JPH0572039A - Correcting method for spectrum of fluorescence spectrophotometer and fluorescence spectrophotometer with spectrum correcting function - Google Patents

Abstract

PURPOSE:To make it possible to perform highly accurate spectrum correction in a broad measuring range by utilizing an existing reference light source or sub-reference light source used for the wavelength-characteristic measurement on the fluorescence side, and obtaining the wavelength characteristic on the exciting side. CONSTITUTION:An optical quantum meter wherein rhodamine B is contained is set at the position of a sample cell 7. A fluorescence wavelength is set, and a spectroscope 2 on the exciting side undergoes wavelength scanning. The exciting-side wavelength characteristic in a wavelength region lambda1 (e.g. 200-600nm) is measured. The light from a sub-reference light source is made incidention a fluorescence-side spectroscope 9. The fluorescence-side wavelength characteristic in a wavelength region lambda2 (e.g. 500-900nm) is measured. Then, a diffusing element is set at the position of the cell 7. Both wavelengths on the exciting side and the fluorescence side are scanned at the same time for the whole wavelength region (200-900nm). The spectrum value, which is the product of both wavelengths is measured. The spectrum value and both wavelength characteristics are utilized, and the spectrum correcting functions for the fluorescence-side wavelength characteristic in the region lambda1, the exciting-side wavelength characteristic in the region lambda2 and the whole wavelength region are obtained. The functions are used for the true spectrum measurement and computation of an unknown sample.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a spectrofluorometer used for measuring a spectrum specific to a sample, and more particularly to a technique for correcting its spectrum.

[0002]

2. Description of the Related Art In a spectrofluorometer, a light beam emitted from a light source has its wavelength set by an excitation-side spectroscope, and the monochromatic light having the wavelength set irradiates and excites a measurement sample in a sample cell. The emitted fluorescence is detected via a fluorescence side spectroscope and a fluorescence detector to measure a fluorescence (excitation / emission) spectrum.

The fluorescence spectrum is the intensity Iex of the excitation light.
Since it is proportional to the product of (λ) and the extinction coefficient εex (λ) of the fluorescent molecule, it is necessary to observe the fluorescence spectrum with an ideal measurement system that can obtain a constant excitation light intensity at any wavelength. Is desirable. However, in an actual measurement system, the excitation light intensity Iex (λ) fluctuates with wavelength, and the fluctuation largely depends on the so-called individuality of the device such as the type of the light source or the spectroscope and the secular change of the device. This can be said to be similar not only on the excitation side but also on the fluorescence side, because the sensitivity characteristics of the spectroscope and the detector are unique. Therefore, the fluorescence spectrum appearing on the detector is apparent. In order to obtain a true spectrum, it is necessary to perform spectrum correction in consideration of the above-mentioned device characteristics (also called device function or wavelength characteristic).

Various prior arts of this type of spectrum correction have been proposed, such as the one in which spectrum correction is performed by using a photodiode as disclosed in, for example, Japanese Patent Application Laid-Open No. 2-259533. "Fluorescence measurement" edited by Kinoshita and Mihashi, which has been evaluated as a highly sensitive and reliable spectrum correction technology (Academic Publication Center)
Chapter 2. 4. As discussed in "Apparent" Fluorescence Spectra and "True" Fluorescence Spectra (pages 63 to 70), the photon meter method is used for the excitation side and the photon meter method and the standard light source are used for the fluorescence side. There is a combination of methods.

Here, the spectrum correction described in the above document will be described.

[Correction on the Excitation Side] The photonmeter method used for the correction on the excitation side is 600 nm for a high-concentration rhodamine B solution.
Utilizing the property of exhibiting sufficiently high absorbance in the wavelength range up to. Actually, put the Rhodamine B solution in a triangular cell and set it in the sample holder, and set the wavelength of the spectroscope on the fluorescence side to 61.
It is set to a wavelength side longer than 5 nm (to suppress the influence of the internal blocking effect of light emission), the wavelength of the excitation side spectroscope is scanned, the signal from the photodetector is recorded, and the wavelength characteristic of the excitation light intensity is obtained. Based on this, a correction corresponding to a constant excitation light intensity is performed.

[Correction on Fluorescence Side] In the correction on the fluorescence side, the above-described photon counting method and a method using a standard light source are used together. The method using the standard light source is to use a standard tungsten lamp whose spectral output characteristics are accurately determined based on black body radiation or a secondary standard lamp (sub-standard light source) calibrated based on this lamp. The wavelength characteristic on the fluorescence side is obtained by detecting the radiated light of 1 while scanning the wavelength with the fluorescence side spectroscope. At present, this standard light source method is considered to be the only reliable method for knowing the wavelength characteristic on the fluorescence side in the region longer than 600 nm.

The region of 600 nm or less is covered by the photon measurement method using the Rhodamine B solution. That is, first, the spectral characteristic Iex (λ) of the excitation light is obtained by the procedure of Rhodamine B used on the excitation side. Next, a light diffusing element is set at the position of the sample holder, and the excitation side and the fluorescence side spectroscopes are scanned at the same wavelength while guiding the excitation light, and the output H (λi) of the detector is measured. From this ratio of H (λi) and Iex (λ), the wavelength characteristic on the fluorescence side of 600 nm or less can be obtained.

[0009]

In the above-mentioned prior art, on the fluorescence side, the measurement wavelength range of a usual spectrofluorophotometer is obtained by the combined use of the photon meter method using Rhodamine B and the method using a standard light source. The wavelength characteristic of the photometer for (200-900 nm) can be obtained, but there is no reliable and effective method other than the photon-meter method using Rhodamin B on the excitation side, so that it is outside the range of 200-600 nm. For, the wavelength characteristics cannot be obtained. Therefore, as a whole of the apparatus, it is impossible to perform sample analysis in the wavelength range of 600 nm or more with sufficient spectrum correction.

The present invention has been made in view of the above points, and it is an object of the present invention to provide a spectrofluorometer with a highly accurate wavelength characteristic of, for example, 600 nm or more on the excitation side as well as the fluorescence side. It is an object of the present invention to provide a spectrum correction technique which can be obtained by using an existing standard light source or sub-standard light source used for measuring the wavelength characteristic of the side, and which can widen the measurement target wavelength and thus the measurement range of the sample.

[0011]

In order to achieve the above object, the present invention basically proposes the following spectrum correction technique.

That is, when performing spectrum correction of a spectrofluorometer equipped with a light source, an excitation-side spectroscope, a fluorescence-side spectroscope, a fluorescence detector, and a calculator for calculating a measurement result from a fluorescence signal value (fluorescence spectrum value) In addition, the wavelength characteristic of the fluorescence side optical system in a predetermined wavelength range (for example, a wavelength range of 600 nm or more or a value close thereto) is obtained by using a standard light source or a sub-standard light source, and the excitation is performed for the predetermined wavelength range. Side spectroscope and fluorescence side spectroscope simultaneously measure both fluorescence wavelengths on the excitation side and the fluorescence side to measure the fluorescence spectrum, and use the measured fluorescence spectrum value and the standard light source (or sub-standard light source) The wavelength characteristic of the excitation side optical system in the predetermined wavelength region is calculated by the ratio calculation with the wavelength characteristic of the fluorescence side optical system obtained in the above, and the excitation side optical system is obtained as described above. Determining the spectral correction function based on the length properties and fluorescence side wavelength characteristic (referred to as first means for solving problems).

Further, as an application thereof, in addition to the above first means for solving the problems, a wavelength range other than the above-mentioned predetermined range (for example, a wavelength range of 600 nm or less) is used by using an optical quantum meter method in combination.
We propose a method to obtain the wavelength characteristic of.

That is, when performing the spectrum correction of the spectrofluorophotometer as described above, first, the wavelength region λ of the measurement range is divided into the first and second wavelength regions λ1 and λ2 (λ1 <λ2). .. Here, the regions λ1 and λ2 are arbitrarily selected, and for example, in a later-described embodiment, λ1 is 200 to 50.
0 nm and λ2 are set to 500 to 900 nm. Then, on the premise of the above-mentioned regions λ1 and λ2, a step of obtaining the wavelength characteristic Fex1 (λEX) of the excitation side optical system in the first wavelength region λ1 by the photon counting method, the fluorescence side optical system in the second wavelength region λ2 For obtaining the wavelength characteristic Fem2 (λEM) of the above using a standard light source or a sub-standard light source, b, the excitation side spectroscope and the fluorescence side spectroscope for all wavelength regions of the first wavelength region λ1 and the second wavelength region λ2 Step c in which both the excitation side and the fluorescence side wavelengths are simultaneously scanned with each other to measure the fluorescence spectrum, and the measurement value F1 (corresponding to the first wavelength region λ1 among the fluorescence spectrum values measured in the step c) λ
EX, λEM), the measured value F corresponding to the second wavelength region λ2
2 (λEX, λEM) are divided and measured value F1 (λEX,
λEM) and the wavelength characteristic Fex1 (λE
X), the wavelength characteristic Fem1 (λEM) of the fluorescence side optical system in the first wavelength region λ1 is obtained by the ratio calculation, and the measured value F
2 (λEX, λEM) and the wavelength characteristic F obtained in the step b
The second wavelength region λ2 is calculated by the ratio calculation with em2 (λEM).
Of the wavelength characteristic Fex2 (λEX) of the excitation-side optical system, and the excitation side and the fluorescence side of each of the first and second wavelength regions λ1 and λ2 based on the wavelength characteristics obtained by the steps a to d. Spectral correction function Gex1 (λEX), Ge
m1 (λEM) and Gex2 (λEX), Gem2 (λE
M) is obtained (this is the second means for solving the problem).

Further, as an application thereof, a correction function for each of the wavelength regions λ1 and λ2 obtained by the second problem solving means, that is, Gex1 (λEX), Gem1 (λ
EM) and Gex2 (λEX), Gem2 (λEM) are stored, and the correction functions Gex1 (λEX), Gex1 (λEX), according to the wavelength regions λ1, λ2 to which the actually measured fluorescence spectrum values belong.
Gem1 (λEM) or Gex2 (λEX), Gem
We propose a spectrofluorophotometer with a spectrum correction function, which is provided with a means for reading out any one of 2 (λEM) and performing a correction calculation of an apparent spectrum value (this will be referred to as a third means for solving problems).

[0016]

Operation of the first problem-solving means: When light emitted from the standard light source or the sub-standard light source is scanned by the spectroscope of the fluorescence side optical system in the predetermined wavelength region λ and measured by the fluorescence detector, the result is , Wavelength characteristics of standard light source (sub-standard light source) Fc (λE
M) becomes the product of the wavelength characteristic Fem (λEM) of the fluorescence side optical system, and Fc (λEM) is known, so that Fem (λEM) is obtained by the ratio calculation. Further, the fluorescence spectrum value obtained by simultaneous scanning while making both wavelengths on the excitation side and the fluorescence side coincide with each other in the predetermined wavelength region λ is F (λEX, λ
EM). This fluorescence spectrum value F (λEX, λE
M) includes the wavelength characteristic Fex (λEX) of the excitation side optical system in addition to the wavelength characteristic Fem (λEM) of the fluorescence side optical system, which is expressed by the following equation.

[0017]

Fex (λEX) × Fem (λEM) = F (λEX, λEM) That is, Fem (λEM) and F (λEX, λEM)
Is known, the pump side wavelength characteristic Fex (λEX) in the wavelength region λ can be defined as F by satisfying the above equation.
It can be calculated by the ratio calculation of (λEX, λEM) and Fem (λEM). Then, a method using a standard light source or a sub-standard light source that is reliable at 600 nm or more and
By using the formula, we know the wavelength characteristics of the excitation side optical system of 600 nm or more, which could not be measured by the conventional photon measurement method.
As a result, based on the excitation-side wavelength characteristic and the fluorescence-side wavelength characteristic of 600 nm or more, both the excitation-side and fluorescence-side correction functions Ge
x (λEX) and Gem (λEM) can be obtained (normally, the correction function is represented by the reciprocal of the wavelength characteristic).

Operation of the second means for solving the problem ... Excitation-side wavelength characteristic Fex1 of the first wavelength region λ1 obtained in steps I to C
(ΛEX), fluorescence-side wavelength characteristic Fe of the second wavelength region λ2
m2 (λEM), fluorescence spectrum values F1 (λEX, λEM), F2 (λE) in all wavelength regions λ1 and λ2 to be measured
X, λEM) can all be obtained by using the existing highly reliable method.

The above-mentioned known actual measurement data Fex1 (λE
X), Fem2 (λEM), (λEX, λEM), F2
The following expression holds for (λEX, λEM) and the unknown fluorescence-side wavelength characteristic Fem1 (λEM) of the first wavelength region λ1 and the excitation-side wavelength characteristic Fex2 (λEX) of the second wavelength region λ2.

[0020]

[Expression 3] About λ1 Fex1 (λEX) × Fem1
(ΛEM) = F1 (λEX, λEM) About λ2 Fex2 (λEX) × Fem2 (λE
M) = F2 (λEX, λEM) From the above formula, F1 (λE
X, λEM) and Fex1 (λEX) ratio calculation
Of the fluorescence side wavelength characteristic Fem1 (λEM) in the wavelength region λ1 of F2 (λEX, λEM) and Fem2 of known data.
The wavelength characteristic Fex2 (λEX) of the excitation-side optical system in the second wavelength region λ2 is obtained by the ratio calculation with (λEM).

Then, by obtaining all of the above wavelength characteristics, the correction function Gex1 on the excitation side of the first wavelength region λ1 is obtained.
(ΛEX), the correction function Gem1 (λEM) on the fluorescence side, and the correction function Gex2 (λE on the excitation side of the second wavelength region λ2.
X), the correction function Gem2 (λEM) on the fluorescence side can be obtained as follows.

[0022]

For λ1, Gex1 (λEX) = Fex1 (λA) / Fex1 (λEX) Gem1 (λEM) = Fem1 (λA) / Fem1 (λEM) ・ For λ2 Gex2 (λEX) = Fex2 (λA) / Fex2 (ΛEX) Gem2 (λEM) = Fem2 (λA) / Fem2 (λEM) λA: A value appropriately selected with reference to one of the apparent spectral values in order to correct the relative shape of the spectrum. Among the elements described in the second means for solving problems, λ2, Fex2 (λEX), Fem2 (λE
M), Gex2 (λEX), and Gem2 (λEX) are respectively λ and Fex (λE) described in the first means for solving problems.
X), Fem (λEM), Gex (λEX), Gem
This corresponds to (λEX).

According to this means for solving the problem, compared with the conventional photon-meter method and the method using the standard light source (or the sub-standard light source), only the excitation side / fluorescence side spectrum correction of 200 to 600 nm can be conventionally performed. What was not 200-90
Enables spectral correction of excitation side and fluorescence side up to 0 nm.

Operation of the third means for solving problems ... Spectral correction function Gex1 (λ
EX), Gem1 (λEM) and Gex2 (λEX), G
By reading em (λEM) from the storage unit according to the wavelength used for spectrum measurement, the true spectrum can be calculated by automatic spectrum data processing.

[0025]

An embodiment of the present invention will be described with reference to the drawings.

FIG. 1 is a flow chart showing an example of the spectrum correction of the present invention, FIG. 2 is an explanatory view for obtaining wavelength characteristics of an apparatus used for the spectrum correction, FIG. 3 is a waveform explanatory view corresponding to FIG. 2, and FIG. [Fig. 3] is a configuration diagram of a spectrofluorophotometer to which the present invention is applied.

First, a spectrofluorometer will be described with reference to FIG.

In FIG. 4, the light beam emitted from the light source 1 enters the excitation side spectroscope 2. The set wavelength of the excitation side spectroscope 2 can be changed by the pulse motor 3. The operation of the pulse motor 3 is preprogrammed in the computer 4 and is controlled via the interface 5. The wavelength of the excitation side spectroscope 2 is set by keying in the desired excitation wavelength on the operation panel 6. The monochromatic light extracted by the excitation side spectroscope illuminates the measurement sample 8 in the sample cell 7, and the fluorescence emitted from the sample 8 enters the fluorescence side spectroscope 9. The fluorescence side spectroscope 9 is driven by the pulse motor 10, and its operation is the pulse motor 3.
Is controlled by the computer 4 in the same manner as. The fluorescence of the wavelength selected by the fluorescence-side spectroscope 9 enters the detector 11 and is converted into an electric signal. The electrical signal is converted into a digital signal by the analog-digital converter 12. On the other hand, part of the monochromatic light extracted from the excitation-side spectroscope 2 enters the monitor detector 14 via the beam splitter 13 for monitoring the light source light amount, and is converted into an electric signal. This electrical signal is also used by the analog-digital converter 12
Can be converted into a digital signal. The digital signal (S) from the detector 11 and the digital signal (M) from the monitor detector 14 are sent to the computer 4 and the ratio (S /
M) is calculated, and this ratio is stored in the computer 4 as the fluorescence intensity at each wavelength.

The computer 4 is equipped with a Rhodamine B, which will be described later.
Wavelength characteristic (excitation side wavelength characteristic) of the excitation side optical system in the first wavelength region λ1 (where λ1 is 200 to 600 nm) obtained using the photon meter 15 (FIG. 2) according to The second wavelength region λ2 (λ2 is 500 to 900 n) obtained by using the calibrated secondary lamp 16
m) the wavelength characteristic of the optical system on the fluorescence side (wavelength characteristic of the fluorescence side), and the first relation based on a predetermined relational expression using these wavelength characteristics.
Wavelength region λ1 fluorescence side wavelength characteristic, second wavelength region λ2
Excitation side wavelength characteristic and, by extension, spectrum correction function Gex1
(ΛEX), Gem1 (λEM), Gex2 (λE
X) and Gem2 (λEM) are stored in the computer 4. Each wavelength range λ obtained
The wavelength characteristics and the correction function of the excitation side and the fluorescence side of 1, λ2 are
It is stored in the RAM in the computer 4 and used for true spectrum measurement and calculation of an unknown sample.

Next, the procedure for obtaining the wavelength characteristics and the spectrum correction function of the device will be described with reference to FIGS.

As shown in steps S1 to S4 of FIG.
The procedure for obtaining the wavelength characteristic and the spectrum correction function is roughly classified into four stages.

In step S1, Rhodamine B is used to measure the wavelength characteristic on the excitation side in the first wavelength region λ1 (200 to 600 nm in this example). 2 (a) corresponds to this, in which the triangular cell (photometer) 15 containing rhodamine B is set at the sample cell position, and the fluorescence wavelength is set to 6
It is fixed at 40 nm. At this time, the excitation side spectroscope 2
When the excitation spectrum for 200 to 600 nm is measured by scanning the wavelength of the wavelength, the result becomes the excitation side wavelength characteristic Fex1 (λEX) of the wavelength region λ1 as shown in FIG. 3 (a), which is stored in the computer 4. ..

In step S2, the sub-standard light source 16 causes the second wavelength region λ2 (in this example, 500 to 900 n).
The wavelength characteristic on the fluorescence side for m) is measured. That is, as shown in FIG. 2B, the light beam emitted from the sub-standard light source 16 is made incident on the fluorescence side spectroscope 9. Fluorescence side spectroscope 9
When the fluorescence spectrum is measured by scanning the wavelength from 500 to 900 nm with the result, the result is the wavelength characteristic Fc (λEM) of the sub-standard light source 16 and the wavelength characteristic Fem2 (λ of the fluorescence side optical system.
EM). Sub-standard light source 16 used here
Is a tungsten lamp, and its wavelength characteristics have been confirmed to match the equation for black body radiation at 2995 ° K. Therefore, Fc (λE
M) is known, and Fem2 (λEM) as shown in FIG. 3B can be obtained by the ratio calculation, and this is stored in the computer 4.

In step S3, a spectrum obtained by scanning both excitation-side and fluorescence-side wavelengths at the same wavelength and at the same speed for all wavelength ranges λ1 and λ2 is measured. That is, (c) of FIG.
In this case, as shown in FIG.
Set 7. Since the diffusing element 17 itself does not have wavelength dependence, the result is as shown in the following equation:
And the wavelength characteristic of the fluorescence side spectroscope 9. The measurement result is shown in FIG.

[0035]

## EQU5 ## For λ1 <500 nm, Fex1 (λE
X) × Fem1 (λEM) = F1 (λEX, λEM) For λ2> 500 nm Fex2 (λEX) × Fe
m2 (λEM) = F2 (λEX, λEM) λEX = λEM Among the above, Fex1 (λEX) and Fem2 (λEM) have already been obtained by the above operation, so
Fem1 (λEM) and Fex2 (λEX) are obtained by the ratio calculation of 1 (λEX, λEM) and F2 (λEX, λEM).

In step 4, from the wavelength characteristics obtained by the above operation, the entire wavelength range λ of each of the excitation side and the fluorescence side is determined.
Spectral correction function Gex1 (λE
X), Gem1 (λEM) and Gex2 (λEX), G
Determine em2 (λEM).

These spectrum correction functions are for obtaining the true spectrum by multiplying the apparent spectrum measured for the unknown sample. The spectrum obtained by the fluorometer is an arbitrary unit on the vertical axis,
The meaning referred to as a correction spectrum here does not give an absolute unit for the intensity, but means to correct the relative shape of the spectrum. However, it is not preferable that the value after correction is significantly different from the value before correction, and therefore the value of the correction function is normalized so that it becomes close to 1, and the value of the appropriate wavelength becomes 1. In this case, 500
nm value [Fex1 (500), Fex2 (500),
Fem1 (500), Fem2 (500)] standardizes each function, and a correction function is calculated by the following equation.

[0038]

For λ1 (500 nm or less) Gex1 (λEX) = Fex1 (500) / Fex1 (λEX) Gem1 (λEM) = Fem1 (500) / Fem1 (λEM) ・ For λ2 (500 nm or more) Gex2 (λEX) = Fex2 (500) / Fex2 (λEX) Gem2 (λEM) = Fem2 (500) / Fem2 (λEM) The spectral correction function obtained as described above is stored in the computer 4.

After the unknown sample is measured in step 5, it is used to obtain a corrected spectrum by the following formula.

[0040]

## EQU00007 ## Regarding .lambda.1 Ic (.lambda.EX, .lambda.EM) = I
(ΛEX, λEM) × Gex1 (λEX) × Gem1
(ΛEM) For λ2 Ic (λEX, λEM) = I (λE
X, λEM) × Gex2 (λEX) × Gem2 (λE
M) In the above formula, Ic is the corrected spectrum and I is the spectrum before the correction.

According to the present embodiment, the wavelength characteristics on both the excitation side and the fluorescence side are obtained by a highly reliable method over 200 to 900 nm, and the spectrum correction functions on both the excitation side and the fluorescence side based on this are obtained in the wavelength range. 200-500
nm and 500 to 900 nm are finely set and the fluorescence spectrum value can be accurately corrected and calculated, and the true spectrum is automatically obtained by computer data processing.

[0042]

As described above, according to the present invention, the wavelength characteristics on the excitation side and the fluorescence side of the spectrofluorometer, and thus the spectrum correction function based on the wavelength characteristics, are obtained more widely than before, and a method with high reliability is used. Therefore, the measurement range of the sample can be expanded while maintaining high accuracy.

[Brief description of drawings]

FIG. 1 is a flowchart showing an example of spectrum correction of the present invention.

FIG. 2 is an explanatory diagram for obtaining wavelength characteristics of a device used for the spectrum correction.

FIG. 3 is a waveform explanatory diagram corresponding to FIG.

FIG. 4 is a configuration diagram of a spectrofluorophotometer to which the present invention is applied.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Light source, 2 ... Excitation side spectroscope, 3 ... Excitation side pulse motor, 4 ... Computer (spectrum correction means), 5 ... Interface, 6 ... Operation panel, 7 ... Sample cell, 8 ...
Measurement sample, 9 ... Fluorescence side spectroscope, 10 ... Fluorescence side pulse motor, 11 ... Detector, 12 ... A / D converter, 13 ... Beam splitter, 14 ... Monitor detector, 15 ... Photoquantum meter, 1
6 ... Sub-standard light source, 17 ... Diffusing element.

Claims (6)

[Claims] 1. When performing spectrum correction of a spectrofluorometer equipped with a light source, an excitation-side spectroscope, a fluorescence-side spectroscope, a fluorescence detector, and a calculator that calculates a measurement result from a fluorescence signal value (fluorescence spectrum value). In addition, the wavelength characteristics of the fluorescence side optical system in a predetermined wavelength region are obtained by using a standard light source or a light source calibrated by a standard light source (sub-standard light source), and the excitation side spectroscope, fluorescence in the predetermined wavelength region. The excitation side and the fluorescence side are simultaneously scanned by the side spectroscope while measuring the fluorescence spectrum at the same time, and the measured fluorescence spectrum value and the fluorescence side obtained by using the standard light source or the sub-standard light source. The wavelength characteristic of the excitation side optical system in the predetermined wavelength region is calculated by a ratio calculation with the wavelength characteristic of the optical system, and the spectrum is calculated based on the wavelength characteristic of the excitation side optical system and the wavelength characteristic of the fluorescence side optical system. A spectral correction method for a spectrofluorophotometer, which comprises defining a correction function. 2. The tungsten lamp is used as the standard light source or the sub-standard light source according to claim 1, wherein the predetermined wavelength region includes a region longer than 600 nm, and both the excitation side and the fluorescence side have the same wavelength. A spectral correction method for a spectrofluorometer, which comprises setting a light diffusing element on the optical path for fluorescence spectrum measurement by simultaneous scanning while performing. 3. When performing spectrum correction of a spectrofluorometer equipped with a light source, an excitation-side spectroscope, a fluorescence-side spectroscope, a fluorescence detector, and a calculator that calculates a measurement result from a fluorescence signal value (fluorescence spectrum value). In addition, the wavelength region λ of the measurement range is divided into first wavelength regions λ1 and λ2 (λ1 <λ2), and the wavelength characteristic Fex1 of the excitation side optical system in the first wavelength region λ1
Step (a) of obtaining (λEX) by the photon measurement method, wavelength characteristic Fem2 of the fluorescence side optical system in the second wavelength region λ2
Step (b) for obtaining (λEM) by using a standard light source or a light source (sub-standard light source) calibrated by the standard light source, the excitation side spectroscope for all wavelength regions of the first wavelength region λ1 and the second wavelength region λ2 A step c of simultaneously scanning the fluorescence side spectroscope while matching both wavelengths on the excitation side and the fluorescence side to measure the fluorescence spectrum, which corresponds to the first wavelength region λ1 of the fluorescence spectrum values measured in the step c. Measured value F1 (λEX, λEM) of
The measured value F2 (λEX, λE corresponding to the second wavelength region λ2
M), and the wavelength characteristic Fem of the fluorescence-side optical system in the first wavelength region λ1 is calculated from the ratio calculation of the measured value F1 (λEX, λEM) and the wavelength characteristic Fex1 (λEX) obtained in step a.
1 (λEM) is obtained, while the measured value F2 (λEX, λE
M) and the wavelength characteristic Fem2 (λEM) obtained in the step B
And the wavelength characteristic Fex2 (λEX) of the excitation-side optical system in the second wavelength region λ2 by the ratio calculation with the first and second wavelength regions λ1 based on the respective wavelength characteristics obtained in the steps a to d. , Λ2 for each of the excitation-side and fluorescence-side spectral correction functions Gex1 (λEX), Gem1 (λE
M) and Gex2 (λEX) and Gem2 (λEM) are determined, and a spectrum correction method for a spectrofluorometer. 4. The method according to claim 3, wherein Rhodamine B is used as the fluorescent screen of the photon meter in the step b, a tungsten lamp is used as the standard light source or the sub-standard light source in the step b, and the first wavelength region is 600 nm or less. 20
It is set from a range of 0 nm or more, and the second wavelength region includes a range of 900 nm or less in the first wavelength region or more (the first and second regions may partially overlap). A spectral correction method for a spectrofluorophotometer, characterized in that: 5. The spectrum correction method for a spectrofluorophotometer according to claim 3 or 4, wherein in the step C, a light diffusing element is set on an optical path to perform spectrum measurement. 6. A spectrofluorometer comprising a light source, an excitation-side spectroscope, a fluorescence-side spectroscope, a fluorescence detector, and a calculator for calculating a measurement result from a fluorescence signal value (fluorescence spectrum value), wherein an excitation-side optical system is provided. And a means for correcting the apparent fluorescence spectrum value influenced by the wavelength characteristic of the fluorescence side optical system, and this spectrum correcting means changes the wavelength range λ of the measurement range to the first wavelength range λ1 and the second wavelength range λ2. Divided into (λ1 <λ
2), correction functions Gex1 (λEX) and Gem1 (λ) obtained in advance by the following relational expressions for each of these wavelength regions λ1 and λ2.
EM) and Gex2 (λEX) and Gem2 (λEM) are stored, and for λ1 Fex1 (λEX) × Fem
1 (λEM) = F1 (λEX, λEM) for λ2 Fex2 (λEX) × Fem2 (λE
M) = F2 (λEX, λEM) where Fex1 (λEX): wavelength characteristic of the excitation side optical system in the first wavelength region λ1 obtained by the photon counting method F1 (λEX, λEM): first wavelength region λ1 In the above, the spectral measurement values obtained by simultaneously scanning the wavelengths of the excitation-side spectroscope and the fluorescence-side spectrograph while matching the excitation-side wavelength and the fluorescence-side wavelength are Fem1 (λEM): F1 (λEX, λEM) and Fex.
1 (λEX, λEM) wavelength characteristic of the fluorescence side optical system of the first wavelength region λ1 obtained by the ratio calculation with 1 (λEX, λEM) Fem2 (λEX): using a standard light source or a light source (sub-standard light source) calibrated by the standard light source Obtained wavelength characteristic of the fluorescence side optical system in the second wavelength region λ2 F2 (λEX, λEM): Excitation side spectroscope and fluorescence side spectroscope while matching the excitation side wavelength and the fluorescence side wavelength in the second wavelength region λ2 Measurement values obtained by simultaneously scanning wavelengths of Fex2 (λEM): F2 (λEX, λEM) and Fem
Wavelength characteristics of the excitation side optical system in the second wavelength region λ2 obtained by the ratio calculation with 2 (λEX, λEM). For λ1, Gex1 (λEX) = Fex1 (λA) / Fex1 (λEX) Gem1 (λEM) = Fem1 About (λA) / Fem1 (λEM) · λ2 Gex2 (λEX) = Fex2 (λA) / Fex2 (λEX) Gem2 (λEM) = Fem2 (λA) / Fem2 (λEM) where Gex1 (λEX): λ1 excitation Correction spectrum function of side optical system Gem1 (λEM): Correction spectrum function of fluorescence side optical system of λ1 Gex2 (λEX): Correction spectrum function of excitation side optical system of λ2 Gem2 (λEM): Correction of fluorescence side optical system of λ2 Spectral function λA: In order to correct the relative shape of the spectrum, one of the apparent spectral values was selected as a reference. And said storage .lambda.1, measurement wavelength region of the fluorescence spectrum values measured one of the compensation spectrum function of .lambda.2 .lambda.1,
A spectrofluorometer with a spectrum correction function, which is set to read out according to λ2 and perform correction calculation of an apparent spectrum value.