JPH0210136A - Method and instrument for measuring fluorescence - Google Patents

Abstract

PURPOSE:To measure the fluorescence with high accuracy by performing each different modulation to a stimulating light for stimulating a fluorescent substance and a reference light for stimulating no fluorescent substance and irradiating the fluorescent substance, receiving a fluorescence and a scattered light generated thereby by one photodetector and separating them electrically. CONSTITUTION:A light beam from a stimulating liquid light source 1 is led to a wave length selector 1b, converted to the stimulating light in which wavelength which can stimulate a fluorescent substance has the center wavelength, and a light beam from a reference light light source 2 is converted to a reference light through a wavelength selector 2b. Subsequently, the stimulating light and the reference light are led to exter nal modulators 1d, 2d, each different intensity modulation is performed and a sample chamber 3 is irradiated. A fluorescence and a scattered light generated thereby are led to a wavelength selector 3c, and led to a photodetector 4 after a stimulating light component is eliminated. Its output is supplied to demodulators 4a, 4b to which synchro nizing signals are supplied from the external modulators 1d, 2d, subjected to synchro nous detection, becomes a DC signal corresponding to a scattered stimulating light component, a fluorescent component and a scattered reference light component, respec tively and supplied to a correction arithmetic part 5 and amplitude E of the fluorescent component is calculated.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to a method and apparatus for measuring fluorescence, and more specifically, the present invention relates to a method and apparatus for measuring fluorescence, and more specifically, irradiates a fluorescent substance with excitation light, and in a direction different from the direction of the excitation light, The present invention relates to a method and apparatus for measuring the intensity of excited fluorescent light.

<Prior Art> Conventionally, in order to measure fluorescence intensity, a fluorescence photometer has been provided which is composed of a light source, an excitation wavelength selector, a sample chamber, a fluorescence wavelength selector, and a photodetector. When measuring fluorescence using a fluorescence photometer with this configuration, in addition to the fluorescence component to be measured, blind fluorescence components such as scattered light are guided to the photodetector, and moreover, the fluorescence component is guided to the photodetector. The intensity is 10 of the excitation light intensity
Since the level is about -6 times higher, the fluorescence intensity measurement sensitivity and the fluorescence intensity measurement accuracy are reduced. In particular, when the sample has a low concentration or is a highly scattering material, when the excitation light is not sufficiently monochromatic, or when the excitation light wavelength and the fluorescence wavelength are close to each other, excitation light removal may be difficult. If the wavelength selection for this purpose is insufficient, the proportion of the blind fluorescent component increases, resulting in a significant decrease in the sensitivity and accuracy of fluorescence intensity measurement.

In consideration of such problems, it has been proposed to measure the offset caused by the blind fluorescence component separately and correct the fluorescence measurement value using the offset measurement value to improve the accuracy of fluorescence intensity measurement. .

Further, a fluorescence measuring device having a configuration shown in Japanese Patent Application Laid-Open No. 82-2711438 has been provided.

This fluorescence measurement device divides the scattered light and fluorescence generated by irradiating the sample chamber with excitation light into two parts, and guides one of the lights through a fluorescence wavelength selector to a photodetector to increase the fluorescence intensity. Obtain an electrical signal I em corresponding to . and,
The other light is guided to a photodetector through a scattering wavelength selector to obtain an electrical signal Isc corresponding to the intensity of the scattered light. Then Iem−fβ(λex, λem)/α(λe
x)llsc (where α(λex) is the proportion of the light emitted from the sample chamber that is incident on the photodetector through the scattering wavelength selector, and β(λex, λe+++) is the proportion of the light emitted from the sample chamber. , the rate of light incident on the photodetector through the fluorescence wavelength selector, λex is the scattered light wavelength, λam
is the fluorescence wavelength), it is possible to improve the measurement accuracy of the fluorescence intensity.

<Problems to be Solved by the Invention> In the former fluorescence measurement device, in addition to the operation for measuring fluorescence, an operation for obtaining an offset measurement value is required, making the overall operation complicated. Not only that, but the conditions for obtaining offset measurements are not completely the same as those for fluorescence measurements, so there is no guarantee that accurate offset measurements will be obtained, and as a result, fluorescence measurements There is a problem that accuracy may be reduced. Further, if a drift of the offset value occurs, the offset measurement value becomes an inaccurate value, and there is a problem that the accuracy of fluorescence measurement further deteriorates.

In the latter type of fluorescence measuring device, it is possible to simplify the operation and improve the accuracy of fluorescence measurement. There is a problem in that a photodetector, a large number of wavelength selectors, etc. are required, and the overall configuration of the light receiving optical system becomes complicated. In particular, the excitation light wavelength and fluorescence wavelength cannot be set freely, but are determined by the type of fluorophore to be measured, so the difference between the two wavelengths may be relatively small. In this method, it is necessary to use a wavelength selector with extremely steep selection characteristics, so there is a problem that the accuracy of fluorescence measurement decreases depending on the selection characteristics of the wavelength selector. Furthermore, since the level of fluorescence is extremely low, a photomultiplier with extremely high sensitivity is used as a photodetector, so even though the characteristics of both photomultipliers must be the same, it is necessary to adjust the applied voltage to a certain degree. Since sensitivity easily and widely fluctuates due to fluctuations in There is also.

<Object of the invention> This invention was made in view of the above problems,
It is an object of the present invention to provide a method and apparatus for measuring fluorescence that can simplify the light receiving optical system and significantly improve the accuracy of fluorescence measurement.

Means for Solving the Problems> In order to achieve the above object, the fluorescence measurement method of the present invention uses excitation light of a wavelength that can excite a fluorescent substance and reference light of a wavelength that does not excite a fluorescent substance. , a fluorescent substance is irradiated with different modulations through almost the same optical path, the fluorescent light and the scattered light are received by one photodetector in a direction shifted by a predetermined angle from the irradiated light optical path, and the fluorescent light and the scattered light are received by a single photodetector. During demodulation based on the output electrical signal, the signal is separated into a signal corresponding to the fluorescence and excitation light scattering components and a signal corresponding to the reference light scattering component,
This is a method of obtaining a signal corresponding only to fluorescence based on both signals.

However, it is preferable that the excitation light and the reference light are irradiated onto the fluorescent substance through the same optical path.

To achieve the above object, the fluorescence measurement device of the present invention includes an excitation light source that emits excitation light with a wavelength that can excite a fluorescent substance, and a reference light that emits a wavelength that does not excite the fluorescent substance. a reference light source, a modulation means for applying different modulation to each light beam, and a single photodetector for receiving scattered light from a fluorescent substance, to which the light emitted from both light sources is irradiated through substantially the same optical path. , demodulating means for separating a signal corresponding to the excitation light scattering component and a signal corresponding to the reference light scattering component based on the electrical signal output from the photodetector; and calculation means for obtaining a signal corresponding only to light.

However, the modulation means may be one that modulates the light emitted from the light source to convert it into pulsed light; It may be possible to obtain

Further, the demodulation means includes a first demodulation means that performs a demodulation operation in synchronization with a modulation means for the pump light, and a second demodulation means that performs a demodulation operation in synchronization with the modulation means for a reference destination. It is preferable.

With the above fluorescence measurement method, when a fluorescent substance is irradiated with excitation light and the intensity of the excited fluorescence is measured, the excitation light and fluorescence with a wavelength that can excite the fluorescent substance are used. Reference light with a wavelength that does not excite the substance is modulated differently and is irradiated onto the fluorescent substance through almost the same optical path, so scattered light and excited fluorescent light are generated due to the irradiation with the excitation light. is emitted in a direction different from the excitation light optical path, and only scattered light is emitted in a direction different from the reference light optical path due to the irradiation with the reference light. Then, by receiving the above emitted light with one photodetector, a corresponding electric signal is obtained, and upon demodulation, a signal corresponding to the scattered light based on the fluorescent light and the excitation light and a signal corresponding to the scattered light based on the reference light are generated. By performing a correction operation on the signal corresponding to the scattered light based on the fluorescent light and the excitation light using the signal corresponding to the scattered light based on the reference light,
A signal corresponding only to fluorescence can be obtained.

That is, since the signals are separated when demodulating the electrical signals output from the photodetector without using an optical filter, the signal separation accuracy can be improved, and as a result, accurate offset measurement values can be obtained. Since the correction calculation can be performed based on the obtained value, the fluorescence intensity can be calculated with extremely high accuracy regardless of the type of fluorescent substance.

With the fluorescence measuring device having the above configuration, when a fluorescent substance is irradiated with fluorescent light and the intensity of the excited fluorescent light is measured, excitation light from the excitation light source has a wavelength that can excite the fluorescent substance. At the same time, the reference light source emits reference light with a wavelength that does not excite the fluorescent substance, and the modulation means modulates each light differently and irradiates the fluorescent substance. Excited fluorescent light, scattered light caused by the excitation light, and scattered light caused by the reference light are emitted from the substance.

Then, by receiving the fluorescent light and scattered light with a single photodetector, an electrical signal corresponding to the fluorescent light and scattered light can be obtained.The obtained electrical signal is then supplied to the demodulating means. to remove the modulation component, and
A signal corresponding to the excitation light scattering component and a signal corresponding to the reference light scattering component can be separated. Therefore, by supplying both separated signals to the calculation means, the scattered light signal caused by the excitation light is removed based on the scattered light signal caused by the reference light, and finally a signal corresponding only to the fluorescence is generated. Obtainable.

That is, since the signals are separated when demodulating the electrical signals output from the photodetector without using an optical filter, the signal separation accuracy can be improved, and as a result, accurate offset measurement values can be obtained. Since the correction calculation can be performed based on the obtained value, the fluorescence intensity can be calculated with extremely high accuracy regardless of the type of fluorescent substance.

If the modulation means modulates the light emitted from the light source to convert it into intensity-modulated light, even if the light source is difficult to modulate directly, the fluorescent material Any type of light source can be used since the light irradiated into the room can be intensity modulated.

Further, when the modulation means modulates the light source to obtain intensity-modulated output light, there is no need to arrange a chopper or the like for modulation in the optical path, and the optical The system configuration can be simplified and the light source can be modulated electrically, so
The degree of freedom in modulation can be increased, and changes in modulation methods can be easily handled.

Furthermore, when the demodulation means has a first demodulation means that performs a demodulation operation in synchronization with a modulation means for the pump light, and a second demodulation means that performs a demodulation operation in synchronization with the modulation means for a reference destination. In this case, a signal to be subjected to a correction calculation can be obtained from the first demodulation means, and a signal to be subjected to a correction calculation can be obtained from the second demodulation means. Therefore, an identification circuit for identifying the type of signal is not required, and the configuration can be simplified.

<Example> Hereinafter, embodiments will be described in detail with reference to the accompanying drawings showing examples.

FIG. 1 is a schematic block diagram showing an embodiment of the fluorescence measurement device of the present invention, which includes an excitation light source (1) and a reference light source (1).
2), a sample chamber (3) containing a sample containing a fluorescent substance, and an optical path (la) that guides the emitted light from each light source to the sample chamber (3).
) A wavelength selector (Lb) (2b), a light projection optical system (lc) (2c), arranged in this order in (2a),
An external modulator (1d) (2d), a photodetector (4) that receives light emitted from the sample chamber (3) in a direction different from the above optical path, and a photodetector that detects the light emitted from the sample chamber (3). Vessel (4)
The light receiving optical system (3a) arranged in this order on the optical path (3a) leading to
3b), a wavelength selector (3c), a demodulator (4a) (4b) that performs predetermined demodulation processing by inputting the electrical signal output from the photodetector (4), and a double modulator (4a) ( 4b
), and a correction calculation section (5) which receives the output signal from the. The demodulators (4a) (4b) are
The demodulation operation is controlled by the synchronization signal output from the external modulator (+, d) (2d).

More specifically, the excitation light source (1) and the reference light source (2) may be a DC-lit discharge lamp such as a tungsten lamp, a halogen-filled tungsten lamp, a xenon lamp, or a mercury lamp, or may be a xenon flash lamp. It may be a pulse-lit discharge lamp such as
Various lasers such as semiconductor lasers may be used, or LEDs may be used.

It is preferable that these light sources have a stabilized power supply or are stabilized by monitoring the amount of light emitted and giving negative feedback.

The wavelength selector (lb) (2b) (3c) may be an optical filter such as an interference filter, a colored glass filter, a diffraction grating, a wavelength selective mirror, or a combination thereof; For 2b), the optimum wavelength can be selected as the excitation light and the reference light, and for the wavelength selector (3c), the fluorescent light and the scattered reference light can be selectively made incident on the photodetector (4). good.

The projection optical system (1, c) (2c) includes a condenser lens, a projection lens, a mirror, a prism, an aperture pinhole,
It may be a slit or the like.

The external modulator (Id) (2d) may be an electro-optical chopper, a mechanical chopper, etc. using the Pockels-Kerr effect, a liquid crystal, an acousto-optic effect, etc. It is only necessary to perform intensity modulation on the excitation light and the reference light. However, in cases where direct modulation is easy, such as with semiconductor light sources, or in the case of pulsed light sources, an external modulator (
1d) (2d) is unnecessary.

The light receiving optical system (3b) may be a condenser lens or the like.

The photodetector (4) may be a photodiode, phototransistor, phototube, photomultiplier tube, or the like, and can be appropriately selected depending on the level of fluorescence intensity.

The operation of the fluorescence measuring device having the above configuration is as follows.

The light emitted from the excitation light source (1) is guided to the wavelength selector (1b) arranged in the optical path (1a), and is converted into excitation light having a central wavelength that can excite a fluorescent substance. . In addition, the light emitted from the reference light source (2) is guided to the wavelength selector (2b) disposed in the optical path (2a), so that the wavelength component that can excite the fluorescent substance becomes a reference light with a significantly low level. converted.

The excitation light and the reference light are each transmitted through a projection optical system (l
c) External modulator (td) through (2c) (2d)
are guided, and subjected to mutually different intensity modulation (for example, the modulation layer wave numbers are f ax and f ref , respectively). Specifically, intensity modulation of several tens of H2 to several tens of KHz is performed using a chopper or the like.

In this way, the intensity-modulated excitation light and reference light are irradiated into the sample chamber (3), so that fluorescent light with an intensity determined by the excitation light intensity and the amount of fluorescent substance present is excited, and the excitation light is Scattered light corresponding to the light intensity and the reference light intensity is generated.

The fluorescent light and scattered light are guided to the wavelength selector (3c) through the light receiving optical system (3b) arranged in the optical path (3a), where the excitation light component is removed, and the excitation light component is directly transmitted to the photodetector (4). guided by. Therefore, from the photodetector (4), the scattered excitation light component E sinωex, the fluorescence component F
An electrical signal on which the SlnωeXs scattered reference light component Rsinωrer and the noise component ε are superimposed is output.

(However, ωex is the angular frequency corresponding to the excitation light modulation frequency feX, and ωref is the angular frequency corresponding to the reference light modulation frequency f ref.) Both of the above electrical signals are supplied to the demodulators (4a) and (4b). , each demodulator (4a) (4b) is supplied with the synchronization signal output from the external modulator (1d) (2d), so the demodulator (4a) performs synchronized detection. DC signals corresponding to the scattered excitation light component and fluorescent light component are output, and the demodulator (4b) performs synchronous detection to output a DC signal corresponding to the scattered reference light component. Both DC signals are then supplied to the correction calculation section (5), and since the ratio between the amplitude E of the scattered excitation light component and the amplitude R of the scattered reference light component is a constant determined by the fluorescence measuring device, If this constant is initialized in advance in the correction calculation section (5), the amplitude E of the fluorescent component can be calculated based on this constant and both DC signals.

As is clear from the above explanation, the photodetector (4) outputs an electrical signal in which the fluorescent light component and the scattered light component are superimposed, but the excitation light modulation frequency and the reference light modulation frequency are By performing a demodulation operation based on the base, it is possible to accurately separate the DC signal corresponding to the scattered reference light component and the DC signal corresponding to the scattered excitation light component and the fluorescent light component. Only the DC signal corresponding to can be calculated with high precision.

<Example 2> FIG. 2 is a schematic diagram showing a more specific example of the fluorescence measuring device of the present invention, in which a halogen lamp (11) is used as an excitation light source, and an LED is used as a reference light source. (
21) is used, and FIT C (Pluorescejn js) is used as the fluorescent substance housed in the sample chamber (31).
(41)
are using.

To explain in more detail, excitation light emitted from a halogen lamp (11) is guided to a slit (13) through a condensing lens system (12), and is subjected to intensity modulation (modulation frequency is f ax) by a chopper (14). After that, it is collimated by a collimator lens (15), and passed through an optical filter (
16) The optimum wavelength component (
The light is monochromated into wavelength components shorter than about 490 nm (in FIG. 3), and then irradiated into the sample chamber (31) through the dichroic mirror (17).

Then, L driven by the LED drive circuit (22)
The intensity-modulated reference light (the modulation frequency is f ref and is set to a value sufficiently different from the above modulation frequency f ax) emitted from the E D (21) is collimated by the collimator lens (23). The optical filter (24) converts the beam into a parallel beam, and the optical filter (24) converts the wavelength component not shorter than the maximum fluorescence wavelength of the fluorescent material (the maximum fluorescence wavelength of FITC is approximately 520 nm as shown by the broken line in Figure 3). nrrl, it is monochromated into a wavelength component of approximately 520 to 550 nm), which is reflected by the dichroic mirror (17) and passes through the same optical path as the excitation light into the sample chamber (
31).

Also, with the sample chamber (31) as a reference, the light emitted in a direction substantially perpendicular to the optical path of the irradiation light is collected using a condensing lens system (32).
The excitation light is guided to the short wavelength cut filter (33) through the filter, and the excitation light is removed and guided to the photomultiplex (41).

Furthermore, the electrical signal output from the photomultiple (4) is amplified by a preamplifier (42) and then supplied to bandpass filters (43a) and (43b) whose center frequencies are respectively set to f eXs f ref. The output signals from each bandpass filter (43a) (43b) are supplied to multipliers (44a) (44b) to which synchronization signals of frequencies f Bx and f ref are supplied, respectively. The output signals from 44a and 44b are respectively supplied to low pass filters (45a and 45b), and the output signals from the low pass filters (45a and 45b) are passed through an A/D converter (46) to a microcomputer as a correction calculation section. The information is supplied to the computer (51).

The operation of the fluorescence measuring device having the above configuration is as follows.

The excitation light emitted from the halogen lamp (11) is subjected to intensity modulation at a frequency of facsimile by a chopper (14), is made monochromatic by an optical filter (16) so that the maximum wavelength is approximately 490 nm, and then is passed through a dichroic mirror. (17
) and is irradiated into the sample chamber (31), so that fluorescent light with an intensity corresponding to the amount of FITC existing inside the sample chamber (31) and irradiated with the excitation light is generated. At the same time, scattered excitation light is generated.

In addition, L E driven by the LED drive circuit (22)
D (21) emits reference light that has been intensity-modulated at a frequency f ref, is monochromated by an optical filter (24) so that the maximum wavelength is approximately 520 nm, and is then monochromated by a dichroic mirror (17). Since it is reflected and irradiated into the sample chamber (31) through the same optical path as the excitation light, scattered reference light is generated.

The fluorescent light, scattered excitation light, and scattered reference light are guided to a short wavelength cut filter (33) through a condenser lens system (32) to remove the excitation light, and then guided to a photomultiplier (41). Therefore, from the photomultiplier (41), the scattered excitation light component E sinωeX% fluorescent light component F sinω
A current signal on which the el scattered reference light component Rsinωref and the noise ε are superimposed can be output.

After the above current signal is amplified, it is passed through both bandpass filters (
43a) (43b), so in the bandpass filter (43a), the scattered excitation light component E si
Signals corresponding to nωex and the fluorescence component F sinωex are obtained, and a signal corresponding to the scattered reference light component Rsinωref' is obtained in the bandpass filter (43b). Therefore, by supplying each of the signals mentioned in section 2 to the multipliers (44a) and (44b), a signal with a frequency component equal to the synchronizing signal frequency and a high frequency signal can be obtained.
By supplying only signals with frequency components equal to the synchronization signal frequency, that is, (E0F)/2 components and R/
2 components can be extracted as a DC signal.

If the DC signals corresponding to the components of the DC signal (E+F)/2 and R/2 are converted into digital data and then supplied to the microcomputer (51), the ratio of E and R can be determined in advance as a constant unique to the device. Since it is set, the value of F can be calculated accurately.

To summarize the above, two light sources are required to emit light to irradiate the sample chamber (31).
Only one optical system is required to guide the scattered light and fluorescent light from. In particular, it has extremely high sensitivity for measuring fluorescent light with an extremely weak light level, but it can also use only one photomultiplier, which is extremely unstable, and moreover, Since two types of signals are generated at the current signal processing stage to correct errors caused by scattered light components, the photomultiplier (4
It is possible to perform highly accurate correction without considering the poor stability of 1), and as a result, the accuracy of the obtained fluorescence intensity can be significantly improved.

<Embodiment 3> FIG. 4 is a schematic diagram showing still another embodiment of the fluorescence measuring device, and the difference from the embodiment of FIG. 2 is that one white light source (61) is used as the light source, By splitting the light emitted from the white light source (61) into two by a beam splitter (64) and reflecting only one of the lights by a mirror (65), a wavelength selector (62a) in which mutually different selection wavelengths are set.
) (62b) and external modulators (63a) and (63b) set with mutually different modulation frequencies in this order,
After that, only one of the lights is reflected by a mirror (66) and both lights are guided to the same optical path by a beam splitter (67), and the chopper (14>) is omitted. The configuration of the parts is the same.

Therefore, in this embodiment, not only can the number of light sources be reduced to one, but also the accuracy of fluorescence measurement can be significantly improved as in the above embodiment.

<Embodiment 4> Fig. 5 is a schematic diagram showing still another embodiment of the fluorescence measuring device, and the difference from the embodiment shown in Fig. 4 is that a white light source (61
) A pair of wavelength selectors (62a) and (62b) each having different selection wavelengths are provided in order to obtain whitening light having a specific maximum wavelength from the light emitted from the wavelength selector (62a) and (62b). The only difference is that a drive unit (not shown) for positioning the light beam in the optical path and a chopper (14) are provided, and the configuration of other parts is the same.

Therefore, in this embodiment, the wavelength selector doubles as an external modulator, and pulse modulation is selected to selectively irradiate the sample chamber (31) with the excitation light and reference light as external modulation. It means that it was done. As a result, the optical system for irradiating the sample chamber (31) with excitation light and reference light can be further simplified, and the accuracy of fluorescence measurement can be significantly improved as in the above embodiment.

Note that this invention is not limited to the above embodiments; for example, by employing pulse modulation, it is possible to separate both lights in the time domain, and by using a monochromatic light source, it is possible to perform wavelength selection. vessel (lb) (2b) (16
) (24) ([12a) (82b) can be omitted and the external modulator (1d) (2d) (14) (63
a) (63b) can be omitted, and various design changes can be made without changing the gist of the invention.

<Effects of the Invention> As described above, the first invention uses excitation light with a wavelength that can excite a fluorescent substance and reference light with a wavelength that does not excite a fluorescent substance.
The fluorescent material is irradiated through almost the same optical path with different modulations, and the fluorescent light and scattered light from the fluorescent material are combined into one.
After the light is received by two photodetectors, the signals caused by both lights are electrically separated and the fluorescence intensity is calculated.
It is said that it is possible to perform highly accurate fluorescence measurements regardless of the scattering properties of the fluorescent substance, and it is also possible to perform even more accurate fluorescence measurements by eliminating the effects of variations in the characteristics of the photodetector. It has a unique effect.

The second invention is to irradiate the fluorescent substance with excitation light of a wavelength that can excite the fluorescent substance and reference light of a wavelength that does not excite the fluorescent substance through substantially the same optical path in a state in which mutually different modulations are applied. After the fluorescent light and scattered light from the fluorescent substance are received by one photodetector, the signals caused by both lights are electrically separated to calculate the fluorescent light intensity. Highly accurate fluorescence measurements can be performed regardless of scattering properties, and the effects of fluctuations in the characteristics of the photodetector can be eliminated.
This has the unique effect of making it possible to perform highly accurate fluorescence measurements.

The third invention is that even if the light source is difficult to directly modulate, the light irradiated onto the fluorescent material can be intensity-modulated, so any type of light source can be used. It has a unique effect.

The fourth invention is that there is no need to dispose a shutter for modulation in the optical path, the configuration of the optical system can be simplified, and the light source can be modulated electrically. This has the unique effect of increasing the degree of freedom and being able to easily deal with changes in the modulation method.

The fifth invention provides the two
Since different types of signals can be obtained, there is no need for an identification circuit for identifying the type of signal, and the unique effect is that the configuration can be simplified.

[Brief explanation of the drawing]

FIG. 1 is a schematic block diagram showing an embodiment of the fluorescence measuring device of the present invention, FIG. 2 is a schematic diagram showing a more specific embodiment of the fluorescence measuring device of the present invention, and FIG. 3 is a diagram of the FITC. FIGS. 4 and 5 are diagrams illustrating fluorescence excitation efficiency and fluorescence wavelength distribution; FIGS. 4 and 5 are schematic diagrams showing still other embodiments of the fluorescence measuring device, respectively. (1)...Excitation light source, (2)...Reference light source, (
3) (31)...Sample chamber, (4)...Photodetector, (
5)...Correction calculation unit, (1d) (2d) (63a) (63b)-=external modulator, (4a) (4b)...demodulator, (11)...
・Halogen lamp, (14)... Chopper, (21)
...LED. (41)...Photomaru

Claims (1)

[Claims] 1. In a fluorescence measurement method in which a fluorescent substance is irradiated with excitation light and the intensity of the excited fluorescence is measured, the excitation light of a wavelength that can excite the fluorescent substance and the fluorescent substance are used. A fluorescent substance is irradiated with reference light of a non-exciting wavelength and modulated in different ways through almost the same optical path, and the fluorescent light and scattered light are detected by one photodetector ( 4)(
41), and during demodulation based on the electrical signals output from the photodetectors (4) and (41), the signal is separated into a signal corresponding to the fluorescent light and excitation light scattering components and a signal corresponding to the reference light scattering component. A method for measuring fluorescence, characterized in that a signal corresponding only to fluorescence is obtained based on both signals. 2. In a fluorescence measuring device that irradiates a fluorescent substance with fluorescent light and measures the intensity of the excited fluorescent light, an excitation light source (1) (1b) that emits excitation light with a wavelength that can excite the fluorescent substance. )(1
1) (16) (61) (62a), and a reference light source (2) (2b) that emits reference light of a wavelength that does not excite the fluorescent substance.
(21) (24) (61) (62b), and modulation means (1d) (2d) (14) (2
2) (63a) (63b), one photodetector (4) (41) that receives the scattered light from the fluorescent material, which is irradiated with the light emitted from both light sources through almost the same optical path; Detector(
4) Demodulation means (4a) (4b) (43a) (4) for separating a signal corresponding to the excitation light scattering component and a signal corresponding to the reference light scattering component based on the electrical signal output from (41).
3b) (44a) (44b) (45a) (45b) and
Demodulation means (4a) (4b) (43a) (43b) (44
a) (44b), (45a), and (45b) for obtaining a signal corresponding only to the fluorescent light based on both the signals separated by (5) and (51). measuring device. 3. Modulation means (1d) (2d) (14) (63a) (6
3b) modulates the light emitted from the light sources (1), (2), (11), and (61) to convert it into intensity-modulated light. Light measurement device. 4. The fluorescence measuring device according to claim 2, wherein the modulating means (22) modulates the light source (21) to obtain intensity-modulated output light. 5. The demodulation means modulates the excitation light (1d) (1
4) First demodulation means (4a) (43a) (44a) (45a) that performs demodulation operation in synchronization with (63a) and demodulation in synchronization with modulation means (2d) (22) (63b) for reference light. The second demodulating means (4b) (43b) (
44b) (45b).