JP5654509B2 - Fluorescence detection apparatus and fluorescence detection method - Google Patents

Description

  The present invention relates to a fluorescence detection apparatus and a fluorescence detection method for detecting fluorescence emitted when a measurement object is irradiated with laser light.

There are known a fluorescence detection apparatus and a fluorescence detection method for irradiating a measurement target with laser light, receiving fluorescence emitted from the measurement target, and acquiring information on the measurement target.
A flow cytometer using a fluorescence detection apparatus and a fluorescence detection method allows measurement objects such as cells, DNA, RNA, enzymes, and proteins labeled with a fluorescence reagent to flow through a sheath liquid. By irradiating the measurement object with laser light, the fluorescent dye applied to the measurement object emits fluorescence. The flow cytometer can acquire information on the measurement object by detecting this fluorescence.

  Also known are a fluorescence detection apparatus and a fluorescence detection method for calculating a fluorescence relaxation time (fluorescence lifetime) by irradiating a measurement object with laser light intensity-modulated at a predetermined frequency and receiving fluorescence emitted from the measurement object. (Patent Document 1).

JP 2006-226698 A

  In a conventional fluorescence detection apparatus, fluorescence emitted from a measurement object is amplified and demodulated after being converted into an electrical signal by being incident on a photoelectric conversion element. As the photoelectric conversion element, a photomultiplier tube is often used because of its excellent amplification factor. By the way, a general photomultiplier tube has an excellent electron amplification factor and a low quantum efficiency (for example, about 25% or less). Therefore, when the number of photons incident on the photomultiplier tube is small, The number of photons that can be converted into is further reduced. In this case, since the intensity of the fluorescence converted into the electrical signal becomes weak, the fluorescence electrical signal is buried in another electrical signal, which makes it difficult to extract and may be removed as noise. Therefore, when the intensity of the fluorescence is low, there is a possibility that the measurement accuracy of the fluorescence relaxation time obtained by the fluorescence detection apparatus is lowered.

  Therefore, an object of the present invention is to provide a fluorescence detection apparatus and a fluorescence detection method that can acquire a highly accurate fluorescence relaxation time.

One embodiment of the present invention is a fluorescence detection device that detects fluorescence emitted when a measurement target is irradiated with laser light.
The fluorescence detection device is
A laser light source unit for irradiating the measurement object with intensity-modulated laser light;
An optical element that divides a fluorescent optical signal emitted when the laser beam is irradiated onto the measurement object into two, and converts the intensity of the laser light into a first optical signal and a second optical signal; A first optical amplifier that amplifies the first optical signal by performing a first stimulated emission with a bias signal using a signal in phase with the modulation signal of the first modulation signal, and a modulation for intensity-modulating the laser light A second optical amplifier that amplifies the second optical signal by performing second stimulated emission with a bias signal using a signal that is phase-shifted by 90 degrees with respect to the signal; and A light receiving unit comprising: a first light receiving element that receives light and outputs a first electric signal; and a second light receiving element that receives the second optical signal and outputs a second electric signal;
A process of calculating a phase difference of the fluorescence with respect to the modulation signal using the first electric signal and the second electric signal output from the light receiving unit, and obtaining a fluorescence relaxation time of the fluorescence from the phase difference Part.

Another aspect of the present invention is a fluorescence detection method for detecting fluorescence emitted when a measurement object is irradiated with laser light.
The fluorescence detection method is:
Irradiating the measurement object with intensity-modulated laser light; and
Dividing the fluorescence optical signal emitted when the measurement object is irradiated with the laser light into two to be a first optical signal and a second optical signal;
Amplifying the first optical signal by performing first stimulated emission with a bias signal using a modulation signal for intensity-modulating the laser light;
Amplifying the second optical signal by performing second stimulated emission with a bias signal using a signal phase-shifted by 90 degrees with respect to a modulation signal for intensity-modulating the laser light;
Receiving a first optical signal obtained by performing the first stimulated emission and outputting a first electrical signal;
Receiving a second optical signal obtained by performing the second stimulated emission and outputting a second electrical signal; and
Calculating the phase difference of the fluorescence with respect to the modulation signal using the output first electric signal and the second electric signal, and obtaining the fluorescence relaxation time of the fluorescence from the phase difference. .

  According to the above-described fluorescence detection apparatus and fluorescence detection method, a highly accurate fluorescence relaxation time can be acquired.

It is a schematic block diagram which shows an example of the flow cytometer using the fluorescence detection apparatus of 1st Embodiment. It is a figure which shows an example of a structure of a laser light source part. It is a figure which shows an example of a structure of a 2nd light-receiving part. It is a figure which shows an example of a structure of a signal processing part. It is a figure which shows an example of a structure of a control part. It is a figure which shows an example of a structure of an analyzer. It is a figure explaining an example of the flow of the fluorescence detection method of this embodiment. It is a figure explaining the modification of the 2nd light-receiving part shown in FIG. It is a figure explaining an example of the composition of the oscillator contained in the flow cytometer using the fluorescence detection device of a 2nd embodiment.

Hereinafter, a flow cytometer to which the fluorescence detection apparatus and the fluorescence detection method of the present invention are applied will be described in detail.
<First Embodiment>
(Configuration of flow cytometer)
First, with reference to FIG. 1, the structure of the flow cytometer of 1st Embodiment is demonstrated. FIG. 1 is a schematic configuration diagram illustrating an example of a flow cytometer according to the present embodiment. The flow cytometer can acquire information on the measurement target 12 by irradiating the measurement target 12 with laser light and receiving fluorescence emitted from the measurement target 12 irradiated with the laser light.
The flow cytometer includes a flow cell 10, a laser light source unit 20, a first light receiving unit 30, a second light receiving unit 40, a control unit 50, an analysis device 60, and an output unit 70. Further, a container 16 for collecting the measurement object 12 is disposed downstream of the flow cell 10. Hereinafter, each configuration will be described in detail.

  A measurement object 12 such as a cell, DNA (Deoxyribonucleic Acid), RNA (Ribonucleic Acid), enzyme, protein or the like flows inside the flow cell 10 surrounded by a sheath liquid. As will be described later, since the laser light source unit 20 irradiates the measurement object 12 with laser light and acquires information on the measurement object 12 from the fluorescence emitted at that time, the measurement object 12 has a fluorescent dye 14. It is given in advance. As the fluorescent dye 14, for example, CFP (Cyan Fluorescent Protein), YFP (Yellow Fluorescent Protein) or the like is used. Within the flow cell 10, the measurement object 12 surrounded by the sheath liquid flows into the flow cell 10 as a thin liquid flow by receiving hydrodynamic narrowing.

The laser light source unit 20 has a wavelength in the visible light band of 350 nm to 800 nm, for example, and irradiates the measurement object 12 with laser light whose intensity is modulated using a predetermined modulation signal.
As shown in FIG. 2, the laser light source unit 20 includes a laser light source 21, a lens system 22, and a laser driver 23.
The laser light source 21 emits a CW (continuous wave) laser beam having a constant intensity after intensity modulation.
The lens system 22 focuses the laser light emitted from the laser light source 21 on a predetermined measurement point (measurement field) in the flow cell 10.
The laser driver 23 is electrically connected to a control unit 50 to be described later, and is configured to modulate the intensity of the laser light with the frequency (modulation frequency) of the modulation signal supplied from the control unit 50.
The laser light source unit 20 may use one laser light source or a plurality of laser light sources. In the case where a plurality of laser light sources are used, it is preferable that the laser light emitted from the plurality of laser light sources is synthesized using a dichroic mirror or the like to form laser light emitted toward the measurement field.

  As a light source for emitting laser light, for example, a semiconductor laser can be used. The output of the laser beam is, for example, 5 mW to 100 mW. Also, the modulation frequency has a period that is slightly longer than the fluorescence relaxation time, for example, 10 MHz to 200 MHz.

The first light receiving unit 30 is disposed on the opposite side of the laser light source unit 20 with the measurement field of the flow cell 10 as a reference. The first light receiving unit 30 receives forward scattered light that is generated when the measurement target 12 that passes through the measurement field of the flow cell 10 is irradiated with laser light.
The first light receiving unit 30 includes, for example, a photoelectric converter such as a photodiode. The photoelectric converter converts the received forward scattered light into an electrical signal.
The electrical signal converted by the photoelectric converter of the first light receiving unit 30 is output to the analyzer 60, and the electrical signal is used as a trigger signal for notifying the timing when the measurement object 12 passes through the measurement field of the flow cell 10. It is done.
In addition, the first light receiving unit 30 includes, for example, a lens system (not shown) that focuses forward scattered light on the photoelectric converter, and a front surface on the measurement object 12 side of the lens system so that laser light does not directly enter the photoelectric converter. And a shielding plate (not shown) provided on the surface.

The second light receiving unit 40 is arranged in a direction perpendicular to the emission direction of the laser light emitted from the laser light source unit 20 and perpendicular to the moving direction of the measurement object 12 in the flow cell 10. ing. The second light receiving unit 40 is an optical amplifier that converts fluorescence emitted from the measurement object 12 when the measurement object 12 passing through the measurement field of the flow cell 10 is irradiated with laser light and side scattered light of the laser light. Amplifies and receives light.
As shown in FIG. 3, the second light receiving unit 40 includes a lens system 41, a dichroic mirror 42, a half mirror 43, bandpass filters (BPF) 44a and 44b, optical amplifiers 45a and 45b, and a signal processing unit. 46a, 46b, a power splitter 47, and a 90-degree phase shifter 48.
The lens system 41 condenses the light incident on the second light receiving unit 40.
The dichroic mirror 42 is a mirror that reflects the light in the wavelength region of the side scattered light of the laser light among the light transmitted through the lens system 41 and transmits the light in the wavelength region including the fluorescent wavelength region. The light in the wavelength region of the side scattered light of the laser light is reflected by the dichroic mirror 42 and then absorbed by the light absorbing element 42a.
The half mirror 43 is a mirror that distributes fluorescence in two directions by transmitting a part of the fluorescence transmitted through the dichroic mirror 42 and reflecting the remaining fluorescence. Note that a beam splitter may be used instead of the half mirror 43.
The BPFs 44a and 44b are filters that are provided in front of the optical amplifiers 45a and 45b, respectively, and transmit only fluorescence in a predetermined wavelength band. Note that the wavelength band of fluorescence to be transmitted is set corresponding to the wavelength band of fluorescence emitted by the fluorescent dye 14. A band reject filter may be used instead of the BPFs 44a and 44b.

The optical amplifiers 45a and 45b are, for example, semiconductor optical amplifiers, and amplify incident fluorescence optical signals as will be described later. The optical amplifiers 45a and 45b are arranged so that the optical path lengths of the light from the lens system 41 to the entrance are the same. As will be described later, when optical amplification is performed by each of the optical amplifiers 45a and 45b, the optical amplifiers 45a and 45b perform stimulated emission with a bias signal using the modulation signal, thereby causing a phase difference of fluorescence with respect to the modulation signal. This is because an optical signal including the above information is output.
The optical amplifiers 45 a and 45 b are electrically connected to the control unit 50 via the power splitter 47 and are biased with a modulation signal transmitted from the control unit 50. Further, the optical amplifier 45 b is connected to the power splitter 47 through a 90-degree phase shifter 48. The modulated signal transmitted from the control unit 50 is distributed by the power splitter 47. The optical amplifier 45a is supplied with a signal having the same phase as the modulation signal transmitted from the control unit 50. On the other hand, the phase of the signal supplied to the optical amplifier 45 b is shifted by 90 degrees with respect to the modulation signal transmitted from the control unit 50 by the 90-degree phase shifter 48. As a result, the atoms or molecules of the laser medium constituting each of the optical amplifiers 45a and 45b are excited by the modulation signal. When the fluorescence is incident, the atoms or molecules of the laser medium are stimulated to emit, and the fluorescence incident on the optical amplifiers 45a and 45b is amplified.
By using the optical amplifiers 45a and 45b, the fluorescence optical signal can be amplified before the fluorescence optical signal is converted into an electrical signal.

Next, amplification of the fluorescence optical signal will be described in detail. The amplification of the fluorescence optical signal is performed by causing the optical amplifiers 45a and 45b to perform stimulated emission of light using a modulation signal and a signal obtained by phase shifting the modulation signal by 90 degrees as a bias signal. Hereinafter, a description will be given of the case where the optical signal of the fluorescence is amplified using the optical amplifier 45a.
The atoms or molecules of the laser medium constituting the optical amplifier 45a, when absorbing the energy of the modulation signal, transition from the ground state to the excited state, emit light (spontaneous emission) after a certain time, and return to the ground state again. In addition, when an optical signal (fluorescence signal) having the same frequency as the modulation signal is incident, the excited atoms or molecules emit light (stimulated emission) in a chain reaction toward the same direction. Probabilities of spontaneous emission, absorption, or stimulated emission per unit time of atoms or molecules of the laser medium are represented by A, B ₁₂ W, and B ₂₁ W, respectively. Here, W is the energy density of incident light, and A, B ₁₂ , and B ₂₁ are probabilities of state transitions. Further, when a group of atoms or molecules is in a thermal equilibrium state, since B ₁₂ = B ₂₁ , each of B ₁₂ and B ₂₁ is simply represented as B hereinafter.
The density of atoms or molecules in the ground state (the occupied position number) N _1, when the density of the atoms or molecules of the excited state (occupied quantiles) was N _2, differential equation representing the time variation of N _1, N ₂ ( The rate equation is expressed as the following equation (1). In the following formula (1), the time variation of the density of excited atoms or molecules (left side of formula (1)) is the frequency of spontaneous emission of excited atoms or molecules (first on the right side of formula (1)). ) And the frequency of occurrence of stimulated emission (the second term on the right side of Equation (1)).

また、Ｎ_１＋Ｎ_２＝Ｎとすると、式（１）は、以下の式（２）のように示される。

When N ₁ + N ₂ = N, the expression (1) is expressed as the following expression (2).

  Here, when the voltage of the bias signal of the optical amplifier 45a is changed at the same angular frequency ω as that of the modulation signal, the probabilities A and B are expressed by A = acosωt and B = bcosωt, respectively. Since a and b are probability values, a and b ≦ 1. In addition, since the fluorescence is emitted by the laser light whose intensity is modulated by the modulation signal, the incident light (fluorescence) incident on the optical amplifier 45a is also modulated by the angular frequency ω. Then, the energy density W of the incident light is expressed by W = wcos (ωt + θ). W is the intensity of incident light. By substituting the values of A, B, and W into Equation (2) and using the trigonometric addition theorem, Equation (2) is expressed as Equation (3) below.

  In Equation (3), t is the time for incident light to pass through the optical amplifier 45a. Also,

としたとき、式（３）の一般解は、定数変化法を用いることにより、以下の式（６）のように示される。

Then, the general solution of the equation (3) is expressed as the following equation (6) by using the constant change method.

なお、式（６）において、∫ｐ（ｔ）ｄｔは、式（４）より以下の式（７）のように示される。

In Expression (6), ∫p (t) dt is expressed as Expression (7) below from Expression (4).

ここで、変調信号の変調周波数は、例えば、１０^８Ｈｚ程度であることから、角周波数ω＞＞１となる。また、入射光の強度ｗは、例えば、数ｍＷ〜数十ｍＷ程度である。これにより、（ａ／ω）・ｓｉｎωｔの値と、（ｂｗ／２ω）・ｓｉｎ（２ωｔ＋θ）の値は、極めて小さくなることから、ほぼ影響が無いと考えられる。したがって、式（７）は、以下の式（８）のように近似することができる。

Here, since the modulation frequency of the modulation signal is, for example, about 10 ⁸ Hz, the angular frequency ω >> 1. Further, the intensity w of the incident light is, for example, about several mW to several tens mW. As a result, the value of (a / ω) · sin ωt and the value of (bw / 2ω) · sin (2ωt + θ) are extremely small, so it is considered that there is almost no influence. Therefore, the equation (7) can be approximated as the following equation (8).

  Further, ∫dt′q (t ′) exp (∫p (t) dt) in the equation (6) is expressed as the following equation (9).

ここで、入射光が進行する方向の光増幅器４５ａの長さは、数ｃｍ〜数十ｃｍ程度であることから、ｔ＜＜１とすることができる。したがって、式（９）のｅｘｐ｛（ｂｗｃｏｓθ）ｔ｝は、以下の式（１０）のように近似することができる。

Here, since the length of the optical amplifier 45a in the direction in which the incident light travels is about several centimeters to several tens of centimeters, it can be set to t << 1. Therefore, exp {(bw cos θ) t} in equation (9) can be approximated as in the following equation (10).

さらに、Ｎの値はアボガドロ数程度の大きさと考えられることから、式（９）は、以下の式（１１）のように近似することができる。

Furthermore, since the value of N is considered to be as large as the Avogadro number, Equation (9) can be approximated as Equation (11) below.

したがって、式（６）の右辺第２項は、以下の式（１２）のように示される。

Therefore, the second term on the right side of Equation (6) is expressed as in Equation (12) below.

  Therefore, Expression (6) is expressed as Expression (13) below using Expression (12).

そして、光増幅器４５ａから出力される蛍光は、Ｎ_２の時間微分で表すことができることから、式（１３）を用いて以下の式（１４）のように示される。

Since the fluorescence output from the optical amplifier 45a can be expressed by time differentiation of N ₂ , the following expression (14) is obtained using the expression (13).

式（１４）より、光増幅器４５ａから出力される蛍光の光信号には、入射光（蛍光）の周波数と変調信号の周波数との加算周波数を成分とする高周波成分と、入射光の周波数と変調信号の周波数との差分周波数を成分とする低周波成分とが含まれている。すなわち、変調信号を用いたバイアス信号で誘導放出が行われることにより、光増幅器４５ａは、入射光すなわち蛍光の光信号と、変調信号とをミキシングした結果を得ることができる。また、蛍光の光信号と、蛍光の光信号と同じ周波数を有する変調信号とをミキシングした結果が得られることによって、レーザ光によって変調された蛍光を復調することができる。さらに、蛍光の光信号は、Ｎｂ／２（＞１）倍に増幅されている。
このようにして、光増幅器４５ａは、変調信号を用いたバイアス信号で誘導放出を行うことにより、蛍光の光信号の増幅し、これによりミキシングした結果を得ることができる。なお、光増幅器４５ａから出力された蛍光の光信号の低周波成分は、後述する信号処理部４６ａにおいて、変調信号に対する蛍光の位相差の情報である実数部成分（Ｒｅ成分）として得られる。
また、光増幅器４６ｂは、制御部５０から送信された変調信号に対して９０度位相シフトした信号を用いたバイアス信号で誘導放出を行うことにより、光増幅器４５ａと同様に、蛍光の光信号を増幅する。このとき、光増幅器４６ｂは、蛍光の光信号とバイアス信号とをミキシングした結果を得ることができる。なお、光増幅器４５ｂから出力された蛍光の光信号の低周波成分は、後述する信号処理部４６ｂにおいて、変調信号に対する蛍光の位相差の情報である虚数部成分（Ｉｍ成分）として得られる。

From the equation (14), the fluorescence optical signal output from the optical amplifier 45a has a high frequency component whose component is an addition frequency of the frequency of the incident light (fluorescence) and the frequency of the modulation signal, and the frequency and modulation of the incident light. A low frequency component having a difference frequency from the signal frequency as a component is included. That is, by performing stimulated emission with a bias signal using a modulation signal, the optical amplifier 45a can obtain a result of mixing the incident light, that is, the fluorescence optical signal, with the modulation signal. Further, by obtaining a result of mixing the fluorescence optical signal and the modulation signal having the same frequency as the fluorescence optical signal, it is possible to demodulate the fluorescence modulated by the laser light. Further, the fluorescence optical signal is amplified by Nb / 2 (> 1) times.
In this way, the optical amplifier 45a can amplify the fluorescence optical signal by performing stimulated emission with the bias signal using the modulation signal, and thereby obtain a mixed result. The low-frequency component of the fluorescence optical signal output from the optical amplifier 45a is obtained as a real part component (Re component), which is information on the phase difference of the fluorescence with respect to the modulation signal, in the signal processing unit 46a described later.
The optical amplifier 46b performs stimulated emission with a bias signal using a signal shifted by 90 degrees with respect to the modulation signal transmitted from the control unit 50, and thus the fluorescence optical signal is transmitted in the same manner as the optical amplifier 45a. Amplify. At this time, the optical amplifier 46b can obtain the result of mixing the fluorescence optical signal and the bias signal. The low-frequency component of the fluorescence optical signal output from the optical amplifier 45b is obtained as an imaginary part component (Im component), which is information on the phase difference of the fluorescence with respect to the modulation signal, in the signal processing unit 46b described later.

  Next, the configuration of the signal processing units 46a and 46b will be described with reference to FIG. FIG. 4 is a diagram illustrating an example of the configuration of the signal processing units 46a and 46b. The signal processing unit 46a converts the fluorescent optical signal output from the optical amplifier 45a into an electrical signal, and the signal processing unit 46b converts the fluorescent optical signal output from the optical amplifier 45b into an electrical signal. As illustrated in FIG. 4, the signal processing units 46 a and 46 b include a BPF 461, a photoelectric converter 462, a low-pass filter (LPF) 463, and an A / D converter 464.

The BPF 461 is a filter that is provided in front of the photoelectric converter 462 and transmits a fluorescent optical signal in a predetermined wavelength band. Thereby, stray light can be suppressed. A band reject filter may be used instead of the BPF 461.
The photoelectric converter 462 is, for example, a photodiode or a photomultiplier tube, and has a light receiving element that receives an optical signal, converts the optical signal into an electrical signal, and outputs the electrical signal. Here, the photodiode has a characteristic that the quantum efficiency is superior to that of the photomultiplier tube, but the amplification factor is inferior. For this reason, it is difficult to use a photodiode for a photoelectric converter in the conventional configuration in which fluorescence is detected using a photoelectric converter that amplifies a fluorescent light signal. On the other hand, in the present embodiment, since the fluorescent optical signal is amplified by the optical amplifiers 45a and 45b, the optical signal need not be amplified by the photoelectric converter. Thereby, in this embodiment, the photodiode which was difficult to use for the photoelectric converter in the prior art can be used for the photoelectric converter 462.
The LPF 463 removes a high frequency component having a component of an addition frequency of the frequency of the modulation signal and the frequency of the fluorescence signal from the fluorescence electric signal output from the photoelectric converter 462, and the frequency of the modulation signal and the frequency of the fluorescence signal. Is a filter for passing a low frequency component having a difference frequency as a component. As a result, the real part component (Re component) of the fluorescent electrical signal is output from the LPF 463 of the signal processing unit 46a, and the imaginary part component (Im component) of the fluorescent electrical signal is output from the LPF 463 of the signal processing unit 46b. The Further, by outputting a low-frequency signal from the LPF 463, a flow cytometer can be configured using a low-frequency circuit that is easier to manufacture than a high-frequency circuit in signal processing after the LPF 463.
Note that when a low-speed light receiving element is used for the photoelectric converter 462, the high frequency component of the electric signal is naturally removed by the light receiving element. For example, after the photoelectric conversion process, the high frequency of the electric signal is obtained using a filter for the electric signal. There is no need to perform the process of removing the components. Thereby, since the number of parts of signal processing parts 46a and 46b can be reduced, the manufacturing cost of a flow cytometer can be reduced as a result.
The A / D converter 464 of the signal processing unit 46a converts the Re component of the fluorescent electrical signal output from the LPF 463 into digital data. The A / D converter 464 of the signal processing unit 46b converts the Im component of the fluorescence signal output from the LPF 463 into digital data. Each of the converted digital data is supplied to the analysis device 60.

  Next, the configuration of the control unit 50 will be described with reference to FIG. FIG. 5 is a diagram illustrating an example of the configuration of the control unit 50. The control unit 50 controls the modulation frequency of the modulation signal. As shown in FIG. 5, the control unit 50 includes an oscillator 51, a power splitter 52, and amplifiers (AMP) 53 and 54.

The oscillator 51 generates and outputs a sine wave signal having a predetermined frequency. The sine wave signal output from the oscillator 51 is used as a modulation signal. The frequency of the sine wave signal is, for example, 10 to 200 MHz.
A sine wave signal (modulation signal) having a predetermined frequency output from the oscillator 51 is distributed to the two amplifiers 53 and 54 by the power splitter 52. The modulation signal amplified by the amplifier 53 is output to the laser light source unit 20. The modulation signal amplified by the amplifier 54 is output to the second light receiving unit 40. The modulation signal amplified by the amplifier 54 is output to the second light receiving unit 40 because the modulation signal is used as a bias signal for the optical amplifiers 45a and 45b of the second light receiving unit 40 as described above.

Next, the configuration of the analyzer 60 will be described with reference to FIG. FIG. 6 is a diagram illustrating an example of the configuration of the analysis device 60. The analysis device 60 is a computer mainly composed of a CPU, and calculates the phase difference of the fluorescence with respect to the modulation signal using the electrical signals output from the signal processing units 46a and 46b of the second light receiving unit 40, The fluorescence relaxation time of fluorescence is determined from this phase difference. The analysis device 60 includes a phase difference calculation unit 61 and a fluorescence relaxation time calculation unit 62. The phase difference calculation unit 61 and the fluorescence relaxation time calculation unit 62 are modules formed by executing a computer-executable program.
The analyzer 60 is an example of a processing unit in the present invention.

The phase difference calculation unit 61 uses the Re component data received from the signal processing unit 46 a of the second light receiving unit 40 and the Im component data received from the signal processing unit 46 b of the second light receiving unit 40 to tan ^−1. By calculating (Im / Re) (Im is the value of the data of the Im component and Re is the value of the data of the Re component), the phase difference θ of the fluorescence with respect to the modulation signal is calculated. The calculated phase difference θ is transmitted to the fluorescence relaxation time calculation unit 62.
The phase difference calculation unit 61 may calculate the corrected phase difference θ ₁ instead of the calculated phase difference θ and transmit the phase difference θ ₁ to the fluorescence relaxation time calculation unit 62. Here, the corrected phase difference θ ₁ is obtained by calculating θ−θ ′ using the phase difference θ and the correction phase θ ′. The correction phase θ ′ is stored and held in advance in a storage device (not shown) provided in the analysis device 60, and is obtained as follows. That is, the fluorescence of the measurement object 12 is measured using a known fluorescent dye that emits fluorescence with a known fluorescence relaxation time as the measurement object 12. At this time, the correction phase θ ′ is determined so that the fluorescence relaxation time τ obtained from the corrected phase difference θ−θ ′ matches the known fluorescence relaxation time of the fluorescent dye. Thus, the correction phase θ ′ is a correction amount for calibrating so that the measurement result matches the known fluorescence relaxation time.
The fluorescence relaxation time calculation unit 62 uses the phase difference θ received from the phase difference calculation unit 61 (or the corrected phase difference θ ₁ ) to determine the fluorescence relaxation time τ according to τ = tan θ / (2πf). The reason why the fluorescence relaxation time τ can be obtained according to the above formula is that fluorescence follows a relaxation response with a substantially first-order lag.

The output unit 70 is, for example, a display device, a printer, or the like, and outputs the fluorescence relaxation time or the like as a result of calculation by the analysis device 60.
The above is the schematic configuration of the flow cytometer of the present embodiment.

(Fluorescence detection method)
FIG. 7 is a diagram for explaining an example of the flow of the fluorescence detection method of the present embodiment. In the fluorescence detection method of the present embodiment, information on the measurement target 12 is acquired by irradiating the measurement target 12 with laser light and receiving fluorescence emitted from the measurement target 12 irradiated with the laser light. Can do.
First, the oscillator 51 of the control unit 50 generates a sine wave signal having a predetermined frequency as a modulation signal (step S1), and supplies the generated modulation signal to the laser light source unit 20 and the second light receiving unit 40 (step S2). ).

Next, when the modulation signal is supplied from the control unit 50, the laser driver 23 of the laser light source unit 20 modulates the intensity of the laser light with the modulation frequency of the modulation signal. The intensity-modulated laser light is emitted from the laser light source 21, and the measurement target 12 passing through the measurement field in the flow cell 10 is irradiated with the laser light (step S3).
On the other hand, the optical amplifiers 45 a and 45 b of the second light receiving unit 40 are biased with a bias signal using the modulation signal supplied from the oscillator 51. When the fluorescence emitted when the measurement object 12 passing through the measurement field is irradiated with the laser light is received by the second light receiving unit 40, the optical amplifiers 45a and 45b amplify the optical signal of the fluorescence (step) S4). As a result, the optical signal including the Re component, which is information on the phase difference of the fluorescence with respect to the modulation signal, and the optical signal including the Im component, which is information on the phase difference of the fluorescence with respect to the modulation signal, are amplified. The optical signals amplified by the optical amplifiers 45a and 45b are converted into digital data by the signal processing units 46a and 46b. At this time, digital data of the Re component and Im component of the fluorescence signal is output.

Next, the phase difference calculation unit 61 of the analysis device 60 calculates the phase difference θ of the fluorescence signal with respect to the modulation signal using the digital data of the Re component and Im component received from the second light receiving unit 40 (step S5). .
And the fluorescence relaxation time calculation part 62 calculates | requires fluorescence relaxation time (tau) using the phase difference (theta) calculated by the phase difference calculation part 61 (step S6).
The output unit 70 outputs information such as the obtained fluorescence relaxation time τ.

As described above, according to this embodiment, since the fluorescence optical signal is amplified using the optical amplifying units 45a and 45b, the fluorescence optical signal is amplified before the fluorescence optical signal is converted into an electrical signal. can do. Thereby, even when the number of incident fluorescence photons is small, the intensity of the fluorescence can be increased. Therefore, after the fluorescence optical signal is converted into an electrical signal, the fluorescence electrical signal is converted into another electrical signal. Extraction is difficult due to being buried, and the possibility of being removed as noise can be reduced. Therefore, even when the intensity of the fluorescence is low, the fluorescence can be detected, and thus a highly accurate fluorescence relaxation time can be acquired.
Further, the optical amplifiers 45a and 45b biased by the bias signal using the modulation signal amplify the fluorescent optical signal, whereby the fluorescent optical signal and the bias signal are mixed. For this reason, for example, it is not necessary to provide a mixer such as a mixer for mixing electric signals. Therefore, it is not necessary to separately provide an apparatus for amplifying an optical signal and a mixer, so that the number of parts can be reduced and the manufacturing cost of the flow cytometer can be reduced.

(Modification)
A modification of the above embodiment will be described with reference to FIG. FIG. 8 is a diagram illustrating a modification of the second light receiving unit illustrated in FIG. 3.
In the above embodiment, the fluorescence optical signal is divided into two and incident on the optical amplifiers 45a and 46b, whereby an optical signal including the Re component of the fluorescence phase difference is output from the optical amplifier 45a, and the fluorescence phase difference is obtained. The optical signal including the Im component is output from the optical amplifier 45b. As shown in FIG. 8, the present modification is different from the above-described embodiment in that a fluorescent light signal is configured to enter one optical amplifier 45a.
The optical amplifier 45 a of this modification is electrically connected to the control unit 50 via the phase shifter 49. The phase shifter 49 transmits the modulation signal received from the control unit 50 to the optical amplifier 45a. When the phase shifter 49 receives a predetermined control signal from the control unit 50 every time a predetermined time (for example, several microseconds) elapses, the phase shifter 49 switches the phase of the modulation signal and transmits it to the optical amplifier 45a. Specifically, the phase shifter 49 transmits a sine wave signal having the same phase as the modulation signal transmitted from the control unit 50 to the optical amplifier 45a, and receives the control signal from the control unit 50. A cosine signal shifted by 90 degrees with respect to the modulation signal is transmitted to the optical amplifier 45a.
Thereby, an optical signal including the Re component of the fluorescence phase difference and an optical signal including the Im component of the fluorescence phase difference can be output from one optical amplifier 45a.
In this modification, since it is not necessary to distribute the fluorescence optical signal into two, it is not necessary to provide the half mirror 43, the BPF 44b, the optical amplifier 45b, the signal processing unit 46b, and the power splitter 47 of the above embodiment. For this reason, the number of parts can be reduced and the manufacturing cost of the flow cytometer can be reduced.

(Second Embodiment)
Hereinafter, a flow cytometer to which the fluorescence detection device and the fluorescence detection method of the second embodiment are applied will be described. The configuration of the flow cytometer of the second embodiment is substantially the same as the configuration of the flow cytometer of the first embodiment. The flow cytometer of the second embodiment is different from the flow cytometer of the first embodiment in that a signal (encoded sequence signal) in which a signal value is encoded is modulated signal and bias for intensity-modulating laser light. It is used as a signal. Specifically, the configuration of the oscillator 51 of the present embodiment is different from the configuration of the oscillator 51 of the first embodiment.

With reference to FIG. 9, the structure of the oscillator 51 of this embodiment is demonstrated. FIG. 9 is a diagram for explaining an example of the configuration of the oscillator 51 included in the flow cytometer of the second embodiment.
The oscillator 51 generates a predetermined encoded sequence signal and supplies the encoded sequence signal to the amplifiers 53 and 54. The oscillator 51 repeatedly generates an encoded sequence signal supplied to the laser light source unit 20 as a modulation signal and an encoded sequence signal supplied to the second light receiving unit 40 as a bias signal for the optical amplifiers 45a and 45b. .
Note that, as an encoded sequence signal, a signal whose signal value is encoded with a predetermined length, and the signal before the shift and the signal after the shift are substantially orthogonal to each other by shifting in the bit direction in the bit unit. A signal configured as described above is used. The bit direction refers to the arrangement direction of signal values. As such an encoded sequence signal, for example, a PN encoded sequence signal is preferably used. The PN encoded sequence signal is preferably a signal using an M-sequence or Gold sequence code, and the M-sequence is particularly preferable in terms of correlation characteristics described later.
In the M series, the oscillator 20 has a shift register code generator, which is an m-stage (m is a natural number) shift register and a logical combination of the states of each stage of the shift register. Is a signal whose length L is expressed by 2m−1. The Gold sequence is obtained by adding two M sequences in synchronization for each bit. Therefore, the phase relationship between the two code generators is invariant, and the length of the generated sequence is the same as the length of the original sequence, but does not become an M sequence.

The PN encoded sequence signal is a 1-bit signal having a value of 0 and 1, and the value of the autocorrelation function that can be shifted by the bit unit in the bit direction is 0 or -1 / n (n is a sequence code described later) Signal).
For example, the PN encoded sequence signal is a signal generated using data of a PN sequence code created as follows.
The order k = 5, the length of the code sequence n = 31, the coefficients h ₁ = 1, h ₂ = 1, h ₃ = 0, h ₄ = 1, h ₅ = 1, and the initial values a ₀ = 1, a _{When 1} = 1, a ₂ = 0, a ₃ = 1, and a ₄ = 0, the PN sequence code C = {a _k } (k is a natural number) is uniquely determined by the recurrence formula shown in the following formula (15). Can be sought.

Further, a reference encoded sequence signal is generated using a sequence code C = {a ₀ , a ₁ , a ₂ ,..., A _n−1 }, and this sequence code C is further converted into q1 bits and bits. An encoded sequence signal is generated using a sequence code T _q1 · C bit-shifted in the direction (T _q1 is an operator that bit-shifts q1 bits in the bit direction). Here, the sequence code T _q1 · C is {a _q1 , a _{q1 + 1} , a _{q1 + 2} ,..., A _{q1 + N−1} }. Further, a coded sequence signal is generated using a sequence code T _q2 · C obtained by shifting the sequence code C by q2 bits (eg, q2 = 2 × q1) and bit-shifting in the bit direction.
Since the sequence codes C, T _q1 · C and T _q2 · C used to generate the encoded sequence signal have characteristics that are orthogonal to each other, the generated encoded sequence signals also have a property to be orthogonal to each other.

More specifically, a sequence code of length n is C = {b ₀ , b ₁ , b ₂ ,..., B _n−1 }, and a sequence code in which the operator T _{q is applied} to the sequence code C. Is C ′ = T _q · C, that is, C ′ = {b _q , b _{q + 1} , b _{q + 2} ,..., B _{q + n−1} }, the correlation function R _cc between the sequence codes C and C ′. _′ (Q) is defined as in the following formula (16). Here, N _A is a number in which the values of the term b _i and the term b _{q + i} (i is an integer between 0 and n−1) in the sequence code match, and N _D is the term b _i and the term b _{q + i} in the sequence code. Is the number of mismatches in the value of. Further, the sum of N _A and N _D is the sequence code length n (N _A + N _D = n). Here, i and q + i are considered as mod (n).

In the PN encoded sequence, the result of adding two sequences by mod (2) for each term has a property of becoming a PN sequence code obtained by cyclically shifting the original PN sequence code, and the value of the PN sequence code is 0. Since the number is one less than the number where the value is 1, N _A −N _D = −1. Thus, the values shown in the following equations (17) and (18) are obtained in the PN sequence code.

From the above equation (17), when the bit shift amount is 0, that is, q = 0 (mod (n)), the value of R _{cc ′} (q) is 1 as shown in equation (17), and there is autocorrelation. . On the other hand, when the bit shift amount is not 0, that is, q ≠ 0 (mod (n)), the value of R _{cc ′} (q) is −1 / n as shown in Expression (18). Here, by increasing the sequence code length n, the value of R _{cc ′} (q) (q ≠ 0) approaches zero.
That is, it can be said that the sequence codes C and C ′ have autocorrelation and substantially orthogonality.
A PN coded sequence signal is a time series signal with such PN sequence code values of 0 and 1.
The oscillator 51 generates such a PN encoded sequence signal.

Next, the configuration of the oscillator 51 of the present embodiment will be described with reference to FIG. In order to increase the resolution of the fluorescence phase difference θ with respect to the modulation signal, it is necessary to extremely narrow the time interval Δt between the data of the encoded sequence signals. That is, it is necessary to reduce the time width for shifting the PN coded sequence signal by 1 bit. Therefore, since the oscillator 51 needs to oscillate a high-speed encoded sequence signal, as shown in FIG. 9, an FPGA (Field Programmable Gate Array) 51a, a parallel-serial (P / S) converter 51b, It is preferable to use 51c, synchronous signal dividers 51d and 51e, and a clock signal generator 51f.
The reason why the parallel / serial converters 51b and 51c are used is to speed up the encoded sequence signal. The clock signal generator 51f is used to control the synchronization or delay time of two encoded series signals generated as serial signals of the parallel / serial converters 51b and 51c and sent to the amplifier 53 and the amplifier 54. It is. The synchronization or delay time is controlled by the fact that the encoded sequence signal sent to the amplifier 54 is mixed with the fluorescent optical signal by the optical amplifiers 45a and 45b in the amplification process using the optical amplifiers 45a and 45b. Thus, a correlation function is created between the optical signals of the fluorescence incident on the optical amplifiers 45a and 45b. The fluorescent optical signal incident on the optical amplifiers 45 a and 45 b is modulated by an encoded sequence signal sent to the amplifier 53.
The oscillator 51 repeatedly generates an encoded sequence signal according to a pulse signal from the analysis device 60 or a pulse signal from a control device (not shown).

The encoded sequence signal supplied to the optical amplifiers 45a and 45b as a bias signal is repeatedly generated by the oscillator 51, and the generated timing is the time interval Δt between the encoded sequence signal data, that is, encoded. Each bit of the sequence signal is shifted in the bit direction. The delay due to the shift at this time is a delay based on the generation timing of the encoded sequence signal supplied to the laser light source unit 20 as a modulation signal.
In the mixing at the time of amplification by the optical amplifiers 45a and 45b, when the delayed encoded series signal is a (t + Δ) and the optical signal of the incident light (fluorescence) is b (t), a (t + Δ) × b ( t) is calculated. Here, Δ is a delay time, and Δ = k · Δt (k is a natural number and represents a shift amount of 1 bit on the data point).

  The LPF 463 of each of the signal processing units 46a and 46b removes the signal component of the high frequency component from the fluorescence signal mixed by the optical amplifiers 45a and 45b with the frequency set according to the encoded sequence signal as a cutoff frequency. Then, the low frequency component signal is passed.

The phase difference calculation unit 61 of the analysis device 60 integrates the low-frequency component signal for one cycle of the encoded sequence signal in synchronization with the generation timing of the encoded sequence signal repeatedly generated by the oscillator 51. To do. Thereby, the value of the correlation function in the delay time Δ can be obtained. By sequentially changing the delay time Δ, a correlation function can be obtained.
Further, the phase difference calculation unit 61 obtains a delay time Δ ₁ that maximizes the value of the correlation function in one period of the encoded sequence signal, and uses the obtained delay time Δ ₁ to obtain 2πf · Δ ₁ ( f is the value of the modulation frequency) to calculate the fluorescence phase difference θ with respect to the modulation signal. The correlation function obtained by the phase difference calculation unit 61 is an autocorrelation function when the circuit band is infinite.
The fluorescence relaxation time calculation unit 62 uses the phase difference θ received from the phase difference calculation unit 61 to determine the fluorescence relaxation time τ according to τ = tan θ / (2πf).

  Thus, according to the present embodiment, the modulation signal is a signal in which the signal value is encoded, and the signal before the shift and the signal after the shift are mutually shifted by shifting in the bit direction in the bit direction. The signal is configured to be substantially orthogonal. For this reason, when the correlation function is obtained by the phase difference calculation unit 61, the value is approximately 0 in a state where there is no correlation. For this reason, the peak position of the correlation function indicating the fluorescence delay time is clearly formed. Therefore, the fluorescence phase difference θ can be obtained with high accuracy, and a highly accurate fluorescence relaxation time can be obtained.

Incidentally, by increasing the SN ratio of the correlation function, in order to facilitate seeking a delay time delta _1, the value other than the maximum value of the correlation function (= -1 / n) is close to 0, i.e. approximately orthogonal It is necessary to realize the property (substantially orthogonal). For this purpose, it is necessary to increase the number of data n in one cycle of the encoded sequence signal. However, by increasing the number of data n, the time T (= n · Δt) corresponding to one cycle increases, and it takes a lot of calculation time to calculate the correlation function in the range of 0 to T delay time. .
Therefore, when fluorescence detection is performed using an encoded sequence signal, it is preferable to increase the residence time of the measurement object 12 in the measurement field. For example, the fluorescence detection apparatus and the fluorescence detection method of the present embodiment are applied to a fluorescence microscope. It is preferable to do.

  As described above, the fluorescence detection apparatus and the fluorescence detection method of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments and modifications, and various improvements and modifications can be made without departing from the gist of the present invention. Of course.

DESCRIPTION OF SYMBOLS 10 Flow cell 12 Measurement object 20 Laser light source part 30 1st light-receiving part 40 2nd light-receiving part 45a, 45b Optical amplifier 50 Control part 51 Oscillator 60 Analyzer 61 Phase difference calculation part 62 Fluorescence relaxation time calculation part 70 Output part

Claims (3)

A fluorescence detection device that detects fluorescence emitted when a measurement object is irradiated with laser light,
A laser light source unit for irradiating the measurement object with intensity-modulated laser light;
An optical element that divides a fluorescent optical signal emitted when the laser beam is irradiated onto the measurement object into two, and converts the intensity of the laser light into a first optical signal and a second optical signal; A first optical amplifier that amplifies the first optical signal by performing a first stimulated emission with a bias signal using a signal in phase with the modulation signal of the first modulation signal, and a modulation for intensity-modulating the laser light A second optical amplifier that amplifies the second optical signal by performing second stimulated emission with a bias signal using a signal that is phase-shifted by 90 degrees with respect to the signal; and A light receiving unit comprising: a first light receiving element that receives light and outputs a first electric signal; and a second light receiving element that receives the second optical signal and outputs a second electric signal;
A process of calculating a phase difference of the fluorescence with respect to the modulation signal using the first electric signal and the second electric signal output from the light receiving unit, and obtaining a fluorescence relaxation time of the fluorescence from the phase difference And having a part,
A fluorescence detection apparatus characterized by the above. The fluorescence detection apparatus according to claim 1 , wherein the optical amplifier outputs an optical signal including information on a phase difference of the fluorescence with respect to the modulation signal. A fluorescence detection method for detecting fluorescence emitted when a measurement object is irradiated with laser light,
Irradiating the measurement object with intensity-modulated laser light; and
Dividing the fluorescence optical signal emitted when the measurement object is irradiated with the laser light into two to be a first optical signal and a second optical signal;
Amplifying the first optical signal by performing first stimulated emission with a bias signal using a modulation signal for intensity-modulating the laser light;
Amplifying the second optical signal by performing second stimulated emission with a bias signal using a signal phase-shifted by 90 degrees with respect to a modulation signal for intensity-modulating the laser light;
Receiving a first optical signal obtained by performing the first stimulated emission and outputting a first electrical signal;
Receiving a second optical signal obtained by performing the second stimulated emission and outputting a second electrical signal; and
Calculating the phase difference of the fluorescence with respect to the modulation signal using the output first electric signal and the second electric signal, and obtaining the fluorescence relaxation time of the fluorescence from the phase difference. ,
A fluorescence detection method characterized by that.

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