JPS62278436A - Fluorescence light measuring method and apparatus - Google Patents

Abstract

PURPOSE:To achieve a highly accurate measurement of fluorescence light, by providing a fluorescence light measuring device with a scattered light measuring section for measuring the intensity thereof to correct the effect of the intensity of scattered lights when greatly affecting the highly accurate measurement and quantitative assay of fluorescence light. CONSTITUTION:Radiation 2 from a light source 1 such as high pressure xenon lamp is focused with a lens 3 to be introduced to an excitation wavelength selector 4. An excitation light 5 analyzed with the excitation wavelength selector 4 is focused with a lens 6 to irradiate a sample 7. Fluorescence and scattered light from the sample 7 are collimated with a lens 8 and the lights are made incident on a dichroic mirror 9 which transmits fluorescence component of the light while reflecting the scattered light component thereof in the direction of 45 deg. to separate the fluorescence 10 from the scattered light 11. These lights are focused with lenses 12 and 13 to be introduced to a fluorescence wavelength selector 14 and a scattered light wavelength selector 15 to analyze and then, the intensities of the fluorescence light and the scattered light are measured respectively with photodetectors 16 and 17 such as photoelectric multiplier tube. The measured values thus obtained are sent to a correction computing section 18 to calculate the correct intensity of fluorescence using a specified formula and results are recorded on a recorder 19.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a fluorescence measurement method and apparatus, and is particularly applicable to a sample with a strong scattered light intensity and a fluorescence measurement method where the scattered light intensity changes over time. The present invention relates to a method and apparatus suitable for easy fluorescence measurement of samples.

[Conventional technology]

A fluorometer is generally used as a device for measuring fluorescence intensity and the like. This device basically consists of five parts: a light source, an excitation wavelength selector, a sample chamber, a fluorescence wavelength selector, and a photodetector.

Usually, the light source is a lamp that emits light in the ultraviolet and visible range,
The light is passed through an excitation wavelength selector such as a monochromator and separated into light having a wavelength suitable for exciting the sample, and the light is irradiated onto the sample. The resulting fluorescence is passed through a fluorescence wavelength selector such as a monochromator and received by a detector. However, in actual fluorescence measurement, various light components are detected in addition to the target fluorescence component. In particular, when the concentration of the sample is low or when the sample is a highly scattering substance, blind fluorescent components such as scattered light increase relatively, hindering highly sensitive and highly accurate fluorescence measurements. Therefore, various efforts have been made to perform fluorescence measurements with high sensitivity and precision.

For example, "The Review of Scientific Instruments, 33. (1962) pp. 1213-1215.
ntificInstruments, 33 (19
62) How to compensate for fluctuations in light source intensity as discussed in pp. 1213-1215) and
82 (1958) pp. 1002-1004 (
Nature 182 (1958) ppio02
-1004), highly accurate fluorescence measurements were carried out using a method of manually correcting the intensity distribution of a light source.

[Problem that the invention seeks to solve]

In the conventional example, high accuracy is achieved mainly by correcting temporal fluctuations in the light source and characteristics of the devices themselves such as the light source and spectrometer. However, in actual fluorescence measurement, blind fluorescence also contaminates the target fluorescence component, impairing the measurement accuracy and quantification of fluorescence intensity. Blind fluorescence includes ■scattered and reflected light,
■Raman scattered light and its secondary light, ■So-called stray light due to abnormal reflection in optical systems such as spectrometers, ■Others,
Fluorescence from solvents, fluorescent cells, etc. is considered.

Components other than the desired fluorescence wavelength (scattered light, etc.) can be reduced using a filter or monochromator, but
Because the tail of the scattered light component overlaps with the fluorescence wavelength, it cannot be completely removed and is detected as blind fluorescence. These are particularly problematic when the sample has a low concentration or is a highly scattering material. Blind fluorescence.

When a standard sample that is completely identical except for not having the desired fluorescent component can be prepared, the fluorescence intensities of the sample and the standard sample can be alternately measured and subtracted.

However, preparing such standard samples is generally repeated. In particular, samples such as aggregated silkworm white matter, biological membranes, and polymers are highly scattering substances, and the intensity of scattered light varies from sample to sample or even in the same sample depending on the measurement area, or when the sample is in solution. If the scattered light intensity changes over time due to sedimentation of scatterers such as itself or other dust or air bubbles, or if the scattered light intensity changes due to differences in the drying state of powder or solid samples in an infiltrated state, When the intensity of scattered light changes due to changes in sample concentration, such as when the phosphor is a polymer, it is extremely difficult to prepare standard samples for these changes. In cases such as those mentioned above,
Scattered light is a major factor that hinders highly accurate and quantitative measurement of fluorescence, but in the conventional example, sufficient consideration has not been given to the removal of such scattered light.

In view of the above-mentioned problems, an object of the present invention is to provide a method in which the intensity of scattered light is different from that of fluorescence when the VS constant sample is a scatterer, when the scattered light intensity differs from sample to sample, and when the scattering rate changes within the measurement time. In cases where high precision measurement/quantitative measurement is significantly affected,
[Means for solving the problem] The above purpose is to provide a method and apparatus for correcting the influence and performing highly accurate fluorescence measurement [Means for solving the problem]. This is achieved by providing an optical system for detecting scattered light, a scattered light detector, and an arithmetic unit that calculates and corrects the scattered light intensity included in the fluorescence intensity determined by 'lIA.

(Operation) Using an optical system for detecting scattered light, the intensity of scattered light generated on the sample surface and inside is measured by a scattered light detector.

In addition, the ratio of the transmitted scattered light intensity to the total scattered intensity in the scattered light detection system and the ratio of the transmitted scattered light intensity to the total scattered intensity at the fluorescence wavelength in the fluorescence measurement system are determined by the combination of the scattering wavelength and the fluorescence wavelength. Determine as a constant. Therefore, the correction calculation unit uses this information to calculate the scattered light intensity included in the fluorescence intensity from the measured scattered light intensity and fluorescence intensity, and subtracts it from the fluorescence intensity (
This allows accurate fluorescence intensity to be calculated and output.

(Example) An example of the present invention will be described below with reference to one drawing.

FIG. 1 is a block diagram of a fluorescence measuring device according to the present invention. Emitted light 2 from a light source 1 such as a high-pressure xenon lamp or a high-pressure mercury lamp is focused by a lens 3, and an excitation wavelength selector 4 such as a monochromator, double monochromator, or interference filter is used.
lead to. The excitation light 5 separated by the excitation wavelength selector 4 is focused using a lens 6 and irradiated onto the sample 7. The excitation wavelength selector 4 is adjusted so that the excitation light 5 has a wavelength suitable for exciting the sample 7. Fluorescence emitted from the sample 1
0 and the scatterer 11 using the lens 8 are incident on the dichroic ink mirror 9 from the direction of 45'', which reflects the scattered light component and transmits the fluorescent component, and the fluorescent light 10 and the scattered light 11 are collimated using the lens 8. Further, each of the lights is focused by a lens 12 and 13, guided to a fluorescence wavelength selector 14 and a scattering wavelength selector 15, and after being separated into spectra, the fluorescence is detected by a photodetector 16 and 17 such as a photomultiplier tube. The fluorescence intensity and the scattered light intensity are measured.The fluorescence intensity and the scattered light intensity are corrected by the calculation unit 18.
The correct fluorescence intensity is calculated and recorded by the recorder 19.

The correction calculation unit 18 performs the following calculation. First, the scattered light collimated by the lens 8 is reflected by the dichroic ink mirror 9, passes through the lens 13 and the scattered wavelength selector 15, and is detected by the photodetector 17. Let α(λ
ex) (λe = wavelength of scattered light). Also, among the scattered light, it passes through the dichroic mirror 9 and the lens 12
．． The ratio of light passing through the fluorescence wavelength selector 14 adjusted to transmit light of the fluorescence wavelength and measured by the photodetector 16 is β(λ
. . .

λ. , )(λe,: fluorescence wavelength). α(λθxLβ
(Once the scattered light wavelength and fluorescence wavelength are determined, λeX and λeJ are values unique to the device, so once the calibration is performed, subsequent calibration is unnecessary. Note that these calibration data are stored in the memory 20. Based on the above data, the fluorescence intensity (including a slight scattered light intensity) measured by the photodetector 16 is
sIon and the scattered light intensity measured by the photodetector 17 is I 5eater , the correction calculation unit calculates . This value becomes the true fluorescence intensity excluding the mixed scattered light component.

In this example, a dichroic mirror 9 is used to separate fluorescence and scattered light, but since fluorescence and scattered light wavelength selectors 14 and 15 are also used, a half mirror is used instead of the dichroic mirror. can be used. Of course, in that case α(λeX) lβ
The value of (λear λe,) is a value specific to that case. In addition, as the fluorescence and scattered light wavelength selector 14.15, 2
Although a monochromator is effective, a scattered light wavelength selector 15
is when the scattered light intensity is much larger than the fluorescence intensity.

An interference filter or the like is also sufficient.

Further, in this embodiment, the fluorescent light and the scatterer are collected and detected from the same direction, but it is also possible to detect them from different directions using different optical paths. In this case, since the scattered light intensity generally changes depending on the detection angle, it is also necessary to correct for the difference in direction.

Figure 2 shows the effect of using the measuring device shown in Figure 1.21! FIG. 2(a), which shows a q constant diagram, shows the dependence of fluorescence intensity on fluorescein concentration when a fluorescein solution containing minute glass spheres as a scatterer is used as a sample. In this measurement, the excitation light wavelength and scattered light wavelength were set to 485n.
m, and the fluorescence wavelength was 520 nm. Curve A in the same figure is the 8 (q constant value) when this device is used, and curve B is the photodetector 1 when no calculation is performed in the correction calculation section.
This is the output value of 6. In this way, by passing the signal through the correction calculation section, it was possible to improve the linearity, especially on the low concentration side. Furthermore, FIG. 2(b) shows the change in fluorescence intensity when a sample prepared by dissolving a fluorine solution into silicon powder is gradually dried. The curve C is the K11l constant value when the correction calculation is performed using this device, and the curved line is the measured value when the correction calculation is not performed. By using the scattered light detector and the correction calculation unit, it is possible to eliminate the influence of the scattered light intensity, which increases due to drying of the sample, on the fluorescence intensity, and moreover, it is possible to correct the fluorescence at the same time as detection.

According to this embodiment, the scattered light is detected at the same time as the fluorescence is detected, and the correction calculation unit performs the correction, so that the measurement time can be shortened. Furthermore, since the excitation optical system and fluorescence detection section of this embodiment are almost the same as those of a normal fluorometer, it can also be applied to fluorescence measurements of normal samples such as samples with low scattered light intensity.

〔Effect of the invention〕

According to the present invention, in the case of a sample with a large scattered light magnetism in fluorescence measurement, when the scattered light intensity differs from sample to sample, when the scattered light intensity changes depending on the measurement part of the same sample, and when the scattered light intensity changes during measurement. In the case of samples where the scattering photometer changes, etc., the intensity of the scattered light component mixed in as blind fluorescence within the measured fluorescence intensity can be calculated in real time, allowing for more accurate fluorescence measurements. It can be performed.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the configuration of an embodiment of the present invention;
FIG. 2 is a measurement diagram showing the effects of the present invention.

Claims (1)

[Scope of Claims] 1. A method for measuring fluorescence using a fluorescence measurement device having an excitation light source section for exciting a fluorophore and a fluorescence measurement section for measuring fluorescence generated by the excitation, comprising: A scattered light measuring section for measuring scattered light is provided in the device to measure the scattered light intensity, and a scattered light component mixed in the fluorescence intensity measured by the fluorescence measuring section is calculated based on the scattered light intensity. Fluorescence measurement method characterized by correction. 2. Excitation light source section for exciting the fluorophore In a fluorescence measurement device having a fluorescence measurement section for measuring the fluorescence generated by the excitation, a scattered light comprising an optical system for measuring the scattered light and a photodetector. A correction calculation unit that calculates and corrects a scattered light component mixed in the fluorescence intensity measured by the fluorescence measurement unit based on the measurement unit and the scattered light intensity measured by the scattered light measurement unit. Fluorescence measuring device.