Optical measuring device and optical measuring method

Abstract

To provide an optical measuring device and an optical measuring method capable of properly removing a noise component caused by irradiation light itself.SOLUTION: An optical measuring device 1 comprises: a timing generator 70; a light source 11; a detection optical system 20 which detects detection light including fluorescent light and scattered light, and output a detection signal; a frequency converting circuit 45 which multiplies the detection signal by a second frequency signal input from the timing generator 70 to generate a difference frequency signal which is a signal having a frequency difference between a frequency associated with a first frequency signal and a frequency associated with a second frequency signal; and a cancel circuit 60 which removes a signal component corresponding to the scattered light from the difference frequency signal on the basis of the difference frequency signal and a third frequency signal, wherein the third frequency signal is a signal having a frequency corresponding to a frequency associated with the difference frequency signal.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] One aspect of the present invention relates to an optical measurement device and an optical measurement method. [Background technology]

[0002] Patent Document 1 discloses a technique for generating and subtracting an attenuation signal in order to remove a fluorescence noise component (fluorescence component from the substrate) contained when a fluorescent sample placed on a substrate is irradiated with excitation light and the fluorescence is measured. Specifically, Patent Document 1 generates an attenuation signal having the same phase as the fluorescence from the substrate, and subtracts the attenuation signal from the measured fluorescent signal to remove the fluorescence noise component. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2010-518394 Summary of the Invention [Problem to be solved by the invention]

[0004] As described above, in Patent Document 1, fluorescence noise components arising from areas other than the measurement target portion of the sample are removed from the fluorescence generated by irradiating the sample with excitation light. Here, noise components contained when irradiating the sample with light and detecting the light from the sample include not only noise components caused by light (e.g., fluorescence) generated from the sample (irradiated object), but also light (e.g., scattered light) caused by the irradiated light (e.g., excitation light) itself. The technology in Patent Document 1 does not take into account the case where the irradiated light itself becomes a noise component, and is therefore unable to remove the noise component.

[0005] Even if some method is used to remove the noise components of the irradiated light itself, for example, when the light emission time (light emission duration) from the sample becomes short at the nanosecond level, the circuitry used to process the light must also be able to handle high-speed signals. For example, for high-speed signals of 10 MHz or more, it may be difficult to remove the above-mentioned noise components depending on the processing capacity of the circuitry used.

[0006] One aspect of the present invention has been made in consideration of the above-mentioned situation, and aims to provide an optical measurement device and an optical measurement method that can appropriately remove noise components caused by the irradiated light itself, even when the emission time of the light to be measured is short. [Means for solving the problem]

[0007] An optical measurement device according to one embodiment of the present invention includes a timing generator that outputs a frequency signal, a light source unit that irradiates a first light toward a sample, a light source driving circuit that modulates and drives the light source unit based on the first frequency signal input from the timing generator, a light detection unit that detects detection light including a second light generated from the sample irradiated with the first light and a third light that is caused by the first light and has a phase equal to that of the first light, and outputs a detection signal, a frequency conversion circuit that generates a difference frequency signal that is a signal of a frequency that is a difference between a frequency associated with the first frequency signal and a frequency associated with the second frequency signal by calculating the detection signal and the second frequency signal input from the timing generator, and a signal processing unit that removes a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal input from the timing generator, and the third frequency signal is a signal of a frequency that corresponds to the frequency associated with the difference frequency signal.

[0008] In the optical measuring device according to one aspect of the present invention, the detection light including the second light and the third light is detected, and the detection signal according to the detection light and the second frequency signal are calculated to generate a difference frequency signal between the first frequency signal and the second frequency signal. Conventionally, a method is known in which the phase component of the third light is removed from the detection signal of the detection light including the second light and the third light to remove noise components caused by the irradiation light (first light) itself. Here, when the light emission time to be measured is shortened and the signal processed by the circuit is accelerated, for example, when it becomes a high-speed signal of 10 MHz or more, depending on the processing capacity of the circuit used, the above-mentioned method alone may not be able to adequately remove the noise components. In this regard, in the optical measuring device according to one aspect of the present invention, the second frequency signal is calculated on the detection signal to generate a difference frequency signal between the first frequency signal and the second frequency signal. Such a difference frequency signal is a signal that is sufficiently slow compared to the detection signal. Then, the difference frequency signal and the third frequency signal are input to a signal processing unit, whereby a signal component corresponding to the third light (i.e., a noise component caused by the irradiated light itself) is removed from the difference frequency signal. In this manner, the detection signal is frequency converted, and the noise component caused by the irradiated light itself is removed from the difference frequency signal after frequency conversion, so that even when the emission time of the light to be measured is short, the noise component can be appropriately removed.

[0009] The first light may be excitation light that excites the sample, the second light may be fluorescence emitted by the sample in response to the excitation light, and the third light may include at least one of the excitation light and scattered light of the excitation light. This makes it possible to appropriately remove signal components corresponding to at least one of the excitation light and scattered light of the excitation light from a difference frequency signal corresponding to a detection signal including signal components corresponding to the fluorescence and at least one of the excitation light and scattered light of the excitation light, and to appropriately extract only the signal components corresponding to the fluorescence.

[0010] The frequency of the difference frequency signal may be within a range of 10 Hz to 1 MHz. By setting the frequency of the difference frequency signal within such a range, the frequency related to noise removal is prevented from becoming too high, and the noise component can be removed appropriately.

[0011] The optical measurement device further includes a first frequency filter provided on the input side of the frequency conversion circuit and a second frequency filter provided on the output side of the frequency conversion circuit, and the first frequency filter and the second frequency filter may be band-pass filters. In this way, by providing frequency filters on the input / output sides of the frequency conversion circuit, it is possible to suppress the inclusion of noise in the detection signal, the second frequency signal, and the difference frequency signal. In addition, by using a band-pass filter, it is possible to selectively pass only a desired frequency band, and it is possible to more appropriately suppress the inclusion of noise.

[0012] An optical measurement method according to one embodiment of the present invention includes: irradiating a first light toward a sample based on a first frequency signal; detecting detection light including a second light generated from the sample irradiated with the first light and a third light caused by the first light and having a phase equal to that of the first light, and outputting a detection signal; generating a difference frequency signal that is a signal of a frequency that is a difference between a frequency associated with the first frequency signal and a frequency associated with the second frequency signal by calculating the detection signal and the second frequency signal; and removing a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal, wherein the third frequency signal is a signal of a frequency that corresponds to the frequency associated with the difference frequency signal. Effect of the Invention

[0013] According to one aspect of the present invention, it is possible to provide an optical measurement device and an optical measurement method that are capable of appropriately removing noise components caused by the irradiated light itself, even when the emission time of the light to be measured is short. [Brief description of the drawings]

[0014] [Figure 1]1 is a schematic configuration diagram of an optical measurement device according to an embodiment of the present invention. [Diagram 2] FIG. 4 is a diagram illustrating the phase difference between fluorescent light and scattered light. [Diagram 3] FIG. 13 is a diagram illustrating a method for canceling scattered light. [Figure 4] FIG. 1 is a diagram illustrating a fluorescence measurement technique. [Diagram 5] FIG. 11 is a diagram illustrating the removal of noise components. [Figure 6] FIG. 2 is a diagram showing a schematic diagram of a sample. [Figure 7] FIG. 1 is a diagram for explaining the definition of S / N. [Figure 8] 10A and 10B are diagrams illustrating signals used in a calibration process. [Figure 9] FIG. 11 is a diagram illustrating a calibration process. [Figure 10] FIG. 4 is a diagram for explaining details of the fluorescence intensity in each region of the sample. [Figure 11] 13 is a flowchart showing a calibration process. [Figure 12] FIG. 1 is a diagram illustrating the fluorescence excitation spectra of europium and an organic fluorescent dye (FITC). [Figure 13] 10A and 10B are diagrams for explaining the effect of appropriately removing scattered light (noise). [Figure 14] FIG. 13 is a diagram illustrating a device configuration according to a modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. In each drawing, the same or corresponding parts are denoted by the same reference numerals, and duplicated explanations will be omitted.

[0016] FIG. 1 is a schematic diagram of an optical measurement device 1 according to this embodiment. The optical measurement device 1 is a device that detects light generated from a sample in response to light irradiated onto the sample. In this embodiment, the optical measurement device 1 is described as a fluorescence measurement device that detects fluorescence generated from the sample in response to excitation light irradiated onto the sample. The excitation light is light that excites the sample, and the fluorescence is light that the sample emits in response to the excitation light, and is light with a different wavelength from the excitation light. In this embodiment, the optical measurement device 1 is described as a device that detects fluorescence related to measurement using an immunochromatography method. The immunochromatography method is an immunoassay method that utilizes an antigen-antibody reaction, and is used, for example, to detect influenza viruses.

[0017] As shown in FIG. 1, in a measurement using the immunochromatography method, an immunochromatographic test piece 100 is prepared as a sample. The immunochromatographic test piece 100 contains an immunochromatographic membrane, which is the measurement target, in a reagent holder 101. A capture antibody for a specific antigen (e.g., an antibody for an influenza virus antigen) is fixed to a specific position (measurement target portion) of the immunochromatographic membrane of the immunochromatographic test piece 100. The reagent holder 101 is provided with a sample application window, which is an opening for dropping a sample onto the immunochromatographic membrane, and a measurement window, which is an opening for measuring the measurement target portion to which the capture antibody is fixed. When a sample is dropped onto the sample application window of the reagent holder 101, the antigen in the sample first binds to a detection antibody labeled with a fluorescent reagent, and then undergoes an antigen-antibody reaction with the capture antibody to be trapped. The optical measurement device 1 measures fluorescence intensity by irradiating excitation light onto the immunochromatographic membrane exposed from the measurement window of the immunochromatographic test piece 100 and detecting fluorescence from an antigen-antibody complex (specifically, a fluorescent reagent of an antibody) in the measurement target area. Note that, as the fluorescent reagent, for example, europium, Q-dot (registered trademark), organic fluorescent dyes, etc. can be used. As a fluorescent reagent that emits light (fluorescence) to be measured in a particularly short time (fluorescence lifetime is as short as several nanoseconds to several tens of nanoseconds), an organic fluorescent dye such as FITC (fluorescein isothiocyanate) can be mentioned.

[0018] Here, it is considered that the detection light that is incident on the detection optical system 20 described later in the optical measurement device 1 and detected includes not only fluorescence but also light due to the excitation light itself. For example, such light includes scattered light of the excitation light. For example, such scattered light is a part of the excitation light generated by scattering the excitation light irradiated on the immunochromatographic test piece 100, and is light having a phase substantially equal to that of the excitation light. Since the immunochromatographic membrane and the reagent holder 101 of the immunochromatographic test piece 100 are generally white, the above-mentioned scattered light is likely to occur. In addition, depending on the sample to be measured and the arrangement of the detection optical system, the excitation light itself may be detected. In the following, it is described that the detection light detected in the optical measurement device 1 includes fluorescence and scattered light.

[0019] As shown in FIG. 1, the optical measurement device 1 includes an irradiation optical system 10 (light source section), a detection optical system 20 (light detection section), a light source driving circuit 30, an IV conversion amplifier 40, a frequency conversion circuit 45, a waveform generating circuit 50, a cancellation circuit 60 (signal processing section), a timing generator 70, an AD converter 80, and a CPU 90.

[0020] The irradiation optical system 10 irradiates the immunochromatographic test piece 100 with excitation light. The irradiation optical system 10 has a light source 11, an aperture 12, an excitation light filter 13, and a collimator lens 14. The light source 11 irradiates the immunochromatographic test piece 100 (sample) with excitation light (first light). The light source 11 has a response speed faster than the modulation frequency, and in this embodiment, it is preferable that the light source 11 is compatible with a high-speed drive signal of, for example, 10 MHz or more. Such a light source 11 is, for example, a semiconductor light-emitting element such as an LD (laser diode). In this embodiment, the light source 11 is described as an LD, but is not limited thereto, and may be a light-emitting diode (LED) or a superluminescence diode (SLD). The aperture 12 is a light beam shaping member for shaping the light emitted from the light source 11 into light having a desired light beam cross section. The excitation light filter 13 is a wavelength selection filter that filters the wavelength required for excitation from the excitation light that arrives through the aperture 12. The excitation light filter 13 is an optical filter such as a dielectric multilayer filter or a colored glass filter, and more specifically, a bandpass filter made of a dielectric multilayer filter that transmits only a specific wavelength band (the excitation wavelength of the fluorescent reagent). The collimating lens 14 is a lens that forms an image of the excitation light filtered by the excitation light filter 13 on the immunochromatographic test piece 100 (more specifically, the measurement target portion of the immunochromatographic membrane).

[0021] The detection optical system 20 detects the fluorescence from the immunochromatographic test piece 100. However, in reality, the detection optical system 20 receives detection light, which is light including the fluorescence from the immunochromatographic test piece 100 (fluorescence from the measurement target portion of the immunochromatographic membrane) as well as scattered light caused by the above-mentioned excitation light itself, and detects the detection light. The detection optical system 20 has a photodetector 21, a fluorescence filter 22, and a condenser lens 23. The detection light is condensed by the condenser lens 23 and enters the photodetector 21 through the fluorescence filter 22. The fluorescence filter 22 is a wavelength selection filter provided to suppress light other than the fluorescence from the detection light from the immunochromatographic test piece 100 from reaching the photodetector 21. The fluorescence filter 22 is an optical filter such as a dielectric multilayer filter or a colored glass filter, and more specifically, it is a bandpass filter that combines a dielectric multilayer filter that transmits only a specific wavelength band and a colored glass filter. However, for example, when the excitation light wavelength and the fluorescence wavelength are close to each other, it is difficult for the fluorescence filter 22 to efficiently block only the scattered light having the excitation light wavelength while appropriately transmitting the fluorescence having the fluorescence wavelength. In addition, the characteristics of a dielectric multilayer filter, which is generally used as an efficient wavelength selection filter, change depending on the angle of incidence of light. Therefore, in this embodiment, the fluorescence filter 22 is configured by combining a dielectric multilayer filter and a colored glass filter, and the colored glass filter effectively blocks the scattered light from oblique directions. However, it is difficult to obtain a sufficient effect by wavelength selection alone, and it is difficult to efficiently prevent the entry of scattered light under various conditions. In the following, it is assumed that the detection light reaching the photodetector 21 contains scattered light even when the fluorescence filter 22 is provided.

[0022] The photodetector 21 is an optical sensor that detects the detection light after filtering by the fluorescent filter 22. The photodetector 21 is preferably one that corresponds to a light emission time of, for example, a nanosecond level, and is, for example, an electron tube element such as a photomultiplier tube (PMT). A control circuit (not shown) is connected to the photomultiplier tube, and the control circuit includes a high-voltage power supply and a gain adjustment circuit that adjusts the power supplied to the electron multiplier section. In this embodiment, the photodetector 21 is described as a photomultiplier tube, but is not limited to this, and may be a semiconductor light receiving element such as a SiPM (Silicon Photomultiplier), an avalanche photodiode (APD), or a photodiode (PD), as well as an electron tube, image sensor, or camera using them, as long as it can respond quickly in response to the modulation frequency of the excitation light from the light source 11 described later. More specifically, the photodetector 21 detects detection light including fluorescence (second light) generated from the immunochromatographic test piece 100 (more specifically, the fluorescent reagent of the antigen-antibody complex in the measurement target portion of the immunochromatographic membrane) irradiated with the excitation light, and the above-mentioned scattered light (third light) that is caused by the excitation light and has no phase difference with the excitation light. The photodetector 21 outputs a detection signal corresponding to the detection light to the IV conversion amplifier 40.

[0023] The light source driving circuit 30 is a circuit that modulates and drives the light source 11 by outputting a driving current to the light source 11. The light source driving circuit 30 receives an input of a sine wave-shaped reference frequency signal (first frequency signal) from a timing generator 70 that outputs a frequency signal. The light source driving circuit 30 modulates the frequency of the driving current based on the input reference frequency signal. That is, the light source driving circuit 30 sets the modulation frequency of the light source 11 that outputs excitation light. In response to this, the frequency of the excitation light output from the light source 11 is modulated, and the amount of light (amount of excitation light) from the light source 11 changes in a sine wave shape. The modulation frequency may be determined based on the fluorescence life of the fluorescent reagent used. For example, when europium, which has a fluorescence life of several milliseconds, is used as the fluorescent reagent, the modulation frequency may be about 1 kHz, when Q-dot, which has a fluorescence life of several tens of nanoseconds, is used, the modulation frequency may be about 1 MHz, and when an organic fluorescent dye, whose fluorescence life is several nanoseconds to several tens of nanoseconds, is used, the modulation frequency may be about 10 MHz. In this embodiment, an organic fluorescent dye is used as the fluorescent reagent, and the modulation frequency (the frequency related to the first frequency signal) is 10.01 MHz.

[0024] Generally, the fluorescence lifetime is defined as the time until the fluorescence intensity falls from the peak value to 1 / e (about 37%). Calculating backwards from this definition of the fluorescence lifetime, for example, the optimal modulation frequency is 1 kHz when europium, which has a fluorescence lifetime of several milliseconds, is used, and the optimal modulation frequency is about 100 MHz to 1 GHz when an organic fluorescent dye, which has a fluorescence lifetime of several nanoseconds to several tens of nanoseconds, is used. However, when the signal output derived from the fluorescence was actually measured with respect to the modulation frequency using a europium reagent, it was found that the fluorescence intensity was higher and the ratio of the fluorescence signal to the excitation light was also higher when modulated at a lower frequency than the frequency determined from the fluorescence lifetime. Specifically, the fluorescence lifetime was defined as "the time until the peak value of the fluorescence intensity falls to 1%" instead of 1 / e, and the modulation frequency was calculated from that time, thereby making it possible to increase the fluorescence intensity. In the case of an organic fluorescent dye, the fluorescence lifetime is nanoseconds, and the modulation frequency of the light source 11 determined from this is about 10 MHz (10.01 MHz in this embodiment).

[0025] The IV conversion amplifier 40 converts the current signal (detection signal) output from the photodetection element 21 into a voltage signal. The IV conversion amplifier 40 outputs the detection signal converted into a voltage signal to the frequency conversion circuit 45.

[0026] The frequency conversion circuit 45 multiplies (calculates) the detection signal output from the IV conversion amplifier 40 by a reference sine wave or rectangular wave frequency signal (second frequency signal) input from the timing generator 70, thereby generating a difference frequency signal which is a signal with a frequency (i.e., 10 kHz) which is the difference between the frequency related to the first frequency signal (i.e., 10.01 MHz) and the frequency related to the second frequency signal (here, 10.00 MHz).

[0027] Here, the frequency of the difference frequency signal is described as 10 kHz, but is not limited thereto. For example, the first frequency signal and the second frequency signal may be set so that the frequency of the difference frequency signal is 10 Hz to 1 MHz, preferably 100 Hz to 500 kHz. Here, the frequency of the first frequency signal is described as being 10 kHz higher than the frequency of the second frequency signal, but conversely, the frequency of the second frequency signal may be higher than the frequency of the first frequency signal. That is, the first frequency signal and the second frequency signal may be set so that the frequency of the difference frequency signal generated by multiplying them is 10 Hz to 1 MHz, preferably 100 Hz to 500 kHz. By setting the frequency of the difference frequency signal to 10 Hz or more, it is possible to prevent the frequency of the difference frequency signal from becoming too low to detect high-speed changes in the sample. Furthermore, by setting the frequency of the difference frequency signal to 1 MHz or less, it is possible to reliably prevent the frequency of the difference frequency signal from becoming too high to properly remove noise. The frequency conversion circuit 45 outputs the generated difference frequency signal to the waveform generation circuit 50.

[0028] The waveform generating circuit 50 is a circuit that generates a waveform of a difference frequency signal based on the difference frequency signal output from the frequency conversion circuit 45. The waveform generating circuit 50 receives an input of a reference sine wavelength frequency signal (third frequency signal) from the timing generator 70. The third frequency signal is a signal of a frequency corresponding to (the same as) the frequency related to the difference frequency signal. That is, here, the third frequency signal is a signal of 10 kHz. Moreover, the third frequency signal is a signal indicating the phase of the excitation light (i.e., the scattered light). Such a phase of the excitation light is used in the process in the cancellation circuit 60. The waveform generating circuit 50 outputs information of the generated waveform (difference frequency signal) to the cancellation circuit 60.

[0029] The cancellation circuit 60 is a signal processing unit that removes a signal component corresponding to scattered light from the difference frequency signal based on the waveform (difference frequency signal) generated by the waveform generating circuit 50 and a frequency signal (third frequency signal) input from the timing generator 70. The cancellation circuit 60 removes a signal component corresponding to scattered light from the difference frequency signal based on the phase difference (phase difference) between the fluorescent light and the scattered light. The cancellation circuit 60 receives an input of a reference frequency signal (third frequency signal) from the timing generator 70 to obtain information on the phase of the excitation light (i.e., the scattered light). This enables the cancellation circuit 60 to remove the signal component of the scattered light based on the phase difference between the fluorescent light and the scattered light. Details of the processing by the cancellation circuit 60 will be described with reference to Figs. 2 to 4.

[0030] FIG. 2 is a conceptual diagram for explaining the phase difference between fluorescence and scattered light. As shown in FIG. 2, the sine wave detection light (detection light detected in the light detection unit D) from the sample S irradiated with the sine wave excitation light from the light source unit L contains sine wave scattered light and fluorescence. The excitation light from the light source unit L is not limited to a sine wave, and may be a periodically modulated waveform such as a rectangular wave. In that case, the detection light (scattered light and fluorescence) also has a periodically modulated waveform similar to that of the excitation light. The scattered light is light that has no phase difference with the excitation light, whereas the fluorescence is light generated from the sample S in response to the excitation light, and is detected with a phase delay of about several tens of milliseconds to nanoseconds with respect to the scattered light. The present inventors have focused on such a phase difference and found a method for removing only the scattered light from the detection light and extracting only the fluorescence. In this embodiment, as described above, the scattered light component is removed not from the detection light but from the difference frequency signal obtained by frequency-converting the detection light.

[0031] FIG. 3 is a diagram for explaining a method for removing (canceling) scattered light. FIG. 3 shows only the waveform of the scattered light out of the difference frequency signal. This waveform is equal to the waveform of the excitation light. In FIG. 3, the horizontal axis shows time and the vertical axis shows amplitude. For the waveform according to the phase of the scattered light shown in FIG. 3, for example, if it is separated into time units of 1 / 4 of one period (separating the time domain), and each of the time domains 1 to 4 is integrated, the output of the scattered light in each of the time domains 1 to 4 can be obtained. Here, if the integral values ​​of each of the time domains 1 to 4 are multiplied by a certain multiplier and all are added up, the sum of the outputs can be made zero. That is, the absolute values ​​of the outputs of the time domains 1 to 4 are the same, the amplitude ranges of the time domains 1 and 2 are positive, and the amplitude ranges of the time domains 3 and 4 are negative. As shown in FIG. 3, when the time domain 1 is amplified by multiplier "-1", the output of the time domain 1 becomes a negative value of "positive x negative", when the time domain 2 is amplified by multiplier "+1", the output of the time domain 2 becomes a positive value of "positive x positive", when the time domain 3 is amplified by multiplier "+1", the output of the time domain 3 becomes a negative value of "negative x positive", and when the time domain 4 is amplified by multiplier "-1", the output of the time domain 4 becomes a positive value of "negative x negative". Therefore, when the integral values ​​of the time domains 1 to 4 amplified by a predetermined multiplier are all added together, the values ​​are offset and the sum of the outputs becomes 0. In this way, the signal components corresponding to the scattered light can be removed (the output can be set to 0) by separating them out at a predetermined time interval corresponding to the phase of the scattered light, amplifying each of the separated components, and then combining the amplified components.

[0032] FIG. 4 is a diagram for explaining a fluorescence measurement method. FIG. 4 shows the waveforms of scattered light and fluorescence contained in the difference frequency signal. In FIG. 4, the horizontal axis shows time, and the vertical axis shows amplitude. As described above, the signal components corresponding to the scattered light can be removed (output is set to 0) by separating them in a predetermined time unit corresponding to the phase of the scattered light, amplifying each of the separated components, and synthesizing each of the amplified components. Here, as shown in FIG. 4, since the fluorescence has a phase difference with respect to the scattered light, when it is separated in a predetermined time unit corresponding to the phase of the scattered light, the integral values ​​of each of the time domains 1 to 4 do not become the same value, so that the same multiplier as that of the scattered light is multiplied, each is amplified, and the sum is outputted as a value that is not 0. In this way, by separating the scattered light and the fluorescence in the same time domain, amplifying and synthesizing them, it is possible to detect and output the output intensity of the fluorescence while removing the signal components of the scattered light.

[0033] In this way, the cancellation circuit 60 separates the difference frequency signal in a predetermined time unit according to the phase of the scattered light, amplifies each component of the separated detection signal, and combines the amplified components to remove the signal component according to the scattered light from the difference frequency signal and obtain the signal component of the fluorescence. The cancellation circuit 60 outputs the fluorescence signal, which is a signal from which the signal component according to the scattered light has been removed (i.e., a signal that contains only the signal component of the fluorescence), to the AD converter 80. Note that, although 1 / 4 of one period has been exemplified as the predetermined time unit, this is not limited thereto, and any time unit may be used as long as the signal component according to the scattered light can be removed after the combination. Furthermore, although "+1" and "-1" have been exemplified as the multipliers in the amplification, this is not limited thereto, and any multiplier may be used as long as the signal component according to the scattered light can be removed after the combination.

[0034] The AD converter 80 performs AD conversion on the fluorescence signal output from the cancellation circuit 60, converting it into a digital value and outputting it to the CPU 90. The CPU 90 performs predetermined control and signal processing on the digital signal (fluorescence signal) output from the AD converter 80. The CPU 90 may transfer the signal processing result to an external computer, for example, by serial communication. The CPU 90 may also generate a signal output from the timing generator 70, i.e., a signal that determines the timing of various operations in the optical measurement device 1, and output it to the timing generator 70. An FPGA may be used instead of the CPU 90. Through the above processing, the optical measurement device 1 can remove the effects of scattered light from the detection light and obtain only a signal related to the fluorescence of the fluorescent reagent.

[0035] Next, the removal of the specific signal component (noise) described in the above embodiment will be described more specifically with reference to FIGS.

[0036] FIG. 5 is a diagram for explaining the removal of noise components. FIG. 5(a) shows the intensity of detection light when the signal component (noise) corresponding to the scattered light caused by the excitation light is not removed, and FIG. 5(b) shows the intensity of detection light when the signal component (noise) corresponding to the scattered light caused by the excitation light is removed. In FIG. 5(a) and FIG. 5(b), the vertical axis shows the intensity of detection light, and the horizontal axis shows the channel showing the position in the measurement section 501 which is the measurement area of ​​the sample 500. One channel is, for example, 0.02 mm. FIG. 5(c) is a diagram showing the area of ​​the sample 500 corresponding to the position of the channel in FIG. 5(a) and FIG. 5(b). FIG. 6 is a schematic diagram showing an enlargement of the sample 500 shown in FIG. 5(c). As shown in FIG. 6, the sample 500 has a dripping section 502 where a specimen is dripped, a holding section 503 that holds a detection antibody labeled with a fluorescent reagent, and a measuring section 501 in which a capture antibody is fixed to a measurement target section 504, which are arranged from upstream to downstream. The fluorescent reagent is, for example, FITC. The measuring section 501 is, for example, a part of a white immunochromatographic membrane, and therefore tends to scatter excitation light.

[0037] When a specimen is dropped into the dropping section 502 of such a sample 500, the specimen moves downstream due to capillary action. If a substance to be detected is present in the specimen, the detection antibody in the holding section 503 reacts with the substance to be detected to form a complex, and this complex moves downstream in the measurement section 501. When the complex reaches the measurement object section 504 on the measurement section 501, the complex is captured by the capture antibody in the measurement object section 504, and a complex is formed by the substance to be detected, the detection antibody, and the capture antibody. In this state, excitation light is irradiated while changing the light collection position (channel) on the measurement section 501, which is the measurement area, so that the detection light intensity for each channel can be derived as shown in Figures 5(a) and 5(b). In Figures 5(a) and 5(b), the channel in which the detection light intensity is greater than the others is the channel corresponding to the position of the measurement object section 504 in which the complex is captured.

[0038] As shown in Fig. 5(a), when the signal component (noise) corresponding to the scattered light is not removed, the detected light contains not only the fluorescent light but also the scattered light, so the detected light intensity is high. Such noise increases with increasing the amount of excitation light, so that, as shown in Fig. 5(a), doubling the amount of excitation light also doubles the noise. Generally, a method of increasing the amount of excitation light to increase the amount of fluorescent signal is considered as a method of improving the S / N ratio. However, as described above, in the case of Fig. 5(a) where the noise also increases according to the amount of excitation light, it is difficult to improve the S / N ratio. Furthermore, there is also the problem that the dynamic range is narrowed by increasing the amount of excitation light.

[0039] On the other hand, as shown in FIG. 5(b), when the signal component (noise) corresponding to the scattered light is removed, the detection light contains mostly only fluorescence, and only the signal to be detected (signal based on fluorescence) can be detected. In this case, since the noise is almost zero, even if the amount of excitation light is increased (for example, doubled) as shown in FIG. 5(b), the effect of the excitation light (scattered light) can be canceled to almost zero as long as the photodetector is not saturated, and the noise does not become extremely large. As described above, in the configuration for removing noise as shown in FIG. 5(b), when the excitation light is increased, the noise component can be canceled to almost zero and only the signal component can be increased, leading to an improvement in the S / N ratio. Since the configuration can cancel the effect of the excitation light (scattered light) to almost zero, it is possible to increase the amount of excitation light and increase the multiplication factor of the IV conversion amplifier and the photomultiplier tube. As a result, only the signal component can be increased. For convenience of explanation, it has been described here that the noise components are removed from the detection light. In detail, however, as described above, the noise components are removed from the difference frequency signal obtained by frequency-converting the detection light.

[0040] FIG. 7 is a diagram for explaining the definition of S / N. FIG. 7 shows an example of the intensity of the detected light for each channel (the intensity of the detected light at each position in the measurement area). As shown in FIG. 7, there is a fluctuation component of about ±4 around the intensity of the detected light of 10 counts. Such a fluctuation (standard deviation) of the base light amount is a value obtained by scanning the excitation light on the measurement unit 501 on which no fluorescent substance or the like is applied (or the measurement unit 501 in a wet state similar to the measurement state). Hereinafter, the fluctuation of the base light amount is defined as noise N. In addition, signal S is defined as "a value obtained by subtracting the average value of the noise components excluding the position of the measurement object part 504 in all channels from the peak fluorescence intensity of the measurement object part 504". S / N is defined as a value obtained by dividing the signal S defined above by noise N.

[0041] In the example shown in FIG. 7, the noise N value is offset by about 10 counts. In principle, the noise N value is cancelled to almost 0 by performing a calibration process described later. However, since the background varies according to the noise N value, it is preferable that the signal always be a positive value from the viewpoint of software analysis, and therefore background offset processing is performed. The offset amount is set so that the signal falls within the dynamic range (0 to 4096 counts). The offset amount is set so that the background signal obtained by scanning the measurement unit 501 with the excitation light is always (almost certainly) a positive value while being as small as possible from the viewpoint of the dynamic range. Specifically, the offset amount may be, for example, the average intensity value of the detection light obtained by scanning the measurement unit 501 with no fluorescent material or the like (or the measurement unit 501 in a wet state similar to the measurement state) with the excitation light + 6σ of the average intensity value. In addition, in preparation for the case where an unexpected external noise jumps into the circuit system, an appropriate margin may be added to the offset amount calculated above to obtain the final offset amount. The offset amount is selected so as not to sacrifice the dynamic range and so as not to output a negative signal, and may be, for example, about +20 counts.

[0042] Next, a method for removing the signal components (noise) corresponding to the scattered light will be specifically described. In the optical measurement device 1, a calibration process is performed in the cancellation circuit 60, which is a lock-in circuit, and the signal components (noise) corresponding to the scattered light are removed from the difference frequency signal while taking into account the results of the calibration process.

[0043] Specifically, in the optical measurement method using the optical measurement device 1, first, the optical head of the irradiation optical system 10 is positioned so that the excitation light is irradiated onto a location other than the measurement target portion 504 of the sample 500. Next, scattered light (excitation light components scattered at the measurement portion 501, etc.) caused by the excitation light from the irradiation optical system 10 being irradiated onto the measurement portion 501, etc. is detected by the detection optical system 20. Here, the light detected by the detection optical system 20 is basically only scattered light that does not contain fluorescence at least in the measurement target portion 504, and is calibration process light used for the calibration process.

[0044] The acquisition of the calibration light will be described in more detail. The calibration light may be detected by irradiating an area downstream of the capture antibody fixed on the measurement object portion 504 with excitation light. FIG. 10 is a diagram for explaining the fluorescence intensity in each region of the sample 500. In FIG. 10, the portion indicated as "Line A" indicates the portion of the measurement object portion 504. In the explanation of FIG. 5(a) and FIG. 5(b), etc., the fluorescence intensity other than the measurement object portion 504 is shown as small for convenience of explanation. In reality, as shown in FIG. 10, the fluorescence intensity is likely to be large on the upstream side where the specimen is dropped even if the capture antibody is not present. For this reason, it is preferable that the calibration light, which is desired to be only scattered light, is detected by irradiating an area downstream of the capture antibody fixed on the measurement object portion 504 with excitation light. The calibration light may be acquired using a case for accommodating an immunochromatographic membrane or a separately prepared member for calibration processing, other than the measurement portion 501 in the sample 500.

[0045] Next, a calibration process is performed. Specifically, the cancellation circuit 60 of the optical measurement device 1 performs a calibration process for removing a signal component corresponding to the scattered light from the difference frequency signal based on a calibration signal corresponding to the above-mentioned calibration process light. The calibration process will be described in detail later. After the calibration process is completed, the measurement area (measurement unit 501) of the sample 500 is scanned with the optical head of the irradiation optical system 10 to obtain fluorescence information of the measurement unit 501. Specifically, the cancellation circuit 60 removes a signal component corresponding to the scattered light from the difference frequency signal in consideration of the results of the above-mentioned calibration process, thereby obtaining fluorescence information.

[0046] Next, the calibration process will be described in detail. The cancellation circuit 60 of the optical measurement device 1 is a lock-in circuit using, for example, an FPGA (Field Programmable Gate Array). In the calibration process, the cancellation circuit 60 generates a lock-in switch signal whose phase is shifted with respect to a periodic signal whose period is determined by the operation frequency of the cancellation circuit 60, which is matched to the modulation frequency of the light source 11 (for example, the frequency of a DDS (Direct Digital Synthesizer)) set by the light source drive circuit 30. The cancellation circuit 60, which functions as a lock-in circuit, receives as input a calibration signal, which is a measurement signal, and a switch signal, which is a reference signal, outputs a signal component corresponding to the scattered light, and adjusts the phase of the switch signal so that the voltage value of the signal component corresponding to the scattered light is within a predetermined range (threshold level) close to 0.

[0047] FIG. 8 shows signals used for calibration processing inside the FPGA of the cancellation circuit 60. The periodic signal shown in FIG. 8 is a clock signal that has a period in accordance with the frequency of the DDS as described above. The reference signal is a signal that has an arbitrary phase from the periodic signal (shifted in phase with respect to the periodic signal) and has the same frequency as the periodic signal, and is a trigger for the XY signal described later. The XY signal is a switch signal for lock-in described above, and is a signal that is generated using the reference signal as a trigger. The X signal is a signal that has no phase difference with the reference signal. The Y signal is a signal that is shifted in phase by 90 degrees with respect to the reference signal. In addition to the X signal and the Y signal, the cancellation circuit 60 actually generates an X' signal that is an inverted X signal, and a Y' signal that is an inverted Y signal. The X signal, the Y signal, the X' signal, and the Y' signal are each generated by an independent dedicated circuit. Adjusting the phase of the switch signal so that the voltage value of the signal component corresponding to the scattered light becomes the threshold level means continuing to shift the phase of the reference signal with respect to the periodic signal until the output from the cancellation circuit 60 becomes 0V (or a value close to that).

[0048] FIG. 9 is a diagram for explaining a process of adjusting the output to 0V by shifting the phase of the reference signal with respect to the periodic signal. Now, assume that the initial phase relationship of the periodic signal, the reference signal, and the switch signal is as shown in FIG. 9(a). Then, assume that an integration process is performed in the shaded section in FIG. 9 based on the switch signal, and the output (the voltage value of the signal component corresponding to the scattered light) is not at the threshold level and is a positive value. In this case, as shown in FIG. 9(b), the phase of the reference signal is adjusted to delay the phase of the switch signal. That is, in the calibration process, when the voltage value of the signal component corresponding to the scattered light is not at the threshold level and is a positive value, the cancellation circuit 60 adjusts the phase of the switch signal to delay it.

[0049] Now, suppose that even in the state of Fig. 9(b) where the phase of the switch signal has been adjusted, the integration process for the shaded section results in an output (the voltage value of the signal component corresponding to the scattered light) that is not at the threshold level and is a positive value. In this case, as shown in Fig. 9(c), the phase of the reference signal is adjusted so as to further delay the phase of the switch signal.

[0050] Now, suppose that even in the state of Fig. 9(c) where the phase of the switch signal has been adjusted, the integration process for the shaded section results in an output (the voltage value of the signal component corresponding to the scattered light) that is not at the threshold level and is a positive value. In this case, as shown in Fig. 9(d), the phase of the reference signal is adjusted so as to further delay the phase of the switch signal.

[0051] Now, in the state of Fig. 9(d) where the phase of the switch signal has been adjusted, suppose that as a result of integration processing in the shaded section, the output (the voltage value of the signal component corresponding to the scattered light) is not at the threshold level and is a negative value. In this case, as shown in Fig. 9(e), the phase of the reference signal is adjusted so as to advance the phase of the switch signal. That is, in the calibration processing, when the voltage value of the signal component corresponding to the scattered light is not at the threshold level but is a negative value, the cancellation circuit 60 adjusts the phase of the switch signal to advance it.

[0052] Then, as a result of adjusting the phase of the switch signal to advance, the calibration process is completed when the output (the voltage value of the signal component corresponding to the scattered light) becomes the threshold level (a value within a predetermined range approximating 0) as a result of the integration process of the shaded section, as shown in Figure 9(e).

[0053] When the calibration process is completed, the cancellation circuit 60 receives as input a difference frequency signal corresponding to the detection light containing fluorescent components and scattered light components (excitation light components) and a switch signal whose phase has been adjusted in the calibration process, and removes the signal component corresponding to the scattered light component from the difference frequency signal.

[0054] FIG. 11 is a flow chart showing the calibration process. As shown in FIG. 11, in the calibration process, first, the input to the AD converter 80 is switched to a predetermined offset voltage and a 0 level is stored (step S1). Then, the signal of the cancellation circuit 60 is input to the AD converter 80 by switching the switch (step S2). In this state, the phase of the reference signal is forcibly shifted once (step S3). The output of the cancellation circuit 60 becomes 0V when the phase of the reference signal relative to the periodic signal is 0 degrees and 180 degrees. If the phase coincides with 180 degrees in the initial state, the calibration process is erroneously completed, and the positive and negative of the output signal are inverted, so that the output signal may not be detected depending on the configuration of the subsequent circuit. In this regard, by forcibly shifting the phase of the reference signal at the start, it is possible to prevent the calibration process from being erroneously completed. Also, by matching the phase at the start in this way, the positive and negative of the output signal are fixed. As a result, a sign bit is not required when the output signal is converted into a digital value, and the dynamic range of the AD converter 80 can be used effectively. Furthermore, when it is desired to perform measurement with a negative output, the phase at which calibration is completed may be set to 180 degrees instead of 0 degrees.

[0055] When step S3 is completed, the current input value of the AD converter 80 is recorded (step S4), and a loop process of calibration is executed. First, the current input value of the AD converter 80 is compared with the 0 level to determine whether the input value of the AD converter 80 is smaller than the 0 level (is a negative value) (step S5). If it is determined in step S5 that the input value of the AD converter 80 is a negative value, the phase of the switch signal (i.e., the reference signal) of the cancellation circuit 60 with respect to the periodic signal according to the frequency of the DDS is advanced (step S6). On the other hand, if it is determined in step S5 that the input value of the AD converter 80 is a positive value, the phase of the switch signal (i.e., the reference signal) of the cancellation circuit 60 with respect to the periodic signal is delayed (step S7).

[0056] Then, it is determined whether the input value to the AD converter 80 has reached the threshold level without changing its sign (step S8). If it is determined in step S8 that the threshold level has been reached without changing its sign, the calibration process ends. On the other hand, if it is determined in step S8 that the condition is not satisfied, it is determined whether the sign of the input to the AD converter 80 has changed due to the phase shift (step S9). If it is determined in step S9 that it has not changed, the process of step S4 is performed again, and if it is determined that it has changed, the amount of phase change caused by the control is changed to half the current amount (step S10), and the process of step S4 is performed again. This completes the calibration process.

[0057] Next, the effects of the optical measurement device 1 described above will be described.

[0058] The optical measurement device 1 of this embodiment includes a timing generator 70 that outputs a frequency signal, a light source 11 that irradiates excitation light toward the immunochromatographic test piece 100, a light source driving circuit 30 that modulates and drives the light source 11 based on a first frequency signal input from the timing generator 70, a detection optical system 20 that detects detection light including fluorescence generated from the immunochromatographic test piece 100 irradiated with the excitation light and scattered light that is caused by the excitation light and has the same phase as the excitation light, and outputs a detection signal, a frequency conversion circuit 45 that multiplies the detection signal by the second frequency signal input from the timing generator 70 to generate a difference frequency signal that is a signal with a frequency that is the difference between the frequency related to the first frequency signal and the frequency related to the second frequency signal, and a cancellation circuit 60 that removes a signal component corresponding to the scattered light from the difference frequency signal based on the difference frequency signal and a third frequency signal input from the timing generator 70, and the third frequency signal is a signal with a frequency that corresponds to the frequency related to the difference frequency signal.

[0059] In the optical measurement device 1 according to the present embodiment, detection light including fluorescence and scattered light is detected, and a detection signal corresponding to the detection light is multiplied by a second frequency signal to generate a difference frequency signal between the first frequency signal and the second frequency signal. Conventionally, a method has been known in which a noise component caused by the excitation light itself is removed by removing a phase component of the excitation light (scattered light) from a detection signal of the detection light including fluorescence and scattered light. Here, when the emission time of the light (fluorescence) to be measured is shortened (a fluorescent reagent with a short fluorescence lifetime is used), and the signal to be processed by the circuit is accelerated, for example, when the signal becomes a high-speed signal of 10 MHz or more, depending on the processing capacity of the circuit used, the noise component may not be appropriately removed only by the above-mentioned method.

[0060] FIG. 12 is a diagram for explaining the fluorescence excitation spectrum of europium and an organic fluorescent dye (FITC). FIG. 12(a) shows the wavelength characteristics of the excitation light and fluorescence of europium. FIG. 12(b) shows the wavelength characteristics of the excitation light and fluorescence of an organic fluorescent dye. In FIG. 12(a) and FIG. 12(b), the horizontal axis shows the wavelength, and the vertical axis shows the light intensity. As shown in FIG. 12(a), in the case of europium, which has a relatively long fluorescence life (millisecond level), for example, the wavelength band of the excitation light is around 365 nm, the wavelength band of the fluorescence is around 615 nm, and the modulation speed is about 100 Hz. In this case, the modulation speed is slow and the difficulty of circuit design (processing power required for the circuit) is not high, so it is possible to remove the phase component of the excitation light (scattered light) from the detection signal.

[0061] On the other hand, as shown in Figure 12(b), in the case of an organic fluorescent dye with a relatively short fluorescence lifetime (nanosecond level), for example, the wavelength band of the excitation light is around 488 nm, the wavelength band of the fluorescence is around 520 nm, and the modulation speed is 10 MHz, which is about 100,000 times that of europium. In this case, the modulation speed is fast, and the difficulty of circuit design (the processing power required for the circuit) is high, so it may be difficult to remove the phase component of the excitation light (scattered light) from the detection signal. The difficulty of circuit design is not simply due to the processing power issue, but also due to the susceptibility to noise and the inability to increase the multiplication factor.

[0062] In this regard, in the optical measurement device 1 according to the present embodiment, the detection signal is multiplied by the second frequency signal to generate a difference frequency signal between the first frequency signal and the second frequency signal. Such a difference frequency signal is a signal that is sufficiently slower than the detection signal. Then, the difference frequency signal and the third frequency signal are input to the cancellation circuit 60, so that the signal component corresponding to the third light (i.e., the noise component caused by the excitation light itself) is removed from the difference frequency signal. In this way, the detection signal is frequency converted, and the noise component caused by the excitation light itself is removed from the difference frequency signal after frequency conversion, so that the noise component can be appropriately removed even from a high-speed signal related to an organic fluorescent dye or the like having a relatively short fluorescence lifetime.

[0063] The effect of appropriately removing the scattered light (noise) in this manner will be described with reference to FIG. 13. FIG. 13 is a diagram for explaining the effect of appropriately removing the scattered light (noise). In FIG. 13, the horizontal axis indicates the measurement position, and the vertical axis indicates the light intensity. The thick solid line indicates the result (measurement result of fluorescence and excitation light) of a sample in which an organic fluorescent dye (FITC) is injected as a fluorescent reagent into a micro-channel on the light scatterer in the configuration according to this embodiment, the dashed line indicates the result (measurement result of only excitation light) of a sample in which a dilute solution not containing a fluorescent reagent is injected into a micro-channel on the light scatterer in the configuration according to this embodiment, the thin solid line indicates the result (measurement result of fluorescence and excitation light) of a sample in which an organic fluorescent dye (FITC) is injected as a fluorescent reagent into a micro-channel on the light scatterer in the configuration according to the comparative example, and the dotted line indicates the result (measurement result of only excitation light) of a sample in which a dilute solution not containing a fluorescent reagent is injected into a micro-channel on the light scatterer in the configuration according to the comparative example. The difference between the thick solid line and the dashed line is the amount of fluorescence (signal) in this embodiment, and the difference between the thin solid line and the dotted line is the amount of fluorescence (signal) in the comparative example. As shown by the thin solid line and the dotted line in FIG. 13, in the configuration of the comparative example, the baseline rises due to the background (BKG) of the excitation light (scattered light). In contrast, as shown by the thick solid line and the dashed line in FIG. 13, in the configuration according to this embodiment, the baseline does not rise even if the excitation light is scattered by the light scatterer, so only the minimum offset for software processing (processing to make all signal values ​​positive) is performed. Compared with the measurement results of the configuration according to the comparative example, the noise is about 1 / 10 and the signal is about 6.7 times. In this way, when the noise is removed, the amount of baseline offset is small, so that the amount of excitation light from the light source and the amplification factor of the IV conversion amplifier and the photomultiplier tube can be increased, and the signal intensity can be suitably increased. As a result, the S / N can be improved by about 70 times compared to when the noise is not removed.

[0064] In the optical measurement device 1 according to this embodiment, the first light is excitation light that excites the sample, the second light is fluorescence emitted by the sample in response to the excitation light, and the third light includes at least one of the excitation light and scattered light of the excitation light. This makes it possible to appropriately remove signal components corresponding to at least one of the excitation light and scattered light of the excitation light from a difference frequency signal corresponding to a detection signal including signal components corresponding to the fluorescence and at least one of the excitation light and scattered light of the excitation light, and to appropriately extract only the signal components corresponding to the fluorescence.

[0065] The frequency of the difference frequency signal may be within a range of 10 Hz to 1 MHz. By setting the frequency of the difference frequency signal within such a range, the frequency related to noise removal is prevented from becoming too high, and the noise component can be removed appropriately.

[0066] Although the optical measurement device 1 according to the present embodiment has been described above, the configuration according to the present disclosure is not limited to the above optical measurement device 1. For example, as shown in Fig. 14, frequency filters may be provided on the input side and output side of the frequency conversion circuit 45.

[0067] 14, a band-pass filter 117 (BPF) which is a first frequency filter is provided on the input side (between the IV conversion amplifier 40 and the frequency conversion circuit 45) of the frequency conversion circuit 45, and a band-pass filter 116 (BPF) which is a second frequency filter is provided on the output side (between the frequency conversion circuit 45 and the waveform generation circuit 50) of the frequency conversion circuit 45. In this way, by using the band-pass filter 117, it is possible to selectively pass only a desired frequency band (for example, 10.01 MHz), and it is possible to more appropriately suppress the inclusion of noise and to suppress the above-mentioned rise in the baseline.

[0068] The optical measurement device according to the present disclosure can be applied to various applications. The optical measurement device according to the present disclosure may be applied to, for example, the measurement optical system of a flow cytometer. By applying the optical measurement device according to the present disclosure to the fluorescence measurement in the flow cytometer, it is possible to cancel the excitation light (and scattered light) and selectively obtain only the fluorescence. Normally, in a miniaturized flow cytometer, it is difficult to remove the excitation light, but the optical measurement device according to the present disclosure cancels the excitation light, so that the flow cytometer can be appropriately miniaturized. Similarly, the optical measurement device according to the present disclosure may be applied to a microplate reader, PCR, a microscope, HPLC, or the like.

[0069] Finally, various exemplary embodiments included in the present disclosure are described in [E1] to [E6] below.

[0070] [E1] a timing generator that outputs a frequency signal; a light source unit that irradiates a first light toward the sample; a light source drive circuit that drives the light source unit in a modulated manner based on a first frequency signal input from the timing generator; a light detection unit that detects detection light including a second light generated from the sample irradiated with the first light and a third light caused by the first light and having a phase equal to that of the first light, and outputs a detection signal; a frequency conversion circuit that performs an operation on the detection signal and a second frequency signal input from the timing generator to generate a difference frequency signal that is a signal having a difference frequency between a frequency related to the first frequency signal and a frequency related to the second frequency signal; a signal processing unit that removes a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal input from the timing generator, An optical measurement device, wherein the third frequency signal is a signal having a frequency corresponding to a frequency related to the difference frequency signal.

[0071] [E2] the first light is excitation light that excites the sample, the second light is fluorescence emitted by the sample in response to the excitation light, The third light includes at least one of the excitation light and scattered light of the excitation light. [E1] The optical measurement device described herein.

[0072] [E3] The optical measuring device according to [E1] or [E2], wherein the frequency of the difference frequency signal is within a range of 10 Hz to 1 MHz.

[0073] [E4] The optical measurement device according to any one of [E1] to [E3], further comprising a first frequency filter provided on the input side of the frequency conversion circuit and a second frequency filter provided on the output side of the frequency conversion circuit, wherein the first frequency filter and the second frequency filter are band-pass filters.

[0074] [E5] irradiating a first light toward a sample based on a first frequency signal; detecting a detection light including a second light generated from the sample irradiated with the first light and a third light caused by the first light and having a phase equal to that of the first light, and outputting a detection signal; generating a difference frequency signal, which is a signal having a difference frequency between a frequency related to the first frequency signal and a frequency related to the second frequency signal, by performing an operation on the detection signal and a second frequency signal; removing a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal; An optical measurement method, wherein the third frequency signal is a signal having a frequency corresponding to a frequency related to the difference frequency signal. [Explanation of symbols]

[0075] 1...optical measurement device, 10...irradiation optical system (light source section), 20...detection optical system (light detection section), 30...light source driving circuit, 45...frequency conversion circuit, 60...cancellation circuit (signal processing section), 70...timing generator.

Claims

1. a timing generator that outputs a frequency signal; a light source unit that irradiates a first light toward the sample; a light source drive circuit that drives the light source unit by modulating the light source unit based on a first frequency signal input from the timing generator; a light detection unit that detects detection light including a second light generated from the sample irradiated with the first light and a third light caused by the first light and having a phase equal to that of the first light, and outputs a detection signal; a frequency conversion circuit that performs an operation on the detection signal and a second frequency signal input from the timing generator to generate a difference frequency signal that is a signal having a difference frequency between a frequency related to the first frequency signal and a frequency related to the second frequency signal; a signal processing unit that removes a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal input from the timing generator, An optical measurement device, wherein the third frequency signal is a signal having a frequency corresponding to a frequency related to the difference frequency signal.

2. the first light is excitation light that excites the sample, the second light is fluorescence emitted by the sample in response to the excitation light, The optical measurement device according to claim 1 , wherein the third light includes at least one of the excitation light and scattered light of the excitation light.

3. 2. The optical measurement device according to claim 1, wherein the frequency of the difference frequency signal is within a range of 10 Hz to 1 MHz.

4. 2. The optical measurement device according to claim 1, further comprising a first frequency filter provided on the input side of the frequency conversion circuit and a second frequency filter provided on the output side of the frequency conversion circuit, the first frequency filter and the second frequency filter being bandpass filters.

5. irradiating a first light toward a sample based on a first frequency signal; detecting a detection light including a second light generated from the sample irradiated with the first light and a third light caused by the first light and having a phase equal to that of the first light, and outputting a detection signal; generating a difference frequency signal, which is a signal having a difference frequency between a frequency related to the first frequency signal and a frequency related to the second frequency signal, by performing an operation on the detection signal and a second frequency signal; removing a signal component corresponding to the third light from the difference frequency signal based on the difference frequency signal and a third frequency signal; An optical measurement method, wherein the third frequency signal is a signal having a frequency corresponding to a frequency related to the difference frequency signal.

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