JPH05126746A - Ion concentration quantification method and quantification device - Google Patents

Abstract

PURPOSE:To enable speedily and exactly obtaining the ratio value indicating ion concentration and accurately obtaining the quantity of ion concentration of calsium and the like in the organ of biocell and the like. CONSTITUTION:A fluorescent reagent reacting with a specific chemical in organ of biocell and the like is added to an organ to detect two fluorescence intensity signals and ion concentration is detected with a processed value of the two fluorescence intensity signals. Provided are A/D converters 23, 25 to convert the two fluorescence intensity signals to digital data and a memory means 26 for addressing the two digital data converted in the A/D converter 23, 25, and storing as a lookup table, the value indicating the ion concentration calculated in advance. And provided is a scanning detection means to scan an inspection body of organ and detect the fluorescence intensity signals by turns.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an ion concentration quantifying apparatus and a quantifying method for a specific chemical substance in a tissue such as an ecological cell.

[0002]

2. Description of the Related Art Quantification of ion concentration of a specific chemical substance such as calcium in an ecological cell is an important technique for studying cells.

That is, the stimulus-response reaction of cells is carried out by a mechanism utilizing the concentration gradient of inorganic ions existing in the cells. Among them, calcium ions are one of the information transmitters in the tissues of ecological cells. It is said that it is necessary to regulate calcium ion concentration in order to control cell activity. In other words, quantification of the concentration of ions such as calcium ions is an important research tool that helps clarify physiological functions.

Quantitation of the excited concentration of ions is generally carried out by adding a fluorescent reagent to the tissue and analyzing the emitted fluorescence. For example, a fluorescent reagent called Indo-1 or Fura-2 is used to quantify the calcium ion concentration. In the case of Indo-1, fluorescence emitted by one-wavelength ultraviolet (UV) light excitation is intensity-detected at two different fluorescence wavelengths. In the case of Fura-2, the fluorescence emitted by two different wavelengths of UV excitation is detected for each excitation wavelength.
In each case, the ratio values of the two detected fluorescence intensity signals are not affected by the amount of excitation light or the fading caused by the fluorescent reagent and are proportional to the calcium ion concentration, and the calcium ion concentration can be quantified.

Conventionally, when the ratio value is obtained from the two fluorescence intensity signals as described above, it is executed by an analog divider in a hardware manner, or after A / D conversion, a microcomputer executes a software division to calculate calcium. The quantitative value of the ion concentration was obtained.

[0006]

However, in such a conventional technique, in the former case where the ratio value is calculated in a hardware manner by the analog divider, high accuracy is required due to the limitation of the voltage range in the denominator and the frequency characteristic. It is difficult to calculate, and it is difficult to calculate the ratio at 10 MHz or more, which is necessary to obtain the dynamics, and the performance may be insufficient in terms of accuracy and speed. In the latter, which calculates the ratio value by software, High-precision computation is possible, but it takes computation time and the computation speed is extremely slow.

In recent years, there has been an increasing demand in the biological and medical fields to capture changes in calcium ion concentration distribution in tissues as images in real time. For that purpose, ratio calculation is performed at several MHz to several tens of MHz. Therefore, there is a problem in that the above-described method for obtaining the ion concentration cannot meet such a demand.

The present invention has been made by paying attention to the problems of the prior art as described above. The ratio value representing the ion concentration is quickly and accurately obtained, and ions such as calcium in tissues such as biological cells are found. It is an object of the present invention to provide an ion concentration quantification device and a quantification method that solve the above problems by enabling the concentration to be determined with high accuracy even at several tens of MHz.

[0009]

The gist of the present invention for achieving the above object is to: 1 target a specific chemical substance in a tissue, and to detect two fluorescence intensity signals in response to the chemical substance. A quantification device for ion concentration, wherein a fluorescence reagent to be detected is added to a tissue, and an ion concentration is detected by a calculation processing value from the two fluorescence intensity signals, wherein the two fluorescence intensity signals are respectively digital data (D1, D2). ) A / D converter (23, 25)
And a memory for storing, as a look-up table, a value representing an ion concentration calculated in advance by using two digital data (D1, D2) converted by the A / D converter (23, 25) as an address. An ion concentration quantifying device, comprising: a means (26);

(2) The ion concentration quantifying device according to item (1), further comprising scanning detection means (6, 8) for scanning an object in a tissue to detect fluorescence intensity signals one after another.

3 A method for quantifying an ion concentration of a specific chemical substance in a tissue, wherein a fluorescent reagent that reacts with the chemical substance of interest and detects two fluorescence intensity signals is added to the tissue, A value representing the ion concentration calculated by a predetermined calculation formula corresponding to the two fluorescence intensity signals is stored in advance in the storage means (26) as a lookup table,
A method for quantifying ion concentration is characterized in that the ion concentration is read from the storage means (26) using two fluorescence intensity signals detected from the tissue as addresses.

[0012]

When the fluorescent reagent is added to a tissue such as an ecological cell, the fluorescent reagent reacts with a specific chemical substance in the tissue and two fluorescence intensity signals can be detected. The ion concentration can be detected from the two fluorescence intensity signals by the following arithmetic processing.

Since the two fluorescence intensity signals are analog values, they are converted into digital data by the A / D converters (23, 25). The two digital data converted by the A / D converters (23, 25) become an address signal of the storage means (26), and the storage means (26) is looked up by a value representing an ion concentration calculated in advance. Since it is stored as an up table, the ion concentration of the tissue at that time can be known by reading the stored value of the address determined by the two digital data.

Since only the stored value in the look-up table is read out by the address signal, the operating speed is fast and the stored value is obtained in advance, so that it can be made extremely accurate.

By scanning the subject of the tissue by the scanning detection means (6, 8), the fluorescence intensity signals are detected one after another at high speed in accordance with the scanning, and the quantification of the ion concentration of calcium or the like in the tissue is enhanced even at several tens of MHz. It can be calculated with accuracy, and it is possible to meet the demand for capturing a change in calcium ion concentration distribution in a tissue as an image in real time.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the drawings. 1 and 2 show an embodiment of the present invention.

The ion concentration quantifying device is an illumination optical system 1A.
And an objective system 1B and a data processing system 1C,
Targeting calcium, a specific chemical substance in tissues,
Indo-1 or Fura-2 is added to the tissue as a fluorescent reagent that detects two fluorescence intensity signals in response to calcium ions, and the ion concentration is detected by the arithmetic processing value from these two fluorescence intensity signals. .. In the following, for convenience of explanation, Indo-1 is used, and what scans the subject and displays it on the screen will be described.

The illumination optical system 1A comprises an illumination light source 2, a condenser lens 3 and an excitation filter 4. Illumination light source 2
Is composed of mercury, xenon, UV laser, etc., and the condenser lens 3 is provided so as to be movable in the optical axis direction for focus adjustment of the illumination light source 2. Excitation filter 4 is the fluorescent reagent Indo
It has a characteristic of passing only light having a wavelength of 360 to 380 nm which is an excitation wavelength of -1. However, in the case of UV laser, a beam expander is used instead of the condenser lens 3.

A dichroic mirror 5 is arranged between the objective system 1B and the data processing system 1C, and the UV excitation illumination light a from the illumination optical system 1A is incident on the dichroic mirror 5. The dichroic mirror 5 has a characteristic of reflecting light having a wavelength shorter than 400 nm.

In the objective system 1B, a first relay lens 7, a vertical scanner 8, a second relay lens 9 and an objective lens 10 are sequentially arranged from the horizontal scanner 6 which receives the light reflected by the dichroic mirror 5. Become. The objective lens 10 faces the specimen 11 of the tissue subject.

The horizontal scanner 6 is, for example, an oscillating mirror or a multi-faceted rotating mirror, and has UV excitation illumination light a.
Is to be horizontally scanned at high speed. Vertical scanner 8
Requires only one scan on one screen, so that scanning is slower than that of the horizontal scanner 6. For example, the UV excitation illumination light a is further scanned in the vertical direction by using a vibrating mirror.
The UV excitation light b is dimensionally scanned. Further, the horizontal scanning device 6 and the vertical scanning device 8 are provided with a synchronization control circuit 21.
By doing so, the scanning is repeated in synchronization.

In the first relay lens 7 and the second relay lens 9, a spot is formed by the objective lens 10 so that the sample 11 is two-dimensionally scanned, so that the pupil of the objective lens 10 is scanned by the horizontal scanners 6, 6. It relays to the deflection center position of the vertical scanner 8, and can be omitted when the scanning angle is narrow.

The optical system of the data processing system 1C is arranged on the opposite side of the objective system 1B with the dichroic mirror 5 in between. The optical system receives the fluorescence intensity signal generated in the sample 11 through the dichroic mirror 5 and has wavelength selectivity, and has two directions, a passage direction and a reflection direction, depending on the wavelength of the fluorescence intensity signal. A set of a dichroic mirror 12 for branching fluorescence, a bandpass filter 13, an imaging lens 14, and a photodetector 15 respectively arranged in a reflection direction and a passing direction, and a bandpass filter 16, an imaging lens 17, and a light. It consists of a set of detectors 18.

The bandpass filter 13 has a narrower wavelength selectivity than the dichroic mirror 12 and has a wavelength λ.
1 (for example, 405 nm) is selected and band pass filter 1
6 is set to select the wavelength λ2 (for example, 485 nm). After the photodetectors 15 and 18 that receive and detect the light selected by the bandpass filters 13 and 16, the set of the operational amplifier 22 and the A / D converter 23 and the set of the operational amplifier 24 and the A / D converter 25 are included. Sets are provided.
A synchronization control circuit 21 is connected to the A / D converters 23 and 25, and digital data D1 and D2 synchronized with the horizontal scanner 6 and the vertical scanner 8 are output.

A memory 26 is provided as a storage means after the A / D converters 23 and 25. Digital data D
1 and D2 are set to be the upper and lower addresses of the memory 26. The memory 26 stores, as a lookup table, a value representing the ion concentration calculated in advance by a predetermined calculation formula.

That is, the memory 26 is a ROM or RA.
The ratio value of two fluorescence intensity signals corresponding to the values of the digital data D1 and D2 is stored in the memory space which is M and is accessed by the digital data D1 and D2.
In the case of the ROM, the stored value can only be changed by replacing the ROM itself, but in the case of the RAM, the value can be easily changed by reading from a disk or the like depending on the subject or the fluorescent reagent.

A frame memory 27 for temporarily storing the ratio value read by the memory 26 is provided after the memory 26, and further, a D / A converter 28 and a video signal conversion circuit so that the monitor 30 can observe the data-processed image. And 29 are provided.

Next, the operation will be described.

When Indo-1 is added as a fluorescent reagent to a tissue such as an ecological cell, the fluorescent reagent reacts with a calcium ion which is a specific chemical substance in the tissue to generate two fluorescence intensity signals. The ion concentration is detected from these two fluorescence intensity signals.

In the illumination optical system 1A, the light emitted from the illumination light source 2 is appropriately focused by moving the condenser lens 3 to be adjusted and enters the excitation filter 4, and the excitation filter 4 is the fluorescent reagent Indo-1. Excitation wavelength of 360 to 3
Only the 80 nm light is transmitted and the UV excitation illumination light a is emitted to the dichroic mirror 5.

Since the dichroic mirror 5 reflects light having a wavelength shorter than 400 nm, the UV excitation illumination light a is reflected to the objective system 1B side. In the objective system 1B, the horizontal scanner 6
The UV excitation illumination light a is horizontally scanned at a high speed in the horizontal direction, and the vertical scanner 8 vertically scans the UV excitation illumination light a through the first relay lens 7 to become the UV excitation light b. An image is formed on the specimen 11 of the biological cell subject as a spot which is two-dimensionally scanned from the objective lens 10 through the two relay lenses 9. The pupil of the objective lens 10 is composed of the first relay lens 7 and the second relay lens 9 for horizontal scanning 6,
It is guided to the deflection center position of the vertical scanner 8. The horizontal scanning device 6 and the vertical scanning device 8 are synchronized by the synchronization control circuit 21 and repeat scanning.

Fluorescence is excited from the sample 11 by irradiation with the UV excitation light b, and the excited fluorescence c is converted into the objective lens 10, the second relay lens 9, the first relay lens 7,
The light passes through the horizontal scanner 6, passes through the dichroic mirror 5, and enters the optical system of the data processing system 1C arranged on the opposite side of the objective system 1B with the dichroic mirror 5 in between. When the fluorescence intensity signal generated in the sample 11 passes through the dichroic mirror 5, it enters the dichroic mirror 12, and the dichroic mirror 12 having wavelength selectivity.
Divides the fluorescence into two directions, a passing direction and a reflecting direction, according to the wavelength of the fluorescence intensity signal.

The bandpass filter 13 on the reflection side further narrows the width of wavelength selectivity by the dichroic mirror 12 to select the wavelength λ1 (for example, 405 nm), and the selected light is passed through the imaging lens 14 to the photodetector 15. An image is formed and photoelectrically converted. Similarly, a wavelength λ2 (for example, 485 nm) is selected by the bandpass filter 16 on the transmission side, and the selected light is imaged on the photodetector 18 by the imaging lens 17 and photoelectrically converted. It

After the photodetectors 15 and 18, the operational amplifier 22 amplifies the photoelectric analog signal to a voltage level within the input range of the A / D converter 23 in the subsequent stage, and the A / D converter 23.
The synchronous control circuit 21 converts this photoelectric analog signal into digital data D1 by a clock signal synchronized with the two-dimensional scanning by the horizontal scanner 6 and the vertical scanner 8 and outputs it. Similarly, the operational amplifier 24 amplifies the photoelectric analog signal to a voltage level within the input range of the A / D converter 25 at the subsequent stage, and the A / D converter 25 uses the synchronous control circuit 21 to two-dimensionally amplify the photoelectric analog signal. It is converted into digital data D2 by a clock signal synchronized with scanning and output.

Since the digital data D1 is set to the upper address of the memory 26 and the digital data D2 is set to the lower address of the memory 26, the memory 2 is set.
The memory value at the address position determined by the digital data D1 and D2, which is the address signal of the look-up table constituted by 6, represents the ion concentration calculated in advance by a predetermined calculation formula.

That is, the memory value at the address position of the memory 26 indicates, for example, the ratio value between the fluorescence intensity signal of the fluorescence wavelength λ1 and the fluorescence intensity signal of the fluorescence wavelength λ2, and only the calculation result is stored. It is possible to accurately calculate the ratio value as if the address data is only addressed by the digital data D1 and D2, as if the division was performed, without the need for the sequential calculation. The stored contents of the memory 26 can be recognized as a look-up table, and the ratio calculation time becomes extremely short as the sum of the A / D conversion time and the memory access time, and the sampling cycle of about 20 MHz required for repeated sampling is reduced. Can be easily obtained.

The ratio value read by the memory 26 is temporarily stored in the frame memory 27, converted into an analog signal by the D / A converter 28, and the analog signal is converted into an image signal by the video signal conversion circuit 29. Based on the image signal, the monitor 30 observably displays the data-processed image. Thereby, the calcium ion concentration of the tissue can be visually known.

Since a sampling cycle of about 20 MHz can be easily obtained, the substance of the tissue is closely observed by dynamically observing the calcium ion concentration, which contributes to research and development.

When the subject of the tissue is scanned by the scanning detection means, the fluorescence intensity signals are detected one after another at high speed in accordance with the scanning,
Since a sampling cycle of about 20 MHz can be easily obtained, the quantitative determination of the calcium ion concentration can be dynamically obtained with high accuracy even at several tens of MHz, and the changes in the calcium ion concentration distribution in the tissue can be captured as an image in real time. We can respond to the desire.

Although Indo-1 was used as the fluorescent reagent in the above-mentioned embodiment, it is needless to say that it can be generally applied to the case where two fluorescence intensity signals are obtained and the ion concentration is calculated from them. Absent. As an ion, calcium ion has recently been attracting attention as a research target, but it is also naturally applicable to an ion of another chemical substance and a fluorescent reagent for detecting the ion.

[0041]

According to the ion concentration quantifying apparatus and the quantifying method of the present invention, the ratio value representing the ion concentration is quickly and accurately determined, and the quantification of the ion concentration of calcium or the like in the tissue of the ecological cell can be performed. Since it is possible to obtain the ion concentration with high accuracy even at 10 MHz, it is possible to know the ion concentration quickly and accurately.

If the scanning detection means for scanning the subject of the tissue and detecting the fluorescence intensity signal one after another is provided, it becomes possible to grasp the change in the concentration distribution of calcium ions in the tissue as an image in real time. , Can contribute to the research and development of the biological and medical fields.

[0043]

[Brief description of drawings]

FIG. 1 is a partial block diagram of an ion concentration quantification device according to an embodiment of the present invention.

FIG. 2 is a partial block diagram of an ion concentration quantification device according to an embodiment of the present invention.

[Explanation of symbols]

 1A ... Illumination optical system 1B ... Objective system 6 ... Horizontal scanner 8 ... Vertical scanner 10 ... Objective lens 11 ... Sample 1C ... Data processing system 13, 16 ... Bandpass filter 15, 18 ... Photodetector 21 ... Synchronous control circuit 23, 25 ... A / D converter 26 ... Memory 27 ... Frame memory 28 ... D / A converter 29 ... Video signal conversion circuit 30 ... Monitor

Claims (3)

[Claims] 1. A target value for a specific chemical substance in a tissue, a fluorescent reagent which reacts to the chemical substance and detects two fluorescence intensity signals is added to the tissue, and an arithmetic processing value from the two fluorescence intensity signals is added. An ion concentration quantification device for detecting ion concentration by means of an A / D converter for converting each of the two fluorescence intensity signals into digital data, and 2 for conversion by the A / D converter.
An ion concentration quantification device, comprising: storage means for storing, as a look-up table, a value representing an ion concentration calculated in advance by using one digital data as an address. 2. The ion concentration quantification device according to claim 1, further comprising scanning detection means for scanning a subject of tissue to detect fluorescence intensity signals one after another. 3. A method for quantifying an ion concentration of a specific chemical substance in a tissue, comprising adding a fluorescent reagent which reacts to the chemical substance of interest and detects two fluorescence intensity signals to the tissue, A value representing the ion concentration calculated by a predetermined calculation formula corresponding to the two fluorescence intensity signals is stored in advance in a storage means as a lookup table, and the two fluorescence intensity signals detected from the tissue are used as addresses in the storage means. The method for quantifying ion concentration is characterized in that the ion concentration is read out from.