JP2016148534A - Fluorescence correlation spectroscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence correlation spectroscope that can accurately measure the correlation of materials without being influenced by an after-pulse of an avalanche photodiode.SOLUTION: A fluorescence correlation spectroscope 1 includes: a superconducting single photon detector 30 detecting change with time in intensity of fluorescent light 21 emitted from a material 15a of a sample 15; and a signal processor 50 processing an intensity signal representing the intensity of the fluorescent light 21 detected by the superconducting single photon detector 30, the signal processor 50 having a correlator 55 obtaining at least either one of an autocorrelation function and a cross-correlation function of the intensity signal of the fluorescent light 21.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a fluorescence correlation spectrometer.

  In order to measure the correlation of materials such as cells and molecules in a solution, a fluorescence correlation spectroscopic device is used. A fluorescence correlation spectrometer is an intensity signal of fluorescent light detected by a photodetector that detects fluorescent light emitted from a fluorescent material labeled with a material such as a molecule in a cell or solution. By obtaining at least one of the autocorrelation function and the cross-correlation function, the number and size of materials such as molecules included in the measurement region, the interaction between a plurality of molecules, and the like are obtained. An avalanche photodiode (APD) has been used as a photodetector for fluorescent light in this fluorescence correlation spectroscopic apparatus (see Patent Document 1 and Non-Patent Document 1).

JP-A-2005-43278

R. Rigler, 3 others, "Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion", European Biophysics Journal, Springer-Verlag, August 1993, Vol. 22, p.169-175

  However, in the avalanche photodiode, an undesirable noise called an after pulse is generated. This after-pulse is thought to be caused by charge carriers trapped in the energy level in the band gap during the avalanche phenomenon. Afterpulses typically appear several hundred nanoseconds (submicroseconds) after photon detection. Due to this after-pulse, there has been a problem that the correlation of materials cannot be accurately measured with a fluorescence correlation spectrometer equipped with an avalanche photodiode.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a fluorescence correlation spectrometer capable of accurately measuring the correlation of materials without being affected by the afterpulse of an avalanche photodiode. It is to be.

  The fluorescence correlation spectroscopic device of the present invention includes a superconducting single photon detector that detects temporal changes in the intensity of fluorescent light emitted from a material contained in a sample, and fluorescent light detected by the superconducting single photon detector. A signal processing unit that processes an intensity signal indicating the intensity of the light, and the signal processing unit includes a correlator that obtains at least one of an autocorrelation function and a cross-correlation function of the intensity signal of fluorescent light. In this specification, the fluorescence correlation spectroscopy apparatus includes not only an apparatus using fluorescence correlation spectroscopy (FCS) but also an apparatus using fluorescence cross correlation spectroscopy (FCCS).

  In the fluorescence correlation spectroscopic apparatus of the present invention, a superconducting single photon detector is used to detect temporal changes in the intensity of fluorescent light emitted from a material contained in a sample. Superconducting single photon detectors do not generate afterpulses of avalanche photodiodes. Therefore, according to the fluorescence correlation spectroscopic apparatus of the present invention, the correlation of materials can be accurately measured without being affected by the afterpulse of the avalanche photodiode.

1 is a schematic diagram of a fluorescence correlation spectroscopy apparatus according to Embodiment 1. FIG. (A) is the schematic diagram of the 1st superconducting single photon detector in Embodiment 1 planarly viewed from the side into which the 1st fluorescence light enters. (B) is a schematic sectional view of the first superconducting single photon detector according to the first embodiment, taken along a sectional line IIB-IIB shown in FIG. 2 (A). It is an expansion schematic diagram of the area | region III shown in FIG. (A) is a figure which shows an example of the fluorescence light intensity detected by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 1 when the molecular weight of the material contained in a light irradiation area | region is small. (B) is a figure which shows an example of the fluorescence light intensity detected by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 1 when the molecular weight of the material contained in a light irradiation area | region is large. (A) is a figure which shows an example of the fluorescence light intensity detected by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 1, when there are few materials contained in a light irradiation area | region. (B) is a figure which shows an example of the fluorescence light intensity detected by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 1 when there are many materials contained in a light irradiation area | region. 6 is a graph showing an example of an autocorrelation function of an intensity signal of fluorescent light obtained by the fluorescence correlation spectroscopic device according to Embodiment 1. FIG. It is a figure which shows the graph showing the autocorrelation function of the intensity signal of the fluorescence light of rhodamine B obtained by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 1, and the fluorescence correlation spectroscopy apparatus using the avalanche photodiode of the comparative example. . 10 ⁻² microseconds or more of the autocorrelation function of the intensity signal of the fluorescent light of rhodamine 6G obtained by the fluorescence correlation spectrometer according to the first embodiment and the fluorescence correlation spectrometer using the avalanche photodiode of the comparative example at 10 ² microseconds or less in the time domain, it shows the overall graph. The correlation function value of the autocorrelation function of the intensity signal of the fluorescent light of rhodamine 6G obtained by the fluorescence correlation spectrometer according to the first embodiment and the fluorescence correlation spectrometer using the avalanche photodiode of the comparative example is 1. It is a figure which shows the graph of 00 vicinity. (A) is the schematic diagram of the 1st superconducting single photon detector in the modification of Embodiment 1 planarly viewed from the side into which the 1st fluorescence light enters. FIG. 10B is a schematic cross-sectional view of the first superconducting single photon detector in the modification of the first embodiment, taken along a cross-sectional line XB-XB shown in FIG. 6 is a schematic diagram of a fluorescence correlation spectroscopic device according to Embodiment 2. FIG. It is an expansion schematic diagram of the area | region XII shown in FIG. (A) is a figure which shows an example of the fluorescence light intensity detected by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 2. FIG. FIG. 13B is an enlarged schematic diagram of a region XIIIB shown in FIG. It is a figure which shows the graph of an example of the autocorrelation function of the intensity | strength signal of the fluorescence light obtained by the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 2. FIG. It is a figure which shows the photograph of the material (Q rod) which is a measuring object of the fluorescence correlation spectroscopy apparatus which concerns on Embodiment 2. FIG. FIG. 6 is an enlarged view showing a graph representing an autocorrelation function of an intensity signal of fluorescent light of a Q rod obtained by a fluorescence correlation spectroscopy apparatus according to Embodiment 2 and a fluorescence correlation spectroscopy apparatus using an avalanche photodiode. . 6 is a schematic diagram of a fluorescence correlation spectroscopic device according to Embodiment 3. FIG. It is an expansion schematic diagram of the area | region XVIII shown in FIG.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Unless otherwise described, the same reference numerals are given to the same components and the description will not be repeated.

(Embodiment 1)
With reference to FIG. 1 to FIG. 3, a fluorescence correlation spectroscopy apparatus 1 according to Embodiment 1 will be described.

  The fluorescence correlation spectroscopy device 1 according to the present embodiment includes an optical system 20, a first superconducting single photon detector (SSPD) 30, a refrigerator 40, and a first superconducting single photon detector 30. And a signal processing unit 50 for processing an intensity signal indicating the intensity of the first fluorescent light 21 detected by.

  The optical system 20 includes a first optical system that causes the first excitation light 11 to be incident on the sample 15 including the first material 15a, and a first fluorescent light 21 that is emitted from the first material 15a. And a second optical system incident on the superconducting single photon detector 30. The first material 15a may be a fluorescent material or a material labeled with a fluorescent material. In the present embodiment, examples of the first material 15a include molecules labeled with a fluorescent material, proteins labeled with a fluorescent material, and cells labeled with a fluorescent material.

  The first optical system includes a first light source 10 and an objective lens 14. The first optical system may further include a first filter 12 and a first wavelength selection element 13.

  The first light source 10 emits first excitation light 11 for exciting the first material 15a included in the sample 15 from the ground state to the excited state. In the present embodiment, the first light source 10 is a laser, and the first excitation light 11 is a laser beam. The first light source 10 is not limited to a laser.

  The intensity | strength of the 1st excitation light 11 irradiated to the sample 15 containing the 1st material 15a by the 1st filter 12 is adjusted. An example of the first filter 12 is an ND filter.

  The first wavelength selection element 13 has a property of reflecting the first excitation light 11 and transmitting the first fluorescent light 21 emitted from the first material 15 a included in the sample 15. An example of the first wavelength selection element 13 is a dichroic mirror. The first excitation light 11 is reflected by the first wavelength selection element 13 and directed to the objective lens 14.

The first excitation light 11 is condensed on the sample 15 by the objective lens 14. Referring to FIG. 2, in the sample 15 including the first material 15a, the first excitation light 11 is condensed on the condensing region 11a. The first excitation light 11 may be narrowed down to the diffraction limit of the first excitation light 11 in the sample 15 by the objective lens 14. By condensing the first excitation light 11 in the sample 15 in the narrow condensing region 11a, the amount of the sample 15 included in the condensing region 11a may be set to about femtoliter (10 ⁻¹⁵ L), for example. it can. By collecting the first excitation light 11 using the objective lens 14, a minute measurement region can be obtained.

  The second optical system includes a first confocal optical system 22. In the present embodiment, the first confocal optical system 22 includes a first lens 23 and a first optical fiber 24. The first confocal optical system 22 may be constituted by another optical element such as a pinhole. The second optical system may further include an objective lens 14, a first wavelength selection element 13, a second filter 26, and a first gradient index lens 25.

  The first fluorescent light 21 emitted from the first material 15 a is collected by the objective lens 14. The light that passes through the objective lens 14 and enters the first wavelength selection element 13 includes the first fluorescent light 21 and the first excitation light 11 reflected or scattered by the sample 15. The first wavelength selective element 13 reflects the first excitation light 11 reflected or scattered by the sample 15, and only the first fluorescent light 21 passes through the first wavelength selective element 13.

  The first fluorescent light 21 transmitted through the first wavelength selection element 13 is incident on the second filter 26. The second filter 26 removes light that does not need to be incident on the first superconducting single photon detector 30. An example of the second filter 26 is a band pass filter.

  The first fluorescent light 21 transmitted through the second filter 26 is incident on the first confocal optical system 22. The first fluorescent light 21 is collected by the first lens 23 and enters the incident end 24 f of the first optical fiber 24. Of the first fluorescent light 21 collected by the first lens 23, only light that can be optically coupled to the core of the first optical fiber 24 propagates in the first optical fiber 24. be able to. Of the first fluorescent light 21 collected by the first lens 23, light that cannot be optically coupled to the core of the first optical fiber 24 propagates in the core of the first optical fiber 24. Can not do it. Therefore, the incident end 24f of the first optical fiber 24 acts like a pinhole. The first confocal optical system 22 having the first lens 23 and the first optical fiber 24 is other than the first fluorescent light 21 emitted from the first material 15 a at the focal position of the objective lens 14. Light is prevented from propagating through the first optical fiber 24. An example of the first optical fiber 24 is a multimode fiber having a core with a diameter of 62.5 μm.

  The first optical fiber 24 extends from the outside of the refrigerator 40 to the inside of the refrigerator 40. The first optical fiber 24 causes the first fluorescent light 21 radiated from the first material 15 a in the sample 15 located outside the refrigerator 40, to the first superconductivity located inside the refrigerator 40. The single photon detector 30 can be optically coupled with high efficiency.

  A first gradient index lens 25 is provided at the exit end of the first optical fiber 24. The first fluorescent light 21 propagating through the first optical fiber 24 is condensed on the first superconducting single photon detector 30 by the first gradient index lens 25. The first fluorescent light 21 emitted from the first material 15a in the sample 15 is optically transmitted to the first superconducting single photon detector 30 by the first gradient index lens 25 with high efficiency. Can be combined.

The first superconducting single-photon detector 30 detects a temporal change in the intensity of the first fluorescent light 21 emitted from the first material 15 a included in the sample 15, and detects the first fluorescent light 21. A first intensity signal indicating the intensity is output to the signal processing unit 50. In the present embodiment, the first superconducting single photon detector 30 includes a substrate 31, a mirror 32, a dielectric layer 33, a superconducting thin wire 34, and a pad electrode 35. The first superconducting single photon detector 30 is cooled by the refrigerator 40 to a temperature not higher than the superconducting transition temperature _{Tc of the} superconducting wire 34.

  In the present embodiment, a silicon substrate is used as the substrate 31. The material of the substrate 31 is not limited to silicon.

  A mirror 32 is provided on the substrate 31. The mirror 32 is provided between the substrate 31 and the superconducting thin wire 34. In the present embodiment, the mirror 32 has a dielectric multilayer film that exhibits a reflectance of 99% or more in a wavelength range of 400 nm to 1000 nm. The mirror 32 is not limited to a dielectric multilayer film. The mirror 32 may have a reflectance of 50% or more, preferably 80% or more, more preferably 90% or more. The mirror 32 is located between the substrate 31 and the superconducting thin wire 34. The mirror 32 reflects the first fluorescent light 21 that has not been absorbed by the superconducting thin wire 34 toward the superconducting thin wire 34.

A dielectric layer 33 is provided on the mirror 32. In the present embodiment, the dielectric layer 33 is deposited on the mirror 32 by high frequency sputtering. The dielectric layer 33 may be formed on the mirror 32 by other deposition methods. In the present embodiment, silicon dioxide is used as the dielectric layer 33. The material of the dielectric layer 33 is not limited to silicon dioxide, and may be a dielectric material having a low light absorption rate with respect to the first fluorescent light 21. The dielectric layer 33 preferably has such a thickness that an optical resonator 36 is formed between the superconducting thin wire 34 and the mirror 32. In other words, the superconducting thin wire 34 and the mirror 32 preferably constitute an optical resonator 36. Since the superconducting thin wire 34 and the mirror 32 constitute the optical resonator 36, the first fluorescent light 21 is more easily absorbed by the superconducting thin wire 34. Therefore, according to the fluorescence correlation spectroscopy device 1 of the present embodiment, the detection efficiency of the first superconducting single photon detector 30 can be further improved. The dielectric layer 33 preferably has a thickness d ₃₃ = λ / 4n so that the superconducting thin wire 34 and the mirror 32 constitute an optical resonator 36. Here, n represents the refractive index of the dielectric layer 33, and λ represents the wavelength of the first fluorescent light 21. The thickness of the dielectric layer 33 is, for example, 100 nm.

A superconducting thin wire 34 and a pad electrode 35 are provided on the substrate 31. The provision of the superconducting thin wire 34 on the substrate 31 includes the fact that the superconducting thin wire 34 is not in direct contact with the substrate 31. In the present embodiment, superconducting thin wires 34 and pad electrodes 35 are provided on dielectric layer 33. In the present embodiment, the superconducting thin wire 34 and the pad electrode 35 mainly contain niobium nitride (NbN). The material of the superconducting thin wire 34 is not limited to niobium nitride. As the material of the superconducting thin wire 34, any material exhibiting superconductivity such as niobium (Nb), MgB ₂ (magnesium diboride), yttrium-based copper oxide superconducting material, bismuth-based copper oxide superconducting material is used. May be.

  In the present embodiment, a niobium nitride film is formed on the dielectric layer 33 by direct current reactive sputtering. Thereafter, the niobium nitride film is patterned by electron beam lithography and reactive ion etching to form superconducting thin wires 34 and pad electrodes 35. The superconducting thin wire 34 and the pad electrode 35 may be formed by other methods.

  In order to improve the detection efficiency of the first fluorescent light 21 in the superconducting thin wire 34, the superconducting thin wire 34 of the present embodiment has a meandering shape when viewed from the incident direction of the first fluorescent light 21 ( Meander shape). The shape of the superconducting thin wire 34 when viewed from the incident direction of the first fluorescent light 21 may be other shapes.

  In order to improve the detection efficiency in the first superconducting single photon detector 30, it is necessary to optically couple the first fluorescent light 21 to the first superconducting single photon detector 30 with high efficiency. . Therefore, the shape of the outer periphery 37 of the superconducting thin wire 34 is preferably similar to the cross-sectional shape of the first fluorescent light 21 on the superconducting thin wire 34. Moreover, it is preferable that the outer periphery 37 of the superconducting thin wire 34 is larger than the cross section of the first fluorescent light 21 on the superconducting thin wire 34. Specifically, the superconducting thin wire 34 of the present embodiment has an outer periphery 37 that is a circle having a diameter of 35 μm. The cross section of the first fluorescent light 21 condensed by the first gradient index lens 25 on the superconducting thin wire 34 also has a circular shape. The shape of the outer periphery 37 of the superconducting thin wire 34 is similar to the cross-sectional shape of the first fluorescent light 21 collected by the first gradient index lens 25 on the superconducting thin wire 34. Therefore, the first fluorescent light 21 can be optically coupled to the superconducting thin wire 34 with high efficiency. Further, the first fluorescent light 21 condensed by the first gradient index lens 25 has a diameter of 28 μm on the superconducting thin wire 34. The outer periphery 37 of the superconducting thin wire 34 is larger than the cross section of the first fluorescent light 21 on the superconducting thin wire 34. Therefore, the first fluorescent light 21 can be optically coupled to the superconducting thin wire 34 with high efficiency. The outer peripheral shape of the superconducting thin wire 34 may be any shape other than a circle.

In the present embodiment, the superconducting thin wire 34 has a thickness of 10.5 nm and a width w _{SC of} 150 nm. And if the adjacent superconductive fine wires 34 spaced the 100nm gap _{G SC,} superconducting thin lines 34 are meandering (see FIG. 2 (A), the FIG. 2 (B)). The thickness, width and gap of the superconducting thin wire 34 are not limited to these values.

The operation of the first superconducting single photon detector 30 will be described.
The refrigerator 40 cools the first superconducting single photon detector 30 to a superconducting transition temperature _{Tc of the} superconducting wire 34 or lower. Thereafter, the superconducting fine wires 34 from the pad electrode 35, for applying a bias current I _b having a smaller current density than a superconducting critical current density I _c. Since the current density of the bias current I _b is slightly smaller than the superconducting critical current density I _c, the superconducting fine wires 34 is in a superconducting state. Therefore, the superconducting thin wire 34 has an electric resistance which is zero, and a zero voltage is generated between the pair of pad electrodes 35.

Here, when a single photon having an energy larger than the superconducting gap energy is incident, the single photon is absorbed by a part of the superconducting thin wire 34. The temperature of the region where the single photon is absorbed in the superconducting thin wire 34 is locally increased, and the region where the temperature is locally increased (hot spot region) is in a normal conduction state. Bias current I _b flows through the superconducting fine wires 34 medium to bypass the hot spot area. Since the current density of the bias current I _b in the region other than the hot spot region exceeds a superconducting critical current density I _c, the normal conducting region is spread throughout the width direction of the superconducting fine wires 34 including the hot spot region. Therefore, the superconducting thin wire 34 has an electric resistance larger than zero, and a voltage larger than zero is generated between the pair of pad electrodes 35.

  Since the first superconducting single-photon detector 30 is cooled by the refrigerator 40, the temperature of the hot spot region immediately decreases, and the superconducting wire 34 returns to the superconducting state. When the superconducting thin wire 34 returns to the superconducting state, zero voltage is generated between the pair of pad electrodes 35.

As a result, the fact that a single photon has entered the first superconducting single photon detector 30 can be observed as one voltage pulse (intensity signal) generated between the pair of pad electrodes 35. In the first superconducting single photon detector 30, no afterpulse is generated in the avalanche photodiode. The first superconducting single photon detector 30 can accurately measure the change in intensity of the first fluorescent light 21 without being affected by the afterpulse of the avalanche photodiode. The first current density of the bias current I _b applied to the superconducting single-photon detector 30 is brought closer to the superconducting critical current density I _c, of a single photon of the first superconducting single-photon detectors 30 The detection efficiency can be further increased. The first superconducting single photon detector 30 may have a detection efficiency of 30% or more, more preferably a detection efficiency of 40% or more, and even more preferably a detection efficiency of 70% or more.

  The signal processing unit 50 includes a first correlator 55. The signal processing unit 50 may further include a first low noise amplifier 51 and a first comparator 52.

  The first low noise amplifier 51 is a circuit that amplifies an intensity signal indicating the intensity of the first fluorescent light 21 obtained by the first superconducting single photon detector 30 with low noise.

  The first comparator 52 is a circuit that converts an intensity signal indicating the intensity of the first fluorescent light 21 obtained by the first superconducting single photon detector 30 into a transistor-transistor logic (TTL) signal. . The first comparator 52 may be a circuit that converts an intensity signal indicating the intensity of the first fluorescent light 21 obtained by the first superconducting single photon detector 30 into another signal.

  The first correlator 55 is a circuit that obtains an autocorrelation function of the intensity signal of the first fluorescent light 21. The first correlator 55 performs the calculation represented by the equation (1) on the intensity of the first fluorescent light 21.

Here, I ₁ (t) represents the intensity signal of the first fluorescent light 21 at time t, <I ₁ (t)> represents the time average of the intensity signal of the first fluorescent light 21, and τ represents the correlation. G ₁₁ (τ) represents the autocorrelation function of the intensity signal of the first fluorescent light 21. An example of the first correlator 55 is a digital correlator.

  A method for measuring the correlation of the material (first material 15a) using the fluorescence correlation spectroscopy apparatus 1 of the present embodiment will be described.

  Referring to FIG. 3, the first material 15a such as a molecule contained in the sample 15 such as a solution or a cell performs translational diffusion movement in a random direction (Brownian motion). By this Brownian motion, the first material 15a constantly enters and exits the minute light collection region 11a. The first fluorescent light 21 is emitted only from the first material 15a located in the minute light collection region 11a. For this reason, the intensity of the first fluorescent light 21 detected by the first superconducting single-photon detector 30 is also randomly determined in response to the first material 15a entering and exiting the minute condensing region 11a at random. Shake.

  When the size of the first material 15a such as a molecule is small and the first material 15a diffuses quickly, the time for the first material 15a to pass through the light collection region 11a is shortened. In addition, even when the viscosity of a minute region around the first material 15a such as a molecule is small and the first material 15a diffuses quickly, the time for the first material 15a to pass through the light collection region 11a is shortened. . Therefore, the first fluorescent light 21 detected by the first superconducting single photon detector 30 fluctuates at a short time interval (see FIG. 4A). On the other hand, when the size of the first material 15a such as a molecule is large and the first material 15a diffuses slowly, the time for the first material 15a to pass through the light collection region 11a becomes long. In addition, even when the viscosity of a minute region around the first material 15a such as a molecule is large and the first material 15a diffuses slowly, the time for the first material 15a to pass through the light collection region 11a is long. Become. Therefore, the first fluorescent light 21 detected by the first superconducting single photon detector 30 fluctuates at a long time interval (see FIG. 4B). As a result, the time fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 includes the size (molecular weight) of the first material 15a such as a molecule, The information regarding the environment of the micro area | region around the 1st material 15a, such as these is included.

  When the number of the first materials 15a such as molecules included in the light collection region 11a is small, the first material 15a exists in the light collection region 11a and the first material in the light collection region 11a. There are times when 15a does not exist. When the first material 15a is present in the light collecting region 11a, the first fluorescent light 21 is detected by the first superconducting single photon detector 30. When the first material 15a is not present in the light collection region 11a, the first fluorescent light 21 is not detected by the first superconducting single photon detector 30. For this reason, the amplitude fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 becomes large (see FIG. 5A). On the other hand, when the number of the first materials 15a such as molecules contained in the light collection region 11a is large, the first material 15a is always present in the light collection region 11a. And the ratio of the number of the 1st materials 15a which go in and out of the condensing area | region 11a with respect to the number of the 1st materials 15a which exist in the condensing area | region 11a becomes small. Therefore, the amplitude fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 is reduced (see FIG. 5B). As a result, the fluctuation in the amplitude signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 includes information on the number of first materials 15a such as molecules. Yes.

  As described above, the fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 includes information on the size (molecular weight) of the first material 15a such as a molecule, And the information regarding the environment of the micro area | region around the 1st material 15a and the information regarding the number of the 1st materials 15a, such as a molecule | numerator, are contained. In the first correlator 55, the calculation represented by the equation (1) is performed on the intensity of the first fluorescent light 21 to obtain the autocorrelation function of the intensity signal of the first fluorescent light 21. Time fluctuation of the intensity signal of the first fluorescent light 21 and fluctuation of the amplitude of the intensity signal of the first fluorescent light 21 can be obtained.

  In the graph of the autocorrelation function of the intensity signal of the first fluorescent light 21 shown in FIG. 6, the horizontal axis of the graph represents time, and the vertical axis of the graph represents the correlation function value. The curve of the autocorrelation function of the intensity signal of the first fluorescent light 21 indicates that the probability that the first material 15a detected in the condensing region 11a at a certain time point exists in the condensing region 11a decreases with time. .

τ _T is the correlation time of the translational diffusion movement of the first material 15b. The correlation time τ _T represents the time for the first material 15a to pass through the light collection region 11a. When the size of the first material 15a such as a molecule is small or the viscosity of a micro region around the first material 15a is small, the first material 15a moves in a translational manner at a high speed. The correlation time τ _T representing the time for passing through the material 15a condensing region 11a is shortened. On the other hand, when the size of the first material 15a such as a molecule is large or the viscosity of the micro region around the first material 15a is large, the first material 15a moves in a translational manner at a low speed. The correlation time τ _T that represents the time for passing through the light condensing region 11a of the first material 15a becomes longer. As a result, information on the size of the first material 15a such as a molecule and information on the environment of a micro region around the first material 15a can be obtained from the correlation time τ _T.

When the number of the first materials 15a such as molecules contained in the light collection region 11a is small, the amplitude signal of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 is used. Fluctuations become larger. Therefore, when the number of first materials 15a such as molecules included in the light collection region 11a is small, the maximum correlation function value _GTmax has a large value. On the other hand, when the number of the first materials 15a such as molecules contained in the light collection region 11a is large, the intensity of the first fluorescent light 21 detected by the first superconducting single photon detector 30. The fluctuation of the signal amplitude is reduced. Therefore, when the number of the first materials 15a such as molecules included in the light collection region 11a is large, the maximum value _GTmax of the correlation function value has a small value. As a result, information on the number of first materials 15a such as molecules included in the light collection region 11a can be obtained from the maximum correlation function value _GTmax .

  FIG. 7 shows a graph representing the autocorrelation function of the intensity signal of the fluorescent light of rhodamine B, obtained by the fluorescence correlation spectroscopy apparatus according to the present embodiment and the fluorescence correlation spectroscopy apparatus of the comparative example. In the fluorescence correlation spectroscopy apparatus of the comparative example, an avalanche photodiode is used as a single photon detector. Sample 15 is a solution containing rhodamine B at a concentration of 1.0 μM. The first material 15a is rhodamine B which is a fluorescent material.

  The autocorrelation function of the intensity signal of the fluorescent light of rhodamine B obtained by the fluorescence correlation spectrometer of the comparative example has a large peak in the time region of several hundred nanoseconds (submicroseconds) or less. This large peak is a false peak based on the afterpulse of the avalanche photodiode, and is not a peak based on the first fluorescent light 21 emitted from rhodamine B (first material 15a). Due to the false peak based on the after-pulse of the avalanche photodiode, the comparative fluorescence correlation spectrometer equipped with the avalanche photodiode accurately measures the correlation of materials in the time domain of several hundred nanoseconds (sub-microseconds) or less. Can not do it.

  On the other hand, the autocorrelation function of the intensity signal of the first fluorescent light 21 of rhodamine B (first material 15a) obtained by the fluorescence correlation spectroscopic device according to the present embodiment is several hundred nanoseconds (sub There is no large peak in the time region below (microsecond). In the present embodiment, the first superconducting single photon detector 30 that does not generate an after pulse is used as the single photon detector that detects the first fluorescent light 21 emitted from the first material 15a. Because. By using the first superconducting single photon detector 30 as a single photon detector for detecting the first fluorescent light 21 emitted from the first material 15a, the influence of the afterpulse of the avalanche photodiode is used. It has become clear that the correlation of materials can be accurately measured without being subjected to the above.

  8 and 9 are graphs showing the autocorrelation function of the fluorescent light intensity signal of rhodamine 6G obtained by the fluorescence correlation spectroscopy apparatus according to the present embodiment and the fluorescence correlation spectroscopy apparatus of the comparative example. In the fluorescence correlation spectroscopy apparatus of the comparative example, an avalanche photodiode is used as a single photon detector. Sample 15 is a solution containing rhodamine 6G at a concentration of 1.0 μM. The first material 15a is rhodamine 6G which is a fluorescent material.

The autocorrelation function of the intensity signal of the first fluorescent light 21 of rhodamine 6G (first material 15a) obtained by the fluorescence correlation spectrometer of the comparative example has a time domain of several hundred nanoseconds (submicroseconds) or less. Has a large peak (see FIG. 8). This large peak is a false peak based on the after pulse of the avalanche photodiode, and is not a peak based on the first fluorescent light 21 emitted from the rhodamine 6G (first material 15a). In addition, the bottom of this large peak also affects the autocorrelation function in the time domain of several hundred microseconds (sub-milliseconds) or less. On the other hand, the autocorrelation function of the intensity signal of the first fluorescent light 21 of rhodamine 6G (first material 15a) obtained by the fluorescence correlation spectroscopic device according to the present embodiment is the afterpulse of the avalanche photodiode. Does not have a large peak based on (see FIG. 8). In the fluorescence correlation spectroscopic device according to the present embodiment, the autocorrelation function of the intensity signal of the first fluorescent light 21 emitted from the rhodamine 6G (first material 15a) is analyzed without being affected by the false peak. Therefore, the correlation time τ _T and the number of rhodamine 6G (first material 15a) included in the light collection region 11a can be accurately obtained. The fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 is caused by the translational diffusion movement of rhodamine 6G (first material 15a) and the rhodamine 6G (first This occurs when the material 15a) enters and exits the light collecting region 11a and the rhodamine 6G relaxes from the triplet state to the singlet state. The autocorrelation function of the intensity signal of the first fluorescent light 21 emitted from the rhodamine 6G (first material 15a) is generally given by equation (2).

Here, N represents the average number of molecules of rhodamine 6G (first material 15a) contained in the measurement volume defined by the condensing region 11a and the first confocal optical system 22, and τ _T is a correlation. Represents the time (diffusion time), s represents the ratio between the length of the measurement volume in the optical axis direction and the length of the measurement volume in the direction perpendicular to the optical axis direction, and Q represents the first of rhodamine 6G contained in the sample 15. Represents the ratio of the number of first materials 15a made of rhodamine 6G in the triplet state to the number of materials 15a, and τ ₃₁ represents the relaxation time for relaxation from the triplet state to the singlet state.

  As shown in FIG. 9, the parameter values obtained by fitting the data of the present embodiment and the data of the comparative example using Expression (2) are shown in Table 1. The solid line in FIG. 9 represents a curve obtained by fitting the data of the present embodiment with Expression (2). The dashed-two dotted line of FIG. 9 represents the curve which fitted the data of the comparative example by Formula (2).

As shown in Table 1, the values of these parameters obtained based on the data of the present embodiment are different from the values of these parameters obtained based on the data of the comparative example. In the comparative example, the autocorrelation function of the first fluorescent light 21 emitted from the first material 15a made of rhodamine 6G cannot be accurately analyzed because of the afterpulse of the avalanche photodiode. In contrast, in the present embodiment using the first superconducting single photon detector 30, the first fluorescent light emitted from the rhodamine 6G, which is the first material 15a, is not affected by the afterpulse. 21 autocorrelation functions can be analyzed. Therefore, the correlation time (diffusion time) τ _T , the average number of molecules N, and the like of the first material 15a made of rhodamine 6G contained in the light collection region 11a can be accurately obtained.

  The effects of the fluorescence correlation spectroscopy apparatus 1 according to the present embodiment and the method of measuring the correlation between the materials (first material 15a) will be described.

The fluorescence correlation spectroscopic device 1 according to the present embodiment detects the first superconducting single photon detection that detects the temporal change in the intensity of the first fluorescent light 21 emitted from the first material 15a included in the sample 15. And a signal processing unit 50 for processing an intensity signal indicating the intensity of the first fluorescent light 21 detected by the first superconducting single photon detector 30. The signal processing unit 50 includes a first signal processing unit 50. The first correlator 55 for obtaining the autocorrelation function G ₁₁ (τ) of the intensity signal of the fluorescent light 21 is provided. In the fluorescence correlation spectroscopic device 1 according to the present embodiment, the first superconducting single photon detector 30 is used to detect a temporal change in the intensity of the first fluorescent light 21 emitted from the first material 15a. Is used. The first superconducting single photon detector 30 does not generate afterpulses of the avalanche photodiode. Therefore, according to the fluorescence correlation spectroscopy apparatus 1 of the present embodiment, the correlation of the first material 15a can be accurately measured without being affected by the afterpulse of the avalanche photodiode. By using the fluorescence correlation spectroscopy apparatus 1 according to the present embodiment, for example, it is possible to detect the occurrence of a pathogenic protein in a cell or a change in the microenvironment in the cell in vivo. .

  In the fluorescence correlation spectroscopy device 1 according to the present embodiment, the first superconducting single photon detector 30 includes a substrate 31 and a superconducting thin wire 34 provided on the substrate 31, and the first fluorescent light 21 is The first superconducting single photon detector 30 is arranged so as to be incident from the superconducting thin wire 34 side. Since the first fluorescent light 21 does not pass through the substrate 31, it is possible to prevent the first fluorescent light 21 from being absorbed by the substrate 31. Therefore, according to the fluorescence correlation spectroscopy device 1 according to the present embodiment, the detection efficiency of the first superconducting single photon detector 30 can be improved.

  In the fluorescence correlation spectroscopy apparatus 1 according to the present embodiment, the first superconducting single photon detector 30 further includes a mirror 32 between the substrate 31 and the superconducting wire 34. The first fluorescent light 21 toward the substrate 31 without being absorbed by the superconducting thin wire 34 can be reflected by the mirror 32 toward the superconducting thin wire 34 and absorbed by the superconducting thin wire 34. Therefore, according to the fluorescence correlation spectroscopy device 1 according to the present embodiment, the detection efficiency of the first superconducting single photon detector 30 can be improved.

  In the fluorescence correlation spectroscopy device 1 according to the present embodiment, the superconducting thin wire 34 and the mirror 32 constitute an optical resonator 36. Since the superconducting thin wire 34 and the mirror 32 constitute the optical resonator 36, the first fluorescent light 21 is more easily absorbed by the superconducting thin wire 34. Therefore, according to the fluorescence correlation spectroscopy device 1 according to the present embodiment, the detection efficiency of the first superconducting single photon detector 30 can be further improved.

In the method for measuring the correlation of the material (first material 15a) according to the present embodiment, the first time change in intensity of the first fluorescent light 21 emitted from the first material 15a included in the sample 15 is measured. And the autocorrelation function G ₁₁ (τ) of the intensity signal indicating the intensity of the first fluorescent light detected by the first superconducting single photon detector 30. And getting ready. In the method of measuring the correlation of the material (the first material 15a) according to the present embodiment, the first change is performed in order to detect the temporal change in the intensity of the first fluorescent light 21 emitted from the first material 15a. A superconducting single photon detector 30 is used. The first superconducting single photon detector 30 does not generate afterpulses of the avalanche photodiode. Therefore, according to the method of measuring the correlation of the first material 15a according to the present embodiment, the correlation of the first material 15a can be accurately measured without being affected by the afterpulse of the avalanche photodiode. it can. By using the method for measuring the correlation of materials according to the present embodiment, for example, detection of pathogenic proteins in cells or changes in intracellular microenvironments in vivo Can do.

  Referring to FIGS. 10A and 10B, a first superconducting single photon detector 30a, which is a modification of the first superconducting single photon detector 30, is shown. The modified first superconducting single photon detector 30 a does not include the mirror 32. In the first superconducting single photon detector 30a of the modification, the dielectric layer 33 can be formed by oxidizing the surface of the substrate 31. According to the modified first superconducting single photon detector 30a, the structure and the manufacturing method of the first superconducting single photon detector 30a can be simplified, so that the manufacturing cost can be reduced. it can.

(Embodiment 2)
With reference to FIG.11 and FIG.12, the fluorescence correlation spectroscopy apparatus 2 which concerns on Embodiment 2 is demonstrated. The fluorescence correlation spectroscopy device 2 of the present embodiment basically has the same configuration as the fluorescence correlation spectroscopy device 1 of the first embodiment shown in FIGS. 1 to 3 and can obtain the same effects. The main differences are as follows.

  The optical system 20 b of the fluorescence correlation spectroscopy device 2 according to the present embodiment includes a second polarization selection element 27. The optical system 20b of the fluorescence correlation spectroscopic device 2 according to the present embodiment radiates from the first optical system that causes the first excitation light 11 to enter the sample 15 including the first material 15b, and the first material 15b. And a second optical system that causes the first fluorescent light 21 to be incident on the first superconducting single-photon detector 30. More specifically, in the optical system 20 b, the second optical system that causes the first fluorescent light 21 to enter the first superconducting single photon detector 30 includes the second polarization selection element 27. In the present embodiment, the second polarization selection element 27 transmits only light of a specific polarization component out of the first fluorescent light 21 emitted from the first material 15b in the sample 15. The polarization direction of the second polarization selection element 27 may be changed in an arbitrary direction within a plane intersecting the optical path of the first fluorescent light 21. The second polarization selection element 27 can be disposed at any location in the second optical system that causes the first fluorescent light 21 to be incident on the first superconducting single photon detector 30. In the present embodiment, the second polarization selection element 27 is disposed between the second filter 26 and the first lens 23. Examples of the second polarization selection element 27 include a polarizer and a polarization beam splitter.

  The optical system 20 b of the fluorescence correlation spectroscopy device 2 according to the present embodiment includes a first polarization selection element 17. More specifically, in the optical system 20 b, the first optical system that makes the first excitation light 11 incident on the sample 15 including the first material 15 b includes the first polarization selection element 17. In the present embodiment, the first polarization selection element 17 transmits only light of a specific polarization component in the first excitation light 11 emitted from the first light source 10. The polarization direction of the first polarization selection element 17 may be changed in any direction within the plane intersecting the optical path of the first excitation light 11. The first polarization selection element 17 can be disposed at an arbitrary position in the first optical system that causes the first excitation light 11 to enter the sample 15. In the present embodiment, the first polarization selection element 17 is disposed between the first filter 12 and the first wavelength selection element 13. In the present embodiment, the first light source 10 is a laser light source, and the first excitation light 11 is laser light polarized in the x direction. The first polarization selection element 17 transmits x-polarized light and blocks y-polarized light. Examples of the first polarization selection element 17 include a polarizer and a polarization beam splitter. The first polarization selection element 17 may not be arranged. For example, when the first light source 10 is a laser light source and the first excitation light 11 is a laser light, the first excitation light 11 is polarized light, and therefore the first polarization selection element 17 is used. May not be provided.

  In the present embodiment, the direction in which the first fluorescent light 21 emitted from the first material 15b in the sample 15 travels toward the first superconducting single photon detector 30 is defined as the z direction. . The direction in which the first excitation light 11 emitted from the first light source 10 travels toward the first wavelength selection element 13 is defined as the y direction. A direction orthogonal to the z direction and the y direction is defined as an x direction.

  A method for measuring the correlation of the first material 15b using the fluorescence correlation spectroscopy apparatus 2 of the present embodiment will be described. Referring to FIG. 12, first material 15b included in sample 15 measured by fluorescence correlation spectroscopy apparatus 2 according to the present embodiment has a bar shape. The first material 15b may have other shapes including an anisotropic shape and an isotropic shape.

Referring to FIG. 12, first material 15b such as a molecule contained in sample 15 such as a solution or a cell performs translational diffusion movement in a random direction (Brownian movement) and rotational rotation movement in a random direction. To do. The rotational diffusion movement of the first material 15b such as a molecule is affected by the shape (anisotropy of shape, size, etc.) of the first material 15b, the viscosity of a minute region around the first material 15a, and the like. . The rotational diffusion movement of the first material 15b is reflected in a change in the polarization direction of the first fluorescent light 21 emitted from the first material 15b. Therefore, in the present embodiment, the second polarization selection element 27 is disposed in the second optical system that causes the first fluorescent light 21 to enter the first superconducting single photon detector 30, thereby The change in the intensity of the first fluorescent light 21 accompanying the rotational diffusion movement of the first material 15b is measured. By measuring the change in intensity of the first fluorescent light 21 accompanying the rotational diffusion movement of the first material 15b, information on the shape (anisotropy, size, etc.) of the first material 15b and the first It is possible to obtain information on the environment of a minute region around the material 15a. The correlation time τ _R in the rotational diffusion motion of the first material 15b such as a molecule is proportional to the cube of the size of the first material 15b such as a molecule. In contrast, the correlation time τ _T in the translational diffusion motion of the first material 15b such as a molecule described in the first embodiment is proportional to the cube of the size of the first material 15b such as the molecule. Therefore, by using the fluorescence correlation spectroscopy device 2 according to the present embodiment, the size of the first material 15b such as a molecule can be measured with high accuracy.

  The rotational diffusion movement of the first material 15b is faster than the translational diffusion movement of the first material 15b. Therefore, the time change of the intensity of the first fluorescent light 21 radiated from the first material 15b measured by the first superconducting single photon detector 30 is relatively shown in FIG. The first time based on the time variation of the intensity of the first fluorescent light 21 based on the translational diffusion motion of the slow first material 15b and the relatively fast rotational diffusion motion of the first material 15b shown in FIG. 13B. The intensity of the fluorescent light 21 is changed with time.

  As described above, the fluctuation of the intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 includes information on the size (molecular weight) of the first material 15a such as a molecule, Information on the environment of the micro region around the first material 15a, information on the number of the first materials 15a such as molecules, and the shape (anisotropy and size of the shape) of the first material 15a such as molecules Etc.) is included. In the first correlator 55, the calculation represented by the equation (1) is performed on the intensity of the first fluorescent light 21 to obtain the autocorrelation function of the intensity signal of the first fluorescent light 21. Time fluctuation of the intensity signal of the first fluorescent light 21 and fluctuation of the amplitude of the intensity signal of the first fluorescent light 21 can be obtained.

Referring to FIG. 14, the rotational diffusion motion of first material 15b is shorter than the auto-correlation function based on the translational diffusion motion of first material 15b because it is faster than the translational diffusion motion of first material 15b. An autocorrelation function based on the rotational diffusion movement of the first material 15b appears in the time domain. τ _R is a width in which the autocorrelation function based on the rotational diffusion motion of the first material 15b takes a value of G _Tmax + G _Rmax × e ⁻¹ , and is a correlation time of the rotational diffusion motion of the first material 15b. _GRmax is the maximum value of the correlation function value based on the rotational diffusion motion of the first material 15b.

  The sample 15 used in the present embodiment is a solution containing a Q rod at a concentration of 100 nM. FIG. 15 shows a TEM image of the Q rod, which is the first material 15b. The Q rod is a rod-like semiconductor fluorescent material in which a shell made of cadmium sulfide (CdS) is formed around a core made of cadmium selenide (CdSe). The dimension of the Q rod used in the present embodiment has a length of 20 μm in the long axis direction and a length of 4 μm in the short axis direction.

  FIG. 16 shows a graph representing the autocorrelation function of the intensity signal of the fluorescence light of the Q rod, obtained by the fluorescence correlation spectroscopy apparatus according to the present embodiment and the fluorescence correlation spectroscopy apparatus of the comparative example. In the fluorescence correlation spectroscopy apparatus of the comparative example, an avalanche photodiode is used as a single photon detector. The two-dot chain line (APD) in FIG. 16 shows the autocorrelation function obtained by the fluorescence correlation spectrometer of the comparative example. An alternate long and short dash line (SSPD1) in FIG. 16 represents an autocorrelation function obtained by the fluorescence correlation spectroscopy apparatus of the first embodiment. A solid line (SSPD2x) in FIG. 16 represents an autocorrelation function obtained by the fluorescence correlation spectroscopy device 2 of the present embodiment in which the second polarization selection element 27 is arranged to transmit x-polarized light. A solid line (SSPD2y) in FIG. 16 represents an autocorrelation function obtained by the fluorescence correlation spectroscopy device 2 of the present embodiment in which the second polarization selection element 27 is arranged to transmit y-polarized light.

  When the fluorescence correlation spectroscopy apparatus of the comparative example is used, the autocorrelation function of the rotational diffusion motion of the Q rod (first material 15b) cannot be observed (see APD in FIG. 16). The autocorrelation function of the rotational diffusion motion of the Q rod (first material 15b) located in the time region shorter than the autocorrelation function of the translational diffusion motion of the Q rod (first material 15b) is the afterpulse of the avalanche photodiode. This is because it is hidden by the peak of the autocorrelation function.

Even using the fluorescence correlation spectroscopy apparatus 1 according to the first embodiment, the autocorrelation function of the rotational diffusion motion of the Q rod (first material 15b) cannot be clearly observed (see SSPD1 in FIG. 16). In the fluorescence correlation spectroscopic device 1 according to Embodiment 1, since the first superconducting single photon detector 30 is used, the autocorrelation function of the translational diffusion motion of the Q rod (first material 15b) is used. The autocorrelation function of the rotational diffusion motion of the Q rod (first material 15b) located in the short time region is not obscured by the peak of the autocorrelation function of the afterpulse of the avalanche photodiode. However, in the fluorescence correlation spectroscopy device 1 according to the first embodiment, the second polarization selection element 27 is disposed in the optical path for allowing the first fluorescent light 21 to enter the first superconducting single photon detector 30. Absent. Therefore, the first superconducting single photon detector 30 cannot detect a change in the polarization direction of the first fluorescent light 21 emitted from the Q rod (first material 15b). As a result, even when the fluorescence correlation spectroscopic device 1 according to the first embodiment is used, the rotation diffusion time τ _R that characterizes the rotational diffusion motion of the Q rod (first material 15b) and the maximum correlation function value due to the rotational diffusion motion. The value _GRmax cannot be determined.

On the other hand, by using the fluorescence correlation spectroscopy device 2 according to the present embodiment, an autocorrelation function based on the rotational diffusion motion of the Q rod (first material 15b) can be clearly obtained around 1 microsecond. Yes (see SSPD2x and SSPD2y in FIG. 16). In the fluorescence correlation spectroscopic device 2 according to the present embodiment, since the first superconducting single photon detector 30 is used, the autocorrelation function of the translational diffusion motion of the Q rod (first material 15b) is used. The autocorrelation function of the rotational diffusion motion of the Q rod (first material 15b) located in the short time region is not obscured by the peak of the autocorrelation function of the afterpulse of the avalanche photodiode. Further, in the fluorescence correlation spectroscopy device 2 according to the present embodiment, the second polarization selection element 27 is arranged in the optical path for allowing the first fluorescent light 21 to enter the first superconducting single photon detector 30. Yes. Therefore, the first superconducting single photon detector 30 can detect a change in the polarization direction of the first fluorescent light 21 emitted from the Q rod (first material 15b). As a result, by using the fluorescence correlation spectroscopy device 2 according to the present embodiment, the rotation diffusion time τ _R that characterizes the rotation diffusion motion of the Q rod (first material 15b) and the maximum correlation function value due to the rotation diffusion motion. The value _GRmax can be determined.

  The effect of the fluorescence correlation spectroscopy apparatus 2 of the present embodiment and the method of measuring the correlation between the materials (first material 15b) will be described.

  The fluorescence correlation spectroscopy apparatus 2 of the present embodiment further includes a second polarization selection element 27, and the second polarization selection element 27 causes the first fluorescent light 21 to be converted into the first superconducting single photon detector 30. Arranged in the incident optical path. Since the second polarization selection element 27 is disposed in the optical path where the first fluorescent light 21 enters the first superconducting single photon detector 30, the first fluorescence emitted from the first material 15b. A change in the polarization direction of the light 21 can be detected by the first superconducting single photon detector 30. As a result, by using the fluorescence correlation spectroscopy device 2 according to the present embodiment, the correlation of the first material such as the rotational diffusion of the first material 15a can be obtained without being affected by the afterpulse of the avalanche photodiode. It can be measured accurately. By using the fluorescence correlation spectroscopy apparatus 2 according to the present embodiment, for example, it is possible to detect the occurrence of pathogenic proteins in cells or changes in the intracellular microenvironment in vivo. .

  In the method of measuring the correlation of the material (first material 15b) of the present embodiment, the first fluorescent light 21 is incident on the second polarization selection element 27 and is emitted from the second polarization selection element 27. The method further includes causing the first fluorescent light 21 to be incident on the first superconducting single photon detector 30. Since the first fluorescent light 21 emitted from the second polarization selection element 27 is incident on the first superconducting single photon detector 30, the first fluorescent light 21 emitted from the first material 15b A change in polarization direction can be detected by the first superconducting single photon detector 30. As a result, the method of measuring the correlation of the first material 15b according to the present embodiment allows the first material such as rotational diffusion of the first material 15a without being affected by the afterpulse of the avalanche photodiode. Can be accurately measured. By using the method of measuring the correlation of the first material 15b according to the present embodiment, for example, in vivo, pathogenic proteins are generated in the cell, or the microenvironment in the cell is changed. Can be detected.

(Embodiment 3)
With reference to FIG.17 and FIG.18, the fluorescence correlation spectroscopy apparatus 3 which concerns on Embodiment 3 is demonstrated. The fluorescence correlation spectroscopy device 3 of the present embodiment basically has the same configuration as the fluorescence correlation spectroscopy device 1 of the first embodiment shown in FIGS. 1 to 3 and can obtain the same effects. The main differences are as follows.

  According to the fluorescence correlation spectroscopy device 3 of the present embodiment, the correlation of a plurality of materials included in the sample 15 can be measured. More specifically, according to the fluorescence correlation spectroscopy device 3 of the present embodiment, the cross-correlation between a plurality of materials (the first material 15a and the second material 15c) included in the sample 15 can be measured. it can. In addition, according to the fluorescence correlation spectroscopy device 3 of the present embodiment, the autocorrelation of each of a plurality of materials (first material 15a and second material 15c) included in the sample 15 can be measured simultaneously. The second material 15c is a material different from the first material 15a. The first material 15a and the second material 15c may be a fluorescent material or a material labeled with a fluorescent material. Examples of the first material 15a and the second material 15c include molecules labeled with a fluorescent material, proteins labeled with a fluorescent material, and cells labeled with a fluorescent material.

  The fluorescence correlation spectroscopy device 3 according to the present embodiment includes an optical system 20c, a first superconducting single photon detector (SSPD) 30, a second superconducting single photon detector (SSPD) 30c, The first intensity signal indicating the intensity of the first fluorescent light 21 detected by the refrigerator 40 and the first superconducting single photon detector 30 and the second superconducting single photon detector 30c are detected. And a signal processing unit 50c for processing a second intensity signal indicating the intensity of the second fluorescent light 21c.

  The optical system 20c includes a first optical system and a second optical system. The first optical system causes the first excitation light 11 and the second excitation light 11c to enter the sample 15 including the first material 15a and the second material 15c. The second optical system causes the first fluorescent light 21 radiated from the first material 15a to enter the first superconducting single-photon detector 30, and the second radiated from the second material 15c. The fluorescent light 21c is incident on the second superconducting single photon detector 30c.

  The first optical system of the optical system 20c of the fluorescence correlation spectroscopic device 3 according to the present embodiment includes a second light source in addition to the optical elements included in the first optical system of the optical system 20 of the first embodiment. 10c and the optical multiplexer 19 are further included. The first optical system of the present embodiment may further include a first mirror 18.

  The second light source 10c emits second excitation light 11c for exciting the second material 15c included in the sample 15 from the ground state to the excited state. The wavelength of the second excitation light 11 c is different from the wavelength of the first excitation light 11. In the present embodiment, the second light source 10c is a laser, and the second excitation light 11c is a laser beam. The second light source 10c is not limited to a laser. The second excitation light 11 c is reflected by the first mirror and then combined with the first excitation light 11 in the optical multiplexer 19. An example of the optical multiplexer 19 is a dichroic mirror that transmits the first pumping light 11 and reflects the second pumping light 11c.

  The first pumping light 11 and the second pumping light 11 c combined by the optical multiplexer 19 are incident on the first filter 12. The first filter 12 adjusts the intensities of the first excitation light 11 and the second excitation light 11c irradiated on the sample 15 including the first material 15a and the second material 15c. An example of the first filter 12 is an ND filter.

  The first excitation light 11 and the second excitation light 11 c that have passed through the first filter 12 enter the first wavelength selection element 13. The first wavelength selection element 13 reflects the first excitation light 11 and the second excitation light 11 c, and reflects the first fluorescent light 21 and the sample 15 emitted from the first material 15 a included in the sample 15. It has a property of transmitting the second fluorescent light 21c emitted from the included second material 15c. The first excitation light 11 and the second excitation light 11 c are reflected by the first wavelength selection element 13 and directed to the objective lens 14.

The first excitation light 11 and the second excitation light 11 c are collected on the sample 15 by the objective lens 14. Referring to FIG. 18, in the sample 15 including the first material 15a and the second material 15c, the first excitation light 11 and the second excitation light 11c are condensed on the condensing region 11a. The first excitation light 11 and the second excitation light 11c may be narrowed down to the diffraction limit of the first excitation light 11 and the second excitation light 11c in the sample 15 by the objective lens 14. By condensing the first excitation light 11 and the second excitation light 11c in the sample 15 onto the narrow light collection region 11a, the amount of the sample 15 contained in the light collection region 11a is reduced to, for example, femtoliter (10 ⁻¹⁵ L). By collecting the first excitation light 11 and the second excitation light 11c using the objective lens 14, a minute measurement region can be obtained.

  The second optical system of the optical system 20c of the present embodiment includes a second wavelength selection element 28, a second wavelength selection element 28 in addition to the optical elements included in the second optical system of the optical system 20 of the first embodiment. The confocal optical system 22c. In the present embodiment, the second confocal optical system 22c includes a second lens 23c and a second optical fiber 24c. The second confocal optical system 22c may be configured by other optical elements such as pinholes. The second optical system of the present embodiment may further include a second mirror 29 and a second gradient index lens 25c.

  The first fluorescent light 21 emitted from the first material 15 a and the second fluorescent light 21 c emitted from the second material 15 c are collected by the objective lens 14. The light that passes through the objective lens 14 and enters the first wavelength selection element 13 includes the first fluorescent light 21, the second fluorescent light 21 c, and the first excitation light reflected or scattered by the sample 15. 11 and second excitation light 11c. The first excitation light 11 and the second excitation light 11c reflected or scattered by the sample 15 are reflected by the first wavelength selection element 13, and only the first fluorescence light 21 and the second fluorescence light 21c are the first. 1 wavelength selection element 13 is transmitted.

  The first fluorescent light 21 and the second fluorescent light 21 c that have passed through the first wavelength selection element 13 are incident on the second filter 26. The second filter 26 removes light that does not need to be incident on the first superconducting single photon detector 30 and the second superconducting single photon detector 30c. An example of the second filter 26 is a band pass filter.

  The first fluorescent light 21 and the second fluorescent light 21 c that have passed through the second filter 26 enter the second wavelength selection element 28. The second wavelength selection element 28 transmits the first fluorescent light 21 and reflects the second fluorescent light 21c. An example of the second wavelength selection element 28 is a dichroic mirror.

  The second fluorescent light 21c reflected by the second wavelength selection element 28 is reflected by the second mirror 29 and then enters the second confocal optical system 22c. In the present embodiment, the second confocal optical system 22c includes a second lens 23c and a second optical fiber 24c. The second fluorescent light 21c is collected by the second lens 23c and enters the incident end 24cf of the second optical fiber 24c. Of the second fluorescent light 21c collected by the second lens 23c, only the light that can be optically coupled to the core of the second optical fiber 24c propagates in the second optical fiber 24c. be able to. Of the second fluorescent light 21c collected by the second lens 23c, light that cannot be optically coupled to the core of the second optical fiber 24c propagates in the core of the second optical fiber 24c. Can not do it. Therefore, the incident end 24cf of the second optical fiber 24c acts like a pinhole. The second confocal optical system 22c having the second lens 23c and the second optical fiber 24c is other than the second fluorescent light 21c emitted from the second material 15c at the focal position of the objective lens 14. Light is prevented from propagating through the second optical fiber 24c. An example of the second optical fiber 24c is a multimode fiber having a core with a diameter of 62.5 μm.

  The second optical fiber 24 c extends from the outside of the refrigerator 40 to the inside of the refrigerator 40. By the second optical fiber 24c, the second fluorescent light 21c radiated from the second material 15c in the sample 15 located outside the refrigerator 40 is converted into the second superconductivity located inside the refrigerator 40. The single photon detector 30c can be optically coupled with high efficiency.

  A second gradient index lens 25c is provided at the exit end of the second optical fiber 24c. The second fluorescent light 21c propagating through the second optical fiber 24c is condensed on the second superconducting single photon detector 30c by the second gradient index lens 25c. By the second gradient index lens 25c, the second fluorescent light 21c emitted from the second material 15c in the sample 15 is optically transmitted to the second superconducting single photon detector 30c with high efficiency. Can be combined.

  The second superconducting single photon detector 30c detects the intensity of the second fluorescent light 21c emitted from the second material 15c included in the sample 15, and indicates the intensity of the second fluorescent light 21c. The intensity signal is output to the signal processing unit 50c. The second superconducting single photon detector 30c has the structure shown in FIGS. 2A and 2B or FIGS. 10A and 10B.

  The signal processing unit 50c according to the present embodiment includes a second correlator 55c and a third correlator 56 in addition to the circuits included in the signal processing unit 50c according to the first embodiment. The signal processing unit 50c of the present embodiment may further include a second low noise amplifier 51c and a second comparator 52c.

  The second low noise amplifier 51c is a circuit that amplifies the intensity signal of the second fluorescent light 21c detected by the second superconducting single photon detector 30c with low noise.

  The second comparator 52c is a circuit that converts the intensity signal of the second fluorescent light 21c detected by the second superconducting single photon detector 30c into a transistor-transistor logic (TTL) signal. The second comparator 52c may be a circuit that converts an intensity signal indicating the intensity of the second fluorescent light 21c obtained by the second superconducting single photon detector 30c into another signal.

  The second correlator 55c is a circuit that obtains an autocorrelation function of the intensity signal of the second fluorescent light 21c detected by the second superconducting single photon detector 30c. The third correlator 56 includes an intensity signal of the first fluorescent light 21 detected by the first superconducting single photon detector 30 and a second signal detected by the second superconducting single photon detector 30c. This circuit obtains a cross-correlation function with the intensity signal of the fluorescent light 21c. The second correlator 55c performs the calculation represented by the expression (3) on the intensity signal of the second fluorescent light 21c, and the third correlator 56 calculates the intensity signal of the first fluorescent light 21. And the calculation represented by Expression (4) is performed on the intensity signal of the second fluorescent light 21c.

Here, I ₂ (t) represents the intensity signal of the second fluorescent light 21c at time t, <I ₂ (t)> represents the time average of the intensity signal of the second fluorescent light 21c, and τ is a correlation. G ₂₂ (τ) represents the autocorrelation function of the intensity signal of the second fluorescent light 21c, and G ₁₂ (τ) represents the intensity signal of the first fluorescent light 21 and the intensity of the second fluorescent light 21c. Represents the cross-correlation function with the signal. A digital correlator can be exemplified as the second correlator 55c and the third correlator 56.

  A method for measuring the correlation between the first material 15a and the second material using the fluorescence correlation spectroscopy apparatus 3 of the present embodiment will be described.

  Referring to FIG. 18, the first material 15a and the second material 15c such as molecules contained in a sample 15 such as a solution or a cell are translated and diffused in random directions (Brownian motion). By this Brownian motion, the first material 15a and the second material 15c constantly enter and exit the minute condensing region 11a. The first fluorescent light 21 is emitted only from the first material 15a located in the minute condensing region 11a, and the second fluorescent light 21c is emitted only from the second material 15c located in the minute condensing region 11a. Is done. Therefore, the first fluorescent light detected by the first superconducting single photon detector 30 in response to the first material 15a and the second material 15c entering and exiting the minute condensing region 11a at random. The intensity of 21 and the intensity of the second fluorescent light 21c detected by the second superconducting single photon detector 30c also fluctuate randomly.

  Similar to the first embodiment, the first correlator 55 obtains the autocorrelation function of the intensity signal of the first fluorescent light 21 emitted from the first material 15a included in the sample 15. From the autocorrelation function of the intensity signal of the first fluorescent light 21, information on the size of the first material 15a, information on the environment of the minute region around the first material 15a, and the first included in the light collection region 11a. Information on the number of materials 15a can be obtained. In addition, the autocorrelation function of the intensity signal of the second fluorescent light 21c emitted from the second material 15c included in the sample 15 is obtained by the second correlator 55c. From the autocorrelation function of the intensity signal of the second fluorescent light 21c, information on the size of the second material 15c, information on the environment of the minute region around the second material 15c, and the second included in the light collection region 11a. Information on the number of the materials 15c can be obtained.

  The signal processing unit 50c in the fluorescence correlation spectroscopic device 3 according to the present embodiment has the intensity signal of the first fluorescent light 21 emitted from the first material 15a and the second fluorescent light emitted from the second material 15c. A third correlator 56 is provided for obtaining a cross-correlation function with the intensity signal 21c. The third correlator 56 can measure the simultaneity of the intensity fluctuation of the first fluorescent light 21 and the intensity fluctuation of the second fluorescent light 21c. The simultaneous fluctuation of the intensity fluctuation of the first fluorescent light 21 and the intensity fluctuation of the second fluorescent light 21c radiates the first material 15a that emits the first fluorescent light 21 and the second fluorescent light 21c. This represents the degree to which the second material 15c to be present exists temporally and spatially.

  From the amplitude of the cross-correlation function between the intensity signal of the first fluorescent light 21 emitted from the first material 15a and the intensity signal of the second fluorescent light 21c emitted from the second material 15c, the first material The degree of coupling between 15a and the second material 15c can be obtained. For example, as the amplitude of the cross-correlation function between the intensity signal of the first fluorescent light 21 and the intensity signal of the second fluorescent light 21c increases, the degree of coupling between the first material 15a and the second material 15c increases. The smaller the amplitude of the cross-correlation function between the intensity signal of the first fluorescent light 21 and the intensity signal of the second fluorescent light 21c, the smaller the degree of coupling between the first material 15a and the second material 15c.

  The signal processing unit 50c according to the present embodiment uses the first correlator 55 to obtain the amplitude of the cross-correlation function between the intensity signal of the first fluorescent light 21 and the intensity signal of the second fluorescent light 21c. And an arithmetic circuit 58 that divides by the amplitude of the autocorrelation function of the intensity signal of the first fluorescent light 21 and the amplitude of the autocorrelation function of the intensity signal of the second fluorescent light 21c obtained by the second correlator 55c. May be. By such an arithmetic circuit 58, the degree of coupling between the first material 15a and the second material 15c can be obtained more accurately.

  From the correlation time of the cross-correlation function between the intensity signal of the first fluorescent light 21 emitted from the first material 15a and the intensity signal of the second fluorescent light 21c emitted from the second material 15c, the first The average time during which the material 15a and the second material 15c are combined, or a material structure in which the first material 15a and the second material 15c are combined, such as a molecular structure, stays in the minute light collection region 11a. Average time can be obtained.

  The effects of the fluorescence correlation spectroscopy device 3 of the present embodiment and the method of measuring the correlation between a plurality of materials (the first material 15a and the second material 15c) will be described.

  The fluorescence correlation spectroscopic device 3 according to the present embodiment includes a plurality of fluorescent lights (first fluorescent light 21, first material 15a, second material 15c) radiated from a plurality of materials (first material 15a, second material 15c). A plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon detector 30c) that detect temporal changes in the intensity of the second fluorescent light 21c); A plurality of fluorescent light (first fluorescent light 21) detected by a plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon detector 30c). And a signal processing unit 50c for processing a plurality of intensity signals indicating the intensity of the second fluorescent light 21c). The signal processing unit 50c includes a plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c). A plurality of correlators (first correlator 5) for obtaining respective autocorrelation functions of a plurality of intensity signals. , A second correlator 55c).

  The first superconducting single photon detector 30 and the second superconducting single photon detector 30c do not generate afterpulses of the avalanche photodiode. Therefore, according to the fluorescence correlation spectroscopy device 3 of the present embodiment, each autocorrelation of the plurality of materials (the first material 15a and the second material 15c) is not affected by the afterpulse of the avalanche photodiode. Functions can be measured simultaneously and accurately. By using the fluorescence correlation spectroscopy device 3 according to the present embodiment, for example, in vivo, a plurality of pathogenic proteins are generated in a cell, or a plurality of changes in a microenvironment in a cell are simultaneously performed. Can be detected.

  The fluorescence correlation spectroscopic device 3 according to the present embodiment includes a plurality of fluorescent lights (first fluorescent light 21, first material 15a, second material 15c) radiated from a plurality of materials (first material 15a, second material 15c). A plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon detector 30c) that detect temporal changes in the intensity of the second fluorescent light 21c); A plurality of fluorescent light (first fluorescent light 21) detected by a plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon detector 30c). And a signal processing unit 50c for processing a plurality of intensity signals indicating the intensity of the second fluorescent light 21c). The signal processing unit 50c includes a plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c). ) Has a correlator (third correlator 56) for obtaining a cross-correlation function of the plurality of intensity signals.

  The plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon detector 30c) do not generate afterpulses of the avalanche photodiode. Therefore, according to the fluorescence correlation spectroscopy device 3 according to the present embodiment, the cross-correlation function of a plurality of materials (the first material 15a and the second material 15c) is not affected by the afterpulse of the avalanche photodiode. Can be measured accurately. According to the fluorescence correlation spectroscopic device 3 according to the present embodiment, the interaction state of a plurality of materials (first material 15a and second material 15c) is not affected by the afterpulse of the avalanche photodiode. It can be measured accurately. By using the fluorescence correlation spectroscopy device 3 according to the present embodiment, for example, the state of interaction of molecules in cells can be detected in vivo (in vivo).

  The method for measuring the correlation between a plurality of materials (first material 15a and second material 15c) according to the present embodiment is based on a plurality of materials (first material 15a and second material 15c) included in the sample 15. ) The time variation of the intensity of the plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c) emitted from a plurality of superconducting single photon detectors (first superconducting single photon detector). 30, detection by a second superconducting single photon detector 30c) and a plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon Obtaining an autocorrelation function of a plurality of intensity signals indicating the intensities of the plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c) detected by the detector 30c).

  The first superconducting single photon detector 30 and the second superconducting single photon detector 30c do not generate afterpulses of the avalanche photodiode. Therefore, according to the method for measuring the correlation between the plurality of materials (first material 15a and second material 15c) according to the present embodiment, the plurality of materials are not affected by the afterpulse of the avalanche photodiode. The autocorrelation functions of (first material 15a and second material 15c) can be simultaneously and accurately measured. By the method of measuring the correlation between a plurality of materials (first material 15a, second material 15c) according to the present embodiment, for example, in vivo, a plurality of pathogenic proteins are present in cells. Occurrence and multiple changes in the microenvironment in the cell can be detected simultaneously.

  The method for measuring the correlation between a plurality of materials (first material 15a and second material 15c) according to the present embodiment is based on a plurality of materials (first material 15a and second material 15c) included in the sample 15. ) The time variation of the intensity of the plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c) emitted from a plurality of superconducting single photon detectors (first superconducting single photon detector). 30, detection by a second superconducting single photon detector 30c) and a plurality of superconducting single photon detectors (first superconducting single photon detector 30, second superconducting single photon Obtaining a cross-correlation function of intensity signals indicating the intensities of the plurality of fluorescent lights (first fluorescent light 21 and second fluorescent light 21c) detected by the detector 30c).

  The first superconducting single photon detector 30 and the second superconducting single photon detector 30c do not generate afterpulses of the avalanche photodiode. Therefore, according to the method for measuring the correlation between the plurality of materials (first material 15a and second material 15c) according to the present embodiment, the plurality of materials are not affected by the afterpulse of the avalanche photodiode. The cross-correlation function of (the first material 15a and the second material 15c) can be accurately measured. According to the method of measuring the correlation between a plurality of materials (first material 15a and second material 15c) according to the present embodiment, a plurality of materials (first materials 15) are not affected by the afterpulse of the avalanche photodiode. It is possible to accurately measure the interaction between the first material 15a and the second material 15c). By the method of measuring the correlation between a plurality of materials (first material 15a, second material 15c) according to the present embodiment, for example, in vivo, the state of interaction of molecules in cells is determined. Can be detected.

  A modification of the present embodiment will be described. The fluorescence correlation spectroscopy device 3 according to the present embodiment includes the first correlator 55 and the second correlator 55c, but the fluorescence correlation spectroscopy device 3 according to the first modification of the present embodiment is , At least one of the first correlator 55 and the second correlator 55c may not be provided. The fluorescence correlation spectroscopy apparatus 3 according to the second modification of the present embodiment may not include the third correlator 56 and the arithmetic circuit 58. The fluorescence correlation spectroscopy device 3 according to the third modification of the present embodiment is arranged between the second wavelength selection element 28 and the first lens 23 and the second wavelength as in the second embodiment. A second polarization selection element 27 may be disposed between the selection element 28 and the second lens 23c. The fluorescence correlation spectroscopic device 3 according to the fourth modification of the present embodiment is arranged between the first wavelength selection element 13 and the second wavelength selection element 28 as in the second embodiment. A polarization selection element 27 may be arranged.

  The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

1, 2, 3 Fluorescence correlation spectroscopic device, 10 1st light source, 10c 2nd light source, 11 1st excitation light, 11a Condensing area | region, 11c 2nd excitation light, 12 1st filter, 13 1st Wavelength selection element, 14 objective lens, 15 sample, 15a, 15b first material, 15c second material, 17 first polarization selection element, 18 first mirror, 19 optical multiplexer, 20, 20b, 20c Optical system, 21 1st fluorescent light, 21c 2nd fluorescent light, 22 1st confocal optical system, 22c 2nd confocal optical system, 23 1st lens, 23c 2nd lens, 24 1st Optical fiber, 24c second optical fiber, 24cf, 24f incident end, 25 first refractive index distribution type lens, 25c second refractive index distribution type lens, 26 second filter, 27 second polarization selection element , 28 second wavelength selection element, 29 second 2 mirrors, 30, 30a first superconducting single photon detector, 30c second superconducting single photon detector, 31 substrate, 32 mirror, 33 dielectric layer, 34 superconducting wire, 35 pad electrode, 36 optical resonator, 37 outer periphery, 40 refrigerator, 50, 50c signal processing unit, 51 first low noise amplifier, 51c second low noise amplifier, 52 first comparator, 52c second comparator, 55 first Correlator, 55c second correlator, 56 third correlator, 58 arithmetic circuit.

Claims (5)

  A superconducting single-photon detector that detects temporal changes in the intensity of fluorescent light emitted from a material contained in the sample, and an intensity signal that indicates the intensity of the fluorescent light detected by the superconducting single-photon detector A signal processing unit for processing the signal, and the signal processing unit includes a correlator that obtains at least one of an autocorrelation function and a cross-correlation function of the intensity signal of the fluorescent light.   The fluorescence correlation spectroscopic apparatus according to claim 1, further comprising a polarization selection element, wherein the polarization selection element is disposed in an optical path where the fluorescent light is incident on the superconducting single photon detector.   The superconducting single photon detector includes a substrate and a superconducting thin wire provided on the substrate, and the superconducting single photon detector is arranged so that the fluorescent light is incident from the superconducting thin wire side. The fluorescence correlation spectroscopic device according to claim 1 or 2, which is arranged.   The fluorescence correlation spectroscopic apparatus according to claim 3, wherein the superconducting single photon detector further includes a mirror between the substrate and the superconducting thin wire.   The fluorescence correlation spectroscopic apparatus according to claim 4, wherein the superconducting thin wire and the mirror constitute an optical resonator.