JP6726859B2 - Fluorescence detector and control method thereof - Google Patents

Description

The present invention relates to a fluorescence detector that detects fluorescence emitted from a sample by irradiating the sample with excitation light and a control method thereof.

In the field of biotechnology, a fluorescence detection method for detecting biomolecules such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or protein present in a sample is known. In this fluorescence detection method, a fluorescence detector that detects fluorescence generated from a sample by irradiating a sample labeled with a fluorescent molecule with excitation light to excite the sample is used.

As the above-mentioned fluorescence detector, a so-called multi-color type fluorescence detector that collectively detects a plurality of types of biomolecules labeled with a plurality of types of fluorescent molecules having different center wavelengths is known (for example, patents References 1 and 2). This type of fluorescence detector includes a plurality of light emitting elements that emit excitation lights having mutually different center wavelengths.

In this type of fluorescence detector, the optimum central wavelength of excitation light to be irradiated on the sample differs depending on the type of fluorescent molecule labeled on the sample. Therefore, by causing the plurality of light emitting elements to sequentially emit light, the plurality of excitation lights having different center wavelengths are sequentially irradiated to the sample, and a plurality of types of biomolecules can be collectively detected. At this time, the light emission of the light emitting elements other than the light emitting element that is emitting light among the plurality of light emitting elements is completely turned off.

International Publication No. 2004/063731 International Publication No. 2013/191135

In recent years, it has been demanded to shorten the detection time by the fluorescence detector. However, in the above-described conventional fluorescence detector, when the light-emitting element is made to emit light, it takes time for the output of the light-emitting element to stabilize, so there is a problem that it is difficult to switch the light emission of a plurality of light-emitting elements at high speed. Occurs.

Therefore, the present invention provides a fluorescence detector capable of switching the light emission of a plurality of light emitting elements at high speed and a manufacturing method thereof.

A method for controlling a fluorescence detector according to one aspect of the present invention is a method for controlling a fluorescence detector that detects fluorescence generated from the sample by irradiating the sample with excitation light, the first light-emitting element and Emitting a first pumping light and a second pumping light having different center wavelengths from the second light-emitting element in the order of a first rated output and a second rated output, respectively; Irradiating light and the second excitation light in sequence to sequentially receive the first fluorescent light and the second fluorescent light having different central wavelengths generated from the sample by a light receiving element to generate a light reception signal, In the step of emitting, when the first excitation light is emitted from the first light-emitting element at the first rated output, the second light-emitting element includes the second rated output. When the second excitation light is emitted at a lower output than the second emission light and the second excitation light is emitted from the second light emission element at the second rated output, the first light emission element is The first excitation light is emitted at an output lower than the first rated output.

Note that these comprehensive or specific aspects may be realized by a recording medium such as a system, a method, an integrated circuit, a computer program, or a computer-readable CD-ROM, and the system, the method, the integrated circuit, the computer program. And may be realized by any combination of recording media.

In the method for controlling the fluorescence detector according to one aspect of the present invention, the light emission of the plurality of light emitting elements can be switched at high speed.

FIG. 3 is a diagram showing an appearance of a fluorescence detector according to the first embodiment. FIG. 3 is a diagram schematically showing the configuration of the fluorescence detector according to the first embodiment. FIG. 3 is a diagram showing transmission characteristics of the excitation filter according to the first embodiment. FIG. 6 is a diagram showing transmission characteristics of the dichroic mirror according to the first embodiment. FIG. 3 is a diagram showing transmission characteristics of the absorption filter according to the first embodiment. 3 is a flowchart showing the operation of the fluorescence detector according to the first embodiment. It is a figure which shows the light emission sequence of each light emitting element of the fluorescence detector which concerns on an Example. It is a figure which shows the light emission condition of each light emitting element of the fluorescence detector which concerns on an Example. It is a figure which shows the transmission characteristic of the dichroic mirror of the fluorescence detector which concerns on an Example. It is a figure which shows the light emission sequence of each light emitting element of the fluorescence detector which concerns on a comparative example. It is a figure which shows the detection result of the fluorescence signal by the fluorescence detector which concerns on an Example. It is a figure which shows the crosstalk ratio of the fluorescence signal of FIG. It is a figure which shows the detection result of the fluorescence intensity by the fluorescence detector which concerns on an Example and a comparative example. FIG. 6 is a diagram schematically showing a configuration of a fluorescence detector according to a second embodiment. FIG. 6 is a plan view showing a light receiving element of the fluorescence detector according to the second embodiment.

A method for controlling a fluorescence detector according to one aspect of the present invention is a method for controlling a fluorescence detector that detects fluorescence generated from the sample by irradiating the sample with excitation light, the first light-emitting element and Emitting a first pumping light and a second pumping light having different center wavelengths from the second light-emitting element in the order of a first rated output and a second rated output, respectively; Irradiating light and the second excitation light in sequence to sequentially receive the first fluorescent light and the second fluorescent light having different central wavelengths generated from the sample by a light receiving element to generate a light reception signal, In the step of emitting, when the first excitation light is emitted from the first light-emitting element at the first rated output, the second light-emitting element includes the second rated output. When the second excitation light is emitted at a lower output than the second emission light and the second excitation light is emitted from the second light emission element at the second rated output, the first light emission element is The first excitation light is emitted at an output lower than the first rated output.

According to this aspect, for example, when the first light-emitting element emits the first excitation light at the first rated output, the second light-emitting element outputs the first excitation light at a lower output than the second rated output. It emits 2 excitation lights. Thereby, when the second light emitting element emits the second excitation light at the second rated output in the next sequence, the case where the light emission of the second light emitting element is completely turned off in the immediately preceding sequence In comparison, the output of the second light emitting element can be stabilized in a short time. As a result, light emission of the first light emitting element and light emission of the second light emitting element can be switched at high speed.

For example, in the step of emitting, when the first excitation light is emitted from the first light emitting element at the first rated output, the second light emitting element outputs the second rated output. When the second excitation light is emitted with an output of 28% or less and the second excitation light is emitted from the second light emitting element with the second rated output, the first light emitting element is The first excitation light may be emitted at an output that is 28% or less of the first rated output.

For example, the method for controlling the fluorescence detector may further include, when the first excitation light is emitted from the first light emitting element at the first rated output, the second light emission signal included in the second light reception signal. When the signal component corresponding to the fluorescence is removed by calculation and the second excitation light is emitted from the second light emitting element at the second rated output, the first fluorescence of the received light signal is detected. It may be configured to include a step of removing a signal component corresponding to the calculation.

According to this aspect, it is possible to generate a more reliable light reception signal.

Further, the fluorescence detector according to one aspect of the present invention is a fluorescence detector that detects fluorescence generated from the sample by irradiating the sample with excitation light, and the first fluorescence emitting the first excitation light. A light-emitting element, a second light-emitting element that emits second excitation light having a central wavelength different from that of the first excitation light, and when the first light-emitting element emits light, By irradiating the sample with the first excitation light, the first fluorescence generated from the sample is received, and when the second light-emitting element emits light, the second light-emitting element emits light. By irradiating the sample with the second excitation light, a light-receiving element that receives second fluorescence having a center wavelength different from that of the first fluorescence generated from the sample, the first light-emitting element, and the The first light-emitting element and the second light-emission so that the first excitation light and the second excitation light are sequentially emitted from the second light-emitting element at the first rated output and the second rated output, respectively. A control unit for controlling an element, wherein the control unit emits the first excitation light at the first rated output from the first light emitting element, and the second light emitting element. Is emitting the second excitation light at an output lower than the second rated output, and when emitting the second excitation light at the second rated output from the second light emitting element, The first light emitting element controls the first light emitting element and the second light emitting element so as to emit the first excitation light with an output lower than the first rated output.

According to this aspect, when the first light emitting element emits the first excitation light at the first rated output, the second light emitting element outputs the second excitation light at an output lower than the second rated output. Emitting excitation light. Thereby, when the second light emitting element emits the second excitation light at the second rated output in the next sequence, the case where the light emission of the second light emitting element is completely turned off in the immediately preceding sequence In comparison, the output of the second light emitting element can be stabilized in a short time. As a result, light emission of the first light emitting element and light emission of the second light emitting element can be switched at high speed.

Note that these comprehensive or specific aspects may be realized by a recording medium such as a system, a method, an integrated circuit, a computer program, or a computer-readable CD-ROM, and the system, the method, the integrated circuit, the computer program. Alternatively, it may be realized by any combination of recording media.

Hereinafter, embodiments will be specifically described with reference to the drawings.

It should be noted that each of the embodiments described below shows a comprehensive or specific example. Numerical values, shapes, materials, constituent elements, arrangement positions and connection forms of constituent elements, steps, order of steps, and the like shown in the following embodiments are examples, and are not intended to limit the present invention. Further, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claim indicating the highest concept are described as arbitrary constituent elements.

(Embodiment 1)
[1-1. Configuration of fluorescence detector]
First, the configuration of the fluorescence detector 2 according to the first embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is a diagram showing an appearance of the fluorescence detector 2 according to the first embodiment. 1A is a diagram showing the front side of the fluorescence detector 2, and FIG. 1B is a sectional view taken along the line I-I of FIG. 1A. FIG. 2 is a diagram schematically showing the configuration of the fluorescence detector 2 according to the first embodiment.

As shown in FIG. 1, the fluorescence detector 2 is a so-called multi-color type fluorescence detector that detects the fluorescence generated from the sample 4 by irradiating the sample 4 with excitation light to excite it. The fluorescence detector 2 includes a housing 6 and an optical system 8 arranged inside the housing 6.

The sample 4 is a sample to be detected by the fluorescence detector 2, and is, for example, a micro TAS (Total Analysis Systems) chip. The sample 4 contains a plurality of types of biomolecules labeled with a plurality of types of fluorescent molecules having different central wavelengths. Specifically, the sample 4 is a) a first fluorescent molecule (for example, a central wavelength of 455 nm) that is excited by being irradiated with a first excitation light (described later) to generate a first fluorescence (for example, a central wavelength of 455 nm). (Pacific Blue) labeled biomolecule, and b) a second fluorescent molecule that is excited by being irradiated with a second excitation light (described later) to generate a second fluorescence (for example, a central wavelength of 512 nm) ( For example, a biomolecule labeled with BODIPY-FL) and c) a third fluorescence that is excited by being irradiated with a third excitation light (described later) to generate a third fluorescence (for example, a central wavelength of 580 nm). A biomolecule labeled with a molecule (for example, TAMRA), and d) fourth fluorescence that is excited by irradiation with fourth excitation light (described later) to generate fourth fluorescence (for example, center wavelength 680 nm). A biomolecule labeled with a molecule (for example, ATTO665) is included.

As shown in FIG. 2, the optical system 8 of the fluorescence detector 2 includes a first light emitting element 10, a second light emitting element 12, a third light emitting element 14, a fourth light emitting element 16, and a fourth light emitting element 16. The first lens 18, the second lens 20, the third lens 22, the fourth lens 24, the first dichroic mirror 26, the second dichroic mirror 28, and the third dichroic mirror 30. , A fifth lens 32, an excitation filter 34, a dichroic mirror 36, a sixth lens 38, an absorption filter 40, a seventh lens 42, and a light receiving element 44.

The first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 are laser diodes that emit monochromatic laser light having different central wavelengths as excitation light. Specifically, the first light emitting element 10 emits violet laser light having a first central wavelength λ1 (for example, 405 nm) as first excitation light. The second light emitting element 12 emits blue laser light having a second center wavelength λ2 (for example, 470 nm) as second excitation light. The third light emitting element 14 emits green laser light having a third center wavelength λ3 (for example, 528 nm) as third excitation light. The fourth light emitting element 16 emits red laser light having a fourth central wavelength λ4 (for example, 632 nm) as fourth excitation light.

The first lens 18, the second lens 20, the third lens 22 and the fourth lens 24 are respectively the first light emitting element 10, the second light emitting element 12, the third light emitting element 14 and the fourth light emitting element 10. It is a collimating lens that converts laser light from the light emitting element 16 from divergent light to parallel light.

Each of the 1st dichroic mirror 26, the 2nd dichroic mirror 28, and the 3rd dichroic mirror 30 is an optical element which reflects only the light of a specific center wavelength, and transmits the light of other center wavelengths. Specifically, the first dichroic mirror 26 reflects only a green laser beam and transmits a laser beam of a color other than green. The second dichroic mirror 28 reflects only blue laser light and transmits laser light of colors other than blue. The third dichroic mirror 30 reflects only the purple laser light and transmits the laser light of a color other than purple.

The red laser light (fourth excitation light) from the fourth light emitting element 16 passes through each of the first dichroic mirror 26, the second dichroic mirror 28, and the third dichroic mirror 30. The green laser light (third excitation light) from the third light emitting element 14 is reflected by the first dichroic mirror 26, and then passes through the second dichroic mirror 28 and the third dichroic mirror 30. The blue laser light (second excitation light) from the second light emitting element 12 is reflected by the second dichroic mirror 28 and then passes through the third dichroic mirror 30. The purple laser light (first excitation light) from the first light emitting element 10 is reflected by the third dichroic mirror 30.

The fifth lens 32 is a condenser lens for condensing the laser light emitted from the third dichroic mirror 30.

The excitation filter 34 is a so-called multi-bandpass type filter that transmits only light in a plurality of wavelength bands. The excitation filter 34 is arranged between the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, the fourth light emitting element 16 and the dichroic mirror 36. FIG. 3 is a diagram showing the transmission characteristics of the excitation filter 34 according to the first embodiment. 3A is a graph showing the transmission characteristics of the excitation filter 34, and FIG. 3B is a graph showing the transmission characteristics of the first excitation light, the second excitation light, and the third excitation light, which are limited by the excitation filter 34. 4C is a graph showing each wavelength band of the excitation light and the fourth excitation light, and FIG. 3C is a graph showing the absorption/fluorescence intensity in the sample 4.

In the example shown in FIG. 3, the excitation filter 34 is a laser of the first wavelength band B1, the second wavelength band B2, the third wavelength band B3, and the fourth wavelength band B4 (B1<B2<B3<B4). Only light is transmitted. Thereby, the excitation filter 34 limits the wavelength band of the first excitation light from the first light emitting element 10 to the first wavelength band B1. Moreover, the excitation filter 34 limits the wavelength band of the second excitation light from the second light emitting element 12 to the second wavelength band B2. Moreover, the excitation filter 34 limits the wavelength band of the third excitation light from the third light emitting element 14 to the third wavelength band B3. Further, the excitation filter 34 limits the wavelength band of the fourth excitation light from the fourth light emitting element 16 to the fourth wavelength band B4.

The dichroic mirror 36 is a so-called multi-band pass type dichroic mirror that reflects only light having a plurality of central wavelengths and transmits light having other central wavelengths. FIG. 4 is a diagram showing the transmission characteristics of the dichroic mirror 36 according to the first embodiment. 4A is a graph showing the transmission characteristics of the dichroic mirror 36, and FIG. 4B is a graph showing the absorption/fluorescence intensity of the sample 4.

In the example shown in FIG. 4, the dichroic mirror 36 has a laser light having a first center wavelength λ1 (first pump light), a laser light having a second center wavelength λ2 (second pump light), and a third center. Only the laser light having the wavelength λ3 (third excitation light) and the laser light having the fourth central wavelength λ4 (fourth excitation light) are reflected, and the laser light having the other central wavelengths and the fluorescent light (for example, the first fluorescent light) , Second fluorescence, third fluorescence and fourth fluorescence).

The sixth lens 38 is a condenser lens for condensing the laser light reflected by the dichroic mirror 36 and the fluorescence generated from the sample 4.

The absorption filter 40 is a so-called multi-bandpass type filter that transmits only light in a plurality of wavelength bands. The absorption filter 40 is arranged between the dichroic mirror 36 and the light receiving element 44. FIG. 5 is a diagram showing transmission characteristics of the absorption filter 40 according to the first embodiment. 5A is a graph showing the transmission characteristics of the absorption filter 40, and FIG. 5B is a graph showing the first fluorescence, the second fluorescence, and the third fluorescence restricted by the absorption filter 40. 5 is a graph showing each wavelength band of the fourth fluorescence, and FIG. 5C is a graph showing absorption/fluorescence intensity in the sample 4.

In the example shown in FIG. 5, the absorption filter 40 uses the fluorescence of the fifth wavelength band B5, the sixth wavelength band B6, the seventh wavelength band B7, and the eighth wavelength band B8 (B5<B6<B7<B8). Only transparent. Thereby, the absorption filter 40 limits the wavelength band of the first fluorescence generated from the sample 4 to the fifth wavelength band B5. Further, the absorption filter 40 limits the wavelength band of the second fluorescence generated from the sample 4 to the sixth wavelength band B6. Further, the absorption filter 40 limits the wavelength band of the third fluorescence generated from the sample 4 to the seventh wavelength band B7. Further, the absorption filter 40 limits the wavelength band of the fourth fluorescence generated from the sample 4 to the eighth wavelength band B8.

The seventh lens 42 is a condenser lens for condensing the fluorescence that has passed through the absorption filter 40.

The light receiving element 44 receives the fluorescence (the first fluorescence, the second fluorescence, the third fluorescence, and the fourth fluorescence) from the seventh lens 42, thereby receiving a light reception signal (fluorescence signal) indicating the fluorescence intensity. To generate.

As shown in FIG. 1, the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, the fourth light emitting element 16, the dichroic mirror 36, and the light receiving element 44 are the dichroic mirror 36. The reflected excitation light (the first excitation light, the second excitation light, the third excitation light, and the fourth excitation light) and the fluorescence (first fluorescence, second fluorescence, The third fluorescence and the fourth fluorescence are arranged on the same plane (YZ plane).

As shown in FIG. 2, the fluorescence detector 2 further includes a control unit 46. The control unit 46 causes the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 to have a predetermined rated output (for example, a light amount of 100%) (first rated output). And an example of the second rated output) and repeatedly emit light in this sequence.

At this time, the control unit 46 causes the second light emitting element 12, the third light emitting element 14 other than the first light emitting element 10 and the third light emitting element 14 when the first light emitting element 10 is emitting light at a predetermined rated output. The fourth light emitting element 16 is caused to emit light with an output lower than a predetermined rated output, specifically, an output that is equal to or more than a light emission threshold value (e.g., less than 1% of light amount) and 28% (e.g., 28% of light amount) of a predetermined rated output. .. In addition, the controller 46 causes the first light emitting element 10, the third light emitting element 14, and the third light emitting element 14 other than the second light emitting element 12 when the second light emitting element 12 emits light at a predetermined rated output. The light emitting element 16 of No. 4 emits light with an output of not less than the light emission threshold and not more than 28% of the predetermined rated output.

Further, the control unit 46 causes the first light emitting element 10, the second light emitting element 12, and the second light emitting element 12 other than the third light emitting element 14 when the third light emitting element 14 emits light at a predetermined rated output. The light emitting element 16 of No. 4 emits light with an output of not less than the light emission threshold and not more than 28% of the predetermined rated output. Further, the control unit 46 causes the first light emitting element 10, the second light emitting element 12, and the second light emitting element 12 other than the fourth light emitting element 16 when the fourth light emitting element 16 is emitting light at a predetermined rated output. The light-emitting element 14 of No. 3 is caused to emit light with an output of not less than the light-emission threshold and not more than 28% of the predetermined rated output.

Further, the control unit 46 receives the light receiving signal generated by the light receiving element 44. At this time, the control unit 46 detects the signal component corresponding to the first fluorescence in the received light signal when the first light emitting element 10 is emitting light at a predetermined rated output, and Of these, the signal components corresponding to the second fluorescence, the third fluorescence, and the fourth fluorescence are removed by calculation. In addition, the control unit 46 detects the signal component corresponding to the second fluorescence in the received light signal while causing the second light emitting element 12 to emit light at a predetermined rated output, and The signal components corresponding to the first fluorescence, the third fluorescence and the fourth fluorescence are removed by calculation.

In addition, the control unit 46 detects the signal component corresponding to the third fluorescence in the received light signal while causing the third light emitting element 14 to emit light at a predetermined rated output, and The signal components corresponding to the first fluorescence, the second fluorescence, and the fourth fluorescence are removed by calculation. In addition, the control unit 46 detects the signal component corresponding to the fourth fluorescence in the received light signal while causing the fourth light emitting element 16 to emit light at a predetermined rated output, and The signal components corresponding to the first fluorescence, the second fluorescence, and the third fluorescence are removed by calculation.

[1-2. Operation of fluorescence detector]
Next, the operation of the fluorescence detector 2 (control method of the fluorescence detector 2) will be described with reference to FIGS. 2 and 6. FIG. 6 is a flowchart showing the operation of the fluorescence detector 2 according to the first embodiment.

As shown in FIG. 6, first, the first sequence (S1 to S3) is executed. The control unit 46 causes the first light emitting element 10 to emit light with a predetermined rated output (S1). At this time, the control unit 46 causes the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 other than the first light emitting element 10 to have a light emission threshold value or more and 28% or less of a predetermined rated output. Make the output emit light.

As shown in FIG. 2, the first excitation light from the first light emitting element 10 is reflected by the third dichroic mirror 30, and then passes through the fifth lens 32 to enter the excitation filter 34. At this time, the excitation filter 34 limits the wavelength band of the first excitation light to the first wavelength band B1 (see FIG. 3). The first excitation light that has passed through the excitation filter 34 is reflected by the dichroic mirror 36, then passes through the sixth lens 38, and is applied to the sample 4.

When the first fluorescent molecule contained in the sample 4 is excited by the first excitation light, the sample 4 emits the first fluorescence. The first fluorescence emitted from the sample 4 passes through the sixth lens 38 and the dichroic mirror 36, and then enters the absorption filter 40. At this time, the absorption filter 40 limits the wavelength band of the first fluorescence to the fifth wavelength band B5 (see FIG. 5). The first fluorescence that has passed through the absorption filter 40 passes through the seventh lens 42 and is received by the light receiving element 44 (S2). As a result, the light receiving element 44 generates a light reception signal based on the received first fluorescence.

As described above, since the second light emitting element 12, the third light emitting element 14 and the fourth light emitting element 16 other than the first light emitting element 10 emit light with a relatively low light amount, In addition to the signal component corresponding to the first fluorescence, the signal components corresponding to the second fluorescence, the third fluorescence, and the fourth fluorescence are slightly included. Therefore, the control unit 46 removes the signal components corresponding to the second fluorescence, the third fluorescence, and the fourth fluorescence in the light reception signal by calculation, and thereby the signal corresponding to the first fluorescence in the light reception signal. Only the component can be detected.

After that, the control unit 46 lowers the output of the first light emitting element 10 from the predetermined rated output (S3), and causes the first light emitting element 10 to emit light with an output that is equal to or higher than the light emission threshold and equal to or lower than 28% of the predetermined rated output.

Next, the second sequence (S4 to S6) is executed. The control unit 46 causes the second light emitting element 12 to emit light with a predetermined rated output (S4). At this time, the control unit 46 causes the first light emitting element 10, the third light emitting element 14, and the fourth light emitting element 16 other than the second light emitting element 12 to have a light emission threshold value or more and 28% or less of a predetermined rated output. Make the output emit light.

As shown in FIG. 2, the second excitation light from the second light emitting element 12 is reflected by the second dichroic mirror 28, and then transmitted through the third dichroic mirror 30 and the fifth lens 32 to be excited. It is incident on the filter 34. At this time, the excitation filter 34 limits the wavelength band of the second excitation light to the second wavelength band B2 (see FIG. 3). The second excitation light that has passed through the excitation filter 34 is reflected by the dichroic mirror 36, then passes through the sixth lens 38, and is applied to the sample 4.

When the second fluorescent molecule contained in the sample 4 is excited by the second excitation light, the sample 4 emits the second fluorescent light. The second fluorescence emitted from the sample 4 passes through the sixth lens 38 and the dichroic mirror 36, and then enters the absorption filter 40. At this time, the absorption filter 40 limits the wavelength band of the second fluorescence to the sixth wavelength band B6 (see FIG. 5). The second fluorescence that has passed through the absorption filter 40 passes through the seventh lens 42 and is received by the light receiving element 44 (S5). As a result, the light receiving element 44 generates a light reception signal based on the received second fluorescence.

As described above, since the first light emitting element 10, the third light emitting element 14 and the fourth light emitting element 16 other than the second light emitting element 12 emit light with a relatively low light amount, In addition to the signal component corresponding to the second fluorescence, the signal components corresponding to the first fluorescence, the third fluorescence, and the fourth fluorescence are slightly included. Therefore, the control unit 46 removes the signal components corresponding to the first fluorescence, the third fluorescence, and the fourth fluorescence in the light reception signal by calculation, and thereby the signal corresponding to the second fluorescence in the light reception signal. Only the component can be detected.

After that, the control unit 46 lowers the output of the second light emitting element 12 from the predetermined rated output (S6), and causes the second light emitting element 12 to emit light with an output that is equal to or higher than the light emission threshold and equal to or lower than 28% of the predetermined rated output.

Next, the third sequence (S7 to S9) is executed. The control unit 46 causes the third light emitting element 14 to emit light with a predetermined rated output (S7). At this time, the control unit 46 causes the first light emitting element 10, the second light emitting element 12, and the fourth light emitting element 16 other than the third light emitting element 14 to have a light emission threshold value or more and 28% or less of a predetermined rated output. Make the output emit light.

As shown in FIG. 2, the third excitation light from the third light-emitting element 14 is reflected by the first dichroic mirror 26, and then is reflected by the second dichroic mirror 28, the third dichroic mirror 30, and the fifth dichroic mirror 30. The light passes through the lens 32 and enters the excitation filter 34. At this time, the excitation filter 34 limits the wavelength band of the third excitation light to the third wavelength band B3 (see FIG. 3). The third excitation light that has passed through the excitation filter 34 is reflected by the dichroic mirror 36, then passes through the sixth lens 38, and is applied to the sample 4.

When the third fluorescent molecule contained in the sample 4 is excited by the third excitation light, the sample 4 emits the third fluorescence. The third fluorescence generated from the sample 4 passes through the sixth lens 38 and the dichroic mirror 36, and then enters the absorption filter 40. At this time, the absorption filter 40 limits the wavelength band of the third fluorescence to the seventh wavelength band B7 (see FIG. 5). The third fluorescence that has passed through the absorption filter 40 passes through the seventh lens 42 and is received by the light receiving element 44 (S8). As a result, the light receiving element 44 generates a light reception signal based on the received third fluorescence.

As described above, since the first light emitting element 10, the second light emitting element 12 and the fourth light emitting element 16 other than the third light emitting element 14 emit light with a relatively low light amount, In addition to the signal component corresponding to the third fluorescence, the signal components corresponding to the first fluorescence, the second fluorescence, and the fourth fluorescence are slightly included. Therefore, the control unit 46 removes the signal components corresponding to the first fluorescence, the second fluorescence, and the fourth fluorescence of the light reception signal by calculation, thereby making a signal corresponding to the third fluorescence of the light reception signal. Only the component can be detected.

After that, the control unit 46 lowers the output of the third light emitting element 14 from the predetermined rated output (S9), and causes the third light emitting element 14 to emit light with an output that is equal to or more than the light emission threshold and equal to or less than 28% of the predetermined rated output.

Next, the fourth sequence (S10 to S12) is executed. The control unit 46 causes the fourth light emitting element 16 to emit light with a predetermined rated output (S10). At this time, the control unit 46 causes the first light emitting element 10, the second light emitting element 12, and the third light emitting element 14 other than the fourth light emitting element 16 to have a light emission threshold value or more and 28% or less of a predetermined rated output. Make the output emit light.

As shown in FIG. 2, the fourth excitation light from the fourth light emitting element 16 passes through the first dichroic mirror 26, the second dichroic mirror 28, the third dichroic mirror 30, and the fifth lens 32. After that, the light enters the excitation filter 34. At this time, the excitation filter 34 limits the wavelength band of the fourth excitation light to the fourth wavelength band B4 (see FIG. 3). The fourth excitation light that has passed through the excitation filter 34 is reflected by the dichroic mirror 36, then passes through the sixth lens 38, and is applied to the sample 4.

When the fourth fluorescent molecule contained in the sample 4 is excited by the fourth excitation light, the sample 4 emits the fourth fluorescence. The fourth fluorescence emitted from the sample 4 passes through the sixth lens 38 and the dichroic mirror 36, and then enters the absorption filter 40. At this time, the absorption filter 40 limits the wavelength band of the fourth fluorescence to the eighth wavelength band B8 (see FIG. 5). The fourth fluorescence that has passed through the absorption filter 40 passes through the seventh lens 42 and is received by the light receiving element 44 (S11). As a result, the light receiving element 44 generates a light reception signal based on the received fourth fluorescence.

As described above, since the first light emitting element 10, the second light emitting element 12, and the third light emitting element 14 other than the fourth light emitting element 16 emit light with a relatively low light amount, In addition to the signal component corresponding to the fourth fluorescence, the signal components corresponding to the first fluorescence, the second fluorescence, and the third fluorescence are slightly included. Therefore, the control unit 46 removes the signal components corresponding to the first fluorescence, the second fluorescence, and the third fluorescence from the received light signal by calculation, thereby obtaining a signal corresponding to the fourth fluorescence of the received light signal. Only the component can be detected.

After that, the control unit 46 lowers the output of the fourth light emitting element 16 from a predetermined rated output (S12), and causes the fourth light emitting element 16 to emit light with an output that is equal to or higher than the light emission threshold and equal to or lower than 28% of the predetermined rated output.

When the detection of the first to fourth fluorescences has not been completed (NO in S13), steps S1 to S12 described above are executed again. When the detection of the first to fourth fluorescences is completed (YES in S13), the operation of the fluorescence detector 2 is completed.

[1-3. effect]
As described above, in the fluorescence detector 2 according to the first embodiment, for example, when the first light emitting element 10 is emitting light at a predetermined rated output, the second light emitting element other than the first light emitting element 10 is used. The light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 are caused to emit light with an output lower than a predetermined rated output (with a light amount larger than 0%). Thereby, when the second light emitting element 12 is made to emit light at a predetermined rated output in the next sequence, the light emission of the second light emitting element 12 is completely turned off (light amount 0%) in the immediately preceding sequence. Compared with the case, the output of the second light emitting element 12 can be stabilized in a short time, and the stable second fluorescence can be generated from the sample 4. As a result, the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16, which are the targets for emitting light with a predetermined rated output, can be switched at high speed.

Note that, for example, when the second light emitting element 12 is emitting light at a predetermined rated output, the first light emitting element 10, the third light emitting element 14, and the fourth light emitting element other than the second light emitting element 12 are included. It is preferable that 16 emit light at an output that is 28% or less of a predetermined rated output. At this time, when the first light emitting element 10, the third light emitting element 14, and the fourth light emitting element 16 other than the second light emitting element 12 are caused to emit light with an output exceeding 28% of the predetermined rated output, For example, when the third fluorescent molecule (TAMRA) contained in the sample 4 is excited by the third excitation light (green laser light) from the third light emitting element 14, the third fluorescent molecule is generated from the third fluorescent molecule. The crosstalk ratio of the fluorescence signal of the third fluorescence becomes large.

Further, as described above, in the fluorescence detector 2 according to the first embodiment, the so-called multi-bandpass type dichroic mirror 36 is used. Accordingly, even when a plurality of types of fluorescent molecules are labeled on the sample 4, one dichroic mirror 36 can reflect a plurality of types of excitation light for exciting the plurality of types of fluorescent molecules. In addition, a plurality of types of fluorescence generated from a plurality of types of fluorescent molecules can be transmitted. As a result, the turret and the motor for rotating the turret described in the background art section can be omitted, and the fluorescence detector 2 can be miniaturized.

As an example of the size of the housing 6, as shown in FIG. 1, the width W of the housing 6 can be about 17 mm, the depth D can be about 74 mm, and the height H can be about 39 mm.

[1-4. Examples and Comparative Examples]
Examples and comparative examples of the fluorescence detector will be described below with reference to FIGS. 7 to 13.

FIG. 7 is a diagram showing a light emission sequence of each of the light emitting elements 10, 12, 14 and 16 of the fluorescence detector 2 according to the example. FIG. 8 is a diagram showing emission conditions of the light emitting elements 10, 12, 14 and 16 of the fluorescence detector 2 according to the example. FIG. 9 is a diagram showing the transmission characteristics of the dichroic mirror 36 of the fluorescence detector 2 according to the example. FIG. 10 is a diagram showing a light emission sequence of each of the light emitting elements 10, 12, 14 and 16 of the fluorescence detector 2 ′ according to the comparative example.

FIG. 11 is a diagram showing a detection result of a fluorescence signal by the fluorescence detector 2 according to the example. FIG. 12 is a diagram showing the crosstalk ratio of the fluorescence signal of FIG. FIG. 13 is a diagram showing the detection results of the fluorescence intensity by the fluorescence detectors 2 and 2 ′ according to the example and the comparative example. FIG. 13A is a diagram showing a fluorescence intensity detection result by the fluorescence detector 2 according to the example, and FIG. 13B is a diagram showing a fluorescence intensity detection result by the fluorescence detector 2′ according to the comparative example. It is a figure and (c) is a figure which shows the variation rate of the fluorescence intensity by the fluorescence detectors 2 and 2'according to an example and a comparative example.

[1-4-1. Example]
The fluorescence detector 2 shown in FIG. 1 was used as the fluorescence detector according to the example. The case 6 used was formed by shaving aluminum.

As the first light emitting element 10, an LED (Light Emitting Diode) (LZ1-00UB00, manufactured by LED ENGIN) that emits violet light having a central wavelength of 405 nm was used. As the second light emitting element 12, an LED (manufactured by OSRAM, LBW5SN-GYHZ-25) that emits blue light having a center wavelength of 470 nm was used. As the third light emitting element 14, an LED (LTRAM5SN-KYLY-25 manufactured by OSRAM) that emits green light having a center wavelength of 528 nm was used. As the fourth light-emitting element 16, an LED (OSRAM, LRW5SN-JYKY-1) that emits red light having a central wavelength of 632 nm was used.

In addition, the fourth light emitting element 16 (red: 632 nm), the third light emitting element 14 (green: 528 nm), the second light emitting element 12 (blue: 475 nm) and the first light emitting element 10 (purple: 405 nm). , The sequence “1”, “2”, “3”, “4”,... Shown in FIG. 7 was repeatedly emitted with a light amount of 100% (rated output). The light emitting period of each of the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 was 500 msec. In FIG. 7, “High” means that the light output of the light emitting element is 100%, and “Low” means that the light output of the light emitting element is 28% or less. For example, in the sequence “1” shown in FIG. 7, the fourth light emitting element 16 is caused to emit light with a light amount of 100% (High), and the first light emitting element 10 and the second light emitting element 12 other than the fourth light emitting element 16 are emitted. The third light emitting element 14 was made to emit light with a light amount of 28% or less (Low).

In addition, the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 were caused to emit light by the LED dimming circuit under the light emitting conditions shown in FIG. The LED dimming circuit is a circuit that controls the amount of current flowing through the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 by D/A converter control. .. For example, when the fourth light emitting element 16 was made to emit light at a high level, the input current was 700 mA, the voltage was 2.48 V, the power consumption was 1.74 W, and the light amount was 100%. When the fourth light emitting element 16 was caused to emit light at a low level, the input current was 100 mA, the voltage was 1.9 V, the power consumption was 0.19 W, and the light amount was 15%.

As the dichroic mirror 36, a multi-bandpass type dichroic mirror (FF409/493/573/652-Di01-25x36 manufactured by Semrock) having the transmission characteristics shown in FIG. 9 was used. As the dichroic mirror 36, one cut into a size of 12×18 mm was used.

As the excitation filter 34, a multi-bandpass type filter (FF01-392/474/554/635-25 manufactured by Semrock) was used. As the absorption filter 40, a multi-bandpass type filter (manufactured by Semrock, FF01-432/515/595/730-25) was used. As the excitation filter 34 and the absorption filter 40, those cut to a diameter of 12 mm were used.

Plano-convex lenses (Edmund's #45-228) were used as the first lens 18, the second lens 20, the third lens 22, and the fourth lens 24. As the fifth lens 32, the sixth lens 38, and the seventh lens 42, achromatic lenses (#65-549, manufactured by Edmund) were used.

As the light receiving element 44, a photodiode (S2386-44K manufactured by Hamamatsu Co.) was used. The fluorescence signal from the light receiving element 44 is amplified by a photosensor amplifier (Hamamatsu Co., C9329) with a multiplication factor of 1E+7V/A and then A/D converted, and the voltage value representing the magnitude of the fluorescence signal is taken into a personal computer. It is.

As sample 4, a Q-Probe color compensation kit (manufactured by Nittetsu Sumikin Environmental Co., Ltd.) was used. 200 μL of the fluorescent reagent was injected into the microtube, and the fluorescent reagent was irradiated with excitation light to detect fluorescence. As a first fluorescent molecule constituting Q-Probe, Pacific Blue (hereinafter, also referred to as “dye B”) that is excited by violet light is used, and as a second fluorescent molecule, BODIPY-FL (hereinafter, “BODIPY-FL”) that is excited by blue light is used. , "Dye G"), TAMRA (hereinafter also referred to as "dye R") that is excited by green light as the third fluorescent molecule, and ATTO665 that is excited by red light as the fourth fluorescent molecule. (Hereinafter, also referred to as “dye S”) was used.

[1-4-2. Comparative example]
A fluorescence detector 2′ shown in FIG. 1 was used as the fluorescence detector according to the comparative example.

The fourth light emitting element 16 (red: 632 nm), the third light emitting element 14 (green: 528 nm), the second light emitting element 12 (blue: 475 nm) and the first light emitting element 10 (purple: 405 nm) are shown in FIG. Sequences “1”, “2”, “3”, “4”,... Shown in 10 were repeatedly emitted with a light amount of 100% (rated output). The light emitting period of each of the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 was 500 msec. In FIG. 10, “High” means that the output of the light emitting element is 100% of the light amount (rated output), and “Off” means that the output of the light emitting element is 0% of the light amount (no light emission). To do. For example, in the sequence “1” shown in FIG. 10, the fourth light emitting element 16 is caused to emit light with a light amount of 100% (High), and the first light emitting element 10 and the second light emitting element 12 other than the fourth light emitting element 16 are emitted. Further, the light emission of the third light emitting element 14 was completely turned off.

The conditions of the comparative example other than the above are the same as those of the example, and thus the description thereof is omitted.

[1-4-3. result]
The detection result of the fluorescence signal by the fluorescence detector 2 according to the example was as shown in FIGS. 11 and 12.

As apparent from FIGS. 11 and 12, for example, when the fourth light emitting element 16 is caused to emit light with a light amount of 100%, the fourth fluorescent molecule (dye S) contained in the sample 4 emits the fourth light. By being excited by the fourth excitation light (red light) from the element 16, the fluorescence signal (voltage value) of the fourth fluorescence generated from the fourth fluorescent molecule was increased. At this time, when the crosstalk ratio of the fluorescence signal of the fourth fluorescence is 100.0%, the first fluorescent molecule (dye B), the second fluorescent molecule (dye G) and the third fluorescent molecule contained in the sample 4 The crosstalk ratios of the fluorescence signals of the first fluorescence, the second fluorescence, and the third fluorescence generated from the respective fluorescent molecules (dye R) were 1.4%, 3.4%, and 1.9%. ..

Further, when the third light emitting element 14 is caused to emit light with a light amount of 100%, the third fluorescent molecule (dye R) contained in the sample 4 causes the third excitation light (green) from the third light emitting element 14 to be emitted. Excitation), the fluorescence signal of the third fluorescence generated from the third fluorescent molecule increased. At this time, when the crosstalk ratio of the fluorescence signal of the third fluorescence is 100.0%, the first fluorescent molecule (dye B), the second fluorescent molecule (dye G), and the fourth fluorescent molecule (dye G) contained in the sample 4 are included. The crosstalk ratios of the fluorescence signals of the first fluorescence, the second fluorescence and the fourth fluorescence generated from the respective fluorescent molecules (dye S) were 9.7%, 26.4% and 0.7%. ..

Further, when the second light emitting element 12 is caused to emit light with a light amount of 100%, the second fluorescent molecule (dye G) contained in the sample 4 emits the second excitation light (blue) from the second light emitting element 12. Excitation), the fluorescence signal of the second fluorescence generated from the second fluorescent molecule increased. At this time, when the crosstalk ratio of the fluorescence signal of the second fluorescence is 100.0%, the first fluorescent molecule (dye B), the third fluorescent molecule (dye R), and the fourth fluorescent molecule (dye R) contained in the sample 4 are included. The crosstalk ratios of the fluorescence signals of the first fluorescence, the third fluorescence, and the fourth fluorescence generated from the respective fluorescent molecules (dye S) were 3.8%, 0.2%, and 0.7%. ..

When the first light emitting element 10 is caused to emit light with a light amount of 100%, the first fluorescent molecule (dye B) contained in the sample 4 emits the first excitation light (purple light from the first light emitting element 10 (purple). Excitation), the fluorescence signal of the first fluorescence generated from the first fluorescent molecule becomes large. At this time, when the crosstalk ratio of the fluorescence signal of the first fluorescence is 100.0%, the second fluorescent molecule (dye G), the third fluorescent molecule (dye R), and the fourth fluorescent molecule (dye G) contained in the sample 4 are used. The crosstalk ratios of the fluorescence signals of the second fluorescence, the third fluorescence, and the fourth fluorescence generated from the fluorescent molecules (dye S) of 0.8%, 0.0%, and 1.5%, respectively. ..

Furthermore, the detection results of the fluorescence intensity by the fluorescence detectors 2 and 2'according to the examples and the comparative examples were as shown in FIG.

As is clear from (a) and (c) of FIG. 13, in the fluorescence detector 2 according to the example, there is almost no variation in the peak value of the detected fluorescence intensity, and the variation rate of the fluorescence intensity is 0.2. %Met.

On the other hand, as is clear from FIGS. 13B and 13C, in the fluorescence detector 2′ according to the comparative example, the peak value of the detected fluorescence intensity varies, and the variation rate of the fluorescence intensity is 4%. It was 0.1%.

From the above results, it was confirmed that the fluorescence detector 2 of the example has an effect of generating stable fluorescence from the sample 4.

(Embodiment 2)
Next, the configuration of the fluorescence detector 2A according to the second embodiment will be described with reference to FIGS. 14 and 15. FIG. 14 is a diagram schematically showing the configuration of the fluorescence detector 2A according to the second embodiment. FIG. 15 is a plan view showing the light receiving element 44A of the fluorescence detector 2A according to the second embodiment.

As shown in FIG. 14, in the fluorescence detector 2A according to the second embodiment, the light receiving element 44A has a filter array 48. As shown in FIG. 15, a plurality of pixels 50 are arranged in a matrix on the light receiving surface of the light receiving element 44A. The filter array 48 has a plurality of filter sets 52 arranged corresponding to the plurality of pixels 50, respectively.

Each of the plurality of filter sets 52 has a first filter 52a, a second filter 52b, a third filter 52c, and a fourth filter 52d. The first filter 52a is a so-called bandpass type filter that limits the wavelength band of the first fluorescence transmitted through the dichroic mirror 36 when the first light emitting element 10 emits light with a predetermined rated output. The second filter 52b is a so-called bandpass type filter that limits the wavelength band of the second fluorescence transmitted through the dichroic mirror 36 when the second light emitting element 12 emits light with a predetermined rated output. The third filter 52c is a so-called bandpass type filter that limits the wavelength band of the second fluorescence transmitted through the dichroic mirror 36 when the third light emitting element 14 emits light with a predetermined rated output. The fourth filter 52d is a so-called bandpass type filter that limits the wavelength band of the fourth fluorescence transmitted through the dichroic mirror 36 when the fourth light emitting element 16 emits light with a predetermined rated output.

Accordingly, since the first filter 52a, the second filter 52b, the third filter 52c, and the fourth filter 52d are arranged for one pixel 50, the fluorescence received by one pixel 50 The spectral width can be reduced. Therefore, it is possible to suppress the excitation of other fluorescent molecules by the fluorescence generated from the specific fluorescent molecule.

(Modification)
Although the fluorescence detector and the control method thereof according to one or more aspects have been described above based on the above-described embodiment, the present invention is not limited to this embodiment. As long as it does not depart from the spirit of the present invention, various modifications made by those skilled in the art may be applied to the present embodiment, or an embodiment constructed by combining components in different embodiments or modifications may be one or more. It may be included in the range of the aspect.

In each of the above embodiments, the first light emitting element 10, the second light emitting element 12, the third light emitting element 14 and the fourth light emitting element 16 are laser diodes. Etc.

In each of the above embodiments, both the excitation filter 34 and the absorption filter 40 are arranged, but the present invention is not limited to this, and only one of the excitation filter 34 and the absorption filter 40 may be arranged, or the excitation filter Both 34 and the absorption filter 40 may be omitted.

In each of the above-described embodiments, the rated outputs of the first light emitting element 10, the second light emitting element 12, the third light emitting element 14, and the fourth light emitting element 16 are all the same output (light amount 100%). However, the output is not limited to this, and the outputs may be different from each other. For example, the first rated output of the first light emitting element 10 and the second rated output of the second light emitting element 12 may be different.

In addition, in each of the above-described embodiments, each component may be configured by dedicated hardware, or may be realized by executing a software program suitable for each component. Each component may be realized by a program execution unit such as a CPU or a processor reading and executing a software program recorded in a recording medium such as a hard disk or a semiconductor memory.

The present invention also includes the following cases.

(1) Each of the above devices can be specifically realized by a computer system including a microprocessor, a ROM, a RAM, a hard disk unit, a display unit, a keyboard, a mouse, and the like. A computer program is stored in the RAM or the hard disk unit. Each device achieves its function by the microprocessor operating according to the computer program. Here, the computer program is configured by combining a plurality of instruction codes indicating instructions to the computer in order to achieve a predetermined function.

(2) Part or all of the constituent elements of each of the above devices may be composed of one system LSI (Large Scale Integration). The system LSI is a super-multifunctional LSI manufactured by integrating a plurality of constituent parts on one chip, and specifically, is a computer system including a microprocessor, ROM, RAM and the like. .. A computer program is stored in the ROM. The system LSI achieves its function by the microprocessor loading the computer program from the ROM to the RAM and performing operations such as calculation according to the loaded computer program.

(3) Part or all of the constituent elements of each of the above devices may be configured with an IC card that can be attached to and detached from each device or a single module. The IC card or module is a computer system including a microprocessor, ROM, RAM and the like. The IC card or the module may include the above-mentioned super-multifunctional LSI. The IC card or module achieves its function by the microprocessor operating according to the computer program. This IC card or this module may be tamper resistant.

(4) The present invention may be realized by the method shown above. Further, these methods may be realized by a computer program realized by a computer, or may be realized by a digital signal including the computer program.

The present invention also provides a computer-readable recording medium such as a flexible disk, a hard disk, a CD-ROM, a MO, a DVD, a DVD-ROM, a DVD-RAM, and a BD (Blu-ray (registered trademark)). ) Disc), may be realized by recording in a semiconductor memory or the like. Further, it may be realized by a digital signal recorded on these recording media.

In the present invention, the computer program or the digital signal may be transmitted via a telecommunication line, a wireless or wired communication line, a network represented by the Internet, data broadcasting and the like.

Further, the present invention is a computer system including a microprocessor and a memory, the memory stores a computer program, and the microprocessor may operate according to the computer program.

Alternatively, the program or the digital signal may be recorded on a recording medium and transferred, or the program or the digital signal may be transferred via a network or the like to be implemented by another independent computer system.

(5) The above embodiments and the above modifications may be combined.

The method for controlling the fluorescence detector of the present invention can be applied to, for example, a fluorescence detection method for detecting fluorescence generated from a sample by irradiating the sample with excitation light.

2, 2′, 2A Fluorescence detector 4 Sample 6 Housing 8 Optical system 10 First light emitting element 12 Second light emitting element 14 Third light emitting element 16 Fourth light emitting element 18 First lens 20 Second Lens 22 Third lens 24 Fourth lens 26 First dichroic mirror 28 Second dichroic mirror 30 Third dichroic mirror 32 Fifth lens 34 Excitation filter 36 Dichroic mirror 38 Sixth lens 40 Absorption filter 42 7 lens 44, 44A light receiving element 46 control section 48 filter array 50 pixel 52 filter set 52a first filter 52b second filter 52c third filter 52d fourth filter

Claims (4)

By irradiating a sample with excitation light, a method for controlling a fluorescence detector for detecting fluorescence generated from the sample,
Emitting a first pumping light and a second pumping light having different center wavelengths from the first light emitting element and the second light emitting element, respectively, in order of a first rated output and a second rated output,
By irradiating the sample with the first excitation light and the second excitation light in order, the light receiving element sequentially receives the first fluorescence and the second fluorescence having different central wavelengths generated from the sample. Generating a received light signal,
In the step of issuing,
When the first excitation light is emitted from the first light emitting element at the first rated output, the second light emitting element outputs the second excitation light at an output lower than the second rated output. Emits the excitation light of
When the second excitation light is emitted from the second light emitting element at the second rated output, the first light emitting element outputs the first excitation light at an output lower than the first rated output. A method for controlling a fluorescence detector that emits excitation light. In the step of issuing,
When the first light emitting element emits the first excitation light at the first rated output, the second light emitting element outputs at 28% or less of the second rated output. Emits a second excitation light,
When the second excitation light is emitted from the second light emitting element at the second rated output, the first light emitting element outputs at 28% or less of the first rated output. The method for controlling the fluorescence detector according to claim 1, wherein the fluorescence detector emits the first excitation light. The method for controlling the fluorescence detector further comprises
When the first excitation light is emitted from the first light emitting element at the first rated output, the signal component corresponding to the second fluorescence in the received light signal is removed by calculation, and When the second light-emitting element emits the second excitation light at the second rated output, the method includes a step of calculating and removing a signal component corresponding to the first fluorescence in the received light signal. The method for controlling the fluorescence detector according to claim 1. A fluorescence detector for detecting fluorescence emitted from the sample by irradiating the sample with excitation light,
A first light emitting element which emits a first excitation light;
A second light emitting element that emits second excitation light having a central wavelength different from that of the first excitation light;
When the first light-emitting element emits light, the first excitation light from the first light-emitting element is applied to the sample to receive the first fluorescence generated from the sample, When the second light emitting element emits light, the second excitation light from the second light emitting element is applied to the sample, so that the first fluorescence generated from the sample and the central wavelength A light receiving element for receiving a second fluorescent light having a different
The first excitation light and the second excitation light are sequentially emitted from the first light emitting element and the second light emitting element at a first rated output and a second rated output, respectively. A light emitting element and a control unit for controlling the second light emitting element,
The control unit is
When the first excitation light is emitted from the first light emitting element at the first rated output, the second light emitting element outputs the second excitation light at an output lower than the second rated output. Emits the excitation light of
When the second excitation light is emitted from the second light emitting element at the second rated output, the first light emitting element outputs the first excitation light at an output lower than the first rated output. A fluorescence detector that controls the first light-emitting element and the second light-emitting element so as to emit the excitation light.

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