Optical measurement device and optical measurement method

Abstract

An optical measurement device 1 comprises: a timing generator 70; a light source 11; a detection optical system 20 that detects detection light including fluorescence and scattered light, and outputs a detection signal; a frequency conversion circuit 45 that multiplies the detection signal by a second frequency signal inputted from the timing generator 70 to generate a difference frequency signal that is a signal of a frequency difference between a frequency related to a first frequency signal and a 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, on the basis of the difference frequency signal and a third frequency signal. The third frequency signal is a signal of a frequency corresponding to a frequency related to the difference frequency signal.

Description

Optical measurement device and optical measurement method

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

[0002] Patent Document 1 discloses a technique for generating and subtracting an attenuation signal to remove a fluorescence noise component (fluorescence component from the substrate) contained in a fluorescence sample placed on a substrate when the fluorescence is measured by irradiating the fluorescence sample with excitation light. Specifically, Patent Document 1 generates an attenuation signal that is in phase with the fluorescence from the substrate, and subtracts the attenuation signal from the measured fluorescence signal to remove the fluorescence noise component.

[0003] Special Publication No. 2010-518394

[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 a sample with excitation light. Here, noise components contained when irradiating a sample with light and detecting light from the sample may include not only noise components originating from light (e.g., fluorescence) generated from the sample (the irradiated object), but also light (e.g., scattered light) originating from the irradiated light (e.g., excitation light) itself. The technology in Patent Document 1 does not take into account cases where the irradiated light itself becomes a noise component, and is therefore unable to remove such noise components.

[0005] Furthermore, even when noise components of the irradiated light itself are removed by some method, if the light emission time (emission duration) from the sample becomes short, for example, on the order of nanoseconds, 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 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.

[0007] An optical measurement device according to one aspect 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 drive 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 second light generated from the sample irradiated with the first light and third light that is caused by the first light and has the same phase as the first light, and outputs a detection signal, a frequency conversion circuit that generates a difference frequency signal that is a signal with a frequency that is the difference between the frequency associated with the first frequency signal and the 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, wherein the third frequency signal is a signal with a frequency that corresponds to the frequency associated with the difference frequency signal.

[0008] In an optical measurement device according to one aspect of the present invention, detection light including second and third light is detected, and a detection signal corresponding to the detection light and a second frequency signal are calculated to generate a difference frequency signal between the first frequency signal and the second frequency signal. Conventionally, a method has been known in which noise components caused by the irradiated light (first light) itself are removed by removing the phase component of the third light from the detection signal of the detection light including the second and third light. However, if the emission time of the light to be measured is shortened and the signal processed by the circuit becomes faster, for example, a high-speed signal of 10 MHz or higher, depending on the processing capacity of the circuit used, the above-described method alone may not be able to adequately remove the noise components. In this regard, in an optical measurement device according to one aspect of the present invention, a second frequency signal is calculated from 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 significantly slower than 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 way, 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 the noise component can be appropriately removed even when the emission time of the light to be measured is short.

[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 that includes 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 component 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 this range, the frequency involved in noise removal can be prevented from becoming too high, and noise components can be removed appropriately.

[0011] The optical measurement device may further include 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. By providing frequency filters on the input / output sides of the frequency conversion circuit in this manner, noise can be suppressed from being included in the detection signal, the second frequency signal, and the difference frequency signal. Furthermore, by using a band-pass filter, only a desired frequency band can be selectively passed, thereby more appropriately suppressing noise from being included.

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

[0013] According to one aspect of the present invention, it is possible 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.

[0014] FIG. 1 is a schematic configuration diagram of an optical measurement device according to an embodiment of the present invention. FIG. 2 is a diagram explaining the phase difference between fluorescence and scattered light. FIG. 3 is a diagram explaining a scattered light cancellation method. FIG. 4 is a diagram explaining a fluorescence measurement method. FIG. 5 is a diagram explaining the removal of noise components. FIG. 6 is a diagram schematically showing a sample. FIG. 7 is a diagram explaining the definition of S / N. FIG. 8 is a diagram explaining signals used in calibration processing. FIG. 9 is a diagram explaining calibration processing. FIG. 10 is a diagram explaining details of fluorescence intensity in each region of the sample. A flowchart showing calibration processing. FIG. 11 is a diagram explaining fluorescence excitation spectra of europium and an organic fluorescent dye (FITC). FIG. 12 is a diagram explaining the effect of appropriately removing scattered light (noise). FIG. 13 is a diagram explaining the device configuration according to a modified example.

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In each drawing, the same or corresponding parts are designated by the same reference numerals, and redundant 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 will be 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 emitted by the sample in response to the excitation light, and is light of a different wavelength from the excitation light. In addition, in this embodiment, the optical measurement device 1 will be described as a device that detects fluorescence related to measurement using immunochromatography. The immunochromatography 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 measurements using immunochromatography, an immunochromatographic test strip 100 is prepared as a sample. The immunochromatographic test strip 100 contains an immunochromatographic membrane, which serves as 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) on the immunochromatographic membrane of the immunochromatographic test strip 100. The reagent holder 101 is provided with a sample application window, which is an opening for dripping the sample onto the immunochromatographic membrane, and a measurement window, which is an opening for measuring the measurement target portion where the capture antibody is fixed. When a sample is dripped 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, resulting in the antigen being 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 the antigen-antibody complex (more specifically, the antibody fluorescent reagent) in the measurement target area. Examples of the fluorescent reagent that can be used include europium, Q-dot (registered trademark), and organic fluorescent dyes. Examples of fluorescent reagents that emit light (fluorescence) with a short emission time (a fluorescence lifetime of several nanoseconds to several tens of nanoseconds) include organic fluorescent dyes such as FITC (fluorescein isothiocyanate).

[0018] Here, the detection light incident on and detected by the detection optical system 20 (described later) in the optical measurement device 1 may include not only fluorescence but also light resulting from the excitation light itself. Examples of such light include scattered light of the excitation light. Such scattered light is, for example, a portion of the excitation light generated when the excitation light is irradiated onto the immunochromatographic test strip 100 and scattered, and is light having a phase substantially equal to that of the excitation light. The immunochromatographic membrane and reagent holder 101 of the immunochromatographic test strip 100 are generally white, which makes the above-mentioned scattered light more likely to occur. Furthermore, depending on the sample being measured and the arrangement of the detection optical system, the excitation light itself may also be detected. In the following description, the detection light detected by the optical measurement device 1 is assumed to include fluorescence and scattered light.

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

[0020] The illumination optical system 10 irradiates excitation light toward the immunochromatographic test piece 100. The illumination optical system 10 includes a light source 11, an aperture 12, an excitation light filter 13, and a collimating lens 14. The light source 11 irradiates excitation light (first light) toward the immunochromatographic test piece 100 (sample). The light source 11 has a response speed faster than the modulation frequency. In this embodiment, a light source compatible with high-speed drive signals, for example, 10 MHz or higher, is preferred. 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 beam shaping member for shaping the light emitted from the light source 11 into light having a desired beam cross section. The excitation light filter 13 is a wavelength-selective filter that filters out wavelengths required for excitation from the excitation light that reaches the light source 100 via 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 focuses the excitation light filtered by the excitation light filter 13 onto the immunochromatographic test piece 100 (more specifically, the measurement target portion of the immunochromatographic membrane).

[0021] The detection optical system 20 detects fluorescence from the immunochromatographic test strip 100. However, in reality, the detection optical system 20 receives detection light, which is light including not only fluorescence from the immunochromatographic test strip 100 (fluorescence from the measurement target portion of the immunochromatographic membrane) but also scattered light caused by the excitation light itself, and detects the detection light. The detection optical system 20 includes 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 via the fluorescence filter 22. The fluorescence filter 22 is a wavelength-selective filter provided to prevent light other than fluorescence from reaching the photodetector 21 in the detection light from the immunochromatographic test strip 100. The fluorescence filter 22 is an optical filter such as a dielectric multilayer filter or a colored glass filter, and more specifically, is a bandpass filter that combines a dielectric multilayer filter and a colored glass filter that transmits only a specific wavelength band. 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. Furthermore, dielectric multilayer filters, which are generally used as efficient wavelength-selective filters, have characteristics that 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, so that scattered light from oblique directions is effectively blocked by the colored glass filter. However, wavelength selection alone is still insufficient to achieve a sufficient effect, and it is difficult to efficiently prevent the entry of scattered light under various conditions. In the following description, it is assumed that scattered light is still included in the detection light reaching the photodetector element 21 even with the fluorescence filter 22.

[0022] The photodetector element 21 is an optical sensor that detects the detection light filtered by the fluorescence filter 22. The photodetector element 21 is preferably one that can emit light at a nanosecond level, for example, and is 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 element 21 is described as a photomultiplier tube, but is not limited thereto. As long as it can respond quickly to the modulation frequency of the excitation light from the light source 11 (described later), the photodetector element 21 may be a semiconductor light-receiving element such as a silicon photomultiplier (SiPM), an avalanche photodiode (APD), or a photodiode (PD), or an electron tube, image sensor, or camera using such a semiconductor light-receiving element. Specifically, the photodetector element 21 detects detection light that includes fluorescence (second light) generated from the immunochromatographic test piece 100 (specifically, the fluorescent reagent of the antigen-antibody complex in the measurement target portion of the immunochromatographic membrane) irradiated with 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 element 21 outputs a detection signal corresponding to the detection light to the I-V conversion amplifier 40.

[0023] The light source drive circuit 30 is a circuit that modulates and drives the light source 11 by outputting a drive current to the light source 11. The light source drive circuit 30 receives a reference sine-wave frequency signal (first frequency signal) from a timing generator 70 that outputs a frequency signal. The light source drive circuit 30 modulates the frequency of the drive current based on the input reference frequency signal. That is, the light source drive 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 (excitation light amount) from the light source 11 changes in a sinusoidal manner. The modulation frequency may be determined based on the fluorescence lifetime of the fluorescent reagent used. For example, when europium, which has a fluorescence lifetime of several milliseconds, is used as the fluorescent reagent, the modulation frequency may be approximately 1 kHz. When Q-dot, which has a fluorescence lifetime of several tens of nanoseconds, is used, the modulation frequency may be approximately 1 MHz. When an organic fluorescent dye, whose fluorescence lifetime is several nanoseconds to several tens of nanoseconds, is used, the modulation frequency may be approximately 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 it takes for the fluorescence intensity to drop to 1 / e (approximately 37%) from its peak value. Working backward from this definition of fluorescence lifetime, it is believed that the optimal modulation frequency is 1 kHz when using europium, which has a fluorescence lifetime of several milliseconds, and that the optimal modulation frequency is approximately 100 MHz to 1 GHz when using an organic fluorescent dye, whose fluorescence lifetime is several nanoseconds to several tens of nanoseconds. However, when the fluorescence-derived signal output versus modulation frequency was actually measured using a europium reagent, it was found that modulation at a frequency lower than that determined by the fluorescence lifetime resulted in higher fluorescence intensity and a greater ratio of the fluorescence signal to the excitation light. Specifically, by defining the fluorescence lifetime as "the time it takes for the fluorescence intensity to drop to 1% of its peak value" rather than as 1 / e and calculating the modulation frequency from that time, it was possible to increase the fluorescence intensity. For organic fluorescent dyes, the fluorescence lifetime is nanoseconds, and the modulation frequency of the light source 11 determined from this is approximately 10 MHz (10.01 MHz in this embodiment).

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

[0026] The frequency conversion circuit 45 multiplies (calculates) the detection signal output from the I-V 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) that is the difference between the frequency associated with the first frequency signal (i.e., 10.01 MHz) and the frequency associated with the second frequency signal (10.00 MHz in this case).

[0027] Here, the frequency of the difference frequency signal is described as 10 kHz, but this is not limiting. 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. Although the frequency of the first frequency signal is described as being 10 kHz higher than the frequency of the second frequency signal, 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 higher, it is possible to prevent the frequency of the difference frequency signal from becoming too low and thus making it impossible to detect rapid changes in the sample. Furthermore, by setting the frequency of the difference frequency signal to 1 MHz or lower, it is possible to reliably prevent the frequency related to noise removal from becoming too high and making it impossible to properly remove noise. The frequency conversion circuit 45 outputs the generated difference frequency signal to the waveform generation circuit 50 .

[0028] The waveform generation circuit 50 generates the waveform of the difference frequency signal based on the difference frequency signal output from the frequency conversion circuit 45. The waveform generation circuit 50 receives a reference sine wavelength frequency signal (third frequency signal) from the timing generator 70. The third frequency signal is a signal with a frequency corresponding to (the same as) the frequency of the difference frequency signal. That is, in this example, the third frequency signal is a 10 kHz signal. The third frequency signal is also a signal indicating the phase of the excitation light (i.e., scattered light). This phase of the excitation light is used in processing in the cancellation circuit 60. The waveform generation circuit 50 outputs information on the generated waveform (difference frequency signal) to the cancellation circuit 60.

[0029] The cancellation circuit 60 is a signal processing unit that removes signal components corresponding to scattered light from the difference frequency signal based on the waveform (difference frequency signal) generated by the waveform generation circuit 50 and the frequency signal (third frequency signal) input from the timing generator 70. The cancellation circuit 60 removes signal components corresponding to scattered light from the difference frequency signal based on the phase difference (phase difference) between the fluorescence and scattered light. The cancellation circuit 60 acquires information about the phase of the excitation light (i.e., scattered light) by receiving a reference frequency signal (third frequency signal) from the timing generator 70. This enables the cancellation circuit 60 to remove signal components of scattered light based on the phase difference between the fluorescence and 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 illustrating the phase difference between fluorescence and scattered light. As shown in FIG. 2 , the sinusoidal detection light (detected by the light detection unit D) from the sample S irradiated with sinusoidal excitation light from the light source unit L contains sinusoidal scattered light and fluorescence. The excitation light from the light source unit L is not limited to a sinusoidal waveform, but may have a periodically modulated waveform such as a rectangular wave. In this case, the detection light (scattered light and fluorescence) also has a periodically modulated waveform similar to that of the excitation light. While the scattered light has no phase difference with the excitation light, the fluorescence is light generated from the sample S in response to the excitation light and is detected with a phase delay of several tens of milliseconds to nanoseconds relative to the scattered light. The inventors focused on this phase difference and discovered 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 a difference frequency signal obtained by frequency-converting the detection light.

[0031] FIG. 3 is a diagram illustrating a technique for removing (canceling) scattered light. FIG. 3 shows only the waveform of the scattered light from the difference frequency signal. This waveform is identical to the waveform of the excitation light. In FIG. 3, the horizontal axis represents time and the vertical axis represents amplitude. If the waveform corresponding to the phase of the scattered light shown in FIG. 3 is separated into time units of, for example, 1 / 4 of one period (separating the time domains), and each of time domains 1 to 4 is integrated, the scattered light output for each of time domains 1 to 4 can be obtained. Here, if the integral values ​​for each of time domains 1 to 4 are each multiplied by a certain multiplier and then all are added together, the total output can be made zero. That is, the absolute values ​​of the outputs of time domains 1 to 4 are the same, and the amplitude ranges of time domains 1 and 2 are positive, while the amplitude ranges of time domains 3 and 4 are negative. However, as shown in FIG. 3, when time domain 1 is amplified by a multiplier of "-1", the output of time domain 1 becomes a negative value (positive x negative), when time domain 2 is amplified by a multiplier of "+1", the output of time domain 2 becomes a positive value (positive x positive), when time domain 3 is amplified by a multiplier of "+1", the output of time domain 3 becomes a negative value (negative x positive), and when time domain 4 is amplified by a multiplier of "-1", the output of time domain 4 becomes a positive value (negative x negative). Therefore, when all the integral values ​​of time domains 1 to 4 amplified by a predetermined multiplier are added together, the values ​​cancel each other out, 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 in predetermined time units 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 illustrating a fluorescence measurement technique. It shows the waveforms of scattered light and fluorescence contained in the difference frequency signal. In FIG. 4, the horizontal axis represents time, and the vertical axis represents amplitude. As described above, the signal components corresponding to scattered light can be removed (output set to zero) by separating them into predetermined time intervals corresponding to the phase of the scattered light, amplifying each of the separated components, and combining the amplified components. Here, as shown in FIG. 4, since fluorescence has a phase difference with respect to scattered light, when separated into predetermined time intervals corresponding to the phase of the scattered light, the integral values ​​of time regions 1 to 4 do not become the same. Therefore, the fluorescence is amplified by the same multiplier as for scattered light, and the sum of the amplified values ​​outputs a non-zero value. In this way, by separating, amplifying, and combining the scattered light and fluorescence into the same time interval, it is possible to detect and output the fluorescence output intensity while removing the scattered light signal component.

[0033] In this way, the cancellation circuit 60 separates the difference frequency signal in predetermined time units corresponding to the phase of the scattered light, amplifies each of the separated components of the detection signal, and combines the amplified components to remove the signal component corresponding to the scattered light from the difference frequency signal and obtain the signal component of the fluorescence. The cancellation circuit 60 outputs a fluorescence signal, which is a signal from which the signal component corresponding to the scattered light has been removed (i.e., a signal containing 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 corresponding to the scattered light can be removed after combination. Furthermore, while "+1" and "-1" have been exemplified as multipliers for amplification, this is not limited thereto and any multiplier may be used as long as the signal component corresponding to the scattered light can be removed after 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 results to an external computer, for example, via serial communication. The CPU 90 may also generate signals output from the timing generator 70, i.e., signals that determine the timing of various operations in the optical measurement device 1, and output them to the timing generator 70. Note that 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 signals 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 in more detail with reference to FIGS.

[0036] FIG. 5 is a diagram illustrating the removal of noise components. FIG. 5(a) shows the intensity of detected light when signal components (noise) corresponding to scattered light due to excitation light are not removed, and FIG. 5(b) shows the intensity of detected light when signal components (noise) corresponding to scattered light due to excitation light are removed. In FIGS. 5(a) and 5(b), the vertical axis represents the intensity of detected light, and the horizontal axis represents the channel indicating the position in the measurement unit 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 region of the sample 500 corresponding to the channel positions in FIGS. 5(a) and 5(b). FIG. 6 is a schematic diagram illustrating an enlarged view of the sample 500 shown in FIG. 5(c). As shown in FIG. 6, the sample 500 includes a dripping unit 502 onto which a specimen is dripped, a holding unit 503 holding a detection antibody labeled with a fluorescent reagent, and a measurement unit 501 in which a capture antibody is immobilized on a measurement target portion 504, 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, which moves downstream through 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, forming a complex consisting of the substance to be detected, the detection antibody, and the capture antibody. In this state, excitation light is irradiated onto the measurement section 501, which is the measurement area, while changing the focusing position (channel), thereby deriving the detected light intensity for each channel as shown in Figures 5(a) and 5(b). In Figures 5(a) and 5(b), the channel with the higher detected light intensity is the channel corresponding to the position of the measurement object section 504 where the complex is captured.

[0038] As shown in Figure 5(a), when the signal component (noise) corresponding to scattered light is not removed, the detected light contains not only fluorescent light but also scattered light, resulting in a high intensity of the detected light. Furthermore, this noise increases with increasing the amount of excitation light, so as shown in Figure 5(a), doubling the amount of excitation light also roughly doubles the noise. A commonly considered method for improving the S / N ratio is to increase the amount of fluorescence signal by increasing the amount of excitation light. However, as described above, in a configuration where noise also increases with the amount of excitation light, as in Figure 5(a), it is difficult to improve the S / N ratio. Furthermore, increasing the amount of excitation light also poses the problem of narrowing the dynamic range.

[0039] On the other hand, as shown in FIG. 5( b), when the signal component (noise) corresponding to scattered light is removed, the detected light contains almost only fluorescence, and only the desired signal (signal based on fluorescence) can be detected. In this case, since the noise is almost zero, even if the excitation light intensity is increased (e.g., 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 that performs noise removal as shown in FIG. 5( b), increasing the excitation light intensity can increase only the signal component while canceling the noise component to almost zero, leading to an improved S / N ratio. Because this configuration can cancel the effect of the excitation light (scattered light) to almost zero, it is possible to increase the excitation light intensity or increase the multiplication factor of the I-V conversion amplifier or photomultiplier tube. This allows only the signal component to be increased. For the sake of convenience, the noise component has been described as being removed from the detected light. In more detail, however, as described above, the noise component is removed from the difference frequency signal obtained by frequency-converting the detected light.

[0040] FIG. 7 is a diagram illustrating the definition of S / N. FIG. 7 shows an example of the detected light intensity for each channel (the detected light intensity at each position in the measurement area). As shown in FIG. 7, there is a fluctuation component of approximately ±4 around 10 counts of detected light intensity. This fluctuation (standard deviation) of the base light intensity is a value obtained by scanning the excitation light over the measurement unit 501, which is not coated with any fluorescent substance or the like (or the measurement unit 501 is wet, similar to the measurement state). Hereinafter, this fluctuation of the base light intensity is defined as noise N. Furthermore, signal S is defined as "the value obtained by subtracting the average value of the noise components in all channels, excluding the position of the measurement unit 504, from the peak fluorescence intensity of the measurement target unit 504." S / N is defined as the 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 approximately 10 counts. In principle, the noise N value is canceled out to approximately 0 by performing the calibration process described below. However, since the background varies depending on the noise N value, it is preferable for the signal to always be a positive value from the perspective 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 as small as possible from the perspective of dynamic range, while ensuring that the background signal obtained by scanning the measurement unit 501 with excitation light is always (almost certainly) a positive value. Specifically, the offset amount may be, for example, the average intensity of the detected light obtained by scanning the measurement unit 501 with excitation light without any applied fluorescent material (or the measurement unit 501 in a wet state similar to the measurement state) plus 6σ of that average intensity. In case of unexpected external noise entering the circuit system, an appropriate margin may be added to the offset amount calculated above to determine the final offset amount. The amount of offset 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 specific description will be given of a method for removing signal components (noise) corresponding to scattered light. 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 scattered light are removed from the difference frequency signal in consideration of 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. The light detected by the detection optical system 20 here is basically only scattered light that does not contain at least fluorescence from the measurement target portion 504, and is calibration process light used in 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 of ​​the sample 500 downstream of the capture antibody immobilized on the measurement target portion 504 with excitation light. FIG. 10 is a diagram illustrating the fluorescence intensity in each region of the sample 500. In FIG. 10, the area labeled "Line A" indicates the measurement target portion 504. For convenience of explanation, in the descriptions of FIGS. 5( a) and 5(b), the fluorescence intensity outside the measurement target portion 504 is shown as low. However, in reality, as shown in FIG. 10, the fluorescence intensity tends to be high upstream of where the sample is dripped, even if the capture antibody is not present. For this reason, the calibration light, which should be limited to scattered light, is preferably detected by irradiating an area of ​​the sample 500 downstream of the capture antibody immobilized on the measurement target portion 504 with excitation light. The light for calibration may be obtained not only from the measurement unit 501 in the sample 500 but also from a case that houses the immunochromatographic membrane or a separately prepared member for calibration.

[0045] Next, a calibration process is performed. Specifically, the cancellation circuit 60 of the optical measurement device 1 performs a calibration process to remove signal components corresponding to scattered light from the difference frequency signal based on a calibration signal corresponding to the calibration process light described above. Details of the calibration process will be described 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, thereby acquiring fluorescence information of the measurement unit 501. Specifically, the cancellation circuit 60 acquires fluorescence information by removing signal components corresponding to scattered light from the difference frequency signal, taking into account the results of the calibration process described above.

[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 that uses, 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 relative to a periodic signal whose period is the operating frequency of the cancellation circuit 60, which is matched to the modulation frequency of the light source 11 set by the light source drive circuit 30 (for example, the frequency of a DDS (Direct Digital Synthesizer)). 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) that approximates zero.

[0047] FIG. 8 shows signals used for calibration processing within the FPGA of the cancellation circuit 60. The periodic signal shown in FIG. 8 is a clock signal whose period is synchronized with the DDS frequency, as described above. The reference signal is a signal of the same frequency as the periodic signal, at an arbitrary phase from the periodic signal (i.e., phase-shifted relative to the periodic signal), and serves as a trigger for the XY signals described below. The XY signals are the lock-in switch signals described above, and are signals generated using the reference signal as a trigger. The X signal is a signal with no phase difference from the reference signal. The Y signal is a signal with a phase shift of 90 degrees relative to the reference signal. In addition to the X and Y signals, the cancellation circuit 60 actually generates an X' signal, which is the inverse of the X signal, and a Y' signal, which is the inverse of the Y signal. The X, Y, X', and Y' signals are each generated by independent, dedicated circuits. 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 relative to the periodic signal until the output from the cancellation circuit 60 becomes 0 V (or a value close to that).

[0048] FIG. 9 is a diagram illustrating a process for adjusting the phase of a reference signal relative to a periodic signal so that the output is 0 V. Assume now that the initial phase relationship between the periodic signal, reference signal, and switch signal is as shown in FIG. 9( a). Then, assume that integration processing 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 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, if the voltage value of the signal component corresponding to scattered light is not at the threshold level and is a positive value during the calibration process, 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 (voltage value of the signal component corresponding to 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 (voltage value of the signal component corresponding to 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 shown in Figure 9(d) after the phase adjustment of the switch signal, let us assume that the integration process for the shaded section results in the output (the voltage value of the signal component corresponding to scattered light) being not at the threshold level but a negative value. In this case, as shown in Figure 9(e), the phase of the reference signal is adjusted to advance the phase of the switch signal. That is, if the voltage value of the signal component corresponding to scattered light is not at the threshold level but a negative value during the calibration process, 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, as shown in Figure 9(e), the calibration process is completed when the output (the voltage value of the signal component corresponding to the scattered light) reaches the threshold level (a value within a predetermined range that approximates 0) as a result of the integration process in the shaded section.

[0053] When the calibration process is completed, the cancellation circuit 60 receives as input a difference frequency signal corresponding to the detection light containing the fluorescent component and the scattered light component (excitation light component) 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 flowchart illustrating the calibration process. As shown in FIG. 11 , the calibration process begins by switching the input to the AD converter 80 to a predetermined offset voltage and storing a zero level (step S1). Then, a signal from the cancellation circuit 60 is input to the AD converter 80 by switching a switch (step S2). In this state, the phase of the reference signal is forcibly shifted (step S3). The output of the cancellation circuit 60 is 0 V when the phase of the reference signal relative to the periodic signal is 0 degrees or 180 degrees. However, if the phase is accidentally matched at 180 degrees in the initial state, the calibration process may be erroneously completed, resulting in an inversion of the positive and negative polarities of the output signal, which may make it impossible to detect the output signal depending on the configuration of downstream circuits. In this regard, forcibly shifting the phase of the reference signal at the start of the calibration process prevents the calibration process from being erroneously completed. Furthermore, by matching the phase at the start in this manner, the positive and negative polarities of the output signal are fixed. As a result, a sign bit is not required when the output signal is converted to a digital value, allowing for effective use of the dynamic range of the AD converter 80. Furthermore, when it is desired to measure with a negative output, the phase at which calibration is completed may be set to 180 degrees instead of 0 degrees.

[0055] Upon completion of step S3, the current input value of the AD converter 80 is recorded (step S4), and a calibration loop process 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 less than the 0 level (i.e., whether it 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 relative to the periodic signal corresponding to the DDS frequency 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 relative 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 the sign has not changed, the process of step S4 is performed again, and if it is determined that the sign has changed, the amount of phase change due to control is changed to half of 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 according to 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 drive 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 a 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 associated with the first frequency signal and the frequency associated with 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, the third frequency signal being a signal with a frequency that corresponds to the frequency associated with 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 the phase component of the excitation light (scattered light) is removed from the detection signal of the detection light including fluorescence and scattered light, thereby removing noise components caused by the excitation light itself. However, if the emission time of the light (fluorescence) to be measured is shortened (e.g., a fluorescent reagent with a short fluorescence lifetime is used), and the signal processed by the circuit is therefore faster, for example, if the signal becomes a high-speed signal of 10 MHz or higher, the above-described method alone may not be able to adequately remove the noise components, depending on the processing capacity of the circuit used.

[0060] FIG. 12 illustrates the fluorescence excitation spectra 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 FIGS. 12(a) and 12(b), the horizontal axis represents wavelength and the vertical axis represents light intensity. As shown in FIG. 12(a), in the case of europium, which has a relatively long fluorescence lifetime (millisecond level), for example, the wavelength band of the excitation light is approximately 365 nm, the wavelength band of the fluorescence is approximately 615 nm, and the modulation speed is approximately 100 Hz. In this case, the modulation speed is slow and the circuit design is not difficult (processing power required for the circuit) and therefore 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 (on the nanosecond level), for example, the wavelength band of the excitation light is approximately 488 nm, the wavelength band of the fluorescence is approximately 520 nm, and the modulation speed is 10 MHz, which is approximately 100,000 times that of europium. In this case, the modulation speed is fast, which increases the difficulty of circuit design (the processing power required for the circuit), and it may be difficult to remove the phase component of the excitation light (scattered light) from the detection signal. Note that the difficulty of circuit design is due not only to processing power issues, but also to 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. This difference frequency signal is a signal that is sufficiently slower than the detection signal. Then, this difference frequency signal and the third frequency signal are input to the cancellation circuit 60, whereby the signal component corresponding to the third light (i.e., the noise component due to the excitation light itself) is removed from the difference frequency signal. In this way, the detection signal is frequency converted, and the noise component due to the excitation light itself is removed from the difference frequency signal after frequency conversion. This allows for appropriate noise component removal even in high-speed signals associated with organic fluorescent dyes, etc., which have a relatively short fluorescence lifetime.

[0063] The effect of appropriately removing scattered light (noise) in this manner will be described with reference to FIG. 13 . FIG. 13 is a diagram illustrating the effect of appropriately removing scattered light (noise). In FIG. 13 , the horizontal axis represents measurement position, and the vertical axis represents light intensity. The thick solid line represents the results (measurement results of fluorescence and excitation light) of a sample measured in the configuration according to this embodiment, in which an organic fluorescent dye (FITC) was injected as a fluorescent reagent into the microchannel above the light scatterer. The dashed line represents the results (measurement results of excitation light only) of a sample measured in the configuration according to this embodiment, in which a dilute solution not containing a fluorescent reagent was injected into the microchannel above the light scatterer. The thin solid line represents the results (measurement results of fluorescence and excitation light) of a sample measured in the configuration according to a comparative example, in which an organic fluorescent dye (FITC) was injected as a fluorescent reagent into the microchannel above the light scatterer. The dotted line represents the results (measurement results of excitation light only) of a sample measured in the configuration according to the comparative example, in which a dilute solution not containing a fluorescent reagent was injected into the microchannel above the light scatterer. The difference between the thick solid line and the dashed line represents the amount of fluorescence (signal) in this embodiment, and the difference between the thin solid line and the dotted line represents the amount of fluorescence (signal) in the comparative example. As shown by the thin solid and dotted lines in Figure 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 and dashed lines in Figure 13, in the configuration of this embodiment, the baseline does not rise even when the excitation light is scattered by the light scatterer, so only a minimum offset is applied for software processing (processing to make all signal values ​​positive). Compared to the measurement results of the configuration of the comparative example, the noise is approximately 1 / 10 and the signal is approximately 6.7 times lower. Thus, when noise is removed, the baseline offset is small, allowing for increased excitation light intensity from the light source and increased amplification factors of the I-V conversion amplifier and photomultiplier tube, thereby favorably increasing signal intensity. As a result, the S / N ratio can be improved by approximately 70 times compared to when noise removal is not performed.

[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 that includes 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 component 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 this range, the frequency involved in noise removal can be prevented from becoming too high, and noise components can be removed appropriately.

[0066] Although the optical measurement device 1 according to this embodiment has been described above, the configuration according to the present disclosure is not limited to the above-described optical measurement device 1. For example, as shown in Fig. 14, frequency filters may be provided on the input and output sides 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 of the frequency conversion circuit 45 (between the I-V conversion amplifier 40 and the frequency conversion circuit 45), and a band-pass filter 116 (BPF) which is a second frequency filter is provided on the output side of the frequency conversion circuit 45 (between the frequency conversion circuit 45 and the waveform generation circuit 50). In this way, by using the band-pass filter 117, it is possible to selectively pass only a desired frequency band (e.g., 10.01 MHz), thereby more appropriately suppressing the inclusion of noise and suppressing 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, for example, to the measurement optical system of a flow cytometer. When applied to fluorescence measurement in a flow cytometer, excitation light (and scattered light) can be canceled to selectively acquire only fluorescence. Typically, miniaturized flow cytometers have difficulty in removing excitation light, but the optical measurement device according to the present disclosure cancels the excitation light, allowing the flow cytometer to be appropriately miniaturized. Similarly, the optical measurement device according to the present disclosure may be applied to a microplate reader, PCR, microscope, HPLC, or the like.

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

[0070] [E1] An optical measurement device comprising: a timing generator that outputs a frequency signal; a light source unit that irradiates a first light toward a sample; a light source drive 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 second light generated from the sample irradiated with the first light and third light that is light caused by the first light and has the same phase as the first light, and outputs a detection signal; a frequency conversion circuit that generates a difference frequency signal that is a signal with a frequency that is the difference between the frequency associated with the first frequency signal and the 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, wherein the third frequency signal is a signal with a frequency that corresponds to the frequency associated with the difference frequency signal.

[0071] [E2] The optical measurement device according to [E1], wherein 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.

[0072] [E3] The optical measurement 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] An 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] An optical measurement method comprising: irradiating a sample with first light based on a first frequency signal; detecting detection light including second light generated from the sample irradiated with the first light and 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 with 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 with a frequency that corresponds to the frequency associated with the difference frequency signal.

[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. An optical measurement device comprising: a timing generator that outputs a frequency signal; a light source unit that irradiates a first light towards a sample; a light source drive 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 which is light 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 which is a signal of a frequency which 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, wherein the third frequency signal is a signal of a frequency which corresponds to the frequency associated with the difference frequency signal.

2. An optical measuring device as described in claim 1, wherein 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.

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

4. An optical measuring device as described in 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 band-pass filters.

5. An optical measurement method comprising: irradiating a first light towards 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 which is a signal of a frequency which 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 a 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 which corresponds to the frequency associated with the difference frequency signal.

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