JP5502124B2 - 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.

2. Description of the Related Art Conventionally, a fluorescence detection device and a fluorescence detection method are known that irradiate a measurement target with laser light, receive fluorescence emitted from the measurement target, and acquire 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.

  Under such circumstances, a fluorescence detection apparatus that calculates a fluorescence relaxation time (fluorescence lifetime) by irradiating a measurement target with laser light whose intensity is modulated at a set modulation frequency and receiving fluorescence emitted from the measurement target; A fluorescence detection method is known (Patent Document 1).

JP 2006-226698 A

  In the fluorescence detection device, an optical filter is used to separate the fluorescence from the scattered light of the laser light before the fluorescence is received by the photoelectric converter. However, in the optical filter, the fluorescence and laser light are not sufficiently separated, and the light receiving element may receive light with the intensity of the scattered light of the laser light that has not been removed still being higher than the fluorescence intensity. The output light reception signal may not be a fluorescence signal with sufficient output.

  Also, a plurality of optical filters can be used to enhance the removal of laser light. However, in this case, since the optical filter has a low transmittance, the intensity of transmitted fluorescence also decreases at the same time. An expensive spectroscope can be used to separate the fluorescence from the scattered light of the laser beam, but the apparatus becomes larger and the cost increases. Further, since the light receiving element simultaneously receives the scattered light of the fluorescence and the laser light, the sensitivity of the light receiving element cannot be increased.

  Therefore, an object of the present invention is to provide a fluorescence detection apparatus and a fluorescence detection method capable of efficiently removing scattered light of a laser beam and calculating a highly accurate fluorescence intensity.

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 that emits laser light whose intensity is time-modulated at a frequency f and irradiates the measurement object;
A light receiving unit that receives fluorescence emitted from the measurement object when the measurement object is irradiated with the laser beam;
When the light receiving unit receives the fluorescence, reference light having the same wavelength component as the fluorescence, the phase of the light being time-modulated with an angular frequency ω _ν , and the intensity being time-modulated with the frequency f is received by the light receiving unit. A reference light source unit including a reference light source that emits the reference light so that the unit receives light simultaneously with the fluorescence;
A filter unit that extracts a component of the angular frequency ω _ν from a light reception signal output from the light receiving unit;
From the signal of the component of the angular frequency ω _ν extracted by the filter unit, using the information of the angular frequency ω _ν , a phase lag with respect to intensity modulation of the laser light of the fluorescence is obtained, and from the obtained phase lag, And a processing unit for obtaining a fluorescence lifetime of the fluorescence.

Specific preferred embodiments are as follows.
The reference light source unit includes a first reference light having an intensity modulation phase in phase with an intensity modulation phase of the laser light, and an intensity modulation phase shifted by 90 degrees relative to the intensity modulation phase of the laser light. 2 reference lights are each emitted as the reference light.
The light receiving unit includes a first light receiving element that receives the first reference light, and a second light receiving element that receives the second reference light whose phase is shifted by 90 degrees with respect to the first reference light.
The filter unit includes a first filter that extracts a component of the angular frequency ω _ν from a first light receiving signal output from the first light receiving element, and a second light receiving signal output from the second light receiving element. a second filter for extracting a component of the angular frequency omega _[nu, a.
Wherein the processing unit, component of said first signal component of the angular frequency omega _[nu taken out by the first filter demodulates using the angular frequency omega _[nu, the angular frequency omega _[nu taken out by the second filter The second signal is demodulated using the angular frequency ω _ν , and the ratio of the demodulation result of the first signal and the demodulation result of the second signal is used to determine the phase delay and to increase the fluorescence lifetime. A fluorescence lifetime calculation unit to be obtained is included.

  In that case, it is preferable that the reference light source unit temporally modulates the phase of the light by temporally changing the optical path length of the reference light from the reference light source to the light receiving element.

  The reference light source unit includes an ultrasonic transducer and a reflective surface mirror provided on the ultrasonic transducer, and the reference light source unit changes the position of the reflective mirror due to vibration of the ultrasonic transducer. It is preferable that the phase of the light is time-modulated by reflecting the incident reference light toward the light receiving element.

  The fluorescence detection device preferably includes an optical amplification unit that amplifies the fluorescence by performing stimulated emission of the light by a bias signal before receiving the fluorescence.

  The fluorescence detection device is, for example, a flow cytometer that performs fluorescence detection using a plurality of inspection target samples that sequentially flow in a line in a pipeline and pass through a laser light irradiation position as measurement objects.

Another embodiment of the present invention is a fluorescence detection method for detecting fluorescence emitted when a measurement target is irradiated with laser light. The method is
Emitting a laser beam whose intensity is time-modulated at a frequency f and irradiating the measurement object;
While receiving the fluorescence emitted from the measurement object when the measurement object is irradiated with the laser light, it has the same wavelength component as the fluorescence, the phase of the light is time-modulated with an angular frequency ω _ν , and the intensity Receiving the reference light time-modulated at the frequency f simultaneously with the fluorescence;
Extracting a signal of the component of the angular frequency ω _ν from the light reception signal obtained by the light reception;
Wherein the signal components of the angular frequency omega _[nu taken out, using the information of the angular frequency omega _[nu, obtains a phase delay with respect to the intensity modulation of the laser beam of the fluorescence from the phase delay determined, of the fluorescent Determining a fluorescence lifetime.

In this case, the fluorescence detection method preferably includes the following steps.
The step of receiving the fluorescence comprises:
(A) receiving, as the reference light, first reference light whose phase of intensity modulation is in phase with the phase of intensity modulation of the laser light;
(B) including a second step of receiving, as the reference light, the second reference light whose intensity modulation phase is shifted by 90 degrees with respect to the intensity modulation phase of the laser light simultaneously with the fluorescence.
The step of extracting the component of the angular frequency ω _ν includes
(C) extracting the component of the angular frequency ω _ν from the first received light signal obtained in the step (A);
A second light receiving signal obtained in step (D) wherein (B), comprising the step of taking out the component of the angular frequency omega _[nu.
The step of obtaining the fluorescence lifetime comprises:
(E) demodulating the first signal of the component of the angular frequency ω _ν extracted in the step (D) using the angular frequency ω _ν ;
(F) demodulating the second signal of the component of the angular frequency ω _ν extracted in the step (E) using the angular frequency ω _ν ;
(G) using the ratio of the demodulation result of the first signal and the demodulation result of the second signal to determine the phase delay and determining the fluorescence lifetime;
including.

  In the above-described fluorescence detection apparatus and fluorescence detection method, the scattered light of the laser light can be efficiently removed and the fluorescence intensity with high accuracy can be calculated.

It is the schematic which shows an example of a structure of the fluorescence detection apparatus which enforces the fluorescence detection method of this embodiment. It is a figure which shows an example of a structure of the laser light source part used for the fluorescence detection apparatus shown in FIG. It is a figure which shows an example of a structure of the 2nd light-receiving part used for the fluorescence detection apparatus shown in FIG. It is a figure which shows an example of a structure of the control part used for the fluorescence detection apparatus shown in FIG. It is a figure which shows an example of a structure of the reference light source part used for the fluorescence detection apparatus shown in FIG. It is a figure which shows an example of a structure of the process part used for the fluorescence detection apparatus shown in FIG. It is a figure which shows an example of the flow of the fluorescence detection method of this embodiment.

Hereinafter, the fluorescence detection apparatus and fluorescence detection method of the present invention will be described in detail.
FIG. 1 is a schematic diagram illustrating an example of a configuration of a fluorescence detection apparatus that performs the fluorescence detection method of the present embodiment.

(Flow cytometer)
Hereinafter, the configuration of the flow cytometer of the present embodiment will be described with reference to FIG. In the flow cytometer, a plurality of samples to be inspected are sequentially flowed in a line in the flow cell, and at this time, each of the samples to be inspected passing through the irradiation position (measurement field) of the laser beam is detected as a measurement object. I do. Specifically, the flow cytometer irradiates the measurement target with laser light, and receives fluorescence emitted from the measurement target irradiated with the laser light, thereby acquiring information on the measurement target. Although this embodiment is an example using a flow cytometer, it is not limited to a flow cytometer. For example, the present invention can also be applied to a fluorescence microscope that irradiates a laser beam with a measurement object stationary.
In the present embodiment, laser light whose intensity is time-modulated at the frequency f is emitted and irradiated on the measurement object. At this time, the light receiving unit receives the fluorescence emitted from the measurement object. At the time of this light reception, the reference light having the same wavelength component as the fluorescence, the phase of the light is time-modulated with an angular frequency (hereinafter referred to as a modulation angular frequency) ω _ν , and the intensity is time-modulated with the frequency f is simultaneously with the fluorescence, The light receiving unit receives light. The light reception signal output from the light receiving unit includes a fluorescence signal and a reference light signal. Flow cytometer, to retrieve information of the fluorescence signal, and the filtering processing on the received signal extraction signal components of the modulation angular frequency omega _[nu, further to the signal component of the modulation angular frequency omega _[nu Te, subjected to demodulation processing using the modulation angular frequency omega _[nu. Furthermore, the flow cytometer obtains the phase delay of the fluorescence emission from the laser light irradiation using the information on the result of the demodulation process, and obtains the fluorescence lifetime of the fluorescence from this phase delay. Thereby, even if the light received by the light receiving unit includes scattered light of the laser light, it is possible to efficiently remove the scattered light of the laser light and calculate the fluorescence intensity with high accuracy.

  The flow cytometer includes a flow cell 10, a laser light source unit 20, a first light receiving unit 30, reference light source units 32 and 34, a second light receiving unit 40, a control unit 50, a processing unit 60, and an output unit 70. And comprising. 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. For example, a flow cytometer causes a cell or the like that has taken in a biological material to which a fluorescent substance such as a fluorescent protein is attached to the inside of the flow cell 10 as a measurement object 12. 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 that has been intensity-modulated using a modulation signal of frequency f.
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 irradiation position (measurement field) in the flow cell 10.
The laser driver 23 is electrically connected to a control unit 50 which will be described later, and is configured to modulate the intensity of laser light by a modulation signal having a frequency f (modulation frequency) 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. When a plurality of laser light sources are used, the laser light emitted from the plurality of laser light sources is combined into one laser beam using a dichroic mirror or the like, and is emitted toward the measurement field. Is preferably formed.

  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. The period of the frequency f of the modulation signal is slightly longer than the fluorescence lifetime (fluorescence relaxation time), and the frequency f of the modulation signal is, 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 of the laser light scattered by the measurement target 12 when the measurement target 12 passing through the measurement field of the flow cell 10 is irradiated with the 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 electric signal converted by the photoelectric converter of the first light receiving unit 30 is output to the processing unit 60, and the electric signal is used as a trigger signal for informing 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 receives and amplifies the fluorescence emitted from the measurement target 12 when the measurement target 12 passing through the measurement field of the flow cell 10 is irradiated with laser light by an optical amplifier. Further, when receiving the fluorescence, the light receiving unit 40 also receives reference light emitted from reference light source units 32 and 34 described later. The reference light will be described later.

FIG. 3 is a diagram illustrating an example of the configuration of the second light receiving unit 40 of the present embodiment.
As shown in FIG. 3, the second light receiving unit 40 includes a lens system 41, a band pass filter (BPF) 42, an optical amplifier 43, a band pass filter 44, a half mirror 45, and photomultiplier tubes 46a and 46b. , Band pass filters 47a and 47b, and a reflection mirror 48.

The lens system 41 condenses the light incident on the second light receiving unit 40.
The BPF 42 is a filter that is provided in front of the optical amplifier 43 and transmits only light having a fluorescent wavelength component. The wavelength band of light to be transmitted is set corresponding to the wavelength band of fluorescence emitted by the fluorescent dye included in the measurement object 12. A band reject filter may be used instead of the BPF 42. The BPF 42 may not sufficiently separate the side scattered light of the laser light having a light intensity stronger than the light intensity of the fluorescence from the fluorescence and may enter the optical amplifier 43.

The optical amplifier 43 is, for example, a semiconductor optical amplifier, and amplifies the incident fluorescent light signal by using the DC bias signal generated by the control unit 50 as a signal for stimulated emission of light.
The optical amplifier 43 is provided to amplify the fluorescence optical signal, is electrically connected to the control unit 50, and receives a DC bias signal transmitted from the control unit 50 as a signal for stimulated emission of light. Thereby, atoms or molecules of the laser medium constituting the optical amplifier 43 are excited by the DC bias signal. When fluorescence is incident, the optical amplifier 43 performs stimulated emission of light having the same wavelength as the fluorescence of atoms or molecules in the laser medium. As a result, the fluorescence incident on the optical amplifier 43 is amplified. In other words, the laser medium is configured so that the optical amplifier 43 stimulates and emits light having a wavelength corresponding to the wavelength of fluorescence emitted from the measurement object 12. Therefore, the side scattered light of the laser light is not amplified. For this reason, the light emitted from the optical amplifier 43 becomes light with extremely strong fluorescence, and the intensity is higher than stray light such as side scattered light of laser light. The optical amplifier 43 is an example of an optical amplification unit in the present invention.

The BPF 44 is a filter that transmits the light-amplified fluorescence and separates the side scattered light of the laser light.
The fluorescence thus obtained is separated by the half mirror 45, and the fluorescence transmitted through the half mirror 45 is received by a photomultiplier tube 46a which is an example of a light receiving element. The fluorescence reflected by the half mirror 45 is received by a photomultiplier tube 46b which is an example of a light receiving element. At this time, the photomultiplier tubes 46a and 46b simultaneously receive the reference light that is time-modulated by the modulation angular frequency ω _ν and the intensity is time-modulated by the frequency f. This point will be described later.

The received light signals output from the photomultiplier tubes 46a and 46b are sent to the bandpass filters 47a and 47b. Bandpass filter 47a, 47b is an example of a filter unit for taking out a component of the modulation angular frequency omega _[nu. As will be described later, the band-pass filters 47a and 47b extract a received light signal component in which the phase of the reference light fluctuates at the modulation angular frequency ω _ν , a component of the frequency modulation frequency f of the intensity of the laser light, and a frequency ω A frequency component obtained by adding / subtracting _{v 2} / (2π) and frequency 2 · ω ₁ / (2π) is removed. The received light signals transmitted through the band pass filters 47a and 47b are sent to the processing unit 60, respectively. That is, the band-pass filters 47a and 47b extract the components of the received light signal that vibrate at the frequency ω _ν / (2π) corresponding to the modulation angular frequency ω _ν , the components of the received light signal with the frequency f, and further the frequency ω _The transmission frequency band is set so as to remove the frequency component obtained by adding / subtracting _{v 2} / (2π) and frequency 2 · ω ₁ / (2π).

  FIG. 4 is a diagram illustrating an example of the configuration of the control unit 50. The control unit 50 controls the frequency f of the modulation signal. As shown in FIG. 4, the control unit 50 includes an oscillator 51, a power splitter 52, amplifiers (AMP) 53, 54 and 56, and a 90-degree phase shifter 55.

In response to an instruction from the processing unit 60, the oscillator 51 generates and outputs a signal having a frequency f, such as a sine wave signal or a pulse signal. The signal output from the oscillator 51 is used as a modulation signal. The frequency of the signal is, for example, 10 to 200 MHz.
The signal (modulation signal) of the frequency f output from the oscillator 51 is sent to the power splitter 52 and sent to the amplifier 53, the amplifier 54, and the 90-degree phase shifter 55. The amplifier 53 amplifies the modulation signal having the frequency f and sends it to the laser driver 23 of the laser light source unit 20. The amplifier 54 amplifies the modulation signal of the frequency f and sends it to the laser driver 32a (see FIG. 5) of the reference light source unit 32. On the other hand, the 90-degree phase shifter 55 delays the phase of the modulation signal of the frequency f by a quarter period (90 degrees) and sends it to the amplifier 56. The amplifier 56 amplifies the modulation signal of the frequency f whose phase is delayed and sends it to the laser driver 34a (see FIG. 5) of the reference light source unit 34. The reference light source units 32 and 34 will be described later.

  FIG. 5 is a diagram illustrating an example of the configuration of the reference light source units 32 and 34 that emit reference light toward the photomultiplier tubes 46a and 46b. The reference light source units 32 and 34 have laser light sources 34a and 34b. Since the configuration of the reference light source unit 32 and the configuration of the reference light source unit 34 are the same, the reference light source unit 32 will be mainly described below, and the description of the reference light source unit 34 will be described in parentheses. Note that the phase of the intensity modulation of the reference light emitted from the laser light source 34b is delayed by 90 degrees from the phase of the intensity modulation of the reference light emitted from the laser light source 34a. This is also maintained when the reference light is received together with the fluorescence by the tubes 46a and 46b. That is, the reference light source units 32 and 34 are arranged so that the phase of the intensity modulation of the reference light received by the photomultiplier tube 46b is delayed by 90 degrees with respect to the phase of the intensity modulation of the reference light received by the photoelectron multiplier tube 46a. Is provided.

The reference light source unit 32 (34) is provided so that the reference light emitted from the reference light source unit 32 (34) enters the photomultiplier tube 46a (46b). The reference light source unit 32 (34) includes a laser driver 32a (34a) and a laser light source 32b (34b). The laser driver 32a (34a) modulates the intensity of the laser light source 32b (34b) according to the modulation signal of the frequency f based on the modulation signal of the frequency f sent from the control unit 50. Drive.
The laser light source 32b (34b) emits, as reference light, laser light obtained by temporally modulating the intensity of light having the same wavelength component as that of fluorescence with the frequency f. The reference light is laser light because the phase of the light is time-modulated. The reference light source unit 32 (34) temporally modulates the phase of light by changing the optical path length from the laser light source 32b (34b) to the photomultiplier tube 46a (46b) over time. In the example shown in FIG. 5, the reference light source unit 32 (34) includes an ultrasonic transducer 32c (34c) that vibrates at a modulation angular frequency ω _ν and a reflection mirror 32d (on the ultrasonic transducer 32c (34c)). 34d). The reference light source unit 32 (34) receives the reference light incident from the laser light source 32b (34b) while the position of the reflection mirror 32d (34d) is fluctuated by the vibration of the ultrasonic transducer 32c (34c). The phase of the light is time-modulated by reflecting the light toward the light-receiving surface (46b). Considering that the measurement time for the measurement object 12 to pass through the measurement field is several tens of microseconds, the frequency ω _ν / (2π) corresponding to the modulation angular frequency ω _ν is in the order of MHz, for example, several MHz. Preferably there is. Incidentally, the ultrasonic transducer 32c and the ultrasonic transducer 34c of the reference light source unit 32, the instruction processing unit 60 is controlled to oscillate at a modulation angular frequency omega _[nu.
As described above, the phase of the modulation signal sent to the laser driver 34a of the reference light source unit 34 is delayed by 90 degrees with respect to the phase of the modulation signal sent to the laser driver 32a. For this reason, the phase of the reference light received by the photomultiplier tube 46b is also delayed by 90 degrees with respect to the reference light received by the photomultiplier tube 46a.
Thus, the laser beams emitted from the reference light source units 32 and 34 are received together with the fluorescence by the photomultiplier tubes 46a and 46b, as shown in FIG. As shown in FIG. 3, the laser beam emitted from the reference light source unit 34 is reflected by the reflection mirror 48 and received by the photoelectron multiplier 46b.

  FIG. 6 illustrates the configuration of the processing unit 60. The processing unit 60 is a computer mainly composed of a CPU 64 and a memory 66, and in accordance with a trigger signal sent from the first light receiving unit 30, signals output from the bandpass filters 47a and 47b of the second light receiving unit 40 are displayed. To calculate the fluorescence intensity. The processing unit 60 includes an AD conversion board 62, a CPU 64, a memory 66, and an input / output port 68. A display 70 a and a printer 70 b are connected to the input / output port 68 as the output unit 70. The memory 66 stores a program. By reading and executing the program stored in the memory 66, the processing unit 60 forms a demodulation unit 70 and a fluorescence lifetime calculation unit 72. That is, the demodulator 70 and the fluorescence lifetime calculator 72 are software modules formed by executing a computer-executable program. Substantially functions of the demodulator 70 and the fluorescence lifetime calculator 72 are performed by the CPU 64.

As described above, the photomultiplier tubes 46a, 46b, in addition to the fluorescence amplified by the optical amplifier 43, for receiving the reference light phase of light is modulated by the modulation angular frequency omega _[nu simultaneously. This reference light is generated by the reference light source unit 32.
The fluorescence is a plane wave, and the fluorescence received by the photomultiplier tube 46a can be represented by a complex number as shown in the following formula (1). The reference light is also a plane wave, and the reference light received by the photomultiplier tube 46a can be represented by a complex number expression as shown in the following equation (2).

At this time, the photomultiplier tubes 46a, since the energy of the light is output as a received signal, the light receiving signal S _a photoelectron multiplier tube 46a is output is proportional to the expression of the following formula (3). By arranging this equation, the following equation (4) is obtained. Furthermore, the reference light has the same wavelength component as fluorescence, since it is omega _p1 = omega _p2, received signal S _a obtained by the photomultiplier 46a is represented by the following formula (5). When formula (5) is described in detail, the following formula (6) is obtained.

In the formula (5), since it is φ = ω ν _t (reference light phase is modulated time modulation angular frequency omega _[nu), the light receiving signal S _a, the frequency f of the (angular frequency omega ₁ = 2 [pi] f) and an AC component that varies component and the modulation angular frequency ω _ν. The band-pass filter 47a has a frequency component obtained by adding / subtracting the frequency f (angular frequency ω ₁ = 2πf) component from the received light signal, and the frequency ω _ν / (2π) and the frequency 2 · ω ₁ / (2π). And the signal component of the frequency component of the modulation angular frequency ω _ν is extracted. Therefore, the light reception signal S _a ′ sent from the band pass filter 47a is expressed as shown in the following equation (7) by removing the first to third terms on the right side of the equation (6).

Similarly, in the reference light received by the photomultiplier tube 46b, the phase of the modulation signal is delayed by 90 degrees as shown in FIG. 4, so cos (ω ₂ t) in the following equation (2) is sin (ω ₂ t). Changed to
According to this change, the light reception signal S _b ′ sent from the bandpass filter 47b is expressed as shown in the following equation (8) by the same processing as the equations (3) to (7).

Such light reception signals S _a ′ and S _b ′ are input to the processing unit 60.
The AD conversion board 62 is activated by a trigger signal from the first light receiving unit 30 and AD converts the light reception signals S _a ′ and S _b ′. The digitized light reception signal S ′ is temporarily recorded in the memory 66 through the input / output port 68.
The demodulator 70 calls the received light signal S ′ from the memory 66 and multiplies cos (ω _v t) separately generated by the demodulator 70 based on the information on the modulation angular frequency ω _ν and the received light signal S _a ′. By further multiplying cos (ω _v t) generated by the demodulator 70 by the received light signal S _b ′, cos (ω _v t) on the right side in the equations (7) and (8) is removed. The calculated value is calculated. That is, the demodulator 70 demodulates the light reception signal S _a ′ and the light reception signal S _b ′. Further, the demodulator 70 obtains the ratio of the demodulation processing result of the received light signal S _a ′ to the demodulation processing result of the received light signal S _b ′. As a result, the demodulator 70 can obtain tan θ where θ is the phase delay of the fluorescence laser light. The demodulator 70 obtains the phase delay θ with respect to the fluorescent laser beam from the tan θ. The obtained phase delay θ is once stored in the memory 66.

The fluorescence lifetime calculation unit 72 calls the phase delay θ stored in the memory 66, and uses this phase delay θ to change the fluorescence lifetime (fluorescence relaxation time) τ to τ = tan θ / (2πf) (f is the modulation signal). Frequency value). The reason why the fluorescence lifetime τ can be obtained according to the above equation is that the fluorescence follows a relaxation response with a substantially first-order lag.
The calculated fluorescence lifetime τ is sent to the display 70a or the printer 70b, displayed on the screen, and printed out. Further, in the flow cytometer of the present embodiment, the measurement object 12 sequentially passes through the measurement field in the flow cell 10, so that the fluorescence lifetime τ is sequentially calculated each time the measurement object 12 passes through the measurement field. Therefore, the processing unit 60 sequentially stores the calculated fluorescence lifetimes τ in the memory 66, and after all the inspections of the measurement object 12 have been completed, all the fluorescence lifetimes τ stored in the memory 66 are called and fluorescence is performed by statistical processing. Can also be analyzed.

(Fluorescence detection method)
FIG. 7 is a diagram illustrating an example of the flow of the fluorescence detection method of the present embodiment.
First, the flow cytometer allows a plurality of inspection target samples including the measurement target 12 to flow in a row in the flow cell 10 together with the sheath liquid. At this time, according to an instruction from the control unit 50, the laser light source unit 20 irradiates the measurement object 12 that has passed through the measurement field with laser light. At this time, each of the reference light source units 32 and 34 emits reference light having the same wavelength component as that of fluorescence, the phase of the light being time-modulated with the modulation angular frequency ω _ν , and the intensity being time-modulated with the frequency f ( Step S10). The phase of intensity modulation of the reference light emitted from the reference light source unit 34 and received is delayed by 90 degrees with respect to the phase of intensity modulation of the reference light emitted from the reference light source unit 32 and received.

Next, the fluorescence emitted from the measurement object 12 when the measurement object 12 is irradiated with the laser light enters the BPF 42 through the lens system 41. The optical amplifier 43 amplifies the fluorescence transmitted through the BPF 42 by performing stimulated emission of light with a DC bias signal (step S20).
The amplified fluorescence passes through the BPF 44, is separated by the half mirror 45, and reaches the photomultiplier tubes 46a and 46b. At this time, since the reference light source units 32 and 34 emit reference light, the separated fluorescence is simultaneously received by the photomultiplier tubes 46a and 46b together with the reference light (step S30).

The light reception signals output from the photomultiplier tubes 46a and 46b upon reception of the fluorescence and the reference light are subjected to bandpass filter processing by the bandpass filters 47a and 47b (step S40). In the band-pass filter processing, the component of the frequency f of the received light signal is removed, and a component having the modulation angular frequency ω _ν used for time modulation of the phase of the reference light as an angular frequency is extracted. This extracted component is sent to the processing unit 60. The processing unit 60 performs a demodulation process using the modulation angular frequency ω _ν that is known information (step S50). That is, a value obtained by removing cos (ω _v t) from the above equation (7) and a value obtained by removing cos (ω _v t) from the equation (8) are calculated by the demodulation process. As is apparent from equation (7) and (8), cos (omega _[nu t) is removed, the value of equation (8) Karakara cos (omega _[nu t) has been removed is a signal of the DC component Therefore, the demodulator 70 calculates the ratio between the value obtained by removing cos (ω _ν t) from Equation (7) and the value obtained by removing cos (ω _ν t) from Equation (8). Thereby, tan θ with respect to the phase delay θ of fluorescence is calculated (step S60). Further, the demodulator 70 calculates the phase delay θ from tan θ.
The fluorescence lifetime calculation unit 72 calculates the fluorescence lifetime (fluorescence relaxation time) τ according to τ = tan θ / (2πf) (f is the value of the frequency of the modulation signal) using the calculated phase delay θ.
The calculated fluorescence lifetime τ is stored in the memory 66 and sent to the display 70a and the printer 70b, displayed on the screen, and printed out.

As described above, in the present embodiment, the fluorescence emitted from the measurement object 12 is amplified by the optical amplifier 43 and the scattered light of the laser light is not amplified. Therefore, the amplified fluorescence is included in the photomultiplier tube 46a and the photomultiplier tube 46b. Is received. At this time, the reference light source units 32 and 34 are configured such that the phase of the intensity modulation is the same as the phase of the intensity modulation of the laser light, and the phase of the intensity modulation is 90 degrees with respect to the phase of the intensity modulation of the laser light. The shifted second reference light is emitted as reference light, respectively. The photomultiplier tube 46a receives the first reference light simultaneously with the fluorescence, and the photomultiplier tube 46b receives the second reference light simultaneously with the fluorescence.
Bandpass filter 47a from the first light receiving signal, takes out the component of the modulation angular frequency omega _[nu, the band-pass filter 47b, the second light receiving signal, takes out the component of the modulation angular frequency omega _[nu.
Demodulator 70 of the processing unit 60, a first signal component of the modulation angular frequency omega _[nu taken out by the band-pass filter 47a, demodulates using the modulation angular frequency omega _[nu, modulation angular taken out by the band-pass filter 47b a second signal component of the frequency omega _[nu, is demodulated using a modulation angular frequency omega _[nu. The demodulator 70 calculates the phase delay θ using the ratio between the demodulation result of the first signal and the demodulation result of the second signal. The fluorescence lifetime calculation unit 72 calculates the fluorescence lifetime from this phase delay θ. Since the flow cytometer has such a configuration, it is possible to efficiently remove the scattered light of the laser light and calculate the fluorescence intensity with high accuracy.

In the present embodiment, for the demodulation processing of the received light signal, the fluorescence signal whose intensity is time-modulated at the frequency f as in the case of the laser light is not used as a reference signal for the frequency f and mixed by the mixer. That is, in this embodiment, a mixing circuit having a mixer is not used. The mixer is likely to have a DC component on the result of mixing. For this reason, when weak fluorescence is received, it is impossible to calculate the phase delay θ and the fluorescence lifetime τ with high accuracy under the influence of the DC component. In the present embodiment, the reference light whose time is modulated with the angular frequency ω _ν is received simultaneously with the fluorescence, and the component of the angular frequency ω _ν is extracted from the received light signal using the bandpass filters 47a and 47b. So do not use a mixer. Therefore, this embodiment does not cause such a conventional problem.

In the present embodiment, the optical amplifier 43 may not be used. Even if the photomultiplier tubes 46a and 46b receive the scattered light of the laser beam, the scattered light of the laser beam is modulated from the received light signal obtained by the received light even if the intensity is time-modulated at the frequency f. band-pass filter 47a takes out the component of the signal of the angular frequency omega _[nu, signal components of the scattered light of the laser light by 47b is removed. For this reason, the scattered light signal of the laser light can be removed from the received light signal without using the optical amplifier 43. However, in order to prevent the scattered light signal of the laser light from being included in the received light signal more effectively, it is preferable to use the optical amplifier 43 as in this embodiment.

  The reference light source units 32 and 34 time-modulate the phase of light by changing the optical path length from the reference light sources 32 and 34 to the photomultiplier tubes 46a and 46b over time. For this reason, the time modulation of the phase of the reference light can be easily performed. For example, as a simple configuration, it includes ultrasonic transducers 32c and 34c and reflecting surface mirrors 32d and 34d provided on the ultrasonic transducers 32c and 34c, and the reflection mirrors 32d and 32d are generated by the vibration of the ultrasonic transducers 32c and 32d. In the state where the position of 32d fluctuates, the reference light incident from the reference light sources 32b and 34b is reflected toward the photomultiplier tubes 46a and 46b. Thereby, the time modulation of the phase of the reference light is easily realized. The configuration of the reference light source units 32 and 34 may be any configuration as long as the phase of the light is time-modulated. However, for a simple configuration, it is provided in the ultrasonic transducers 32c and 34c. A configuration using the reflecting surface mirrors 32d and 34d is preferable.

  As described above, the fluorescence detection device and the fluorescence detection method of the present invention have been described in detail. However, the fluorescence detection device and the fluorescence detection method of the present invention are not limited to the above-described embodiments, and various types can be used without departing from the gist of the present invention. Of course, improvements and changes may be made.

DESCRIPTION OF SYMBOLS 10 Flow cell 12 Measurement object 20 Laser light source part 30 1st light-receiving part 32,34 Reference light source part 32a, 34a Laser driver 32b, 34b Laser light source 32c, 34c Ultrasonic vibrator 32d, 34d Reflection mirror 34 Reference light source 36 Ultrasonic vibration Element 38 Reflection mirror 40 Second light receiving part 41 Lens system 42, 44 Band pass filter 45 Half mirror 46a, 46b Photomultiplier tube 47a, 47b Band pass filter 50 Control part 51 Oscillator 52 Power splitter 53, 54, 56 Amplifier 55 90 degree phase Shifter 60 Processing unit 62 AD conversion board 64 CPU
66 Memory 68 Input / output port 70 Demodulator 72 Fluorescence lifetime calculator 70 Output unit 70a Display 70b Printer

Claims (8)

A fluorescence detection device that detects fluorescence emitted when a measurement object is irradiated with laser light,
A laser light source unit that emits laser light whose intensity is time-modulated at a frequency f and irradiates the measurement object;
A light receiving unit that receives fluorescence emitted from the measurement object when the measurement object is irradiated with the laser beam;
When the light receiving unit receives the fluorescence, reference light having the same wavelength component as the fluorescence, the phase of the light being time-modulated with an angular frequency ω _ν , and the intensity being time-modulated with the frequency f is received by the light receiving unit. A reference light source unit including a reference light source that emits the reference light so that the unit receives light simultaneously with the fluorescence;
A filter unit that extracts a component of the angular frequency ω _ν from a light reception signal output from the light receiving unit;
From the signal of the component of the angular frequency ω _ν extracted by the filter unit, using the information of the angular frequency ω _ν , a phase lag with respect to intensity modulation of the laser light of the fluorescence is obtained, and from the obtained phase lag, And a processing unit for obtaining a fluorescence lifetime of the fluorescence. The reference light source unit includes a first reference light having an intensity modulation phase in phase with an intensity modulation phase of the laser light, and an intensity modulation phase shifted by 90 degrees relative to the intensity modulation phase of the laser light. 2 reference lights are respectively emitted as the reference lights,
The light receiving unit includes a first light receiving element that receives the first reference light, and a second light receiving element that receives the second reference light whose phase is shifted by 90 degrees with respect to the first reference light. ,
The filter unit includes a first filter that extracts a component of the angular frequency ω _ν from a first light receiving signal output from the first light receiving element, and a second light receiving signal output from the second light receiving element. A second filter for extracting the component of the angular frequency ω _ν ,
Wherein the processing unit, component of said first signal component of the angular frequency omega _[nu taken out by the first filter demodulates using the angular frequency omega _[nu, the angular frequency omega _[nu taken out by the second filter The second signal is demodulated using the angular frequency ω _ν , and the ratio of the demodulation result of the first signal and the demodulation result of the second signal is used to determine the phase delay and to increase the fluorescence lifetime. The fluorescence detection apparatus according to claim 1, further comprising a fluorescence lifetime calculation unit to be obtained.   The fluorescence detection apparatus according to claim 1, wherein the reference light source unit temporally modulates the phase of light by changing the optical path length of the reference light from the reference light source to the light receiving element over time.   The reference light source unit includes an ultrasonic transducer and a reflective surface mirror provided on the ultrasonic transducer, and the reference light source unit changes the position of the reflective mirror due to vibration of the ultrasonic transducer. The fluorescence detection apparatus according to claim 1, wherein the phase of the light is time-modulated by reflecting incident reference light toward the light receiving element.   The fluorescence detection according to any one of claims 1 to 4, further comprising an optical amplification unit that amplifies the fluorescence by performing stimulated emission of the light by a bias signal before receiving the fluorescence. apparatus.   The flow cytometer according to any one of claims 1 to 5, wherein the flow cytometer performs fluorescence detection using each of a plurality of inspection target samples that sequentially flow in a line in a pipeline and pass through a laser light irradiation position as a measurement target. The fluorescence detection device according to item. A fluorescence detection method for detecting fluorescence emitted when a measurement object is irradiated with laser light,
Emitting a laser beam whose intensity is time-modulated at a frequency f and irradiating the measurement object;
While receiving the fluorescence emitted from the measurement object when the measurement object is irradiated with the laser light, it has the same wavelength component as the fluorescence, the phase of the light is time-modulated with an angular frequency ω _ν , and the intensity Receiving the reference light time-modulated at the frequency f simultaneously with the fluorescence;
Extracting a signal of the component of the angular frequency ω _ν from the light reception signal obtained by the light reception;
Wherein the signal components of the angular frequency omega _[nu taken out, using the information of the angular frequency omega _[nu, obtains a phase delay with respect to the intensity modulation of the laser beam of the fluorescence from the phase delay determined, of the fluorescent A fluorescence detection method comprising: obtaining a fluorescence lifetime. The step of receiving the fluorescence comprises:
(A) receiving, as the reference light, first reference light whose phase of intensity modulation is in phase with the phase of intensity modulation of the laser light;
(B) a second step of receiving, as the reference light, the second reference light whose intensity modulation phase is shifted by 90 degrees with respect to the intensity modulation phase of the laser light, simultaneously with the fluorescence,
The step of extracting the component of the angular frequency ω _ν includes
(C) extracting the component of the angular frequency ω _ν from the first received light signal obtained in the step (A);
(D) extracting the component of the angular frequency ω _ν from the second received light signal obtained in the step (B),
The step of obtaining the fluorescence lifetime comprises:
(E) a step wherein a first signal component of the angular frequency omega _[nu extracted in step (D) is demodulated using the angular frequency omega _[nu,
(F) demodulating the second signal of the component of the angular frequency ω _ν extracted in the step (E) using the angular frequency ω _ν ;
(G) using the ratio of the demodulation result of the first signal and the demodulation result of the second signal to determine the phase delay and determining the fluorescence lifetime;
The fluorescence detection method of Claim 7 containing this.

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