JP6065517B2 - Fluorescence microscope, fluorescence detection method, and fluorescence detection unit - Google Patents

Description

  The present invention relates to a fluorescence microscope, a fluorescence detection method, and a fluorescence detection unit.

  As a conventional technique for irradiating a sample with excitation light that excites a fluorescent reagent and displaying an image based on fluorescence emitted from the fluorescent reagent, there is a technique described in Patent Document 1, for example. In such a conventional technique, when simultaneously irradiating a sample fluorescent reagent with a plurality of excitation lights, the fluorescence emitted from the fluorescent reagent is separated by an optical filter when it is detected by a detector and guided to different detectors. It is common.

International Publication No. 2005/052668

  When fluorescence emitted from multiple fluorescent reagents is separated by an optical filter and guided to different detectors, it is difficult to completely separate the fluorescence because the wavelength bands of the generated fluorescence are very close or partially overlapping. is there.

  An aspect of the present invention is to provide a fluorescence microscope, a fluorescence detection method, and a fluorescence detection unit capable of separating a plurality of fluorescence received by a detector.

  According to the first aspect of the present invention, an excitation light modulation unit that modulates the intensity of a plurality of excitation lights applied to the sample at the same frequency as the frequency of the reference signal and with a predetermined phase difference with respect to the phase of the reference signal, Phase information is obtained from a relationship between a fluorescence detection unit that detects a plurality of fluorescence generated by the plurality of excitation lights, a phase at a predetermined frequency component extracted from a detection signal by the fluorescence detection unit, and a phase of the reference signal. A phase information generation unit to generate, an amplitude information generation unit for generating amplitude information from the amplitude of a predetermined frequency component extracted from the detection signal by the fluorescence detection unit, and the frequency component is removed from the detection signal by the fluorescence detection unit A component information generation unit that generates component information from the measured components, and calculates an intensity signal of each of the plurality of fluorescences based on the phase information, the amplitude information, and the component information And degree signal calculating section, a fluorescent microscope equipped with is provided.

  According to the second aspect of the present invention, the first irradiation state in which the sample is irradiated with the plurality of excitation lights at the same frequency as the reference signal and at the same phase as the reference signal, and at least one of the plurality of excitation lights An irradiation control unit for instructing a second irradiation state to be irradiated except for one; a fluorescence detection unit for detecting fluorescence generated by the excitation light in the first and second irradiation states; and the first irradiation. Based on the first detection signal from the fluorescence detection unit in the state and the second detection signal from the fluorescence detection unit in the second irradiation state, each intensity signal of the plurality of fluorescences from the first detection signal An intensity signal calculation unit that calculates

  According to the third aspect of the present invention, the plurality of excitation lights to be irradiated onto the sample are respectively modulated in intensity with the same frequency as the frequency of the reference signal and with a predetermined phase difference with respect to the phase of the reference signal. Detecting a plurality of fluorescence generated by the step, and generating phase information from a relationship between a phase of a predetermined frequency component extracted from the detected signal and a phase of the reference signal, and extracting the phase information from the detected signal. Amplitude information is generated from the amplitude of the predetermined frequency component, component information is generated from a component obtained by removing the frequency component from the detected signal, and the plurality of the plurality of information are generated based on the phase information, the amplitude information, and the component information. There is provided a fluorescence detection method for calculating the intensity signals of the respective fluorescences.

  According to the fourth aspect of the present invention, fluorescence detection for detecting a plurality of fluorescence generated by a plurality of excitation lights having the same frequency as the frequency of the reference signal and intensity-modulated with a predetermined phase difference with respect to the phase of the reference signal. A phase information generation unit that generates phase information from a relationship between a phase in a predetermined frequency component extracted from a detection signal by the fluorescence detection unit and a phase of the detection signal, and a detection signal by the fluorescence detection unit An amplitude information generation unit that generates amplitude information from the extracted amplitude of a predetermined frequency component, a component information generation unit that generates component information from a component obtained by removing the frequency component from a detection signal by the fluorescence detection unit, and the phase There is provided a fluorescence detection unit comprising: an intensity signal calculation unit that calculates an intensity signal of each of the plurality of fluorescences based on the information, the amplitude information, and the component information.

  According to the aspect of the present invention, it is possible to separate a plurality of fluorescence received by the fluorescence detection unit by calculation.

It is a figure which shows an example of the fluorescence microscope which concerns on 1st Embodiment. It is a figure explaining the fluorescence microscope which concerns on 1st Embodiment. It is a figure which shows an example of the optical system used for the fluorescence microscope which concerns on 1st Embodiment. It is a figure which shows a detection signal and the fluorescence intensity signal. It is a figure which shows a detection signal and the fluorescence intensity signal. It is a figure which shows a detection signal and the fluorescence intensity signal. It is a figure which shows a detection signal and the fluorescence intensity signal. It is a figure which shows an example of the excitation spectrum of excitation light, and the fluorescence spectrum of the fluorescence which generate | occur | produces with the said excitation light. It is a figure explaining 2nd Embodiment. It is a figure which shows an example of the fluorescence light-receiving unit which concerns on embodiment.

Hereinafter, although an embodiment is described, referring to drawings, it is not limited to this embodiment.
<First Embodiment>
A first embodiment will be described. FIG. 1 shows an example of a microscope system including a fluorescence microscope according to the first embodiment. The microscope system 100 includes a fluorescence microscope 1 that acquires an image of fluorescence generated by excitation light applied to the sample SP, and a personal computer 101 that controls the fluorescence microscope 1. Connected to the personal computer 101 are an input device 102 such as a mouse and a keyboard and a display device 103 such as an LCD (Liquid Crystal Display). The personal computer 101 includes a GUI (Graphical) for allowing a user to input an image acquired by the fluorescence microscope 1 and a setting value of the fluorescence microscope 1 when a built-in CPU (Central Processing Unit) executes the application software 104. User Interface) is displayed on the display device 103. In addition, the personal computer 101 controls the fluorescence microscope 1 based on setting values input by the user operating the input device 102.

  The fluorescence microscope 1 includes an excitation light modulation unit 10, a fluorescence detection unit 20, a phase information generation unit 30, an amplitude information generation unit 40, a component information generation unit 50, an intensity signal calculation unit 60, and a light source 70. Prepare.

  The excitation light modulation unit 10 includes an oscillator 11 that outputs a signal having a predetermined frequency, a phase difference adjustment unit 12 that adjusts a phase difference between a plurality of excitation lights (excitation light EX1, excitation light EX2,..., Excitation light EXn), , 13-n and drive circuits 14-1, 14-2,..., 14-n. The excitation light modulation unit 10 modulates the intensity of a plurality of excitation lights irradiated from the light source L1, the light sources L2,... The oscillator 11 generates a basic sine wave and outputs it to the phase difference adjustment unit 12. The phase difference adjusting unit 12 adjusts the phase of the fundamental sine wave from the oscillator 11 for each excitation light, and outputs the adjusted sine wave to the modulation circuit 13-1 or the like.

  The adjustment by the phase difference adjusting unit 12 is performed by adjusting the phase of other excitation light with respect to one excitation light among the plurality of excitation lights. For example, the basic sine wave signal from the oscillator 11 is used, the excitation light EX1 is used as it is, and the excitation light EX2 is adjusted so as to delay (advance) the phase by π by a 180 ° phase circuit or the like. Good. Further, the excitation light modulation unit 10 is not limited to controlling the driving of the light source 70. For example, the pumping light modulator may be such that the pumping light emitted from the light source 70 is guided to the sample SP by an optical fiber and the optical path length of the optical fiber is adjusted to give a phase difference to the pumping light.

In addition, the phase difference adjustment unit 12 outputs a basic sine wave (hereinafter referred to as a reference signal b) that serves as a reference for the phase of each excitation light to the demodulation circuit 23 and the phase information generation unit 30.
Here, in order to generate the reference signal b with higher accuracy, it is necessary to generate the reference signal b based on a signal having a frequency higher than that of the reference signal b.
The modulation circuit 13-1 or the like modulates the intensity to a predetermined phase based on the output (intensity modulation signals c1, c2,..., Cn) from the phase difference adjustment unit 12, and sends them to the drive circuit 14-1 and the like. Output. The drive circuit 14-1 and the like drive the light source L1 and the like so that the excitation light EX1 and the like are emitted with a phase whose intensity is modulated by the modulation circuit 13-1 and the like. Note that the phase difference adjustment unit 12 includes a storage unit 12a for storing the phase delay amount when used in the second and third embodiments described later.

  The fluorescence detection unit 20 has passed through a photodetector 21 that detects fluorescence generated by excitation light, a current / voltage conversion circuit 22 that converts the output from the photodetector 21 into current / voltage, and a current / voltage conversion circuit 22. And a demodulation circuit 23 that demodulates the output of the photodetector 21 and outputs a detection signal. As the photodetector 21, for example, a photomultiplier tube is used. In FIG. 1, a single photodetector is used as the photodetector 21, but a plurality of (for example, N equal to the number of light sources) photodetectors may be used. When two light sources (light source L1 and light source L2) are used as the light source 70, the light detector 21 emits two fluorescences (fluorescence EM1, EM1,... Generated by the excitation light EX1 and the excitation light EX2 emitted from these light sources. The light intensity combined with the fluorescence EM2) is detected. Therefore, the detection signal that is an output signal from the fluorescence detection unit 20 is a combined output of the intensity signals of the fluorescence EM1 and the fluorescence EM2.

The phase information generation unit 30 outputs the phase of the frequency component (the same frequency component as the reference signal b) extracted from the detection signal by the fluorescence detection unit 20 input from the voltage amplification circuit 42 described later, and the level of the excitation light modulation unit 10. Phase information is generated based on the phase of the reference signal b input from the phase difference adjustment unit 12. This phase information is input to the intensity signal calculation unit 60. For example, a phase difference (phase difference 0, phase difference π, etc.) between the phase of the reference signal b and the phase of the frequency component extracted from the detection signal is generated.
However, the phase difference may be represented by time (0 μs, 30 μs, etc.) in addition to the phase angle (π, 2π, etc.).
Further, when the phase of the reference signal b and any one of the plurality of pump lights are matched (phase difference 0), the phases of the other pump lights are set with reference to the phase of the pump light. Since the phase may be set, the phase of the reference signal b includes the phase of the excitation light serving as a reference.

  FIG. 2 is a diagram for explaining a case where two light sources are used in the fluorescence microscope of the first embodiment shown in FIG. 1, and the same or equivalent components as in FIG. Or simplify. As shown in FIG. 2, when fluorescence EM1 and fluorescence EM2 are generated when the sample SP is irradiated with the excitation light EX1 and the excitation light EX2, an example of phase information generated by the phase information generation unit 30 is “ Phase information: The phase of the reference signal b and the phase of the detection signal by the fluorescence detection unit 20 are the same phase (phase difference = 0), and the phase of the reference signal b and the phase of the detection signal by the fluorescence detection unit 20 are opposite (phase) Phase difference = π) ”.

  The amplitude information generation unit 40 includes a high pass filter 41, a voltage amplification circuit 42, and an AD conversion circuit 43. The high pass filter 41 extracts a frequency component (the same frequency component as the reference signal b) from the detection signal output from the fluorescence detection unit 20. The voltage amplification circuit 42 amplifies the voltage of the frequency component extracted by the high pass filter 41 and then outputs the amplified voltage component to the AD conversion circuit 43 and the phase information generation unit 30. The AD conversion circuit 43 digitizes the frequency component that has undergone voltage amplification, and outputs it to the intensity signal calculation unit 60 as amplitude information of the detection signal (amplitude information in the frequency component) by the fluorescence detection unit 20. The amplitude information is input to the intensity signal calculation unit 60 at a timing based on the pixel clock generated by the pixel clock generation circuit 120.

  The component information generation unit 50 includes a low-pass filter 51, a voltage amplification circuit 52, and an AD conversion circuit 53. The low pass filter 51 extracts a component (mainly a direct current component, hereinafter referred to as a direct current component) obtained by removing a frequency component from the detection signal output from the fluorescence detection unit 20. The voltage amplification circuit 52 amplifies the voltage of the direct current component extracted by the low pass filter 51 and then outputs the amplified voltage to the AD conversion circuit 53. The AD conversion circuit 53 digitizes the voltage-amplified DC component, and component information of the detection signal from the fluorescence detection unit 20 (information on the component obtained by removing the frequency component from the detection signal is mainly DC component information. The light source L1. , Light source L2,..., Even if the output from light source Ln is 100% modulated, the DC component is included in the detection signal. This component information is input to the intensity signal calculation unit 60 at a timing based on the pixel clock generated by the pixel clock generation circuit 120, similarly to the amplitude information.

The intensity signal calculation unit 60 includes phase information input from the phase information generation unit 30, amplitude information (AC component) input from the amplitude information generation unit 40, and component information (DC direct current) input from the component information generation unit 50. And an intensity signal of each of the plurality of fluorescences. The basic principle for calculating the intensity signals of the plurality of fluorescences by the intensity signal calculation unit 60 will be described later.
Further, when adjusting the phases of the plurality of excitation lights EX1 and the like from the plurality of light sources L1 and the like on the basis of the phase of the reference signal b, the detection signals acquired in the preliminary sample observation and the immediately preceding sample observation are used. The phase correction signal a is calculated based on the intensity signals of the plurality of fluorescence EM1 and the like calculated by the intensity signal calculation unit 60, and the phase for performing the intensity modulation of the light source with the phase correction signal a as a correction value is more precise. You may adjust it.
Since the same value can be used for such a phase correction signal a as long as the observation target such as a sample does not change, once calculated, a storage unit (not shown) provided in the intensity signal calculation unit 60 or a phase difference adjustment unit 12 may be used as appropriate when driving the light source L1 or the like.

  The light source 70 outputs a plurality of excitation lights (excitation light EX1, excitation light EX2,..., Excitation light EXn), and N light sources L1, L2, L2, and so on are output for each of the plurality of excitation lights. ..., a light source Ln is provided. Each light source L1 etc. is driven by the respective drive circuit 14-1 etc. and emits excitation light EX1. At this time, the phase of each excitation light EX1 is set by each modulation circuit 13-1. The light source 70 is not limited to a plurality of light sources, and a single light source may be used. For example, excitation light output from a single broadband light source is divided into a plurality of excitation lights having different wavelengths by using an element capable of intensity modulation (for example, AOTF: Acousto-Optic Tunable Filter), and with a predetermined phase difference. It may be output. In the present embodiment, a light source 70 that does not include a modulation circuit and a drive circuit is used. However, a light source that includes a drive circuit and a modulation circuit may be used. When the light source 70 includes a modulation circuit or the like, the excitation light modulation unit 10 does not need to include the modulation circuit 13-1 or the drive circuit 14-1 or the like.

  The fluorescence microscope 1 includes an optical system 110. The optical system 110 irradiates the sample SP with a predetermined phase difference and irradiates the sample SP with a plurality of excitation lights EX1 and the like having the same frequency emitted from the light source L1 and the like by performing optical path division, optical path synthesis, and scanning. The fluorescence EM1 and the like generated according to the light EX1 and the like are guided to the fluorescence detection unit 20. As the optical system 110, for example, a part of components of a confocal microscope (see FIG. 3) disclosed in Japanese Patent Laid-Open No. 11-183806 can be used.

  FIG. 3 shows a specific configuration of the optical system 110. The optical system 110 includes a light splitting device 111, a scanner module 112, a relay lens 113, an objective optical system 114, a stage 115, a fluorescence selection device 116, a condensing lens 117, a pinhole module 118, and the like. Prepare. The light splitting device 111 separates the excitation light EX1 and the like irradiated from the light source 70 onto the sample SP and the fluorescence EM1 and the like generated from the sample SP. The scanner module 112 is a scanning device for two-dimensionally scanning the sample SP with the excitation light EX1 and the like. The relay lens 113 guides the excitation light EX1 and the like to the sample SP. The objective optical system 114 condenses the excitation light EX1 and the like at the sample SP position. The stage 115 supports the sample SP and moves it in three dimensions. The fluorescence selection device 116 removes noise light from the fluorescence EM1 transmitted through the light splitting device 111 and the like. The condensing lens 117 condenses the fluorescence EM1 and the like that have passed through the fluorescence selection device 116. The pinhole module 118 is an opening member for generating a confocal effect.

  The excitation light EX1 and the like emitted from the light source 70 are reflected toward the sample SP by the light splitting device 111, and are irradiated to the sample SP via the scanner module 112, the relay lens 113, and the objective optical system 114. The excitation light EX1 and the like are scanned by the scanner module 112 over all or part of the sample SP. The fluorescence EM1 and the like generated from the sample SP by the excitation light EX1 and the like pass through the light splitting device 111 through the objective optical system 114, the relay lens 113, and the scanner module 112. Then, after the noise light is removed by the fluorescence selection device 116, the fluorescence EM 1 and the like are collected by the condenser lens 117, pass through the scanner module 112, and enter the fluorescence detection unit 20. In the optical system 110, the light splitting device 111, the scanner module 112, the objective optical system 114, the fluorescence selection device 116, and the like are controlled by control devices (not shown). This control device may use the personal computer 101 shown in FIG.

  In addition, the fluorescence microscope 1 is configured as a laser scanning confocal fluorescence microscope, but is not limited thereto. Moreover, it is not limited to a confocal fluorescence microscope. Moreover, although the light source 70 is provided as the fluorescence microscope 1, it is not limited to this, The fluorescence microscope which does not mount the light source 70 may be sufficient. In the fluorescence microscope not equipped with the light source 70, whether the excitation light modulation unit 10 includes the modulation circuit 13-1, etc., the drive circuit 14-1, etc. is arbitrary. As appropriate.

  Next, the basic principle for calculating the fluorescence intensity signal will be described. For simplification of explanation, it is assumed that the plurality of fluorescences are generated with a phase difference of 0 and π with respect to the reference signal b on the assumption that there is no phase lag with respect to the irradiation of the corresponding excitation light. In addition, the plurality of excitation lights are described with an example in which the phase differences are set to 0 and π with respect to the reference signal b and the fluorescence efficiency (that is, the ratio of the fluorescence intensity to the excitation light intensity) is the same. Do. That is, FIGS. 4 to 7 show that the two excitation lights EX1 and EX2 irradiated to the sample SP have the same frequency, the same intensity, and the phase difference π as the reference signal b (the phase difference of the excitation light EX1 with respect to the reference signal b is 0, the reference When intensity modulation is performed with the phase difference of the excitation light EX2 with respect to the signal b as π, each of the fluorescence EM1 and EM2 generated by the excitation light EX1 and EX2 (the phase difference 0 of the fluorescence EM1 with respect to the reference signal b and the reference signal b) 6 is a graph showing the phase difference π) of fluorescence EM2 and the combined output detected by the fluorescence detection unit 20. FIG. 4 to 7, the vertical axis indicates signal intensity, and the horizontal axis indicates time. FIG. 4 shows a case where the fluorescence EM1 and EM2 have the same average intensity and amplitude and are in opposite phases. In this case, only a direct current component is detected as a combined output (detection signal) from the fluorescence detection unit 20. Therefore, it can be seen from this detection signal that the intensity signal of the fluorescence EM1 and the intensity signal of the fluorescence EM2 have the same waveform in opposite phases, and thus the intensity signals of the fluorescence EM1 and the fluorescence EM2 are the same. The intensity signals of the fluorescence EM1 and the fluorescence EM2 are half of the detection signal.

  FIG. 5 shows that the average values (average intensities) of the respective intensity signals in the fluorescence EM1 and EM2 are the same, and the opposite phases (the phase difference 0 of the fluorescence EM1 with respect to the reference signal b and the phase difference π of the fluorescence EM2 with respect to the reference signal b). In this case, an alternating current component as shown in FIG. 5 is detected as a combined output (detection signal) from the fluorescence detection unit 20. Looking at this alternating current component, the phase thereof is in phase with the fluorescence EM1 (the phase opposite to that of the fluorescence EM2 and the phase of the reference signal b). This indicates that the amplitude in the frequency component of the intensity signal of the fluorescence EM1 is larger than that of the fluorescence EM2. Therefore, from this combined output (detection signal), the intensity signal (maximum value of the sine signal) of the fluorescence EM1 is larger than the intensity signal (maximum value of the sine signal) of the fluorescence EM2, and the difference (intensity difference) between the fluorescence EM1 and the fluorescence. It can be seen that the amplitude of the detection signal of EM2. The magnitude of the DC component of the combined output (detection signal) (that is, the center position of the sine wave signal) represents the sum of the DC components of the fluorescences EM1 and EM2.

6, as in FIG. 5, the average values (average intensities) of the respective signal intensities in the fluorescences EM <b> 1 and EM <b> 2 are the same, and the opposite phases (the phase difference 0 of the fluorescence EM <b> 1 with respect to the reference signal b and the fluorescence with respect to the reference signal b) 6 shows a case where the amplitude is different from the phase difference π of EM2, and in this case, an alternating current component as shown in FIG. 6 is detected as a combined output from the fluorescence detection unit 20. Looking at this AC component, the phase is opposite to that of the fluorescence EM1 (in phase with the fluorescence EM2 and opposite to the reference signal b). This represents that the amplitude in the frequency component of the intensity signal of the fluorescence EM2 is larger than that of the fluorescence EM1. Therefore, from this combined output (detection signal), the intensity signal (maximum value of the sine signal) of the fluorescence EM2 is larger than the intensity signal (maximum value of the sine signal) of the fluorescence EM1, and the difference (intensity difference) between the fluorescence EM1 and the fluorescence. It can be seen that this is the amplitude of the combined output (detection signal) of EM2. The magnitude of the DC component of the combined output (detection signal) (that is, the center position of the sine wave signal) represents the sum of the DC components of the fluorescences EM1 and EM2.

In FIG. 7, the average values (average intensities) of the respective signal intensities of the fluorescence EM1 and EM2 are different, and have opposite phases (the phase difference 0 of the fluorescence EM1 with respect to the reference signal b, the phase difference π of the fluorescence EM2 with respect to the reference signal b), 7 shows a case where the amplitudes are different. In this case, an alternating current component as shown in FIG. 7 is detected as a combined output from the fluorescence detection unit 20. Looking at this alternating current component, the phase thereof is in phase with the fluorescence EM1 (the phase opposite to that of the fluorescence EM2 and the phase of the reference signal b). This indicates that the amplitude in the frequency component of the intensity signal of the fluorescence EM1 is larger than that of the fluorescence EM2. Therefore, from this combined output (detection signal), the intensity signal (maximum value of the sine signal) of the fluorescence EM1 is larger than the intensity signal (maximum value of the sine signal) of the fluorescence EM2, and the difference (intensity difference) is different from that of the fluorescence EM1. It turns out that it is the amplitude of the detection signal of fluorescence EM2. The magnitude of the DC component of the combined output (detection signal) (that is, the center position of the sine wave signal) represents the sum of the DC components of the fluorescences EM1 and EM2.

As described above, by setting the phase differences of the plurality of excitation lights to 0 and π with respect to the reference signal b, a plurality of fluorescences are generated in advance with phase differences of 0 and π with respect to the reference signal b, respectively. Thus, the fluorescence signals EM1, EM2 depending on whether the phase of the detection signal and the phase of the reference signal b are the same phase or opposite phase (the phase difference between the phase of the combined output (detection signal) and the phase of the reference signal b). The amplitude of the detection signal represents the difference between the amplitudes of the fluorescence EM1 and EM2, and the average value (average intensity) of the combined output (detection signal) is the DC component of the fluorescence EM1 and EM2. You can see that it represents the sum.

Next, the calculation principle of the fluorescence intensity signal will be described based on mathematical expressions.
[When using two excitation lights]
The emission wavelengths of the two excitation lights (excitation light EX1, excitation light EX2) are λa and λb, respectively, and the excitation light EX1 and the excitation light EX2 have the same frequency as the reference signal b and a predetermined phase difference with respect to the reference signal b ( For example, the phase difference from the excitation light EX1 is 0 and the phase difference from the excitation light EX2 is π, and a predetermined phase difference (for example, the phase difference of the excitation light EX2 with respect to the excitation light EX1 is π). ), The fluorescence EM1 and the fluorescence EM2 generated by the excitation light EX1 and the excitation light EX2, respectively, are input to the fluorescence detection unit 20. Assume that the intensity signals of the fluorescence EM1 and fluorescence EM2 after being converted into electrical signals in the fluorescence detection unit 20 are Sa and Sb, respectively, and that the fluorescence EM1 and fluorescence EM2 have no phase delay with respect to the excitation light irradiation. , Sa, and Sb each have a predetermined phase difference with respect to the reference signal b (the phase difference with the fluorescence EM1 is 0 and the phase difference with the fluorescence EM2 is π), and the combined output (detection) of the fluorescence detection unit 20 Signal) has a phase difference of 0 and π. The combined output (detection signal) has a predetermined phase difference (0 or π) with respect to the reference signal b. That is, Sa and Sb can be expressed by the expressions (1) and (2), respectively.
Sa = Ja · {1 + Ka · cos (θ)} (1)
Sb = Jb · {1 + Kb · cos (θ + π)} (2)
However, Ja and Jb represent the signal intensity determined by the sensitivity coefficient and the fluorescence intensity depending on the fluorescence wavelength of the fluorescence detection unit 20, and Ka and Kb represent the modulation degree (modulation degree 1 corresponds to 100% modulation).

Since the intensity Sa and Sb of the intensity signals of the fluorescence EM1 and fluorescence EM2 detected by the fluorescence detection unit 20 are synthesized and output, if the value of the synthesis output (detection signal) of the fluorescence detection unit 20 is S, S is It can be expressed by equation (3).
S = Sa + Sb = Ja + Ja · Ka · cos (θ)
+ Jb + Jb · Kb · cos (θ + π) (3)
From cos (θ + π) = − cos (θ), equation (3) becomes equation (4).
S = Ja + Jb + (Ja · Ka−
Jb / Kb) / cos (θ) (4)
In equation (4), (Ja + Jb) represents the magnitude of the direct current component of S, and (Ja · Ka−
Jb · Kb) represents the amplitude of the AC component of S.
At this time, (Ja + Jb) and (Ja · Ka−
Jb · Kb) is component information and amplitude information generated in the AD conversion circuit 53 and the AD conversion circuit 43, respectively. When α and β are respectively set, α and β are the expressions (5) and (6), respectively. ).
Ja + Jb = α (5)
Ja ・ Ka-
Jb · Kb = β …… (6)
In Expression (6), since the modulation degrees Ka and Kb are known, Expressions (5) and (6) are linear expressions of two variables. Therefore, the linear expressions of the two variables of Expressions (5) and (6) are used. By solving, Ja and Jb can be determined. Therefore, by generating component information α and amplitude information β in the AD conversion circuit 53 and the AD conversion circuit 43 and outputting them to the intensity signal calculation unit 60, the fluorescence signal EM1 generated by the excitation light EX1 in the intensity signal calculation unit 60. And the intensity signal of the fluorescence EM2 generated by the excitation light EX2.

[When using three excitation lights]
The emission wavelengths of the three excitation lights (excitation light EX1, excitation light EX2, and excitation light EX3) are λ1, λ2, and λ3, respectively. The excitation light EX1, the excitation light EX2, and the excitation light EX3 have the same frequency and the reference signal as the reference signal b. Each of b is a predetermined phase difference (for example, the phase difference with the excitation light EX1 is 0, the phase difference with the excitation light EX2 is 2π / 3, and the phase difference with the excitation light EX3 is 4π / 3). Then, when intensity modulation is performed with a predetermined phase difference (for example, the phase differences of the excitation light EX2 and the excitation light EX3 with respect to the excitation light EX1 are 2π / 3 and 4π / 3, respectively), the excitation light EX1, the excitation light EX2, and the excitation light Fluorescence EM1, fluorescence EM2, and fluorescence EM3 generated by the light EX3 are input to the fluorescence detection unit 20. The intensity of the intensity signals of the fluorescence EM1, fluorescence EM2, and fluorescence EM3 after being converted into electrical signals in the fluorescence detection unit 20 is Sa, Sb, and Sc, respectively, and the fluorescence EM1, fluorescence EM2, and fluorescence EM3 are subjected to excitation light irradiation. Assuming that there is no phase lag, Sa, Sb, and Sc each have a predetermined phase difference with respect to the reference signal b (the phase difference with the fluorescence EM1 is 0, the phase difference with the fluorescence EM2 is 2π / 3, and the fluorescence EM3 And a phase difference of 0, 2π / 3, and 4π / 3 with respect to the combined output (detection signal) of the fluorescence detection unit 20, respectively. Further, the combined output (detection signal) has a predetermined phase difference (any one of 0, 2π / 3, 4π / 3) with respect to the reference signal b. That is, Sa, Sb, and Sc can be expressed by equations (7), (8), and (9), respectively.
Sa = Ja · {1 + Ka · cos (θ)} (7)
Sb = Jb · {1 + Kb · cos (θ + 2π / 3)} (8)
Sc = Jc · {1 + Kc · cos (θ + 4π / 3)} (9)
However, Ja, Jb, and Jc represent the signal intensity determined by the sensitivity coefficient and the fluorescence intensity depending on the fluorescence wavelength of the fluorescence detection unit 27, and Ka, Kb, and Kc are modulation degrees (modulation degree 1 corresponds to 100% modulation). Represents.

Since the signal intensities Sa, Sb, and Sc of the fluorescence EM1, fluorescence EM2, and fluorescence EM3 detected by the fluorescence detection unit 20 are synthesized and output, assuming that the value of the synthesized output (detection signal) of the fluorescence detection unit 27 is S. , S can be represented by the formula (10).
S = Sa + Sb + Sc
= Ja · {1 + Ka · cos (θ)}
+ Jb · {1 + Kb · cos (θ + 2π / 3)}
+ Jc · {1 + Kc · cos (θ + 4π / 3)}
= Ja + Jb + Jc
+ Ja · Ka · cos (θ)
+ Jb · Kb · cos (θ) · cos (2π / 3)
-Sin (θ) · sin (2π / 3)
+ Jc · Kc · cos (θ) · cos (4π / 3)
-Sin (θ) · sin (4π / 3)
= Ja + Jb + Jc
+ Ja · Ka · cos (θ)
+ Jb · Kb · {-(1/2) cos (θ)
-(√3 / 2) sin (θ)}
+ Jc · Kc · {-(1/2) cos (θ)
-(-√3 / 2) sin (θ)}
= Ja + Jb + Jc
+ {Ja · Ka- (1/2) Jb · Kb
− (1/2) Jc · Kc} · cos (θ)
-(√3 / 2) · (Jb · Kb-Jc · Kc) · sin (θ)
= Ja + Jb + Jc + Jp.cos (θ + δ) (10)
Here, Jp and tan δ can be expressed by equations (11) and (12), respectively.
Jp = √ [{Ja · Ka− (1/2) Jb · Kb− (1/2) Jc · Kc} 2
+ {(√3 / 2) · (Jb · Kb−Jc · Kc)} 2] (11)
tan δ = {(√3 / 2) · (Jb · Kb−Jc · Kc) /
{Ja · Ka- (1/2) Jb · Kb- (1/2) Jc · Kc} (12)
On the other hand, the combined output (detection signal) S of the fluorescence detection unit 20 is a composite of signals of a single frequency component, and can be expressed by Expression (13).
S = Acos (θ + δ) + B (13)
Here, A and B can be expressed by equations (14) and (15).
A = √ [{Ja · Ka− (1/2) Jb · Kb− (1/2) Jc · Kc} 2
+ {(√3 / 2) · (Jb · Kb−Jc · Kc)} 2] (14)
B = Ja + Jb + Jc (15)
In Expressions (12) to (15), the modulation degrees Ka, Kb, and Kc are known, and A and B are amplitude information and component information generated in the AD conversion circuit 43 and the AD conversion circuit 43, respectively. Since δ is phase information generated by the phase information generation unit 30, the component information B, amplitude information A, and phase information β are generated by the AD conversion circuit 53, the AD conversion circuit 43, and the phase information generation unit 30, respectively. By outputting to the signal calculation unit 60, the intensity signal calculation unit 60 can calculate the signal intensities Ja, Jb, and Jc of the fluorescences EM1 to EM3.

[When using four excitation lights]
First, the sample SP is irradiated with two of the four excitation lights EX1 and EX2, and the signal intensities Ja and Jb of the fluorescence EM1 and EM2 are calculated by the above-described method of “when two excitation lights are used”. Next, the remaining two of the four excitation lights EX3 and EX4 are irradiated onto the sample SP, and similarly, the intensity signals Jc and Jd of the fluorescence EM3 and EM4 are calculated by the method of “when two excitation lights are used”. To do. Thereby, each intensity signal Ja-Jd of four fluorescence EX1-EX4 is computable. Instead of this method, calculation can be performed by the following method. First, the sample SP is irradiated with three of the four excitation lights EX1 to EX3, and the signal intensities Ja, Jb, and Jc of the fluorescences EM1 to EM3 are calculated by the above-described method of “when using three excitation lights”. To do. Next, the intensity signal Jd of the fluorescence EM4 may be calculated by irradiating the sample SP with the remaining one of the four excitation lights EX4.

[When using 5 or more excitation lights]
If the fluorescence intensity signal is calculated for each of the two excitation lights or for each of the three excitation lights by combining the above-mentioned “when using two excitation lights” and “when using three excitation lights”, five or more Even when excitation light is used, the intensity signal of the fluorescence generated by each excitation light can be calculated.

  As described above, according to the fluorescence microscope of the present embodiment, the intensity signal is based on the phase information by the phase information generation unit 30, the amplitude information by the amplitude information generation unit 40, and the component information by the component information generation unit 50. Since the calculation unit 60 calculates the intensity signals of the plurality of fluorescence EM1 and the like, the plurality of fluorescence received by the fluorescence detection unit 20 can be easily separated. Therefore, it is possible to easily and reliably acquire an image for each fluorescence EM1 or the like. The intensity signals such as the respective fluorescence EM1 calculated by the intensity signal calculation unit 60 are output from the intensity signal calculation unit 60 to the personal computer 101. The personal computer 101 executes the application software 104 to cause the display device 103 to individually display images (two-dimensional images) of the respective fluorescence EM1 acquired by the fluorescence microscope 1.

  FIG. 8 is a diagram illustrating an example of an excitation spectrum of excitation light and a fluorescence spectrum of fluorescence generated by the excitation light. In FIG. 8, YFP (EX) indicates the excitation spectrum of yellow fluorescent protein, YFP (EM) indicates the fluorescence spectrum of yellow fluorescent protein, eGFP (EX) indicates the excitation spectrum of green fluorescent protein, and eGFP (EM) Indicates the fluorescence spectrum of the green fluorescent protein. In FIG. 8, the horizontal axis represents wavelength (nm) and the vertical axis represents fluorescence intensity. In general, the fluorescence spectrum tends to shift to the higher wavelength side with respect to the excitation spectrum. In addition, the two fluorescent spectra in FIG. 8 have overlapping portions near the wavelength of 500 nm. Therefore, in the case of a system in which two fluorescences having such a fluorescence spectrum are separated by an optical filter and led to different detectors, as in the prior art, for example, other fluorescence is taken into each detector, The detection signal of each detector includes a lot of errors.

  The fluorescence microscope 1 according to the first embodiment receives a plurality of fluorescence EM1 and the like collectively by the fluorescence detection unit 20, and calculates the intensity signal Ja and the like of each fluorescence from the detection signal using a calculation formula. The error included in the signal can be reduced. Further, according to the fluorescence microscope 1 of the first embodiment, since one photo detector is used, the manufacturing cost of the fluorescence microscope can be reduced and the configuration of the fluorescence microscope can be simplified. In addition, according to the fluorescence microscope 1 of the first embodiment, it is not necessary to use an expensive spectroscope for optically separating a plurality of fluorescence, so that a fluorescence microscope can be manufactured at a low cost.

Second Embodiment
Next, a second embodiment will be described. In the following description, the same or equivalent components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted or simplified. In the first embodiment, in order to establish the basic principle of calculating the intensity signal Ja of each fluorescence EM1, etc., the plurality of excitation lights EX1 etc. are intensity-modulated with a predetermined phase difference (for example, phase difference π). It is assumed that when the sample SP is irradiated, the respective fluorescence EM1 and the like generated by a plurality of excitation lights have the same phase difference as the excitation light EX1 and the like. However, in reality, depending on the fluorescent reagent and the biological specimen (cell) on which the fluorescent reagent is labeled, there is a delay from when the sample is irradiated with excitation light until fluorescence is generated. Different for each excitation light (there is a difference in time from when the sample is irradiated with the excitation light until fluorescence emission occurs). As a result, even if a plurality of excitation lights are intensity-modulated with a predetermined phase difference and the sample is irradiated, the fluorescence generated by these excitation lights has a phase difference different from that of the excitation light. Furthermore, even when the photodetector 21 and the subsequent signal transmission system have a sensitivity difference depending on the fluorescence wavelength, the phase difference of the plurality of fluorescence intensity signals included in the detection signal is the level of the plurality of excitation lights. It is out of phase. In such a state, as described in the first embodiment, it is not possible to calculate separation of a plurality of fluorescence intensity signals from the detection signal.

  In the second embodiment, the respective excitations generated by the plurality of excitation lights have the desired phase difference and the desired phase difference with respect to the reference signal b. The phase difference adjusting unit 12 arbitrarily adjusts the phase difference between the plurality of excitation lights. As a result, the basic principle described in the first embodiment can be applied, and the signal intensity of each fluorescence can be calculated.

  The adjustment of the phase difference of the plurality of excitation lights EX1 and the like is performed based on the phase delay amount stored in the storage unit 12a of the phase difference adjustment unit 12 illustrated in FIG. The phase delay amount stored in the storage unit 12a stores the data of the phase delay amount of the fluorescence generated from the reagent for each excitation light when the excitation light EX1 or the like is irradiated with respect to the phase of the excitation light EX1 or the like. The However, the data to be stored is not limited to this. For example, phase delay amount data such as how much the phase of each fluorescence is delayed with respect to the reference fluorescence phase may be used. In addition, when the photodetector 21 and the subsequent signal transmission system have a sensitivity difference depending on the fluorescence wavelength, the phase delay amount such as how much the respective phases are delayed with respect to the reference fluorescence phase. Are also stored in the storage unit 12a. That is, for specific fluorescence, both the phase delay amount based on the fluorescence generation and the phase delay amount based on the fluorescence detection are added to adjust the phase of the excitation light.

  When the phase delay amount based on the generation of fluorescence and the phase delay amount based on fluorescence detection are already known, the respective data are stored in the storage unit 12a to adjust the phase with respect to specific excitation light. When the phase delay amount based on the generation of fluorescence is unknown, phase delay amount data is acquired as follows. First, only one of the plurality of excitation lights EX1 to EXn (for example, excitation light EX1) is output, and the phase delay amount is calculated from the phase difference between the detection signal from the fluorescence detection unit 20 and the excitation light EX1. Next, only the excitation light EX2 is output, and similarly, the phase delay amount is calculated from the phase difference between the detection signal from the fluorescence detection unit 20 and the excitation light EX2. By repeating this operation, the phase delay amount for each of the excitation lights EX1 to EXn is calculated and stored in the storage unit 12a. The phase delay amount calculated in this way is data in which the phase delay amount based on the fluorescence generation and the phase delay amount based on the fluorescence detection are added together.

  At this time, if the fluctuation of the waveform of the excitation light EX1 or the like or the fluctuation of the waveform of the detection signal by the fluorescence detection unit 20 occurs, the calculated value of the phase difference may fluctuate and it may be difficult to acquire data. In such a case, when calculating the phase difference between the detection signal from the fluorescence detection unit 20 and the excitation light EX1 or the like, the phase difference between them may be calculated using a signal within a predetermined range. Examples of the predetermined range include a case where the detection signal and the excitation light are cut out for comparison in a specific time range, and a case where several wavelengths are cut out for comparison. This facilitates calculation of the phase difference and improves the accuracy of the phase delay amount. In addition, the phase difference between the excitation light EX1 and the detection signal may fluctuate for each wavelength (with time), and a plurality of wavelengths may be sampled to obtain the average phase difference from each phase difference. Thereby, the error of the phase difference fluctuation is averaged, so that the accuracy of the phase delay amount can be improved.

  Moreover, the output of the detection signal by the fluorescence detection unit 20 may fluctuate. For example, even if a weak signal is used, it is difficult to capture an accurate waveform, and the calculated value of the phase difference includes many errors. Therefore. Of the detection signals, the phase difference may be calculated using a waveform having a predetermined signal intensity. As the predetermined signal intensity, for example, an average signal intensity when other fluorescence is detected is used.

  In order to adjust the phase difference of the excitation light EX1, etc., the phase delay amount data is stored in the storage unit 12a, but instead, the phase difference itself may be used as the correction amount data. In this case, correction amount data regarding π is stored in the storage unit 12a for each excitation light EX1 and the like. When the correction amount data is unknown, the correction amount data is acquired by the above-described method in the same manner as the phase delay amount data.

  The correction amount may be a correction amount for the fluorescence generated by the excitation light. When such a correction amount is stored in the storage unit 12a, the phase difference adjustment unit 12 of the excitation light modulation unit 10 adjusts the phase of each excitation light EX1 and the like as follows to realize a predetermined phase difference To do. First, when a plurality of light sources are used from the first light source to the nth light source, the phase of the excitation light EX1 from the first light source L1 is set to (0 + δ1) on the basis of the phase of the reference signal b, and the excitation from the second light source L2 is performed. The phase of the light EX2 is set to (2π / N + δ2), the phase of the excitation light EX3 from the third light source L3 is set to (4π / N + δ1), and similarly, the phase of the excitation light EXn from the nth light source Ln is {( n-1) · 2π / N + δn}. Here, δ1, δ2,..., Δn are correction amounts related to the phase delay of the fluorescent signal from the fluorescent reagent corresponding to the wavelengths of the light sources L1, L2, L3,. L3 is the total number of Ln.

  When the sample SP is irradiated with the excitation light EX1 to EXn adjusted in this way, the phases of the respective fluorescences EM1 to EMn in the detection signal by the fluorescence detection unit 20 are as follows. When two light sources L1 and L2 are used, the phase of the fluorescence EM1 generated by the excitation light EX1 from the first light source L1 is corrected to 0 by the correction amount δ1 with the phase of the reference signal b as a reference, and the second light source The phase of the fluorescence EM2 generated by the excitation light EX2 from L2 is corrected by the correction amount δ2 to be 2π / 2 = π, and the phase difference between the fluorescence EM1 and the fluorescence EM2 is π. When using the three light sources L1 to L3, the phase of the fluorescence EM1 generated by the excitation light EX1 from the first light source L1 is corrected to 0 by the correction amount δ1 with the phase of the reference signal b as a reference, The phase of the fluorescence EM2 generated by the excitation light EX2 from the two light sources L2 is corrected to 2π / 3 by the correction amount δ2, and the phase of the fluorescence EM3 generated by the excitation light EX3 from the third light source L3 is corrected by the correction amount δ3. To 4π / 3.

  When using the four light sources L1 to L4, the phase of the fluorescence EM1 generated by the excitation light EX1 from the first light source L1 is corrected to 0 by the correction amount δ1 with the phase of the reference signal b as a reference. The phase of the fluorescence EM2 generated by the excitation light EX2 from the two light sources L2 is corrected by the correction amount δ2 to 2π / 4 = π / 2, and the phase of the fluorescence EM3 generated by the excitation light EX3 from the third light source L3 is corrected. 4π / 4 = π / corrected by the amount δ3 and the phase of the fluorescence EM4 generated by the excitation light EX4 from the fourth light source L4 is corrected to 6π / 4 = 3π / 2 by the correction amount δ4. The phase differences of the fluorescence EM1, fluorescence EM2, EM3, and EM4 with respect to the signal b are 0, π / 2, π, and 3π / 2. Thus, the phase delay of the fluorescence signal generated from the sample SP is eliminated by adding the correction amount to the plurality of excitation lights. Therefore, since the phase difference of a plurality (N) of fluorescence is 2π / N, the intensity signal calculation unit 60 can easily calculate the intensity signal of the fluorescence.

  9 shows the phase difference of the plurality of excitation lights EX1, EX2,..., EXn with respect to the reference signal b, the phase correction signal a generated by the intensity signal calculation unit 60 (the phase correction signal a is different for each light source). FIG. 9 is a diagram showing an example of an amplitude modulator that performs fine adjustment control based on a light source and a1, a2,... For example, the amplitude modulator 140 is configured to be included in the phase difference adjustment unit 12 of the excitation light modulation unit 10 of the fluorescence microscope 1 illustrated in FIG. The amplitude modulator 140 shown in FIG. 9 includes signal distributors 142-1, 142-2,..., 142-n that distribute the reference signal b at different output ratios, and 90 ° phase circuits 143-1 and 143-143. , 143-n, adders 144-1, 144-2,..., 144-n, variable gain amplifiers 145-1, 145-2,. Phase difference fine adjustment signal generation units 141-1, 141-2,.

A case where there are two light sources will be described. In the fixed phase circuits 140-1 and 140-2 in the phase difference adjustment unit 12, the amplitude modulator 140 sets the set value (phase difference 0, π) for the reference signal b and the correction amount (δ1, δ2) related to the phase delay. Based on this, a signal b1 (phase difference 0 + δ1 with respect to the reference signal b) and a signal b2 (phase difference π + δ2 with respect to the reference signal b) created by giving a predetermined phase difference are input.
The signal b1 is distributed at a predetermined amplitude ratio in the signal distributor 142-1, by a signal output from the phase difference fine adjustment signal generator 141-1 based on the phase correction signal a1, and the 90 ° phase circuit 143- 1 is input to the adder 144-1.
The signal b2 is distributed at a predetermined amplitude ratio in the signal distributor 142-2 by a signal output from the phase difference fine adjustment signal generation unit 141-2 based on the phase correction signal a2, and the 90 ° phase circuit 143- 2. Input to adder 144-2.
The adder 144-1 adds the signal in phase with the signal b 1 and the signal that has passed through the 90 ° phase circuit 143-1, so that the intensity ratio of the signal that has passed through the 90 ° phase circuit and the signal that has not passed through the phase circuit is increased. Accordingly, the output phase becomes a signal c1 having a phase further finely adjusted from the signal b1.
Similarly, the adder 144-2 adds the signal having the same phase as the signal b2 and the signal that has passed through the 90 ° phase circuit 143-2, so that the signal that has passed through the 90 ° phase circuit and the signal that has not passed through the phase circuit are added. Depending on the intensity ratio, the output phase becomes a signal c2 having a phase further finely adjusted from the signal b2. Specifically, the signals c1 and c2 are the above-described intensity modulation signals of the excitation light.
The variable gain amplifiers 145-1 and 145-2 are for adjusting the final output to a predetermined value, and are controlled based on the outputs from the phase difference fine adjustment signal generation units 141-1 and 141-2. .

The case where there are three or more light sources is the same as the case where there are two light sources. In the following description, the number of light sources is n, but n is used to include two light sources.
A case where there are n light sources will be described. Phase correction signals a1 to an are generated by the intensity signal calculation unit 60 for each light source. The amplitude modulator 140 is a signal generated by giving a predetermined phase difference in the fixed phase circuits 140-1 to 140-n based on a set value (phase difference) with respect to the reference signal b and a correction amount related to the phase delay. b1 to bn are input. For example, when there are three light sources, based on the set values (phase difference 0, 2π / 3, 4π / 3) with respect to the reference signal b and the correction amounts (δ1, δ2, δ3) regarding the phase delay, A signal b1 having a phase difference of 0 + δ1, a signal b2 having a phase difference of 2π / 3 + δ2 with respect to the reference signal b, and a signal b3 having a phase difference of 4π / 3 + δ3 with respect to the reference signal b are input.
The signals b1 to bn are output from the phase difference fine adjustment signal generators 141-1 to 141-n based on the phase correction signals a1 to an in the signal distributors 142-1 to 142-n according to predetermined signals. The signals are distributed at the amplitude ratio and input to the 90 ° phase circuits 143-1 to 143-n and adders 144-1 to 144-n, respectively. The adders 144-1 to 144-n add the signals in phase with the signals b1 to bn and the signals that have passed through the 90 ° phase circuits 143-1 to 143-n according to their intensity ratios. Accordingly, the respective output phases become intensity modulation signals c1 to cn having phases further finely adjusted from the signals b1 to bn.
The variable gain amplifiers 145-1 to 145-n are for adjusting the final output to a predetermined value, and are controlled based on the outputs from the phase difference fine adjustment signal generation units 141-1 to 141-n. .
Note that the 90 ° phase circuits 143-1 to 143-n are set to 90 ° for convenience, but multiples of 0 and 180 ° and circuits having any other phase can be used.

<Third Embodiment>
Next, a third embodiment will be described. In the following description, the same or equivalent components as those of the above-described embodiment are denoted by the same reference numerals, and the description thereof is omitted or simplified. In the third embodiment, as shown in FIG. 1, based on an instruction from the irradiation control unit 60a included in the intensity signal calculation unit 60, the phase difference adjustment unit 12 uses the frequency of the reference signal b for a plurality of light sources L1. The first irradiation state in which the sample SP is irradiated with all of the excitation lights EX1 to EXn at the same frequency and the same phase as the reference signal b, and the second irradiation to be irradiated except at least one of the excitation lights EX1 to EXn. State and do. The fluorescence detection unit 20 detects the fluorescence EM1 and the like generated by the excitation light EX1 and the like in the first irradiation state and the second irradiation state, respectively, and the first detection signal detected in the first irradiation state and the second irradiation state The detected second detection signal is output. Based on the first detection signal and the second detection signal, the intensity signal calculation unit 60 calculates an intensity signal of specific fluorescence.

  For example, when three excitation lights EX1, EX2, and EX3 are used, the combined output (first detection signal) of the fluorescence detector 20 when the sample SP is irradiated with all the excitation lights EX1 to EX3 as the first irradiation state is S. Assuming that the signal components of the fluorescence EM1, EM2, and EM3 are S1, S2, and S3, S = S1 + S2 + S3. On the other hand, when the excitation light EX1 is excluded and the sample SP is irradiated with the excitation light EX2 and EX3 as the second irradiation state, the combined output (second detection signal) of the fluorescence detection unit 20 is S (2, 3). Since S (2,3) = S2 + S3, the signal component S1 of the fluorescence EM1 is calculated by calculating S1 = S−S (2,3).

  Further, when the excitation light EX2 is excluded as the second irradiation state and the sample SP is irradiated with the excitation light EX1 and EX3, the combined output (second detection signal) of the fluorescence detection unit 20 is S (1,3). Since S (1,3) = S1 + S3, the signal component S2 of the fluorescence EM2 is calculated by calculating S2 = S−S (1,3). Similarly, S3 = S−S (1,2) is calculated from the combined output (second detection signal) S (1,2) when the sample SP is irradiated with the excitation light EX1, EX2 as the second irradiation state. The signal component S3 of the fluorescence EM3 is calculated. As the second irradiation state, instead of excluding one excitation light, a plurality of excitation lights (excitation light EX1 and EX2) may be excluded and irradiated. As described above, by calculating based on the first and second detection signals, the intensity signal of the specific fluorescence can be easily calculated, and a plurality of fluorescence received by the fluorescence detection unit 20 can be separated.

<Fluorescent light receiving unit>
Next, the fluorescence light receiving unit according to the embodiment will be described. In the following description, the same or equivalent components as those of the above-described embodiment are denoted by the same reference numerals, and the description thereof is omitted or simplified. As shown in FIG. 10, the fluorescence light receiving unit 200 includes a fluorescence detection unit 20, a phase information generation unit 30, an amplitude information generation unit 40, a component information generation unit 50, and an intensity signal calculation unit 60. The fluorescence light receiving unit 200 is a unit in which the fluorescence detection unit 20 or the like is housed in a single or a plurality of cases, and is separated from the optical system 110 and the light source unit (the light source 70 and the excitation light modulation unit 10) constituting the fluorescence microscope. It is a possible unit. In addition, the fluorescence detection part 20 can be made removable from the housing | casing of a unit so that it can arrange | position to the fluorescence light-receiving part (refer FIG. 2) of the optical system 110. FIG.

  Further, the fluorescence light receiving unit 200 has an input unit for inputting the reference signal b sent from the excitation light modulation unit 10 to the fluorescence detection unit 20, and an input unit for inputting the reference signal b to the phase information generation unit 30. Further, an output unit for outputting the phase correction signal a from the intensity signal calculation unit 60 to the excitation light modulation unit 10 is provided. The intensity signal calculation unit 60 is the same as the fluorescence microscope 1 of the first embodiment in that it includes a connection terminal for connecting to an external personal computer 101.

  In this fluorescence light receiving unit 200, a procedure for detecting a plurality of fluorescence EM1 and the like by the fluorescence detection unit 20 and calculating an intensity signal for each fluorescence EM1 and the like is the same as in the first to third embodiments described above. Moreover, you may provide one or both of the excitation light modulation part 10 and the light source 70 as a fluorescence light-receiving unit. Thus, according to the fluorescence light receiving unit 200 according to the present embodiment, a plurality of fluorescence EM1 and the like can be easily separated into signal intensity for each fluorescence. Further, the fluorescence light receiving unit 200 can be easily used by arranging the fluorescence detection unit 20 in the optical system and controlling the light source unit with respect to the existing fluorescence microscope, and can increase versatility.

  The above embodiment is not limited to the above description, and various modifications can be made without departing from the gist of the present invention. In the above-described embodiment, the sine wave excitation light EX1 and the like are exemplified as the plurality of excitation lights irradiated onto the sample by the excitation light modulation unit 10, but the present invention is not limited to this, and other waveforms such as a rectangular wave are used. The excitation light may be used.

EM1, EM2-EMn ... fluorescence, EX1, EX2-EXn ... excitation light, L1, L2-Ln ... light source, SP ... sample, 1 ... fluorescence microscope, 10 ... excitation light modulation unit, 12 ... phase difference adjustment unit, 12a ... Storage unit, 20 ... fluorescence detection unit, 30 ... phase information generation unit, 40 ... amplitude information generation unit, 50 ... component information generation unit, 60 ... intensity signal calculation unit, 60a ... irradiation control unit, 200 ... fluorescence detection unit

Claims (17)

An excitation light modulator that modulates the intensity of a plurality of excitation lights irradiated on the sample at the same frequency as the frequency of the reference signal and with a predetermined phase difference with respect to the phase of the reference signal;
A fluorescence detection unit for detecting a plurality of fluorescence generated by the plurality of excitation lights;
A phase information generation unit that generates phase information from a relationship between a phase in a predetermined frequency component extracted from a detection signal by the fluorescence detection unit and a phase of the reference signal;
An amplitude information generation unit that generates amplitude information from the amplitude of a predetermined frequency component extracted from the detection signal by the fluorescence detection unit;
A component information generation unit that generates component information from a component obtained by removing the frequency component from the detection signal by the fluorescence detection unit;
An intensity signal calculation unit that calculates an intensity signal of each of the plurality of fluorescences based on the phase information, the amplitude information, and the component information;
A fluorescence microscope.   The fluorescence microscope according to claim 1, further comprising a light source that outputs the plurality of excitation lights.   The fluorescence microscope according to claim 2, wherein a plurality of the light sources are provided so as to output each of the plurality of excitation lights.   The fluorescence according to any one of claims 1 to 3, wherein the phase information is a phase difference between a phase in a predetermined frequency component extracted from a detection signal by the fluorescence detection unit and a phase of the reference signal. microscope.   The fluorescence microscope according to claim 1, wherein the excitation light modulation unit includes a phase difference adjustment unit that adjusts phases of the plurality of excitation lights.   The fluorescence microscope according to claim 5, wherein the phase difference adjustment unit adjusts phases of the plurality of excitation lights with respect to the reference signal. The phase difference adjustment unit includes a plurality of phases of the excitation light such that phases of the plurality of fluorescence have a predetermined phase difference from each other and a predetermined phase difference with respect to the phase of the reference signal. The fluorescence microscope of Claim 6 which adjusts.   The fluorescence microscope according to claim 6, wherein the phase difference adjusting unit adjusts the phase of the excitation light by changing the intensity of the direct current component of the excitation light and the modulation degree of the alternating current component.   9. The phase difference adjusting unit according to claim 5, wherein the phase difference adjustment unit adjusts the phases of the plurality of excitation lights based on a phase delay amount caused by at least one of the generation and detection of the fluorescence. Fluorescence microscope.   The said phase difference adjustment part is provided with the memory | storage part for memorize | storing the said phase delay amount, and adjusts the phase difference of these excitation light based on the said phase delay amount memorize | stored in the said memory | storage part. Fluorescent microscope.   The fluorescence microscope according to claim 10, wherein the storage unit stores a phase delay amount calculated from a phase difference between a detection signal from the fluorescence detection unit and the excitation light when the plurality of excitation lights are individually output. .   The fluorescence microscope according to claim 11, wherein a signal in a predetermined range in the detection signal is used as a detection signal by the fluorescence detection unit.   The fluorescence microscope according to claim 11, wherein an average phase difference is used as a phase difference between a detection signal from the fluorescence detection unit and the excitation light.   The fluorescence microscope according to claim 11, wherein a signal having a predetermined signal intensity in the detection signal is used as a detection signal by the fluorescence detection unit.   When the plurality of light sources are from the first light source to the n-th light source, the excitation light modulation unit sets the phase of the excitation light from the first light source to δ1 with the phase of the reference signal as a reference, and outputs from the second light source The phase of the excitation light is (2π / N + δ2), the phase of the excitation light from the third light source is (4π / N + δ3), and the phase of the excitation light from the Nth light source is {(n-1) · 2π / N + δn} 4. The fluorescence microscope according to claim 3, wherein δ1, δ2, δ3,..., Δn are correction amounts relating to the phase delay of the fluorescence signal from the fluorescence reagent corresponding to the wavelength of the light source. Intensity modulation of each of a plurality of excitation lights irradiating the sample with the same frequency as the frequency of the reference signal and a predetermined phase difference with respect to the phase of the reference signal,
Detecting a plurality of fluorescence generated by the plurality of excitation lights;
Generating phase information from the relationship between the phase of the predetermined frequency component extracted from the detected signal and the phase of the reference signal;
Amplitude information is generated from the amplitude of a predetermined frequency component extracted from the detected signal,
Generating component information from a component obtained by removing the frequency component from the detected signal;
A fluorescence detection method for calculating an intensity signal of each of the plurality of fluorescences based on the phase information, the amplitude information, and the component information. A fluorescence detection unit for detecting a plurality of fluorescence generated by a plurality of excitation lights having the same frequency as the frequency of the reference signal and intensity-modulated with a predetermined phase difference with respect to the phase of the reference signal;
A phase information generation unit that generates phase information from a relationship between a phase in a predetermined frequency component extracted from a detection signal by the fluorescence detection unit and a phase of the detection signal;
An amplitude information generation unit that generates amplitude information from the amplitude of a predetermined frequency component extracted from the detection signal by the fluorescence detection unit;
A component information generation unit that generates component information from a component obtained by removing the frequency component from the detection signal by the fluorescence detection unit;
An intensity signal calculation unit that calculates an intensity signal of each of the plurality of fluorescences based on the phase information, the amplitude information, and the component information;
A fluorescence detection unit comprising:

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