JP2007232716A - Method of measuring oxygen concentration and/or oxygen distribution in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring an oxygen concentration and/or oxygen distribution in a sample. <P>SOLUTION: The method of measuring the oxygen concentration and/or oxygen distribution in the sample comprises a step for distributing a phosphor substance in the sample, and a step for photo-exciting the phosphor substance in the sample and for measuring the change with the lapse of time of intensity of phosphorescence getting luminous thereby. A device using the method is also disclosed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for measuring oxygen concentration and / or oxygen distribution in a sample by measuring the phosphorescence lifetime of a phosphor, and an apparatus used for the method. Specifically, the method includes a step of distributing a phosphor in the sample, and a step of photoexciting the phosphor in the sample and measuring a temporal change in the intensity of phosphorescence emitted from the phosphor. The present invention relates to a method for measuring oxygen distribution and an apparatus used for the method.

  Oxygen is indispensable for living bodies such as humans and animals, and grasping the state of oxygen uptake and consumption is one of means for knowing the health condition and diseases. Current methods include measuring the hemoglobin concentration and oxygen saturation in the blood, and measuring the phosphorescence lifetime using a phosphorescent probe such as Pd porphyrin directly in the microcirculatory blood vessel to thereby measure the oxygen partial pressure in the microvessel. There are known methods for measuring oxygen partial pressure in a local area and applying it to measurement of blood flow velocity. This method is a method of measuring the oxygen concentration in the blood, not the method of measuring the oxygen concentration in the tissue, and it is not possible to measure how oxygen is distributed throughout the tissue. Furthermore, it is impossible to measure whether each cell constituting the tissue takes up oxygen. Living organisms form tissues by gathering a large number of cells, and because living organisms are formed, the health of the cells that make up each tissue / organ can be further improved. It can be understood. However, the above-described method grasps the oxygen state of the blood flowing in the tissue, and is an effective method for searching for the cause of the disease. However, there is currently no method for knowing the oxygen concentration, that is, the activity status of the cells constituting the tissue / organ.

  As a method of measuring the oxygen consumption status of cells, the oxygen concentration in a container in which cells are cultured in a solution and the oxygen concentration in a container containing a cell-free solution are measured and the difference is compared. There is a known method of grasping the oxygen consumption. However, this method can know the amount of oxygen consumed by a large number of cells indirectly in solution, but how much oxygen each cell actually takes up and how it is consumed. I can't figure out. In addition, in order to know the true activity status of cells, it is necessary to measure the amount of individual cells taken up and how oxygen consumes oxygen in the cells.

  (I) As a practical method of knowing the state of a cell by labeling the cell with a fluorescent substance, a diagnostic agent for tumor cells and tumor tissues can be mentioned. In this case, it is necessary to distinguish the somatic cells constituting the normal tissue from the tumor cells and find the presence of the tumor cells. Not only does the fluorescent substance bind only to the tumor cells so that only the tumor cells glow, but also the effects of the fluorescent substance on the normal cells such as side effects must be suppressed. Several methods have been reported to use fluorescent substances to illuminate tumor cells and tumor tissues. However, simply administering a fluorescent substance does not bind the fluorescent substance to normal somatic cells, but only to tumor cells. They cannot be combined, and in some cases have a side effect on normal cells. In Patent Document 6, although a compound that selectively binds to a tumor cell from a normal cell has been created, selective use for a tumor cell can be achieved only by using a fluorescent substance having an action of killing the cell by light irradiation. The problem that must be solved by elucidating the mechanism that binds to cells and clarifying that there is no binding to normal cells, examining the safety of fluorescent substances themselves, and further examining their effects on living bodies Has many. Thus, the present situation is that the creation research of a diagnostic method that solves both safety and effectiveness problems simultaneously continues.

(Ii) As another method, the fluorescence generated from the living tissue is imaged, the density of the normal tissue image measured before diagnosis is normalized, and the tumor tissue is detected based on the difference from the standard. A method is known (see Patent Document 5). This method measures fluorescence generated from a tissue, and is considered to measure fluorescence emitted from a biomolecule produced by a cell constituting the tissue or a biomolecule capable of dispersing the tissue. Usually, the state of cells constituting a tissue is, for example, a cell cycle normal state or a differentiation process in a growth process, cells in various active states, and biomolecules produced or scattered by cells in each state. Is different. Therefore, it is considered difficult to accurately grasp the difference between a normal tissue and a tumor tissue only with the density of an image obtained by photographing fluorescence. In order to solve this problem, a method of directly measuring the state of individual cells constituting the tissue or the tumor cells constituting the tumor tissue can be considered. It is difficult to measure the luminescence of cells. As a method of diagnosing individual cells by distinguishing normal somatic cells from tumor cells, fluorescent labeled antibodies can be used to identify specific proteins present on the surface of tumor cells, specifically expressed proteins, or specific By recognizing the presence of tumor cells.

  (Iii) A diagnostic method is also known by reacting with an enzyme that is highly expressed in tumor cells using a fluorescently labeled antibody or enzyme reaction product or a luminescent reaction product. However, the genes, enzymes, cell surface proteins and intracellular proteins targeted by these methods are specifically expressed or present only in specific tumor cells appearing in specific tissues, or It is expressed at a specific time in the process of cell proliferation. Therefore, each characteristic tumor cell may appear in each organ or tissue, and several types of tumor cells may appear in each organ or tissue. The diagnostic methods using fluorescently labeled antibodies and enzyme reactants described above are effective in determining the presence of specific tumor cells that appear in specific tissues and organs because they are not exhaustive. When the type of tumor cells and the site where the tumor cells are expressed are unknown and the diagnosis is widely performed, it is extremely difficult to determine the presence of tumor cells or tumor tissues (see Patent Documents 7 to 9).

  iv) A fluorescent substance is used when measuring the concentration of a specific substance present in a cell or a cell constituting a tissue (see Non-Patent Document 2). There are two types of luminescent materials: fluorescent materials and phosphorescent materials. Fluorescent materials do not react with oxygen, and phosphors react with oxygen. To measure the concentration of only a specific substance in the cell using the emission intensity of the photoexcited substance, find and use a fluorescent substance that does not react with oxygen and that reacts only with the specific substance to be measured. become. However, in this case, the fluorescence intensity varies depending on the amount of fluorescent material incorporated into the details. Therefore, in order to correctly measure the concentration of a specific substance, it cannot be obtained from the intensity of fluorescence. The concentration can be measured by measuring the disappearance of the molecule excited by the light of the fluorescent substance incorporated into the cell and the disappearance of the molecule, ie, the lifetime of the fluorescence. However, only a method for measuring the concentration of a specific substance present in a cell using a fluorescent material is known, and no method using a phosphorescent material is known, and no method for measuring an oxygen concentration is known.

  v) By applying a method for measuring the concentration of a specific substance present in cells or cells constituting a tissue, the intracellular distribution of the specific substance can also be measured. If the lifetime of the fluorescent substance is measured at several locations in the cell, the distribution of the specific substance to be measured can be known. Furthermore, if the state where the fluorescence emitted from the intracellular fluorescent substance disappears is recorded as an image and the lifetime of the fluorescence in the whole cell is measured, the distribution state can be measured more easily (see Non-Patent Document 4). However, in this case as well, only the method using the fluorescent material is known as in the above-described concentration measurement of the specific material, and the method using the phosphorescent material and the method for measuring the oxygen distribution have not been known.

vi) Methods for measuring oxygen concentration using phosphors that react with oxygen, ie oxygen sensors, are known. The oxygen-reactive substance excited by light absorbs the measurement light when irradiated with the measurement light. The degree of absorption of measurement light decreases as it falls to the ground state (see Non-Patent Document 1). That is, since the lifetime of the excited state is accelerated by the presence of oxygen, the oxygen concentration can be measured as a decrease in the absorption intensity. However, this method has not been applied to the measurement of oxygen concentration of cells constituting cells or tissues. As described above, at present, there is a problem that there is no method for measuring the oxygen concentration of each cell constituting the cell or tissue and the state of the oxygen distribution.
JP 11-37942 A Japanese Patent Publication No. 2003-517342 Japanese Patent Publication No. 2002-534997 Japanese Patent Publication No. 2003-517342 JP 2000-325295 A JP-A-7-336557 JP 2005-315862 A JP 2004-248575 A JP 2004-159600 A “Biosensors and Bioelectronics” 2003 Volume 18 p. 1439-1445. “Analytical Chemistry” 2004 Volume 76 p. 2468-2505. “Scanning” 1992, vol. 14, p. 155-159. “TCVBH1.DOC” February 2003, p. 1-41 (http://www.becker-hickl.de/pdf/tcvghl.pdf). The Institute of Electrical Engineers of Japan, Chemical Sensor System Study Group, September 13, 1998 CS-99, 26-42, p35-38.

  In the current method, a fluorescent substance that does not react with oxygen was used, so that the oxygen concentration in a biological sample could not be measured. An object of the present invention is to make it possible to measure the oxygen concentration in a biological sample by using a phosphor without depending on the distribution of the phosphor. Further, not only the oxygen concentration measurement but also the oxygen concentration measurement result It is an object to make it possible to measure oxygen distribution.

As a result of intensive studies to solve the above problems, the present inventors have measured the phosphorescence lifetime of the phosphor, so that the oxygen concentration and / or oxygen distribution in the biological sample does not depend on the distribution of the phosphor. The present invention has been completed by finding that it can be measured.
That is, the present invention
1. Distributing the phosphor in the sample;
The step of photoexciting the phosphor in the sample and measuring the change over time of the intensity of phosphorescence emitted therefrom.
A method for measuring oxygen concentration and / or oxygen distribution in the sample;
2. The sample is a single cell, cell mass, cell sheet or tissue piece. 2. the described method; 2. The cell is a cancer cell whose basic environment is hypoxic. Described method,
4). 2. The cell is a cell in an ischemic disease or ischemic state. Described method,
5). 1. The phosphorescent substance is a porphyrin compound. Or 4. A method according to any one of
6). 4. The phosphorescent substance is a porphyrin compound having a metal atom. 6. the described method; 5. The phosphor described above in which the phosphor is platinum porphyrin. Described method,
8. Photo-excitation of the phosphor using pulsed light of 400 nm to 700 nm. Or 7. A method according to any one of
9. Optical excitation of the phosphor using a pulsed light of 450 nm to 550 nm. Described method,
10. 1. Use the camera device to image and measure the time course of the intensity of phosphorescence emitted from the photoexcited phosphor. Or 9. A method according to any one of
11. Comprising: means for distributing the phosphor in the sample; a light source for photo-exciting the phosphor; a microscope for observing the sample; and a camera device for imaging and measuring changes in the intensity of phosphorescence over time. 1. Or 10. The apparatus used for the method as described in any one of these.

  The present invention uses a phosphor to measure the oxygen concentration in a biological sample, particularly in a cell, and is characterized by measuring the oxygen concentration in the biological sample based on the phosphor lifetime of the phosphor. This is different from the conventional measurement method in which the concentration of a specific substance in a biological sample is measured by the phosphorescence intensity of the phosphor.

When the concentration of oxygen in a biological sample is to be measured based on the phosphorescence intensity of the phosphor, the phosphorescence intensity changes depending on the distribution state of the phosphor, that is, the concentration at each part of the phosphor. In some cases, the concentration does not reflect, but the phosphorescence lifetime is the same, so it does not depend on the distribution state of the phosphor in each part of the biological sample.
If the degree of decrease in the phosphorescence lifetime due to the decrease in phosphorescence intensity resulting from the reaction between the phosphor and oxygen at each part of the biological sample can be measured, the oxygen concentration at each part of the biological sample can be determined by the distribution state of the phosphor. It can be determined without depending on.

A schematic diagram of the measurement principle of the phosphorescence lifetime in the present invention is shown in FIG.
When a phosphor is photoexcited, phosphorescence is emitted, but the phosphorescence intensity is attenuated with the passage of time and eventually disappears. At this time, by installing a gate or the like that opens at a constant time interval after photoexcitation and measuring the phosphorescence intensity over time, the phosphorescence lifetime can be calculated from the decay of the phosphorescence intensity.
If the phosphorescence lifetime is measured from the change over time of the phosphorescence intensity at each part of the biological sample using the above principle, the oxygen concentration at each part can be measured, thereby measuring the oxygen distribution in the biological sample. You can also.
As described above, the oxygen concentration at each site does not depend on the distribution state of the phosphor in the biological sample.
The temporal change in the intensity of phosphorescence emitted from the phosphor can be measured, for example, by recording with a camera device and analyzing the recorded image, and the recorded image can be measured between images. By performing fitting or the like, the oxygen concentration and / or oxygen distribution in the biological sample can be displayed as an image.
Thus, by culturing cells in a medium in which the phosphorescent substance is seeded, the phosphorescent substance is taken into the cell, the phosphorescent substance is photoexcited, and the time-dependent change in the intensity of phosphorescence emitted from the phosphorescent substance is detected by the camera device. By recording with, it is possible to measure the oxygen distribution in the cell and to visualize the oxygen distribution in the cell.

In the present situation where a method for measuring intracellular oxygen distribution at the cellular level is not known, it is possible to measure intracellular oxygen distribution by the method of the present invention in cytology, biology, biochemistry and medicine. Is expected to have a significant effect. For example, new knowledge can be obtained from oxygen uptake in a single cell, consumption of oxygen, intracellular metabolic process, cellular activation mechanism, function in each organelle, etc. Measure not only single cells but also cell sheets, cell clumps, tissue subsections, and tissues in units of cells that make up these cells, and obtain new academic knowledge between cells. Is expected to have a significant effect on the cell industry.

  For example, in treatments such as regenerative medicine or transplantation, where cells of patients or others are collected and cultured, and then transplanted into the affected area, the cells are cultured by obtaining oxygen distribution information in the cells or tissues. In addition, it is possible to obtain a lot of information such as grasping the health state and activity state of cultured cells and tissues, and can also be used to establish conditions for culturing.

  Moreover, it is expected that not only the active state of cells but also a great effect in the diagnostic field. Tumor tissue is different from normal tissue in hypoxia, and tumor cells survive and proliferate in hypoxia. On the other hand, normal cells cannot proliferate or survive unless there is sufficient oxygen. From these facts, tumor cells can be expected to be fully activated and proliferate with less oxygen uptake than normal cells, and tumor cells can be measured by measuring the oxygen concentration and distribution of the cells and tissues to be measured. Or it is thought that tumor tissue can be found.

In the case of an ischemic disease, the blood is not sufficiently reached and is caused by an oxygen-deficient state. With the current technology, the oxygen distribution in the microvessels is observed, and it is not possible to grasp how oxygen-deficient each individual cell is. Using the present invention, it is possible to find a true oxygen-deficient site in a vascular stenosis site, and to detect an oxygen-deficient site exceeding the limit of the thickness of a blood vessel that can be imaged with current angiography, It is possible to perform an appropriate treatment, and a great benefit can be obtained for the patient.
In the present invention, the active state of the cell can be measured by measuring the oxygen concentration of the cell and the state of oxygen uptake and consumption. It is conceivable that a predicted value can be calculated.

Furthermore, not only in the medical field, but also in the biomass-related industry as a cell-using industry, in the production by fermentation, in the production of organic compounds, proteins, antibodies and genes with cells and bacteria, the activity of the cultured cells and bacteria used in the production It is useful for knowing the state, and can be effectively used in the production by examining the culture conditions of cells and bacteria.
The sample is not limited to a biological sample, and may be a sample in which oxygen is incorporated into the sample in some form.

The phosphorescent substance used in the present invention is a substance that emits phosphorescence when photoexcited, and has a property of disappearing phosphorescence by reacting with oxygen in an excited state.
Examples of the phosphor include phosphors known as photochemical oxygen sensors. Specifically, pyrene derivatives (polycyclic aromatic compounds such as pyrene and perylene, and derivatives thereof), ruthenium complexes, and the like. (Transition metal complexes such as phenanthroline ruthenium chloride), phthalocyanine complexes (metal complexes such as platinum, palladium, zinc) and porphyrin compounds (porphyrin, tetrabenzoporphyrin, tetranaphthoporphyrin, tetraanthraporphyrin, tetranitrotetrabenzoporphyrin) , Octaethylporphyrin, tetrakispentafluorophenylporphyrin, picket fenceporphyrin and the above-mentioned polyphylline compound having a metal atom, and the metal atom includes cobalt, zinc, iron, manganese, tin, And thorium, lanthanum, ruthenium, palladium and platinum), preferably porphyrin compounds (porphyrin, tetrabenzoporphyrin, tetranaphthoporphyrin, tetraanthraporphyrin, tetranitrotetrabenzoporphyrin, octaethylporphyrin, tetrakispenta) Examples thereof include fluorophenylporphyrin, picket fence porphyrin, and the above-mentioned polyphylline compound having a metal atom, and examples of the metal atom include cobalt, zinc, iron, manganese, tin, yttrium, lanthanum, ruthenium, palladium and platinum. And preferably, a porphyrin compound having a metal atom (as the metal atom, cobalt, zinc, iron, manganese, tin, yttrium, orchid) And tantalum, ruthenium, palladium, and platinum.), And platinum porphyrin is preferable.

The sample used in the present invention is not particularly limited as long as it is a measurable sample in which oxygen is incorporated in some form, but examples include biological samples and samples other than biological samples. For example, a sample or the like used for investigating the deterioration state in the inside of the food packaging.
The biological sample is not particularly limited as long as it is a measurable tissue, organ, and cell, and examples thereof include a single cell, a cell mass, a cell sheet, and a tissue piece.
Preferable samples include biological samples, and also include tumor cells, particularly cancer cells whose basic environment is hypoxic and cells that are ischemic or ischemic.

The method for distributing the phosphor used in the present invention in the biological sample can be carried out by adding the phosphor used in the present invention to the biological sample. Does not incorporate phosphor.
Therefore, when the phosphor used in the present invention is incorporated into cells, the cells are usually cultured in a medium to which the phosphor is added, thereby incorporating the phosphor into the cultured cells.
Existing culture conditions can be used as the conditions for the culture.

As a light source for photoexciting the phosphor used in the present invention, pulsed light is preferable, specifically, Nd: YAG laser, Nd: YLF laser, Nd: YVO ₄ laser,
Examples thereof include solid pulse lasers such as Ti: Sapphire laser and glass laser, and gas pulse lasers such as XeCl, KrF, and ArF excimer lasers, preferably Nd: YAG lasers.
The wavelength of the irradiated light is 400 nm to 700 nm, preferably 450 nm to 550 nm, and more preferable wavelength is 532 nm.
For example, an Nd: YAG laser is irradiated with a pulse width of 10 ns in order to photoexcite the phosphor incorporated into the cell.

As a method of measuring the temporal change of phosphorescence intensity, the emitted phosphorescence is recorded as an image using a camera device, and the recorded image is analyzed to measure the temporal change of the phosphorescence intensity. it can.
Although a general optical camera can be used as the camera device, a CCD camera is preferable.
Moreover, when recording phosphorescence as an image using a camera apparatus, it is preferable to use an image intensifying unit (imaging intensifier) or the like for multiplying the emitted phosphorescence.
In addition, when measuring the temporal change of the phosphorescence intensity using the camera device, for example, in the light receiving unit of the camera device, the image is recorded in the camera device in a certain time from the end of light irradiation from the light source. It is preferable to install a gate or the like that opens in a certain time from the end of light irradiation.

The apparatus used for the method for measuring the oxygen concentration and / or oxygen distribution in the sample of the present invention includes, for example, means for distributing the phosphor in the sample, a light source for photoexciting the phosphor, and observing the sample. And a camera device for imaging and measuring changes over time in the intensity of phosphorescence.
As a microscope for observing the sample, an inverted microscope is preferable.
About another structure, the thing similar to the above-mentioned can be used.

An example of an apparatus used in the method for measuring the oxygen concentration and / or oxygen distribution of the present invention will be described with reference to FIG.
The apparatus is an inverted microscope 8 (Nikon: Eclipse TE2000-U), and Nd: YAG pulse laser 7 (LaserCompact-Plus: Diode-Pumped Solid State Laser Model LCS-DTL-314-QT-) as an excitation light source. 20), excitation light having a wavelength of 532 nm, output energy> 20 μJ @ 1 kHz, and 3 μJ @ 10 kHz and a pulse width <10 ns is generated. The excitation light is reflected by the dichroic mirror 3 and irradiated with the sample 1 to be measured. The phosphorescence emitted by photoexcitation is monitored by a gated imaging intensifier 4 (GaAsP image intensifier unit C8600 manufactured by Hmamatsu), and a controller (C7350-275 AquaCosmos) is used. Recording is performed using a digital CCD camera 5 (Digital CCD Camera ORCA-ER C4742-95-12ER manufactured by Hamamatsu). By this recording, the lifetime of phosphorescence emitted can be measured, and the oxygen concentration of the cell and its oxygen distribution can be measured. By synchronizing the gates of the pulse laser 7 and the imaging intensifier 4 with a delay generator and delaying the time from the pulse irradiation until the gate opens little by little, the decline in phosphorescence intensity can be directly measured. By fitting between images obtained by changing the delay time, phosphorescence lifetime distribution, that is, oxygen concentration distribution is imaged.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
In the following examples, measurement was performed using the apparatus shown in FIG.
Example 1
HeLa cells were used as cultured cells.
HeLa cells were cultured in Eagle's MEM medium containing 10% FBS, sodium bicarbonate 2.2 g, 100 units / ml penicillin, 500 μg / ml streptomycin in an atmosphere of 37 ° C. and 5% CO ₂ .
Incorporation of platinum porphyrin (hereinafter referred to as PtTCPP) into HeLa cells was performed by the following method. HeLa cells were subcultured to 1 × 10 ⁴ in a φ35 mm glass base dish, and incubated at 37 ° C. in a 5% CO ₂ atmosphere for 1 day or longer in order to adhere the HeLa cells to the dish. After removing the medium and washing with PBS, 2 ml of FBS-free Eagle MEM medium containing PtTCPP at a predetermined concentration was added and incubated at 37 ° C. in a 5% CO ₂ atmosphere for a predetermined time. After incubation, the medium was removed, washed twice with PBS, and 2 ml of FBS-free Eagle MEM medium was added. afterwards,
It measured using the apparatus shown by FIG.
FIG. 3 shows a photograph of a bright field image of HeLa cells before pulsed light irradiation.
4 to 7 show photographs in which the phosphorescence emission states of HeLa cells at 1 μs, 3 μs, 5 μs, and 7 μs after pulse light irradiation are recorded, respectively. From these photographs, it can be seen that there are sites where the decay of phosphorescence intensity is fast and slow depending on the site of the cells.

Example 2
PtTCPP was taken into MH134 cells by the same method as in Example 1.
Thereafter, measurement was performed using the apparatus shown in FIG. 2, and the resulting image is shown in FIG.
The photograph of the bright-field image of MH134 cell before pulse light irradiation was shown ((a) of FIG. 8).
PtTCPP was distributed throughout the cell ((b) of FIG. 8).
The attenuation of phosphorescence intensity was measured while changing the delay time, and the results were analyzed by fitting between the obtained images and imaging the intracellular oxygen distribution ((c) of FIG. 8). Thus, the method for measuring the oxygen concentration and / or oxygen distribution in the biological sample of the present invention makes it possible to visualize and show the intracellular oxygen concentration distribution.

It is the schematic which shows the measurement principle of the oxygen concentration of this invention. It is the schematic which shows an example of the apparatus used for the method of measuring the oxygen concentration and / or oxygen distribution of this invention. 2 is a photograph of a bright field image of HeLa cells before pulsed light irradiation in Example 1. 2 is a photograph recording the phosphorescence emission state in HeLa cells 1 μs after pulsed light irradiation in Example 1. FIG. FIG. 3 is a photograph of the phosphorescence emission state recorded in HeLa cells 3 μs after pulse light irradiation in Example 1. FIG. 2 is a photograph recording the phosphorescence emission state in a HeLa cell 5 μs after irradiation with pulsed light in Example 1. FIG. 2 is a photograph of the phosphorescence emission state recorded in HeLa cells 7 μs after irradiation with pulsed light in Example 1. FIG. In Example 2, it is a photograph which shows intracellular oxygen distribution measurement using MH134 cell (a) Bright field (b) Fluorescence image (c) Intracellular distribution measurement.

Explanation of symbols

1 Sample 2 Objective Lens 3 Dichroic Mirror 4 Imaging Intensifier with Gate 5 CCD Camera 6 Beam Expander 7 Nd: YAG Pulse Laser 8 Inverted Microscope

Claims (11)

Distributing the phosphor in the sample;
The step of photoexciting the phosphor in the sample and measuring the change over time of the intensity of phosphorescence emitted therefrom.
A method for measuring oxygen concentration and / or oxygen distribution in the sample. The method according to claim 1, wherein the sample is a single cell, a cell mass, a cell sheet, or a tissue piece. The method according to claim 2, wherein the cell is a cancer cell whose basic environment is hypoxic. The method according to claim 2, wherein the cell is a cell in an ischemic disease or ischemic state. The method according to any one of claims 1 to 4, wherein the phosphor is a porphyrin-based compound. 6. The method according to claim 5, wherein the phosphor is a porphyrin compound having a metal atom. The method of claim 6, wherein the phosphor is platinum porphyrin. The method according to claim 1, wherein the phosphor is photoexcited using pulsed light having a wavelength of 400 nm to 700 nm. 9. The method of claim 8, wherein the phosphor is photoexcited using 450 nm to 550 nm pulsed light. 10. A method according to any one of the preceding claims, wherein a camera device is used to image and measure the temporal change in the intensity of phosphorescence emitted from the photoexcited phosphor. A means for distributing the phosphor within the sample, a light source for photoexciting the phosphor, a microscope for observing the sample, and a camera device for imaging and measuring changes in phosphorescence intensity over time. Item 11. An apparatus for use in the method according to any one of Items 1 to 10.