JP2015145829A - Fluorescence signal acquisition device and fluorescence signal acquisition method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To separate generated fluorescence more easily even when a plurality of phosphors are excited at the same time in response to one excitation ray.SOLUTION: A fluorescence signal acquisition device according to the present disclosure includes: a plurality of light sources for irradiating an object to be measured that includes a plurality of phosphors with a plurality of excitation rays modulated by carriers having frequencies different from each other; a plurality of fluorescence detection units for detecting a plurality of fluorescences generated in response to the plurality of excitation rays; a multiband-pass optical filter, located at a prestage of the plurality of fluorescence detection units, for causing the plurality of fluorescences to branch off to a plurality of optical paths and letting fluorescence from the plurality of phosphors whose wavelengths are noncontiguous pass through; and a synchronous detection unit for performing synchronous detection on detection signals detected by the fluorescence detection units and separating fluorescences corresponding to the plurality of phosphors from each other.

Description

  The present disclosure relates to a fluorescence signal acquisition apparatus and a fluorescence signal acquisition method.

  Conventionally, it has been performed in various fields to irradiate a measurement object including a phosphor with excitation light having a specific wavelength and measure fluorescence generated according to the excitation light. Here, when simultaneously measuring fluorescence from a measurement object including a plurality of phosphors, a plurality of excitation lights with specific wavelengths corresponding to each phosphor are irradiated, but a plurality of excitation lights are simply irradiated. In order to detect the generated fluorescence, as many fluorescent detectors as the number of phosphors are required. As a result, a measuring instrument for simultaneously exciting a plurality of phosphors and simultaneously detecting a plurality of fluorescences becomes large.

  On the other hand, for example, in Patent Document 1 and Patent Document 2 below, in order to simultaneously measure fluorescence from a plurality of phosphors, a plurality of excitation lights are modulated in advance at a predetermined frequency, and the generated fluorescence is demodulated. A technique for detecting a plurality of fluorescence simultaneously has been proposed. By using such modulated excitation light, a plurality of fluorescence can be detected by a single fluorescence detector.

JP 11-281893 A JP 2005-91895 A

  However, even when the techniques disclosed in Patent Document 1 and Patent Document 2 are used, when a plurality of phosphors are excited in response to a single excitation light, It is not possible to separate simultaneously excited fluorescence with only one fluorescence detector. Therefore, there has been a demand for a technique capable of separating generated fluorescence even when a plurality of phosphors are excited simultaneously according to a single excitation light.

  Therefore, in the present disclosure, there is provided a fluorescence signal acquisition device and a fluorescence signal acquisition method capable of more easily separating generated fluorescence even when a plurality of phosphors are excited simultaneously according to one excitation light. suggest.

  According to the present disclosure, a plurality of light sources for irradiating a measurement object including a plurality of phosphors with a plurality of excitation lights modulated by carriers having different frequencies, and generated according to the plurality of excitation lights A plurality of fluorescence detectors for detecting a plurality of fluorescence, and a plurality of phosphors that are positioned in front of the plurality of fluorescence detectors and branch the plurality of fluorescences into a plurality of optical paths, and do not have continuous wavelengths A multi-band pass optical filter that transmits fluorescence from each of the filters, and a synchronous detection unit that synchronously detects detection signals detected by the respective fluorescence detection units and separates fluorescence corresponding to the plurality of phosphors. A fluorescence signal acquisition device is provided.

  Further, according to the present disclosure, it is possible to irradiate a measurement object including a plurality of phosphors with a plurality of excitation lights modulated with carriers having different frequencies, and from a plurality of phosphors having non-continuous wavelengths. A plurality of fluorescence generated in response to the plurality of excitation lights is branched into a plurality of optical paths, and the fluorescence branched into the plurality of optical paths is divided into a plurality of fluorescence. There is provided a fluorescence signal acquisition method including detection by a detection unit, and synchronous detection of detection signals detected by the respective fluorescence detection units to separate fluorescence corresponding to the plurality of phosphors. The

  According to the present disclosure, a plurality of fluorescence generated in response to a plurality of excitation lights frequency-modulated in advance are detected by a plurality of fluorescence detectors after being branched into a plurality of optical paths by a multiband pass optical filter, Synchronous detection is performed.

  As described above, according to the present disclosure, it is possible to more easily separate the generated fluorescence even when a plurality of phosphors are excited simultaneously according to one excitation light.

  Note that the above effects are not necessarily limited, and any of the effects shown in the present specification, or other effects that can be grasped from the present specification, together with or in place of the above effects. May be played.

It is explanatory drawing which showed typically the whole structure of the fluorescence signal acquisition apparatus which concerns on 1st Embodiment of this indication. It is the graph which showed the relationship between the wavelength of excitation light, and the wavelength of the fluorescence from fluorescent substance. It is explanatory drawing for demonstrating the relationship between the fluorescent substance in the fluorescence signal acquisition apparatus which concerns on the same embodiment, and the number of fluorescence detection parts. It is explanatory drawing for demonstrating the multiband pass optical filter in the fluorescence signal acquisition apparatus which concerns on the same embodiment. It is explanatory drawing which showed typically an example of the multiband pass optical filter in the fluorescence signal acquisition apparatus which concerns on the embodiment. It is explanatory drawing which showed typically an example of the multiband pass optical filter in the fluorescence signal acquisition apparatus which concerns on the embodiment. It is explanatory drawing which showed typically an example of the synchronous detection part in the fluorescence signal acquisition apparatus which concerns on the embodiment. It is explanatory drawing which showed typically an example of the synchronous detection part in the fluorescence signal acquisition apparatus which concerns on the embodiment. It is explanatory drawing which showed typically the structure of the flow cytometer which is an example of the fluorescence signal acquisition apparatus which concerns on the embodiment. It is explanatory drawing which showed typically the structure of the laser scanning fluorescence microscope which is an example of the fluorescence signal acquisition apparatus which concerns on the embodiment. It is the flowchart which showed an example of the flow of the fluorescence signal acquisition method which concerns on the same embodiment.

  Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In addition, in this specification and drawing, about the component which has the substantially same function structure, duplication description is abbreviate | omitted by attaching | subjecting the same code | symbol.

The description will be made in the following order.
1. First Embodiment (1-1) Configuration of Fluorescence Signal Acquisition Device (1-2) Specific Example 1 of Fluorescence Signal Acquisition Device-1: Flow Cytometer (1-3) Specific Example 2 of Fluorescence Signal Acquisition Device: Laser scanning fluorescence microscope (1-4) Flow of fluorescence signal acquisition method

2. Summary

(First embodiment)
<Configuration of fluorescence signal acquisition device>
[Overall configuration of fluorescence signal acquisition device]
First, the overall configuration of the fluorescence signal acquisition device 1 according to the first embodiment of the present disclosure will be described with reference to FIG. FIG. 1 is an explanatory diagram schematically showing the overall configuration of the fluorescence signal acquisition apparatus according to the present embodiment.

  The fluorescence signal acquisition device 1 according to the present embodiment simultaneously irradiates a measurement object S including a plurality of phosphors with a plurality of excitation lights, and simultaneously detects a plurality of fluorescences generated according to the plurality of excitation lights. It is a device to do.

  In addition, the fluorescence signal acquisition device 1 according to the present embodiment can output information on the detected fluorescence to an arbitrary arithmetic processing device 3. The arithmetic processing unit 3 performs various arithmetic processes and image processing such as visualization of the knowledge about the fluorescence detected by the fluorescence signal acquisition device 1, and uses the fluorescence signal acquisition device 1 to acquire the knowledge about the detected fluorescence. Can be notified.

  The arithmetic processing device 3 may be realized as one function of the fluorescence signal acquisition device 1 or may be various computers or servers connected to the fluorescence signal acquisition device 1. Further, the connection method between the fluorescent signal acquisition device 1 and the arithmetic processing device 3 is not particularly limited, and the fluorescent signal acquisition device 1 and the arithmetic processing device 3 may be directly connected by wire or wirelessly. Alternatively, they may be connected via various networks.

  The measuring object S including a plurality of phosphors is not particularly limited, and may be classified as an organism such as various cells as long as it includes a plurality of phosphors. It may be classified into. Needless to say, the measuring object S including a plurality of phosphors may be various compounds including a plurality of phosphors.

  Moreover, the fluorescent substance contained in the measuring object S may be a phosphor previously held by the measuring object S, or various fluorescent reagents introduced into the measuring object from the outside of the measuring object S It may be something like this.

Then, the structure of the fluorescence signal acquisition apparatus 1 which concerns on this embodiment is demonstrated.
For example, as illustrated in FIG. 1, the fluorescence signal acquisition device 1 according to the present embodiment includes a plurality of light sources 101, a modulation unit 103, a measurement optical system 105, a multiband pass optical filter 107, and a plurality of fluorescence detections. Unit 109 and synchronous detection unit 111 are mainly provided.

  The plurality of light sources 101 emit a plurality of laser beams used to simultaneously excite a plurality of phosphors included in the measurement object S. These laser beams are used as excitation light for exciting the phosphor contained in the measuring object S. The wavelength of the excitation light emitted from the light source 101 is not particularly limited, and a wavelength suitable for exciting the phosphor included in the measurement object S may be selected as appropriate. Further, the number N of the light sources 101 is not particularly limited, and is appropriately set to a number that can simultaneously excite all the phosphors included in the measurement object S.

  The excitation lights emitted from the plurality of light sources 101 are intensity-modulated at different frequencies by a modulation unit 103 described later. In addition, each excitation light emitted from the plurality of light sources 101 is simultaneously emitted to the same region of the measurement object S via the measurement optical system 105 described later.

  Each light source 101 is not particularly limited, and various light sources such as various semiconductor lasers, solid-state lasers, gas lasers, and the like can be used. By using various semiconductor lasers as the light source 101, the fluorescent signal acquisition apparatus 1 can be further downsized.

  The laser used as the light source 101 may be a CW (Continuous Wave) laser or a pulsed laser.

  The modulation unit 103 modulates the intensity of each excitation light emitted from the plurality of light sources 101 using carriers having different frequencies. Here, the number of carrier frequencies used by the modulation unit 103 is the same as the number of light sources 101. That is, when there are N light sources 101, N carriers having N different frequencies are used to intensity-modulate N types of excitation light.

  Here, the fundamental wave of each carrier used in modulation section 103 is preferably separated by a frequency equal to or higher than the bandwidth of the sideband. In addition, the frequency of each carrier is appropriately selected so that the third harmonic and the sideband in one carrier do not overlap with the fundamental and the sideband in another carrier. By selecting the frequency of N carriers in this way, crosstalk between the carriers can be prevented. More preferably, the frequency of each carrier is appropriately selected so that the second harmonic and the sideband in one carrier do not overlap with the fundamental and the sideband in another carrier. By selecting the carrier frequency according to the second harmonic, not the third harmonic, crosstalk between the carriers can be more reliably prevented.

  A more detailed method for selecting the carrier frequency will be described in detail later.

  The detailed configuration of the modulation unit 103 is not particularly limited, and various devices can be used as long as the excitation light having a predetermined wavelength can be intensity-modulated.

  The measurement optical system 105 guides a plurality of excitation lights emitted from a plurality of light sources 101 coaxially and simultaneously to the same region of the measurement object S, and also emits a plurality of fluorescence generated from the measurement object S, This is an optical system that guides light to the multiband pass optical filter 107 coaxially.

  The detailed configuration of the measurement optical system 105 is not particularly limited, and may be configured by appropriately combining various optical elements such as various lenses, various mirrors, pinholes, various filters, and a galvano scan system. By configuring an optical system by combining such various optical elements, for example, a measurement optical system used in a flow cytometer, a measurement optical system used in a laser scanning fluorescence microscope, and the like can be realized. it can.

  The multiband pass optical filter 107 is provided between the measurement optical system 105 and the fluorescence detection unit 109. The multi-bandpass optical filter 107 transmits fluorescence from a plurality of phosphors having non-continuous wavelengths guided coaxially by the measurement optical system 105 and transmits fluorescence having a plurality of wavelengths to a plurality of optical paths. An optical filter to be branched.

  The number of optical paths branched by the multiband pass optical filter 107 is the same as the number of fluorescence detectors 109 provided at the subsequent stage of the multiband pass optical filter 107. The method for setting the number of fluorescence detectors 109 and the detailed configuration of the multiband pass optical filter 107 will be described in detail below.

  Fluorescence branched into a plurality of optical paths by the multiband pass optical filter 107 is detected by a plurality of fluorescence detectors 109, respectively, and information on the intensity of the fluorescence is converted into an electrical signal. The fluorescence detection unit 109 can be configured using various detectors such as a CCD (Charge-Coupled Device), a photomultiplier tube (PMT), and the like. A detection signal related to fluorescence detected by the fluorescence detection unit 109 is output to the subsequent synchronous detection unit 111.

  The synchronous detection unit 111 performs synchronous detection on detection signals output from the plurality of fluorescence detection units 109 based on the carrier frequency used in the modulation unit 103, and separates fluorescence corresponding to each of the plurality of phosphors. To do. A signal corresponding to each fluorescence after separation is output to, for example, the arithmetic processing unit 3 and visualized.

  The synchronous detection unit 111 according to the present embodiment includes a number of demodulators corresponding to the number of phosphors included in the measurement object S. As a demodulator constituting the synchronous detection unit 111, for example, a demodulator including a band pass filter, a multiplier, and a low pass filter can be used. The detailed configuration of the demodulator constituting the synchronous detection unit 111 will be described in detail later.

  The overall configuration of the fluorescence signal acquisition device 1 according to the present embodiment has been described above with reference to FIG.

[Relationship between the number of phosphors contained in the measurement object and the number of phosphor detectors]
Next, the relationship between the number of phosphors included in the measurement object and the number of phosphor detectors will be specifically described with reference to FIGS. 2 and 3. FIG. 2 is a graph showing the relationship between the wavelength of excitation light and the wavelength of fluorescence from the phosphor, and FIG. 3 shows the number of phosphors and fluorescence detectors in the fluorescence signal acquisition apparatus according to this embodiment. It is explanatory drawing for demonstrating this relationship.

  Hereinafter, the measurement object S including the seven types of phosphors (phosphor A to phosphor G) as shown in FIG. 2 is respectively excited with four types of excitation light (405 nm, 488 nm, 561 nm, and 643 nm). A specific description will be given by taking the case of excitation as an example. Note that the number of phosphors and the number of excitation lights used in the fluorescence signal acquisition device 1 according to the embodiment of the present disclosure are not limited to the examples described below.

When the seven types of phosphors as shown in FIG. 2 are excited by four types of excitation light to generate fluorescence, for example, the following combinations of phosphors and excitation light are used.
Phosphor A: excitation light with an excitation wavelength of 405 nm Phosphor B: excitation light with an excitation wavelength of 488 nm Phosphor C and phosphor D: excitation light with an excitation wavelength of 561 nm Phosphor E to phosphor G: excitation light with an excitation wavelength of 643 nm

  In order to separate the fluorescence from phosphor A from other fluorescence, a bandpass filter having a central wavelength of 455 nm / bandwidth of 50 nm can be used, and the fluorescence from phosphor B is separated from other fluorescence. In order to achieve this, a bandpass filter having a center wavelength of 535 nm / bandwidth of 50 nm can be used. Similarly, in order to separate the fluorescence from phosphor C and phosphor D from other fluorescence, it is possible to use a bandpass filter having a center wavelength of 600 nm / bandwidth of 75 nm. Further, in order to separate the fluorescence from the phosphor E from other fluorescence, a bandpass filter having a center wavelength of 665 nm / 30 nm bandwidth is used, and in order to separate the fluorescence from the phosphor F from other fluorescence, In order to separate the fluorescence from the phosphor G from other fluorescence using a bandpass filter having a center wavelength of 720 nm / bandwidth of 60 nm, a bandpass filter having a center wavelength of 785 nm / bandwidth of 60 nm can be used.

  Here, in simultaneous excitation using excitation light of a plurality of wavelengths as is generally performed, in order to separate these seven types of fluorescence, seven fluorescence detection units 109 must be installed, The device becomes large. Therefore, in the fluorescence signal acquisition device 1 according to the present embodiment, frequency-modulated excitation light emitted from the light source 101, a multiband pass optical filter 107 as described in detail below, and a plurality of fluorescence detection units 109 are provided. By using in combination, it is possible to simultaneously detect a plurality of fluorescence while suppressing the number of fluorescence detection units 109. Hereinafter, the technical idea for fluorescence separation in the embodiment of the present disclosure will be described.

  For example, when a phosphor as shown in FIG. 2 is excited by the excitation light shown in FIG. 2, the phosphor A and the phosphor B are associated with one fluorescence for one excitation light. Therefore, in the case of simultaneously detecting the phosphor A and the phosphor B, it is possible to detect these two fluorescences with a single fluorescence detection unit by modulating each excitation light with a carrier having a different frequency. is there. However, when excitation light having a wavelength of 561 nm is used, the phosphor C and the phosphor D are excited simultaneously, and two fluorescences corresponding to the phosphors are generated. Similarly, when excitation light having a wavelength of 643 nm is used, the phosphors E to G are excited at the same time, and three fluorescences corresponding to the phosphors are generated. Therefore, as disclosed in Patent Document 1 and Patent Document 2 described above, the excitation light having the wavelength of 561 nm and the fluorescence C and D are associated with each other, or the excitation light having the wavelength of 643 nm and the fluorescence E˜ are simply modulated by frequency modulation of the excitation light. Although it is possible to associate with fluorescence G, it is not possible to distinguish between fluorescence C and fluorescence D, or between fluorescence E to fluorescence G.

  Therefore, in the fluorescence signal acquisition device 1 according to the present embodiment, by using the multiband pass optical filter 107 as described above, a plurality of fluorescence associated with the same excitation light is branched into separate optical paths. . Thereby, a plurality of fluorescence associated with the same excitation light can be separated and detected by different fluorescence detection units 109. As described above, in the fluorescence signal acquisition device 1 according to the present embodiment, the number of the fluorescence detection units 109 provided in the device (the number M in FIG. 1) is smaller than the number of phosphors to be detected simultaneously (in FIG. 2). In the example, it can be less than 7). Note that the number of light sources 101 provided in the apparatus (number N in FIG. 1) can be set to the minimum necessary number (four in the example of FIG. 2) for exciting the phosphors to be detected simultaneously. .

  Moreover, in the fluorescence signal acquisition apparatus 1 according to the present embodiment, it is only necessary to be able to separate a plurality of fluorescences associated with the same excitation light, so the number of fluorescence detection units 109 provided in the apparatus is the same excitation light. It is preferable to set the maximum number of phosphors excited by. Fluorescence associated with excitation light having different wavelengths can be separated by the degree of modulation of the excitation light. Therefore, the fluorescence signal acquisition device can be obtained by considering the number of fluorescence detection units 109 with the above guidelines. The number of fluorescence detection units 109 provided in 1 can be minimized. As shown in FIG. 3, when the seven types of phosphors shown in FIG. 2 are excited with four types of excitation light, the maximum number of phosphors excited with the same excitation light is three. Therefore, in order to detect the seven phosphors shown in FIG. 2 at the same time, it is preferable to use the three fluorescence detection units 109.

  In other words, by using the multiband pass optical filter 107 after frequency-modulating the excitation light, it becomes possible to simultaneously detect more phosphors by the number of conventional fluorescence detection units 109.

  The relationship between the number of phosphors included in the measurement object and the number of phosphor detectors has been described above with reference to FIGS. 2 and 3.

[Multiband pass optical filter]
Next, the multiband pass optical filter 107 used in the fluorescence signal acquisition apparatus 1 according to the present embodiment will be specifically described with reference to FIGS. 4 to 5B. FIG. 4 is an explanatory diagram for explaining a multiband pass optical filter in the fluorescence signal acquisition apparatus according to the present embodiment, and FIGS. 5A and 5B are multiband pass optics in the fluorescence signal acquisition apparatus according to the present embodiment. It is explanatory drawing which showed an example of the filter typically.

As described with reference to FIGS. 2 and 3, the multiband pass optical filter 107 according to the present embodiment has a function of branching a plurality of fluorescence excited by the same excitation light into different optical paths. Desired. When separating seven types of phosphors as shown in FIGS. 2 and 4, the multiband pass optical filter 107 divides the fluorescence C and the fluorescence D into two different optical paths, and the fluorescence E to the fluorescence F. Is preferably branched into three different optical paths. Therefore, in the example shown in FIG. 4, the multiband pass optical filter 107 transmits any one of the wavelength bands corresponding to λ _A to λ _G in the figure and reflects the other wavelength bands. In addition, it is preferable to guide the wavelength bands λ _C and λ _D and the wavelength bands λ _{E to} λ _G to different fluorescence detection units 109.

The combination of wavelength bands transmitted or reflected by the multiband pass optical filter 107 can be determined as appropriate. For example, as shown in FIG. 4, the multiband pass optical filter 107 has bands λ _A and λ _D. And λ _G are guided to the first fluorescence detection unit 109 (for example, PMT1), the bands λ _B and λ _E are guided to the second fluorescence detection unit 109 (for example, PMT2), and the bands λ _C and λ _What is necessary is just to guide _F to the 3rd fluorescence detection part 109 (for example, PMT3).

  Such a multi-band pass optical filter 107 reflects light in a predetermined wavelength band (more specifically, light in a certain single wavelength band) and transmits a plurality of single waves that transmit light in other wavelength bands. This is realized by arranging band reflection filters in series.

For example, in FIG. 5A, a multiband pass optical filter 107 is formed by combining seven types of single-band reflection filters that reflect any one of the bands λ _{A to} λ _G and transmit light in the other bands. An example of realization is shown. That is, in the example shown in FIG. 5A, seven types of single-band reflection filters 121 are arranged in series while changing the installation angle (the degree of inclination of the filter 121) in the optical path, and fluorescence in a specific wavelength band is emitted. Reflect in a specific direction. As a result, the seven types of single-band reflection filters 121 function as the multiband pass optical filter 107 as a whole, and the seven types of fluorescence can be guided to the three fluorescence detection units 109.

In the example shown in FIG. 5B, seven types of single-band reflection filters that reflect any one of the bands λ _{A to} λ _G and transmit light in the other bands, a band-pass filter 123, The example which implement | achieves the multiband pass optical filter 107 by combining is shown in figure. Even in the case shown in FIG. 5B, the seven types of single band reflection filters 121 and the band pass filter 123 function as a multi-band pass optical filter 107 as a whole, and the seven types of fluorescence are converted into three fluorescence detection units. The light can be guided to 109.

  Note that the multiband pass optical filter 107 shown in FIG. 5A can be realized by using a smaller number of single band reflection filters 121 as compared with the example shown in FIG. This is a preferred form for downsizing. On the other hand, the multiband pass optical filter 107 shown in FIG. 5B is easier to maintain the multiband pass optical filter 107, although the number of single band reflection filters 121 used is larger than that of the example shown in FIG. 5A. It becomes.

  The multiband pass optical filter 107 used in the fluorescence signal acquisition device 1 according to the present embodiment has been described above with reference to FIGS. 4 to 5B.

[Synchronous detection section]
Next, the synchronous detection unit 111 used in the fluorescence signal acquisition apparatus 1 according to the present embodiment will be specifically described with reference to FIGS. 6A and 6B. 6A and 6B are explanatory diagrams schematically illustrating an example of the synchronous detection unit in the fluorescence signal acquisition apparatus according to the present embodiment.

  As described earlier, the synchronous detection unit 111 according to the present embodiment includes a demodulator including a bandpass filter, a multiplier, and a lowpass filter. The demodulator having such a configuration is provided as many as the number of phosphors included in the measurement object S as the whole fluorescence signal acquisition apparatus 1.

  For example, as shown in FIG. 4, when seven types of phosphors are detected by the three fluorescence detection units 109, light including three types of fluorescence is guided to the first fluorescence detection unit 109. Each of the third fluorescence detection units 109 guides light including two types of fluorescence. Accordingly, a 3-channel demodulator capable of separating three types of detection signals is provided at the subsequent stage of the first fluorescence detection unit 109, and two types of detection are performed at the subsequent stage of the second and third fluorescence detection units 109. A two-channel demodulator that can separate signals may be provided. As a result, a demodulator for seven channels as a whole is mounted on the fluorescence signal acquisition device 1 that acquires fluorescence from the phosphor.

FIG. 6A shows a configuration example of a band pass filter 133, a multiplier 135, and a low pass filter 137 for realizing a two-channel demodulator 131. When the respective fluorescence is separated from the input signal MSin including two kinds of fluorescences FL _a and FL _b , as shown in FIG. 6A, a band pass filter 133, a multiplier 135, and a low pass filter 137 are connected in series. Two demodulators connected to may be provided in parallel.

In such a case, by inputting an input signal _{MS in} the band-pass filter BPFa configured for fluorescence FL _a, it extracts an input signal of a portion corresponding to the fluorescence FL _a. Then, the extracted input signal, and inputs to the multiplier together with the carrier frequency f _{C_A} corresponding to fluorescence FL _a obtained from the modulator 101, demodulates the signal relating to fluorescence FL _a. Then, the signal after demodulation, and inputs to the low-pass filter LPFa configured for fluorescence FL _a, baseband fluorescent (i.e., fluorescent FL a from the focused _phosphor) can be obtained.

Similarly, by inputting an input signal _{MS in} the band-pass filter BPFb configured for fluorescence FL _b, to extract the input signal of a portion corresponding to the fluorescence FL _b. Then, the extracted input signal, and inputs to the multiplier together with the carrier frequency f _{C_B} corresponding to fluorescence FL _b obtained from the modulator 101, demodulates the signal relating to fluorescence FL _b. Then, the signal after demodulation, and inputs to the low-pass filter LPFb configured for fluorescence FL _b, baseband fluorescent (i.e., fluorescent FL b from the focused _phosphor) can be obtained.

  FIG. 6B shows a configuration example of a band pass filter 133, a multiplier 135, and a low pass filter 137 for realizing a three-channel demodulator 131. As shown in FIG. 6B, the three-channel demodulator 131 is provided with three demodulators in which a band-pass filter 133, a multiplier 135, and a low-pass filter 137 are connected in series. Since the demodulation processing in each demodulator 131 is the same as that of the two-channel demodulator 131 shown in FIG. 6A, detailed description thereof will be omitted below.

  In the fluorescence signal acquisition device 1 according to the present embodiment, the synchronous detector 111 is configured using the demodulator 131 as illustrated in FIGS. 6A and 6B, so that a plurality of fluorescences from a plurality of phosphors can be transmitted to each other. It becomes possible to separate.

  The synchronous detection unit 111 used in the fluorescence signal acquisition device 1 according to the present embodiment has been described above with reference to FIGS. 6A and 6B.

  In the above description, the case where the synchronous detection unit 111 is configured by a demodulator including a band pass filter, a multiplier, and a low pass filter has been described. However, the configuration of the synchronous detection unit 111 according to the present embodiment is such an example. It is not limited to only. For example, a low-pass filter that passes only signals below the sideband of the maximum modulation frequency among the detection signals detected by each fluorescence detection unit 109, and an output signal from such a filter has a sampling frequency that is at least three times the maximum modulation frequency A digital signal processing unit constituted by an AD conversion unit that performs AD conversion in the above may be provided as the synchronous detection unit 111. By using the digital signal processing unit to digitally process the detection signal of the fluorescence detection unit 109, it is possible to obtain a result equivalent to that of the demodulator.

<Specific Example of Fluorescence Signal Acquisition Device-1: Flow Cytometer>
Next, a flow cytometer is taken as a specific example of the fluorescence signal acquisition apparatus 1 according to the present embodiment, and the fluorescence signal acquisition apparatus according to the present embodiment will be specifically described with reference to FIG. FIG. 7 is an explanatory diagram schematically showing the configuration of a flow cytometer which is an example of the fluorescence signal acquisition apparatus according to the present embodiment.

  The fluorescence signal acquisition apparatus 1 according to the present embodiment as described above can be realized as a flow cytometer that detects fluorescence from a measurement object being conveyed in a predetermined flow path. In such a case, the fluorescence signal acquisition device 1 realized as a flow cytometer uses various cells and the like stained with various phosphors as a measurement object.

  In FIG. 7, the configuration of a flow cytometer in the case where seven types of phosphors as shown in FIG. 4 are excited with four types of excitation light of 405 nm, 488 nm, 561 nm, and 643 nm to detect seven types of fluorescence. Is schematically shown.

In this case, the light source 101 includes a laser (Laser) 1 that emits excitation light of 405 nm, a laser 2 that emits excitation light of 488 nm, a laser 3 of 561 nm, and a laser 4 that emits excitation light of 643 nm. Each of the lasers is intensity-modulated by carrier frequencies f _{c — 1 to} f _{c — 2} input to a driver (Driver).

  The modulation unit 103 includes four types of modulation signal generators (Mod Gen) 1 to 4 and band pass filters (BPF) 1 to 4. The modulation signal (carrier) generated by the modulation signal generator is input to the driver of each laser, and the carrier frequency used for demodulation is output to each demodulator constituting the synchronous detection unit 111 at the subsequent stage. The

  The measurement optical system 105 guides each excitation light emitted from the light source 101 to the measurement object S flowing in the flow path through an optical system composed of various lenses L, a dichroic mirror DM, and the like. To do. Here, as shown in FIG. 7, the excitation light emitted from each laser is guided to the measuring object S on the same axis and irradiated onto the measuring object S at the same time. The seven types of fluorescence generated from the measurement object S are guided coaxially to the multiband pass optical filter (MBPF) 107 by the measurement optical system 105.

  In FIG. 7, one aspheric lens is schematically illustrated as the lens L, but it goes without saying that the measurement optical system 105 may include a plurality of spherical lenses and aspheric lenses. .

As shown in FIG. 5A or FIG. 5B, the multiband pass optical filter (MBPF) 105 is composed of a plurality of single band reflection filters, and branches seven types of fluorescence into three optical paths. Here, as shown in FIG. 4, the fluorescence FL _A , FL _D , and FL _G are branched to a photomultiplier tube (PMT) 1 configured as the fluorescence detection unit 109, and the fluorescence FL _B and FL _E are is branched into PMT2 configured as a fluorescence detector 109, fluorescence _FL C, FL _F is split into PMT3 configured as a fluorescence detector 109.

The synchronous detection unit 111 includes a three-channel demodulator (DEM) 1 and two-channel demodulators DEM2 and DEM3. Fluorescence λ _A , λ _D , λ _G detected by PMT1 is converted into detection signal MS ₁ and input to demodulator DEM1, and fluorescence λ _B , λ _E detected by PMT2 is detected signal MS. ₂ is input to the demodulator DEM2, and the fluorescence λ _C and λ _F detected by the PMT ₃ is converted to a detection signal MS ₃ and input to the demodulator DEM3.

The demodulator 1, a carrier frequency _{f c_1,} f _{c_3,} and _{f c_4} is input, the detection signal MS _1, fluorescence _{FL A,} FL D, is FL _G are separated. Also, the demodulator 2, the carrier frequency _{f c_2,} and _{f c_4} is input, the detection signal MS _2, fluorescence _FL B, the FL _E is separated. Further, carrier frequencies f _{c — 3} and f _{c — 4} are input to the demodulator 3, and the fluorescence FL _C and FL _F are separated from the detection signal MS ₃ . With this configuration, the seven types of fluorescence excited simultaneously are separated from each other.

[Specific example of carrier frequency setting method]
Next, a carrier frequency setting method when a flow cytometer is realized as the fluorescence signal acquisition apparatus 1 will be specifically described.

  In the case of a flow cytometer, the bandwidth required for detection is determined from the minimum size of a measurement object (for example, a cell) to be detected and the conveyance speed of the measurement object. Hereinafter, a method for setting the carrier frequency will be described in detail by taking as an example a case where cells having a minimum size of 0.5 μm are transported in the flow path at a transport speed of 10 m / s.

  When determining the minimum frequency of the carrier, considering the sampling theorem, the carrier is irradiated with two or more wavelengths, preferably three or more wavelengths, while the size of 0.5 μm, which is the minimum size of the measuring object S, is being conveyed. Is required. Accordingly, it is preferable to use a carrier having a repetition period of 0.5 μm / 3 = 0.167 μm or less and having a repetition rate of 1 / 0.167 μm≈60 MHz.

  On the other hand, the frequency when a cell having a size of 0.5 μm is conveyed at 1 μm intervals is 10 MHz. However, when the size of the excitation light of the flow cytometer is 5 μm, for example, the size of 5 μm is 10 m / The time passing by at the speed of s is 0.5 μs. Therefore, from the sampling theorem, 4 MHz, which is twice 1 / 0.5 μs = 2 MHz, may be used as the occupied bandwidth. However, in order to prevent crosstalk more reliably, it is more preferable to set the frequency bandwidth to be three times or more of the frequency bandwidth (in the above case, 2 MHz, for example) determined from the conveyance speed and size.

  When the minimum frequency of the carrier is 60 MHz, if the occupied bandwidth is 10 MHz, the four carrier frequencies are 60, 70, 80, and 90 MHz at the minimum. At this time, the four selected carriers do not overlap with the second harmonic 120 MHz of the carrier of 60 MHz, and thus no crosstalk occurs.

Actually, it is better that many carrier wavelengths are included in the range of 0.5 μm. Therefore, it is preferable that the carrier frequency is as high as possible within the range allowed by the operation speed of the circuit. Therefore, for example, 100 MHz, 110 MHz, 120 MHz, and 130 MHz (Δf = 5 MHz) are preferably selected as the carrier frequencies f _{c — 1 to} f _{c — 4} .

  The carrier frequency setting method when a flow cytometer is realized as the fluorescence signal acquisition apparatus 1 has been specifically described above.

<Specific Example of Fluorescence Signal Acquisition Device-2: Laser Scanning Fluorescence Microscope>
Next, taking a laser scanning fluorescence microscope as a specific example of the fluorescence signal acquisition apparatus 1 according to the present embodiment, the fluorescence signal acquisition apparatus according to the present embodiment will be specifically described with reference to FIG. FIG. 8 is an explanatory diagram schematically showing a configuration of a laser scanning fluorescence microscope which is an example of a fluorescence signal acquisition apparatus according to the present embodiment.

  The fluorescence signal acquisition apparatus 1 according to this embodiment as described above can be realized as a laser scanning fluorescence microscope that scans a measurement object and observes fluorescence from the measurement object. In this case, the fluorescence signal acquisition apparatus 1 realized as a laser scanning fluorescence microscope uses various substances stained with various phosphors as a measurement object.

  In FIG. 8, the seven types of phosphors as shown in FIG. 4 are excited by four types of excitation light of 405 nm, 488 nm, 561 nm, and 643 nm, and a laser scanning fluorescence microscope for detecting seven types of fluorescence is shown. A configuration is schematically shown.

In this case, the light source 101 includes a laser (Laser) 1 that emits excitation light of 405 nm, a laser 2 that emits excitation light of 488 nm, a laser 3 of 561 nm, and a laser 4 that emits excitation light of 643 nm. Each of the lasers is intensity-modulated by carrier frequencies f _{c — 1 to} f _{c — 2} input to a driver (Driver).

  The modulation unit 103 includes four types of modulation signal generators (Mod Gen) 1 to 4 and band pass filters (BPF) 1 to 4. The modulation signal (carrier) generated by the modulation signal generator is input to the driver of each laser, and the carrier frequency used for demodulation is output to each demodulator constituting the synchronous detection unit 111 at the subsequent stage. The

  The measurement optical system 105 converts each excitation light emitted from the light source 101 into various lenses L, dichroic mirror DM, pinhole PH, XY-galvano mirror (XY-gal), objective lens (Obj), notch filter NF. The light is guided to the measuring object S through an optical system composed of the like. Here, as shown in FIG. 8, the excitation light emitted from each laser is guided to the measuring object S on the same axis and is irradiated onto the measuring object S at the same time. The seven types of fluorescence generated from the measurement object S are guided coaxially to the multiband pass optical filter (MBPF) 107 by the measurement optical system 105.

  In FIG. 8, one aspheric lens is schematically illustrated as the lens L, but it goes without saying that the measurement optical system 105 may include a plurality of spherical lenses and aspheric lenses. .

As shown in FIG. 5A or FIG. 5B, the multiband pass optical filter (MBPF) 105 is composed of a plurality of single band reflection filters, and branches seven types of fluorescence into three optical paths. Here, as shown in FIG. 4, the fluorescence FL _A , FL _D , and FL _G are branched to a photomultiplier tube (PMT) 1 configured as the fluorescence detection unit 109, and the fluorescence FL _B and FL _E are is branched into PMT2 configured as a fluorescence detector 109, fluorescence _FL C, FL _F is split into PMT3 configured as a fluorescence detector 109.

The synchronous detection unit 111 includes a three-channel demodulator (DEM) 1 and two-channel demodulators DEM2 and DEM3. Fluorescence λ _A , λ _D , λ _G detected by PMT1 is converted into detection signal MS ₁ and input to demodulator DEM1, and fluorescence λ _B , λ _E detected by PMT2 is detected signal MS. ₂ is input to the demodulator DEM2, and the fluorescence λ _C and λ _F detected by the PMT ₃ is converted to a detection signal MS ₃ and input to the demodulator DEM3.

The demodulator 1, a carrier frequency _{f c_1,} f _{c_3,} and _{f c_4} is input, the detection signal MS _1, fluorescence _{FL A,} FL D, is FL _G are separated. Also, the demodulator 2, the carrier frequency _{f c_2,} and _{f c_4} is input, the detection signal MS _2, fluorescence _FL B, the FL _E is separated. Further, carrier frequencies f _{c — 3} and f _{c — 4} are input to the demodulator 3, and the fluorescence FL _C and FL _F are separated from the detection signal MS ₃ . With this configuration, the seven types of fluorescence excited simultaneously are separated from each other.

[Specific example of carrier frequency setting method]
Next, a method for setting the carrier frequency when a laser scanning fluorescence microscope is realized as the fluorescence signal acquisition apparatus 1 will be specifically described.

  The smaller the resolution of the laser scanning fluorescent microscope, the better. However, the numerical value with the larger optical resolution or electrical resolution is the resolution of the actual laser scanning fluorescent microscope.

Optical resolution _{d o,} using the numerical aperture NA of the objective lens, and a wavelength lambda of the excitation light used for measurement, λ / (NA × 2) , or is estimated at 0.61 × λ / NA. Here, when the use of _{d o} = λ / 2 as NA = 1 for simplicity, the _d o = 250 nm at lambda = 500 nm.

On the other hand, the electrical resolution d _e, when there is no limitation in the band of the photoelectric conversion element such as PMT, are determined by the sampling interval (or pixel spacing). When the beam velocity is v [m / s] and the sampling frequency is f _s [1 / s], the sampling interval is v / f _s [m]. According to the sampling theorem, a resolution twice as high as the sampling interval can be obtained. This is a case where an ideal low-pass filter (LPF) is used. as it comes to estimate _is calculated by _{d e = 3 × (v /} f s).

  The beam velocity v [m / s] can be calculated from the round-trip frequency of the scanning beam and the scanning range. As a representative number, the beam velocity is calculated using a round-trip frequency of 500 Hz and a scanning range of 1 mm. Since the 1 mm range is scanned at 1 ms, which is half the period of 2 ms, the beam velocity is v = 1 [m / s].

In order to match the electrical resolution d _e with the optical resolution d _o , the sampling frequency is f _s = 3 × (v / d _e ) = 3 × (v / d _o ) = 3 × (1 [m / s ] / 250 × 10 ⁻⁹ [m]) = 3 × ^4/1000 × 10 ⁻⁹ [1 / s] = 12 × 10 ⁶ [1 / s] = 12 [MHz], and the frequency band is 1 [m / s] / 250 × 10 ⁻⁹ [m] = ^4/1000 × 10 ⁻⁹ = 4 × 10 ⁻⁶ [1 / s] = 4 [MHz].

  Note that the minimum carrier frequency is preferably set to three or more times the frequency bandwidth, as described above. Accordingly, the minimum carrier frequency is preferably set to 12 MHz or more, which is three times 4 MHz.

  On the other hand, when a plurality of carriers are used, it is preferable that the second harmonic of one carrier does not overlap with another carrier and its sidebands as described above. If the minimum carrier frequency is 12 MHz, and the occupied bandwidth is 8 MHz, which is twice 4 MHz, the sidebands of the four carriers do not overlap. A frequency of 28, 36 MHz can be selected. However, since the second harmonic of the 12 MHz carrier is 24 MHz, it overlaps with the sidebands of the 20 MHz and 28 MHz carriers and causes crosstalk. Therefore, the combinations with the lowest frequency satisfying the condition that the occupied bandwidth is 4 MHz, that is, the interval of at least 8 MHz and the second harmonics do not overlap are 32, 40, 48, and 56 MHz.

  The carrier frequency setting method when a laser scanning fluorescence microscope is realized as the fluorescence signal acquisition apparatus 1 has been specifically described above.

<About fluorescence signal acquisition method>
Next, the flow of the fluorescence signal acquisition method performed by the fluorescence signal acquisition device 1 according to the present embodiment will be briefly described with reference to FIG. FIG. 9 is a flowchart showing an example of the flow of the fluorescence signal acquisition method according to the present embodiment.

  In the fluorescence signal acquisition method according to the present embodiment, first, a plurality of excitation lights intensity-modulated by a carrier having a predetermined frequency are measured via the measurement optical system 105 with respect to the measurement object S including a plurality of phosphors. (Step S101).

  The plurality of fluorescences generated in response to the plurality of excitation lights are branched into a plurality of optical paths by the multiband pass optical filter 107 (step S103).

  Thereafter, the fluorescence branched into the plurality of optical paths is detected by the plurality of fluorescence detection units 109 (step S105), and the fluorescence corresponding to the plurality of phosphors is separated by synchronously detecting the detected detection signals. (Step S107).

  The example of the flow of the fluorescence signal acquisition method according to the present embodiment has been briefly described above with reference to FIG.

(Summary)
As described above, in the fluorescence signal acquisition device and the fluorescence signal acquisition method according to the embodiment of the present disclosure, even if a plurality of phosphors are excited simultaneously according to one excitation light, the generated fluorescence is reduced. It becomes possible to separate more easily. In addition, it is possible to reduce the number of fluorescence detection units used for fluorescence detection. Moreover, since simultaneous irradiation with excitation light is possible, fluorescence from the measurement object can be acquired at higher speed.

  The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the technical scope of the present disclosure is not limited to such examples. It is obvious that a person having ordinary knowledge in the technical field of the present disclosure can come up with various changes or modifications within the scope of the technical idea described in the claims. Of course, it is understood that it belongs to the technical scope of the present disclosure.

  Further, the effects described in the present specification are merely illustrative or exemplary and are not limited. That is, the technology according to the present disclosure can exhibit other effects that are apparent to those skilled in the art from the description of the present specification in addition to or instead of the above effects.

The following configurations also belong to the technical scope of the present disclosure.
(1)
A plurality of light sources for irradiating a measurement object including a plurality of phosphors with a plurality of excitation lights modulated by carriers having different frequencies;
A plurality of fluorescence detectors for detecting a plurality of fluorescence generated in response to the plurality of excitation lights;
A multi-band pass optical filter that is positioned in front of the plurality of fluorescence detection units and branches the plurality of fluorescence into a plurality of optical paths, and transmits fluorescence from a plurality of phosphors having non-continuous wavelengths;
Synchronous detection of the detection signals detected by each of the fluorescence detection units, to separate the fluorescence corresponding to the plurality of phosphors, and a synchronous detection unit,
A fluorescence signal acquisition device comprising:
(2)
The fluorescence signal acquisition device according to (1), wherein the number of the fluorescence detection units is smaller than the number of the plurality of phosphors and is equal to the maximum number of the phosphors excited by one excitation light.
(3)
The multiband pass optical filter is configured such that a plurality of single band reflection filters that reflect light in a predetermined wavelength band and transmit light in other wavelength bands are arranged in series, (1) or The fluorescence signal acquisition device according to (2).
(4)
The fundamental wave of each of the carriers is separated by a frequency equal to or greater than the sideband bandwidth,
The frequency of each of the carriers is selected so that the third harmonic and the sideband in one carrier do not overlap with the fundamental and sidebands in another carrier, any one of (1) to (3) The fluorescence signal acquisition device according to 1.
(5)
The frequency of each of the carriers is selected so that the second harmonic and the sideband in one carrier do not overlap with the fundamental and the sideband in another carrier, any one of (1) to (4) The fluorescence signal acquisition device described in 1.
(6)
The synchronous detector is
A bandpass filter to which a detection signal generated by the fluorescence detection unit is input;
A multiplier to which the detection signal transmitted through the band-pass filter and the carrier are input;
A low-pass filter to which the signal generated by the multiplier is input;
The fluorescent signal acquisition device according to any one of (1) to (5), comprising:
(7)
The fluorescence signal according to any one of (1) to (6), wherein the fluorescence signal acquisition device is a flow cytometer that detects fluorescence from the measurement object being conveyed in a predetermined flow path. Acquisition device.
(8)
The fluorescence signal acquisition device according to (7), wherein the frequency of the carrier is at least three times a frequency bandwidth determined from a conveyance speed of the measurement object and a size of the measurement object.
(9)
The fluorescence signal acquisition device according to any one of (1) to (6), wherein the fluorescence signal acquisition device is a laser scanning fluorescence microscope that scans the measurement object and observes fluorescence from the measurement object. apparatus.
(10)
The frequency of the carrier is at least three times the frequency bandwidth determined from the combination of the optical resolution calculated from the wavelength of the excitation light and the numerical aperture of the objective lens, and the scanning speed of the excitation light. 9) The fluorescence signal acquisition device according to item 9).
(11)
The light source is a laser light source that emits laser light of a predetermined wavelength,
The fluorescence signal acquisition device according to any one of (1) to (10), wherein the fluorescence detection unit is a photomultiplier tube.
(12)
Irradiating a measurement object including a plurality of phosphors with a plurality of excitation lights modulated with carriers having different frequencies;
Branching a plurality of fluorescence generated in response to the plurality of excitation lights into a plurality of optical paths by a multiband pass optical filter that transmits fluorescence from a plurality of phosphors whose wavelengths are not continuous;
Detecting the fluorescence branched into a plurality of optical paths by a plurality of fluorescence detection units;
Synchronously detecting detection signals detected by the respective fluorescence detection units, and separating fluorescence corresponding to the plurality of phosphors;
A fluorescence signal acquisition method comprising:

DESCRIPTION OF SYMBOLS 1 Fluorescence signal acquisition apparatus 101 Light source 103 Modulation part 105 Measurement optical system 107 Multiband pass optical filter 109 Fluorescence detection part 111 Synchronous detection part 121 Single band reflection filter 123, 133 Band pass filter 131 Demodulator 135 Multiplier 137 Low pass filter

Claims (12)

A plurality of light sources for irradiating a measurement object including a plurality of phosphors with a plurality of excitation lights modulated by carriers having different frequencies;
A plurality of fluorescence detectors for detecting a plurality of fluorescence generated in response to the plurality of excitation lights;
A multi-band pass optical filter that is positioned in front of the plurality of fluorescence detection units and branches the plurality of fluorescence into a plurality of optical paths, and transmits fluorescence from a plurality of phosphors having non-continuous wavelengths;
Synchronous detection of the detection signals detected by each of the fluorescence detection units, to separate the fluorescence corresponding to the plurality of phosphors, and a synchronous detection unit,
A fluorescence signal acquisition device comprising:   2. The fluorescence signal acquisition apparatus according to claim 1, wherein the number of the fluorescence detection units is smaller than the number of the plurality of phosphors and is equal to the maximum number of the phosphors excited by one excitation light.   2. The multiband pass optical filter according to claim 1, wherein a plurality of single band reflection filters that reflect light in a predetermined wavelength band and transmit light in other wavelength bands are arranged in series. The fluorescent signal acquisition apparatus described. The fundamental wave of each of the carriers is separated by a frequency equal to or greater than the sideband bandwidth,
The fluorescence signal acquisition device according to claim 1, wherein the frequency of each of the carriers is selected so that a third harmonic and a sideband in one carrier do not overlap with a fundamental and a sideband in another carrier.   The frequency of each said carrier is a fluorescence signal acquisition apparatus of Claim 4 selected so that the secondary harmonic and sideband wave in a certain carrier may not overlap with the fundamental wave and sideband wave in another carrier. The synchronous detector is
A bandpass filter to which a detection signal generated by the fluorescence detection unit is input;
A multiplier to which the detection signal transmitted through the band-pass filter and the carrier are input;
A low-pass filter to which the signal generated by the multiplier is input;
The fluorescence signal acquisition apparatus according to claim 1, comprising:   The fluorescence signal acquisition device according to claim 1, wherein the fluorescence signal acquisition device is a flow cytometer that detects fluorescence from the measurement object being conveyed in a predetermined flow path.   The fluorescence signal acquisition apparatus according to claim 7, wherein the frequency of the carrier is at least three times a frequency bandwidth determined from a conveyance speed of the measurement object and a size of the measurement object.   The fluorescence signal acquisition apparatus according to claim 1, wherein the fluorescence signal acquisition apparatus is a laser scanning fluorescence microscope that scans the measurement object and observes fluorescence from the measurement object.   The frequency of the carrier is at least three times the frequency bandwidth determined from the combination of the optical resolution calculated from the wavelength of the excitation light and the numerical aperture of the objective lens, and the scanning speed of the excitation light. Item 10. The fluorescence signal acquisition device according to Item 9. The light source is a laser light source that emits laser light of a predetermined wavelength,
The fluorescence signal acquisition apparatus according to claim 1, wherein the fluorescence detection unit is a photomultiplier tube. Irradiating a measurement object including a plurality of phosphors with a plurality of excitation lights modulated with carriers having different frequencies;
Branching a plurality of fluorescence generated in response to the plurality of excitation lights into a plurality of optical paths by a multiband pass optical filter that transmits fluorescence from a plurality of phosphors whose wavelengths are not continuous;
Detecting the fluorescence branched into a plurality of optical paths by a plurality of fluorescence detection units;
Synchronously detecting detection signals detected by the respective fluorescence detection units, and separating fluorescence corresponding to the plurality of phosphors;
A fluorescence signal acquisition method comprising:

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