
    　前記信号分配器からの前記複数の出力に基づいて相関関数を計算する相関器と、
    を備える光子検出装置。     A semiconductor laser that outputs laser light while modulating the intensity according to the modulation signal;
    A single photon detector using an avalanche diode as a light receiving element;
    An optical system configured to irradiate the sample with the laser light and direct the light from the sample to the single photon detector;
    A signal distributor for distributing a pulse signal from the single photon detector to a plurality of outputs in synchronization with the modulation signal;
    A correlator for calculating a correlation function based on the plurality of outputs from the signal distributor;
    A photon detection device comprising:
  2. 　請求項１に記載の光子検出装置において、
    　前記半導体レーザは、第１の強度と第２の強度との間で強度変調をしながら前記レーザ光を出力し、
    　前記信号分配器は、前記単一光子検出器からのパルス信号を、前記第１の強度のときのパルス信号である第１の出力と、前記第２の強度のときのパルス信号である第２の出力とに分配することを特徴とする光子検出装置。     The photon detection device according to claim 1,
    The semiconductor laser outputs the laser beam while performing intensity modulation between a first intensity and a second intensity,
    The signal distributor outputs a pulse signal from the single photon detector to a first output that is a pulse signal at the first intensity and a second pulse signal that is at the second intensity. The photon detection device is distributed to the output of
  3. 　請求項１に記載の光子検出装置において、
    　前記半導体レーザは、第１の強度と第２の強度との間で強度変調をしながら前記レーザ光を出力し、
    　前記信号分配器は、前記単一光子検出器からのパルス信号を、前記第１の強度及び前記第２の強度のときのパルス信号である第１の出力と、前記第１の強度のときのパルス信号である第２の出力とに分配することを特徴とする光子検出装置。     The photon detection device according to claim 1,
    The semiconductor laser outputs the laser beam while performing intensity modulation between a first intensity and a second intensity,
    The signal distributor outputs a pulse signal from the single photon detector, a first output which is a pulse signal at the first intensity and the second intensity, and a pulse signal at the first intensity. A photon detection device that distributes to a second output that is a pulse signal.
  4. 　請求項１から３のいずれか一項に記載の光子検出装置を備える蛍光相関分光測定装置。 A fluorescence correlation spectrometer comprising the photon detection device according to any one of claims 1 to 3.
  5. 　請求項１から３のいずれか一項に記載の光子検出装置を備える蛍光相互相関分光測定装置。 A fluorescence cross-correlation spectrometer comprising the photon detection device according to any one of claims 1 to 3.
  6. 　請求項１から３のいずれか一項に記載の光子検出装置を備える動的光散乱測定装置。 A dynamic light scattering measurement device comprising the photon detection device according to any one of claims 1 to 3.
  7. 　請求項１から３のいずれか一項に記載の光子検出装置を備える蛍光顕微鏡。 A fluorescence microscope comprising the photon detection device according to any one of claims 1 to 3.
  8. 　半導体レーザにより、変調信号に従って強度変調しながらレーザ光を出力するステップと、
    　光学系により、前記レーザ光を試料に照射し、前記試料からの光を、アバランシェダイオードを受光素子とする単一光子検出器に導くステップと、
    　前記単一光子検出器により、前記試料からの光を検出するステップと、
    　信号分配器により、前記変調信号に同期して、前記単一光子検出器からのパルス信号を複数の出力に分配するステップと、
    　相関器により、前記信号分配器からの前記複数の出力に基づいて相関関数を計算するステップと、
    を含む光子検出方法。     Outputting a laser beam while modulating the intensity according to a modulation signal by a semiconductor laser;
    Irradiating the sample with the laser beam by an optical system, and guiding the light from the sample to a single photon detector having an avalanche diode as a light receiving element;
    Detecting light from the sample by the single photon detector;
    Distributing a pulse signal from the single photon detector to a plurality of outputs in synchronization with the modulated signal by a signal distributor;
    Calculating a correlation function by a correlator based on the plurality of outputs from the signal distributor;
    A photon detection method comprising:
PCT/JP2016/083010 2015-11-09 2016-11-08 Photon detection device, photon detection method, fluorescence correlation spectroscopy device, fluorescence cross-correlation spectroscopy device, dynamic light scattering device, and fluorescence microscope WO2017082218A1 (en)

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