JPH0324442A - Measuring apparatus for ph - Google Patents

Abstract

PURPOSE:To make an apparatus compact and to make it possible to perform accurate measurement by processing the image through a microscope when fluorescence is emitted from a sample by the projection of light from a light source for a short time. CONSTITUTION:Luminous flux from a light source 1 passes through an exciting filter 3 with the opening and closing of a shutter part 2 for a short time, and the exciting light having a first wavelength is formed. The exciting light is reflected from a dichroic mirror 4 and projected on a sample (a) on a stage 6 through an objective lens 5. The image of the luminous flux of fluorescence emitted from the sample by the exciting light is picked up with a camera 8. The image is stored in the frame memory of an image processing device part 9 through the shutter part 2. Then, the luminous flux from the light source 1 passes through the exciting filter 3 having the exciting wavelength that is different from the first time. Thus the exciting light having the second wavelength is formed. The image of the excited light is picked up and stored in the frame memory of the image processing part 9. The fluorescent surface images which are obtained in this way using two wavelengths undergo operation between the images in the image processing device part 9. Namely, the value of quotient between the two images and the logarithmic value are obtained. The values are converted into pH using a calibration curve which is prepared beforehand. Thus the accurate measurement becomes possible.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Industrial Application Fields The present invention relates to a pH measuring device, and particularly to a pH measuring device suitable for measuring the pH of minute parts such as microorganisms and tissue cells. It is something.

(Conventional technology) Knowing the relationship between cell morphology and intracellular pH, and when cells are present in a certain system, the relationship between their positional information and intracellular pH is important in cell engineering, histochemistry, and physiology. It is becoming necessary in various fields such as

Conventional methods for measuring intracellular pH include pH electrodes and labeled 5. 5-dlseLyl-2,4ox
Methods using azoltdI-ned1ons NMR, a fluorescence photometer, etc. are known. However, in all of these conventional methods, except for the method using the pHm pole, it is possible to obtain the average intracellular pH of a cell population, but it is not possible to obtain the intracellular pH of each individual cell. Further, although it is possible to use a pH electrode for large tissues, it is extremely difficult to apply it to microorganisms, and it is also impossible to measure a large number of cells at once.

Therefore, a method and apparatus for measuring the intracellular pH of a living body from images observed under a W1 fluorescence microscope are expected.

Such a device uses a high-pressure mercury lamp as a light source system and a filter for excitation light of two wavelengths (λ-466n).
m, λ-425nm) to determine the fluorescence intensity generated from the fluorescently stained yeast cell sample, and determine the pH of the yeast cells based on the difference between the two fluorescence intensities, that is, the correlation between ■466-1 425 and pH. There is an example of measuring (PIEBS
: Lett. 156, P. 227~P. 23
0 (1983) 1).

However, with this method, the above-mentioned correlation is not sufficient, and there are also problems such as a decrease in fluorescence intensity, and it is considered difficult to obtain accurate measurement values.

In addition, two monochromators (λ-49
0 nm excitation light and λ-439 nm excitation light), the fluorescence intensities I490 and I439 were determined by photographing the fluorescence generated from the sample containing the fluorescent substance, and
There is also an example of measuring the pH of gastric cells using the correlation between (1490/I439) and pH (Nature vol. 325 447-45
0 (1987)).

However, this method requires the use of two expensive monochromators, which increases the size of the device and the price.

[Summary of the invention]

The present invention aims to solve the above-mentioned problems,
As a light source system for Class 28 excitation light, a system that uses excitation light selected by an excitation light selection filter, that is, a system that uses a light source such as a high-pressure mercury lamp and a filter, is used. This objective is attempted to be achieved by shortening the irradiation time of the light source and further processing the microscopic image when fluorescence is generated from the sample due to this short irradiation.

That is, the pH measuring device according to the present invention includes a light source irradiation means that irradiates a sample containing a fluorescent substance with excitation light of first and second wavelengths selected by an excitation light selection filter, respectively; a means for microscopically imaging the fluorescence generated by each type of excitation light at a specific wavelength; Find the logarithm of the division value of the fluorescence intensity due to the excitation light of the second wavelength, and combine the logarithm and pH.
an image processing means for determining the pH of the sample from the correlation with the
It is characterized by having the following.

How it works: A sample containing a fluorescent substance, for example a cell sample containing a fluorescent substance such as a fluorescently stained cell, is set at a predetermined position in a pHal1 measurement device, and first the excitation light is emitted from a light source such as a high-pressure water hook lamp. When the cell sample is irradiated with excitation light of the first wavelength selected by the selection filter for a short period of 64 r, fluorescence is generated from the cell sample. The fluorescent light produced by this short-time irradiation is photographed microscopically at a specific wavelength to obtain a microscopic image.

By irradiating this excitation light for a short period of time, that is, by shortening the shutter speed, it is possible to prevent a decrease in fluorescence intensity, which is considered to be the cause of errors.

The above-mentioned microscope image is processed by an image processing means, and the first
The intensity of fluorescence from the cell sample due to excitation light with a wavelength of is determined.

Subsequently, in the same manner as in the field a of the excitation light of the first wavelength, the excitation light of the second wavelength is applied to the same cell sample for a short time using the excitation light selection filter and imaged microscopically. A microscopic image of the fluorescence generated from the sample by the short-time irradiation η·I at a specific wavelength is obtained in a state in which a decrease in fluorescence intensity is prevented. This microscopic image is similarly processed by the image processing means to determine the intensity of fluorescence from the cell sample caused by the excitation light of the second wavelength.

The intensity of the fluorescence caused by the excitation light of the first wavelength and the intensity of the fluorescence caused by the excitation light of the second wavelength are determined by the logarithm of the division value of the two by the image processing means, and are created in advance. The pH of the cell can be determined from the correlation between the logarithm value and pH.

Effects> The pn measuring device according to the present invention has the following effects when, for example, a cell sample is used as the sample.

(1) Since we have a means to irradiate the cell sample with the light source in a short interval, we can use a light source system that uses an excitation light selection filter, that is, a light source system that uses excitation light with a broad spectrum. However, even under high magnification using a microscope image, data with high reproducibility can be obtained without the decrease in fluorescent light intensity that causes errors as in the conventional method.

(2) Since not only the fluorescence intensity but also microscopic image data of this fluorescence can be obtained, it is possible to measure even minute parts and to know the pH distribution of various parts in a cell sample.

(3) As the light source system, an inexpensive light source such as a high-pressure mercury lamp and a filter for selecting excitation light can be used, so there is no need to use two expensive monochromators, making the device compact and inexpensive. be able to.

[Specific description of the invention]

The pH measuring device according to the present invention is a device for measuring {15S composition as described above, and (a) applies light emitted from a light source such as a mercury lamp to a sample containing a fluorescent substance such as a fluorescently stained cell. irradiating for a short time with excitation light of a first wavelength selected by an excitation light selection filter; (b) (
(a) obtaining a microscopic image by microscopically imaging fluorescence generated from a sample by short-time irradiation at a specific wavelength; (c) processing the microscopic image obtained in (b) by image processing means; (d) Replace the excitation destination selection filter and shorten the excitation light of the second wavelength to the same sample in the same manner as in (a). (e) (b)
, (c) obtain a microscopic image of fluorescence generated from the sample by short-time irradiation with excitation light of the second wavelength and determine the intensity of the fluorescence; (f) obtain (f) by image processing means ( Find the logarithm of the division value of the intensity of fluorescence due to the excitation light of the first wavelength in C) and the intensity of fluorescence due to the excitation light of the second wavelength in (e), and calculate the logarithm of the division value of the intensity of fluorescence due to the excitation light of the first wavelength in Determining the pH of the sample from the correlation between the numerical value and pH;
The basic structure is to measure the pH of a sample.

Fluorescent substances contained in samples such as cell samples by fluorescent staining include 6-carboxyfluorescein, fluorescein, dimethyl-carboxyfluorescein, and 4-carboxyfluorescein.
Examples include methyl-umbelliferone. Regarding the correlation between fluorescent substances and pH, for example, the ratio of the intensity of each fluorescent light (for example, wavelength - 518 nm) to 2Ff1 excitation light (for example, 488 nm and 441 nm) of 6-carboxyfluorescein and fluorescein is the concentration of the fluorescent substance. It was observed that there was a correlation with pH, independent of (see Figure 2). In Figure 2, the fluorescence intensity of 6-carboxyfluorescein was investigated by changing the wavelength width of excitation light (
The results show that there is a correlation even when the wavelength width is 20 nm.

The pH measuring device according to the present invention applies this principle.

The sample used for the measurement values of the present invention may be any sample as long as it can contain a fluorescent substance, but it is particularly suitable for cell samples, i.e., various types of cells such as microicums and tissue cells, or samples containing these cells, or capillary samples. blood p in blood vessels
Preferably, the method requires measurement of minute portions such as pH distribution in the immobilized iLl body.

Hereinafter, the present invention will be further explained using an example in which the above cell sample is used as a sample.

To perform pH determination using this device. In order to prepare a cell sample containing a fluorescent substance, the fluorescent substance, such as 6-carboxyfluorescein or fluorescein, is first introduced into cells such as larvae or bacteria.

Since this substance is a hydrophobic substance and cannot be incorporated as it is, a form that is easily incorporated, such as esterified non-fluorescent 6-carboxyfluorescein diacetate or fluorescein diacetate, is used. This substance is quickly taken up by cells, and in living cells, the ester moiety is removed by esterase inside the cell to become 6-carboxyfluorescein or fluorescein, which emits fluorescence, and remains inside the cell.

Intracellular 6-carboxyfluorescein or fluorescein also has a fluorescence intensity ratio with respect to two excitation wavelengths depending on pH, and from that value, intracellular pH fJ< is determined.

Hereinafter, the pa measuring device according to the present invention will be specifically explained based on FIG. 1.

This measurement device includes an excitation light source 1, a shutter part 2 (E) through which light from the light source 1 can pass for a short time, and a first or second opening for the light that has passed through the shutter part 2. 2, an excitation filter 3 that shapes excitation light of a specific wavelength, a dichroic mirror 4 and an objective lens 5 provided in the optical path through which the excitation light is irradiated onto the cell sample a, and the cell sample a placed thereon. a stage 6, a fluorescence-side filter 7 that passes light of a specific wavelength for fluorescence from the cell sample a;
A high-sensitivity camera 8 that images the fluorescent light from the filter 7
It has a frame memory which is a storage location for digitized image data, and processes this image data to determine the pH value from the intensity of fluorescent light caused by two kinds of excitation light of the first and second wavelengths. It has an image processing unit 9 for conversion and a display 12 for outputting the image processing results.

In this pH measuring device, the data captured by the camera 8 of the fluorescence emitted from the cell sample a is taken into the frame memory of the image processing device section 9, and then processed for pH conversion in the device section 9. However, if necessary, an image storage unit 10 such as a magnetic tape is provided, and measures are taken so that the image captured by the camera 8 is once stored in the storage unit 10 and then processed by the image processing unit 9. may be completed.

Light source 1 has a wavelength of 40 when the fluorescent substance in cell sample a is 6 to carboxyfluorescein, fluorescein, etc.
Any material having a spectrum in the range of 0 nm to 500 nm may be used, such as high pressure mercury lamps, xenon lamps,
Examples include halogen lamps and incandescent tungsten lamps.

The shutter part 2 is a device having a camera shutter-like structure or a plate-like device that can be opened and closed instantly.
a) Open patent opening is about 1.50 seconds or less, preferably 0.45 seconds
It is necessary to have a function that can set the time to 0.25 seconds or less, more preferably 0.25 seconds or less. Furthermore, it has a function of generating a signal for causing both images to be captured into the frame memory, and (b) this image capture signal is
Within about 0.55 seconds after opening the shutter, preferably 0.55 seconds.
Occurs in 35 seconds or less, more preferably 0.15 seconds or less,
Preference is given to a shutter comprising a device whose constancy is less than or equal to ±0.1 seconds, preferably less than or equal to ±0.02 seconds. The above-mentioned consistency means that the shutter speed and frame memory loading time must be almost the same when creating a calibration curve to find the correlation between the fluorescence intensity ratio and pH and when actually measuring samples. That is,
Consistency is ±0. 1 second or less means that, for example, if you set the image capture time to frame memory to 0.300 seconds after the shutter is released, the image will be captured every time between 0.20 and 0.40 seconds after the shutter is released. This is what happens when the incorporation is carried out (゜).

The shutter part 2 does not necessarily need to be equipped with such a device. That is, images captured within the time that satisfies the condition (a) above are stored in the image storage unit 10, for example, a video tape, and later images that satisfy the condition (b) above are retrieved from the tape. This means that it is possible to perform processing for pH calculation.

The excitation wavelength by the excitation filter 3 is 400 nm to 50 nm.
You only need to choose two wavelengths, high wavelength side and short wavelength side, between 0 nm.
For example, the first excitation wavelength and the second excitation wavelength are 470
nm to about 498 nm and 425 nm to 4 5 1 n
It is a wave in the range of about m (order does not matter),
Preferably, this wavelength width is 20 nm or less.

The fluorescence wavelength by the fluorescence filter 7 is 500 to 600.
The wavelength is preferably in the range of 500 to 560 nm, and preferably the wavelength range is 40 nm or less.

The camera 8 has high sensitivity performance, and is capable of continuously sensing light of 3 x 10' lux or less (specifically, for example, SIT (Sl eye con-1n)).
Tensll'ler Target tube) video camera (RCA)).

When determining pH from the logarithm of the division value of the fluorescence intensity (1) for two types of excitation light, the logarithm is, for example, when the wavelengths of the two types of excitation light are 488 nm and 441 nm, ln(+488/ 1441) or In (1
441/+488).

In addition, in FIG. 1, 11 is a light beam for observation.

The flow of operation of this measuring device is as follows.

The luminous flux from the light source 1 such as a high-pressure mercury lamp is part of the shutter 2
passes through the excitation filter 3 by opening and closing for a short time,
Applying excitation light at a first wavelength.

The excitation light is a dichroic mirror 4 (for example, DM510
(Nikon Corporation), passes through an objective lens 5, and is irradiated onto a sample a on a stage 6.

The luminous flux of fluorescent light generated by the excitation light passes through the objective lens 5 and the dichroic mirror 4, and only the fluorescent light that has passed through the fluorescent filter 7 is imaged by the camera 8.

A signal is generated from the shutter part 2 after a certain period of time after the shutter of the shutter part 2 is opened, and the image of the camera 8 is taken into the frame memory of the image processing unit 9 based on the signal. The shutter is then closed after a certain period of time.

Subsequently, the light beam from the light source 1 passes through the excitation filter 3 of the excitation wavelength, which is a hole for the first time, by opening and closing the shutter part 2 for a short time, and forms excitation light of the second wavelength.

This excitation light is imaged in the same way as the first time, and the image is taken into the frame memory of the image processing section 9. The shutter is then closed after a certain period of time. The fluorescence image obtained in this manner with two wavelengths is calculated by the image processing unit 9, which calculates the division value and its logarithm between the two screens, and calculates the pH value from the calibration curve prepared in advance.
Processing such as arithmetic operations for converting the image into the image is performed, and the result is output as an image on the display 12.

To capture images after capturing the fluorescent light, the image storage unit 10, such as a magnetic tape, stores the images of the shutter openings over time, and later retrieves the images from the specified n! The image processing unit 9 can also calculate the image after j interval.

The flow and principle of image processing are as follows.

(2) Cells loaded with a fluorescent substance are placed on a slide glass, mounted on the measuring device of the present invention, and then irradiated with excitation light of the first wavelength formed by an excitation filter for a constant, short period of time. A fluorescent image captured by a high-sensitivity camera is stored in a frame memory (referred to as image A-1).

(2) While in the state of (2), the excitation light of the first wavelength is emitted at 11 ((), and the image obtained is stored in the frame memory (referred to as image A-2).

(2) After replacing the excitation filter, irradiate the same sample with excitation light of a second wavelength. The resulting fluorescent image is stored in a frame memory (referred to as image B-1).

(2) The image obtained without applying excitation light of the second wavelength to j(aη1) in the state of (2) is stored in the frame memory (referred to as image B-2).

■ Meat from image A-1 on the frame memory {elephant A-2
Subtract each pixel of
) is obtained.

(2) Similarly, each pixel of image B-2 is subtracted from image B-1, and the images resulting from the subtraction (referred to as both images B) are captured.

(2) Dividing images A and B for each pixel and finding the logarithmic value.

(2) Convert this logarithmic value into pH using a calibration curve prepared in advance under the same conditions, and output the result for each pixel. pH can be output in pseudocolor for easier viewing. The image composed of those pixels is p
This becomes one image showing the H distribution.

To prepare a calibration curve, a fluorescent substance, for example 6-carboxyfluorescein, is dissolved in a buffer of known pH, and the measurement method is as described above using the measuring device of the present invention, or using a fluorescence intensity of 1. to measure the fluorescence, p
}Create a calibration curve from the calculation results of I and (image 1 image A/image B).

For more accurate determination, cells loaded with 6-carboxyfluorescein are suspended in a buffer of known pH, and after adding K+ and Nidieridin (ionophore: an antibiotic that eliminates the pH gradient inside and outside the cell), When the pH of the image reaches -1 (I!!i Image A/Image B), calculate and create a calibration curve.

Note that a calibration curve may be created using a buffer to which a crude extract of sample cells is added.

Experimental Example> Experimental Example 1: Performance of pH Measuring Apparatus The above-mentioned pH measuring apparatus of the present invention shown in FIG. 1 was prepared.

Experimental example 2: Measurement of pH standard solution The apparatus of experimental example 1 (excitation filter 425-445 nm
, 470-490nm, fluorescent film 5 2 0
~560 nm) was used for all + determination.

50m containing 50μM 6-carboxyfluorescein
After dropping MES (pH 6.20) buffer onto a slide glass and covering it with a cover glass, the pH was measured at 6.
I went twice. In this method, the shutter speed was set at 0.30 seconds, the image 0.10 seconds after the shutter was released was processed by the image processing unit, and the pH was calculated from a calibration curve created under the same conditions.

As a control, the shutter was opened and closed at any time within 3 seconds, and the opening time peak was 1I: 11! The images between j were similarly processed and the pH was calculated.

The results are shown in Table 1.

Experimental Example 3: Creation of a Calibration Curve Figures 3 and 4 show a calibration curve created using the apparatus of Experimental Example 1 (settings for filter shutter speed and image capture time are as in Experimental Example 2). (The horizontal axis is pH
, the vertical axis is In (average value of high wavelength side excitation image brightness/low wavelength side excitation image brightness XIOO)). 50 mM HEPES (pH 7.13) containing fluorescein or 6-carboxyfluorescein at a final concentration of 50 μM, respectively, MOP
S (pH 6.66), MES (pH 6.20, 5
．． 30), citric acid/disodium phosphate (pH 5.6
0, 4.60, 4.00, 3.40, 3.00) were used. The measurement was performed in the same manner as in Experimental Example 2. Fig. 3 shows a case using fluorescein, and Fig. 4 shows a case using 6-carboxyfluorescein.

Experimental example 4: Creation of calibration curve Part 2 In Figures 5 and 6, the same buffer as in Experimental example 3 was used.
A lQffi line was produced using a fluorophotometer (RF500LC, manufactured by Shimadzu Corporation) using fluorescein and 6-carboxyfluorescein as a fluorescent reagent. At this time, the excitation wavelength width is 431 to 451 nm and 478 to 498 nm.
The measurement was carried out at a fluorescence detection side wavelength width of 500 to 540 nm.

Experimental Example 5: Dispersion in shutter speed and measured viscous The apparatus of Experimental Example 1 (with two filters) was used.

50mM MES containing fluorescein at a final concentration of 50μM
Using a buffer (pH 6.20), variations in the average brightness of images after the anomalous calculation (high wavelength side excitation image/low wavelength excitation image x 100) are shown (Table 2). Note that an image captured 0.10 seconds before the shutter was closed was used. The pH was calculated from a calibration curve created with the shutter open for 0.20 seconds.

Experimental Example 6: M1 determination of yeast intracellular pH The apparatus of Experimental Example 1 was used (settings of filter, shutter speed, image capture interval, etc. were as in Experimental Example 2).

SaceharoIlyces cerevlslae
Yeast extract 1.5%, peptone 3%, glucose 3%
The cells were cultured with shaking at 25°C after [j]. Concentrate at the late stage of logarithmic growth and add 50mM MES buffer (pH 6.
After washing three times at 20<1° C.), fluorescein diacetate or 6-carboxyfluorescein diacetate was added to a final concentration of 1 mM. After standing at 0°C for 30 minutes, add 50mM MES buffer (pH 6.20, 1
After washing three times at 1°C), the fluorescein diacetate added group was washed with 50mM MES buffer (pH 5.30, 1°C).
), and the group containing 6-carpoxyfluorescein diacetate was washed with 50 mM citric acid/disodium phosphate buffer (pH 3.40, 1°C), resuspended in each buffer, and left at 1°C for 90 minutes. After each yeast p
H was measured under the same conditions as in Experimental Example 2.

FIG. 7 schematically shows how the measurement results (image processing results) are output as images on the display. 1 is a pseudo-color display of the logarithm of the division value of the fluorescence intensity of each pixel of the yeast microscopic image (however, only the outline of the yeast microscopic image is shown on the drawing). 2 is an index indicating the correlation between the pseudo color and pH determined from the calibration curve. By correlating the color of the first image with the color of the second index, it is possible to know the pH of each part inside the yeast cell.

No. 1 table

[Brief explanation of drawings]

FIG. 1 shows an embodiment of the pH constant device according to the present invention. Figure 2 shows the fluorescence intensity ratio and pH for two types of excitation light, obtained by all measurements of fluorescence using a fluorophotometer.
FIG. 2 is a diagram showing the correlation between the wavelength width of the excitation light and the influence of the excitation light wavelength width. Figures 3 and 4 show calibration curves prepared using the measuring device according to the present invention.
When using carboxyfluorescein. Figures 5 and 6 show melon lines created using a fluorophotometer. FIG. 7 is a schematic diagram showing an example of an image output on a display when the intracellular pH of yeast in a pH buffer solution is determined as δp1 using the 71pj determination device according to the present invention.

Claims (1)

[Scope of Claims] 1. Light source irradiation means for irradiating a sample containing a fluorescent substance with excitation light of first and second wavelengths selected by an excitation light selection filter for a short time, and each of the above two types. A means for microscopically imaging fluorescence generated by excitation light at a specific wavelength, and determining the intensity of two types of fluorescence from each of the microscopic images, and determining the intensity of fluorescence caused by the excitation light of the first wavelength and the intensity of the second fluorescence. A pH measurement device comprising: an image processing means for determining a logarithm of a value divided by the intensity of fluorescence due to excitation light of a wavelength, and determining the pH of a sample from the correlation between the logarithm and pH. 2. The sample is a cell sample, and the excitation light of the first and second wavelengths and the fluorescent light of a specific wavelength for imaging contain 6-carboxyfluorescein or fluorescein as a fluorescent substance in the cells. It is for
pH measuring device according to claim 1