JPH08178849A - Fluorometry and fluorometer - Google Patents

Abstract

PURPOSE: To provide a fluorometry and fluorometer for measuring fluorescence of sample accurately through a simple and inexpensive constitution. CONSTITUTION: The fluorescent microscope 1 comprises a light source 6, an excitation filter 8 arranged on the optical path of exciting light emitted from the light source 6, a fluorescent filter 26 arranged on the optical path of fluorescence emitted from a sample 10, and a photomultiplier 30 for measuring the fluorescence passed through the filter 26. A sample containing a substance to be inspected is previously given to a fluorescence indicator. The sample 10 is irradiated with an exciting light from the light source 6 through an excitation filter 8 passing the excitation wavelength of the fluorescent indicator selectively. Fluorescence emitted from the sample 10 is passed through the fluorescent filter 26 for selecting the equi-fluorescent wavelength or peak wavelength of the fluorescent indicator and the fluorescent intensity is measured by means of the photomultiplier 30. A determination unit 40 calculates the ratio between two fluorescent intensities and determines the concentration of a substance to be inspected comparing with known fluorescent characteristics.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence metering apparatus and a fluorescence metering method for measuring fluorescence from a sample, and in particular,
The present invention relates to a fluorescence metering apparatus and a fluorescence metering method for quantifying the concentration of a substance to be inspected contained in a sample from a ratio of fluorescence intensities of fluorescence having two different wavelengths.

[0002]

2. Description of the Related Art For example, when quantifying intracellular calcium ion concentration, a predetermined fluorescent indicator (Indo-
It is known that the fluorescence image of the cell emits light having a fluorescence spectrum corresponding to the calcium ion concentration when the cell is irradiated with excitation light (340 nm). The ratio between any two wavelengths of this fluorescence spectrum has a value according to the concentration of calcium ions contained in the cell. Therefore, by measuring the fluorescence image with two wavelengths and obtaining the ratio of the fluorescence intensities between the two wavelengths, the intracellular calcium ion concentration can be quantified.

As a device for measuring two wavelengths of a fluorescent image as described above, for example, a fluorescent microscope device 51 shown in FIG. 6 is known. The fluorescence microscope device 51 irradiates a sample 60 with light from a light source 52 via an excitation filter 54 and a dichroic mirror 56, and emits fluorescence from the sample 60 via a dichroic mirror 56 and a half mirror 58 into a light splitting plate 62. Is being propagated to. One light split by the light splitting plate 62 is detected by the photomultiplier 65 through the absorption filter 64, and the other light is absorbed by the absorption filter 6
It is detected by the photomultiplier 67 through the absorption filter 66 having a transmission wavelength range different from that of No. 4.

The fluorescence intensities of the respective wavelengths detected by the two photomultipliers 65 and 67 are input to the quantification device 70 to calculate the ratio of the respective fluorescence intensities, and this intensity ratio corresponds to the calcium ion concentration. The calcium ion concentration is quantified by collating with known fluorescence intensity characteristics.

[0005]

However, in the conventional device, two photomultipliers are required to perform the two-wavelength simultaneous photometry, and there is a problem that the device becomes large. In addition, since it is necessary to provide two relatively expensive photomultipliers, there is a problem that the device becomes expensive.

Further, it is necessary to adjust the sensitivity of the two photomultipliers so that they have the same sensitivity, which makes it difficult to adjust the sensitivity. Furthermore, the sensitivity of Photomul is easily affected by changes in the environment such as the ambient temperature, and the sensitivity of the two Photomul may be relatively shifted during the photometry. In this case, accurate photometry becomes difficult.

The present invention has been made in view of the above points, and an object thereof is to provide a fluorescence metering apparatus and a fluorescence metering method capable of accurately measuring the fluorescence of a sample with a simple and inexpensive structure.

[0008]

In order to achieve the above object, a fluorescence photometric device according to the present invention is intended to act on a substance to be inspected in a fluorescence photometric device for measuring fluorescence from a sample containing the substance to be inspected. Irradiation means for irradiating the sample with excitation light having the excitation wavelength of the fluorescent indicator administered to the sample, and is arranged on the optical path of the fluorescence emitted from the sample excited by the excitation light, the isofluorescence of the fluorescent indicator A wavelength selecting means for selectively selecting a wavelength or a predetermined wavelength and selectively passing fluorescence having these two wavelengths, and a single photometric means for measuring the fluorescence intensity of the fluorescence selected by the wavelength selecting means. And a quantification means for calculating the ratio of the two fluorescence intensities measured by the photometric means and for quantifying the concentration of the substance to be inspected by collating this ratio with a known fluorescence intensity characteristic. There.

Further, the fluorescence photometric method according to the present invention comprises the steps of administering a fluorescent indicator that acts on the substance to be inspected to a sample containing the substance to be inspected, and exciting light having the excitation wavelength of the fluorescent indicator as described above. The step of irradiating the sample, the step of photometrically measuring the fluorescence emitted from the sample excited by the irradiation of the excitation light with a single photometric device, and the step of photometrically measuring the fluorescence indicator at the beginning and the end of the photometry, etc. The fluorescence intensity of the fluorescence at the fluorescence wavelength is measured, and the fluorescence intensity of the fluorescence at a predetermined wavelength is measured in the middle of the photometry, and the ratio between the fluorescence intensity at the equal fluorescence wavelength and the fluorescence intensity at the predetermined wavelength is calculated. And a step of quantifying the concentration of the substance to be inspected by collating this ratio with a known fluorescence intensity characteristic.

[0010]

According to the fluorescence photometry device and the fluorescence photometry method of the present invention, the excitation light having the excitation wavelength of the substance to be inspected contained in the sample is applied to the sample by the irradiation means. The sample excited by this excitation light emits a fluorescence spectrum corresponding to the concentration of the substance to be inspected in the sample. Then, the fluorescence intensity of this fluorescence spectrum is measured by the photometric means.

The wavelength selecting means provided on the optical path of the fluorescence toward the photometric means has a function of selectively selecting the equi-fluorescence wavelength of the fluorescent indicator or a predetermined wavelength. At the beginning and the end of the photometry, the wavelength selection means selects the equifluorescence wavelength of the fluorescent indicator, and the photometry means measures the fluorescence intensity of the fluorescence at this wavelength. In the middle of photometry, the wavelength selecting means has a predetermined wavelength, for example, a wavelength indicating the peak intensity of the fluorescence spectrum when the fluorescent indicator is bound to the test substance,
The photometric means measures the fluorescence intensity of fluorescence at this wavelength.

The measured fluorescence intensities at the two wavelengths are input to a quantitative means, where the ratio of the fluorescence intensities between the two is calculated, and this ratio of the fluorescence intensities is collated with the known fluorescence intensity characteristics of the sample. , The concentration of the test substance contained in the sample is quantified.

[0013]

Embodiments of the present invention will now be described in detail with reference to the drawings. As shown in FIGS. 1 and 2, a fluorescence photometric device according to an embodiment of the present invention, that is,
The fluorescence microscope apparatus 1 includes a main body 2 including a base portion 2a placed on a desk and an arm portion 2b extending upward from one end of the base portion 2a. A light projecting tube 4 extending in the lateral direction is provided on the upper part of the main body 2, and an epi-illumination light source 6 is provided on one end side of the light projecting tube 4.

An excitation filter 8 is provided in the light projecting tube 4 and on the optical path of the excitation light from the light source 6. The excitation filter 8 has a function of selectively passing only the excitation light having the excitation wavelength of the fluorescent indicator described later to be administered to the sample 10, and is replaceable on the optical path according to the type of the fluorescent indicator used. It is arranged. The light source 6 and the excitation filter 8 constitute an irradiation means.

A dichroic mirror 12 is provided on the downstream side of the excitation filter 8, and the dichroic mirror 1 is provided.
An objective lens 14 is provided on the optical path bent substantially vertically downward by 2. In addition, the main body 2 includes a stage 11 on which the sample 10 is movably mounted along the optical axis of the objective lens 14.

Above the objective lens 14, a single photomultiplier 30 as a photometric means is provided via a lens barrel 20 and a wavelength switching section 25. In the lens barrel 20,
A half mirror 21 is provided to guide a part of the fluorescence from the sample 10 to an eyepiece 22 connected to the lens barrel 20. In the wavelength switching unit 25, a fluorescence filter 26 is provided as a wavelength selection unit that transmits only a predetermined wavelength component of fluorescence from the sample 10. The fluorescence filter 26 is an absorption filter 2 that selectively passes fluorescence of the same fluorescence wavelength of the fluorescence indicator administered to the sample 10.
6a, and an absorption filter 26b that selectively passes the fluorescence peak when the fluorescent indicator binds to the substance to be tested in the sample 10. These filters 26
Both a and 26b are provided so that they can be inserted into and removed from the optical path of fluorescence and can be replaced with other filters.

The photomultiplier 30 is connected to a quantification device 32 as a quantification means for calculating the ratio of fluorescence intensities for two wavelengths and quantifying the concentration of calcium ions based on this ratio.

Fluorescence microscope apparatus 1 constructed as described above
A method for quantifying the concentration of the substance to be inspected in the sample by using will be described with reference to FIG. Here, in particular, a method for quantifying the calcium ion concentration contained in cells as a sample will be described as an example.

First, a predetermined fluorescent indicator is administered to cells to be inspected (step 41). This fluorescent indicator (for example, Indo-1) has a function of binding to intracellular calcium ions and shifting the fluorescence spectrum emitted from the cells.

Then, by the excitation filter 8, the light source 6
Of the excitation light from the fluorescent indicator (340n
Only the excitation light having m) is selectively passed through and the cells are irradiated with this excitation light via the dichroic mirror 12 and the objective lens 14 (step 42). This allows
The excited cells emit light having a fluorescence spectrum that is shifted by the fluorescent indicator.

In the case of measuring fluorescence, first, an absorption filter 26a which passes only the equifluorescence wavelength (455 nm) of the fluorescence indicator is arranged on the fluorescence optical path. Then, the fluorescence intensity S0 of the fluorescence is measured by the photomultiplier 30 (step 43).

Next, an absorption filter 26b that passes only a predetermined wavelength, that is, a wavelength (405 nm) showing the peak intensity of fluorescence when the fluorescent indicator is bound to calcium ions is arranged on the optical path of fluorescence. Then, the fluorescence intensity S1 of the fluorescence is measured by the photomultiplier 30 (step 44).

At the end of the photometry, the absorption filter 26a is arranged again on the fluorescence optical path, and the fluorescence intensity S0 'of the fluorescence is measured by the photomultiplier 30 (step 45). This fluorescence intensity S0 'is used to correct the fluorescence intensity S0 measured in step 43.

Then, the fluorescence intensity S0 and the fluorescence intensity S1 measured by the Photomul 30 are input to the quantification device 32 for quantifying the calcium ion concentration, and the intensity ratio S1 /
S0 is calculated (step 46). The intensity ratio S1 / S0 calculated in step 46 is compared with a known fluorescence intensity characteristic of cells containing calcium ions, or is input into a known conversion formula to quantify the calcium ion concentration (step 47).

As described above, since the fluorescence microscope apparatus of the present invention measures fluorescence using a single photomultiplier, the apparatus can be downsized and the apparatus cost can be reduced as compared with the conventional apparatus. . In addition, by using a single Photomul, the photomul sensitivity can be adjusted easily, and accurate photometry can be performed without the problem of relative shifts in sensitivity during photometry as with conventional devices. Becomes

According to the photometric method of the present invention, the fluorescence intensities S0 and S0 'are measured at the beginning and the end of the photometry, and the fluorescence intensity S1 is measured during the photometry. Therefore, dual-wavelength photometry can be performed without switching the photometric wavelength each time photometry is performed, the time required for wavelength selection can be shortened, and rapid photometry that responds to changes over time of calcium ions is possible.

Next, a modified example of the present invention will be described with reference to FIGS. Since the basic structure is the same as that of the above-mentioned embodiment, the same parts are designated by the same reference numerals, and the description thereof will be omitted. Only different parts will be described.

As shown in FIGS. 4 and 5, the fluorescence microscope apparatus 101 is provided between the light source 6 and the light projecting tube 4 with an excitation wavelength selection device 125 for selectively switching the wavelength of the excitation light. . As the excitation wavelength selection device 125, there is a spectroscope, a continuous interference filter, or an interference filter having a function of changing the transmission wavelength by selecting an arbitrary angle. It has a function of transmitting only its own excitation light. The wavelength switching unit 25 provided on the fluorescence optical path in the above-described embodiment is removed.

Using the device thus constructed, the intracellular calcium ion concentration is quantified. In this case, the isofluorescence wavelength of Fura-2 as a fluorescence indicator is determined prior to the quantitative determination of the calcium ion concentration.

First, two types of samples (solutions) having different calcium ion concentrations are prepared, and Fura-2 is administered to each of these two types of solutions. Then, these solutions having different concentrations are alternately arranged in the fluorescence microscope apparatus 101,
Each is irradiated with excitation light of 360 nm. Graph each fluorescence spectrum obtained from the excited solution,
The isofluorescence wavelength of Fura-2 is determined from the intersection of the two.

Next, the selection wavelength of the excitation wavelength selection device 125 is adjusted so as to match the determined equal fluorescence wavelength,
The cells for measuring the calcium ion concentration are set in the fluorescence microscope apparatus 101. Then, the cells are irradiated with the excitation light, and the intensity of the fluorescence reflected from the cells is measured by the photomultiplier 30.

This fluorescence intensity is input to the quantification device 32 and compared with a known fluorescence intensity characteristic value to quantify the calcium ion concentration of the cell. As described above, after determining the isofluorescence wavelength, the sample is irradiated with the excitation light having the isofluorescence wavelength, and the fluorescence from the sample is measured, whereby more reliable photometry is possible and accurate quantification can be performed. . still,
The present invention is not limited to the above-described embodiments, and can be variously modified without exceeding the scope of the invention.

[0033]

As described above, since the fluorescence photometric device of the present invention has the above-described structure and operation, the number of photometric devices (photomul), which are relatively expensive, is reduced. Therefore, the device can be configured to be relatively small and the device cost can be reduced.

Since it is not necessary to provide a plurality of photometric devices as in the conventional device, it is not necessary to adjust the sensitivities of the photometric devices so that the sensitivities of the photometric devices are matched with each other, and the labor for sensitivity adjustment can be saved. Further, it is possible to solve the problem that the sensitivities of the respective photometric devices relatively shift during the photometry, and more accurate photometry becomes possible.

Furthermore, since the fluorescence intensity of the fluorescence having the same fluorescence wavelength of the fluorescence indicator is measured at the beginning and the end of the photometry and the peak intensity of the fluorescence is measured in the middle of the photometry, the time required for switching the fluorescence filter is reduced. It is possible to reduce the amount, and it is possible to perform rapid photometry corresponding to the change with time of calcium ions.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a fluorescence microscope apparatus according to an embodiment of the present invention.

FIG. 2 is a block diagram showing a device configuration of the fluorescence microscope device of FIG.

FIG. 3 is a diagram showing a quantitative operation of calcium ion concentration using the fluorescence microscope apparatus of FIG. 1.

FIG. 4 is a schematic view showing a modified example of the fluorescence microscope apparatus of the present invention.

5 is a block diagram showing an apparatus configuration of the fluorescence microscope apparatus of FIG.

FIG. 6 is a schematic view showing a conventional fluorescence microscope apparatus.

[Explanation of symbols]

1 ... Fluorescence microscope device, 6 ... Light source, 8 ... Excitation filter,
10 ... Sample, 12 ... Dichroic mirror, 14 ... Objective lens, 26 ... Fluorescent filter, 30 ... Photomaru, 4
0 ... Concentration quantification device.

Claims (2)

[Claims] 1. A fluorescence photometric device for measuring fluorescence from a sample containing a substance to be inspected, wherein the sample is irradiated with excitation light having an excitation wavelength of a fluorescent indicator administered to the sample to act on the substance to be inspected. And an irradiation means arranged on the optical path of fluorescence emitted from the sample excited by the excitation light, and an alternative fluorescence wavelength or a predetermined wavelength of the fluorescence indicator is selectively selected, and fluorescence having these two wavelengths is selected. A wavelength selection means that selectively passes through, a single photometric means that measures the fluorescence intensity of the fluorescence selected by the wavelength selection means, and a ratio of the two fluorescence intensities measured by this photometry means are calculated, A fluorescence photometric device, comprising: a quantitative means for quantifying the concentration of the substance to be inspected by comparing this ratio with a known fluorescence intensity characteristic. 2. A step of administering a fluorescence indicator acting on the test substance to a sample containing the test substance; a step of irradiating the sample with excitation light having an excitation wavelength of the fluorescence indicator; The step of photometrically measuring the fluorescence emitted from the sample excited by the irradiation of light with a single photometric device, and the step of photometrically measuring the fluorescence intensity of the fluorescence at the equal fluorescence wavelength of the fluorescent indicator at the beginning and the end of the photometry. Photometric, and in the middle of the photometry to measure the fluorescence intensity of the fluorescence at a predetermined wavelength, to calculate the ratio of the fluorescence intensity at the equal fluorescence wavelength and the fluorescence intensity at the predetermined wavelength, the ratio of known fluorescence And a step of quantifying the concentration of the substance to be inspected by collating with the intensity characteristic, and a fluorescence photometric method.