Method and apparatus for evaluating cell sample

Abstract

To provide a method for evaluating a cell sample on the basis of a refractive index information distribution of the cell sample labeled with a labeling substance acquired by using light of one wavelength.SOLUTION: Labeling a cell sample with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously, acquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a first refractive index information distribution, causing a refractive index change of the labeling substance labeling the cell sample at the predetermined wavelength, acquiring a refractive index information distribution of the cell sample at the predetermined wavelength again to obtain a second refractive index information distribution, and comparing the first refractive index information distribution with the second refractive index information distribution, A step of evaluating a distribution and / or a surrounding environment of the labeling substance.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method and apparatus for evaluating a cell sample. [Background technology]

[0002] One of the methods for evaluating cell samples is the observation (imaging) of cell samples. Such observation of cell samples can be performed on unlabeled cell samples or on cell samples labeled with a labeling substance, depending on the purpose and target of the evaluation.

[0003] The refractive index is an index that represents the speed of light in a material. The refractive index at a certain position in a cell sample varies depending on the type and density of the material present at that position. Therefore, obtaining refractive index distribution images and phase distribution images, which can provide information about the refractive index distribution in a cell sample, is sometimes used to evaluate cell samples.

[0004] Obtaining information about the refractive index distribution in a cell sample can also be performed using an unlabeled sample. In this case, it is possible to evaluate the morphology of the sample or to evaluate the quality of the sample by comparing the refractive index of each organelle. However, depending on the purpose of the evaluation, the refractive index characteristics of the structure of interest may be obscured by the refractive index characteristics of other structures, making it impossible to achieve the evaluation goal. In such cases, it is effective to label the structure of interest in the cell sample with a labeling substance and then obtain information about the refractive index distribution, which makes it easier to achieve the evaluation goal.

[0005] Patent Document 1 discloses a cell evaluation method that can easily evaluate even unknown cells, the method comprising: a labeling step of labeling a specific site of a cell with a labeling substance having different refractive indices at a first wavelength and a second wavelength; a refractive index distribution acquisition step of acquiring, at the first wavelength and the second wavelength, the refractive index distribution of the cell whose specific site has been labeled in the labeling step; and an analysis step of evaluating the distribution of the specific site in the cell by comparing the refractive index distribution at the first wavelength and the second wavelength. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2023-139787 [Non-patent literature]

[0007] [Non-Patent Document 1] Josefine Liljeruhm et al., "Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology", Journal of Biological Engineering 12:8 (2018). Summary of the Invention [Problem to be solved by the invention]

[0008] In the method described in Patent Document 1, the refractive index distribution of cells obtained at a specific site using light of two wavelengths, a first wavelength and a second wavelength, is compared.

[0009] Generally, the resolution, scattering intensity, and magnitude of chromatic aberration of light differ depending on the wavelength, and therefore, the method described in Patent Document 1 may cause artifacts due to these differences in the refractive index distributions acquired for the two wavelengths.

[0010] Furthermore, since the two wavelengths with different refractive indices differ for each labeling substance, the method described in Patent Document 1 requires the preparation of light sources with two wavelengths suitable for observation for each labeling substance.

[0011] In view of the above problems, the present invention aims to provide a method and apparatus for evaluating a cell sample based on the refractive index information distribution of the cell sample labeled with a labeling substance, which is acquired using light of one wavelength. [Means for solving the problem]

[0012] One aspect of the present invention is "a method for evaluating a cell sample, comprising, in this order: (1) a step of labeling a cell sample with a labeling substance that changes its refractive index at a predetermined wavelength in response to a stimulus and / or spontaneously; a step of acquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a first refractive index information distribution; a step of causing a refractive index change at the predetermined wavelength of the labeling substance that has labeled the cell sample; a step of again acquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a second refractive index information distribution; and a step of comparing the first refractive index information distribution with the second refractive index information distribution to evaluate the distribution of the labeling substance and / or the surrounding environment."

[0013] In the evaluation method of this aspect, a cell sample is labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously, and the distribution of the labeling substance and / or the surrounding environment is evaluated by comparing a first refractive index information distribution and a second refractive index information distribution, which are refractive index information distributions acquired at the same predetermined wavelength before and after the refractive index change. Thus, the evaluation method of this aspect can evaluate the distribution of the labeling substance and / or the surrounding environment based on the refractive index information distribution at a single wavelength, thereby avoiding problems caused by acquiring refractive index information distributions at multiple wavelengths.

[0014] One aspect of the present invention is the evaluation method according to [1], wherein the labeling substance is a substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to at least one stimulus selected from the group consisting of light irradiation, a pH change, a substance concentration change, a temperature change, a solvent change, voltage application, a mechanical stimulus, and gas exposure. According to the evaluation method of this aspect, a cell sample can be evaluated by utilizing the stimulus-responsive and / or spontaneous change in the refractive index at a predetermined wavelength of the labeling substance.

[0015] One aspect of the present invention is "the evaluation method according to [1] or [2], wherein the labeling substance is a substance whose refractive index at a predetermined wavelength changes spontaneously or in response to light irradiation." According to the evaluation method of this aspect, a cell sample can be evaluated by utilizing a simple change in refractive index, that is, a change in the refractive index at a predetermined wavelength in response to light irradiation and / or spontaneously in the labeling substance.

[0016] One aspect of the present invention is "the evaluation method according to any one of [4] to [3], wherein the change in refractive index of the labeling substance is time-dependent." According to the evaluation method of this aspect, a cell sample can be evaluated by utilizing the time-dependent change in refractive index at a predetermined wavelength in the labeling substance. This may enable evaluation of the distribution of the labeling substance and / or the surrounding environment based on the time-dependence of the change in refractive index information distribution.

[0017] One aspect of the present invention is the evaluation method according to any one of [1] to [4], wherein the labeling substance is a substance whose refractive index at a predetermined wavelength increases in response to a stimulus and / or spontaneously. According to the evaluation method of this aspect, a cell sample can be evaluated by utilizing the presence or absence and / or degree of increase in the refractive index at a predetermined wavelength of the labeling substance.

[0018] One aspect of the present invention is "the evaluation method according to any one of [6] to [5], wherein the labeling substance is a chromoprotein." Chromoproteins can be introduced into cell samples easily and with a high degree of freedom using bioengineering techniques, and cause a large change in refractive index. Therefore, the evaluation method of this aspect allows for suitable evaluation of cell samples.

[0019] One aspect of the present invention is "the evaluation method according to any one of [7] to [6], wherein the refractive index information distribution is acquired using quantitative phase imaging, optical diffraction tomography, or optical coherence tomography." According to the evaluation method of this aspect, the refractive index information distribution can be acquired using quantitative phase imaging, optical diffraction tomography, or optical coherence tomography, and therefore the cell sample can be suitably evaluated.

[0020] One aspect of the present invention is "the evaluation method according to any one of [8] to [7], wherein the refractive index information distribution is a refractive index distribution, a phase distribution, or a complex amplitude distribution." According to the evaluation method of this aspect, the distribution of a labeled substance and / or the surrounding environment can be evaluated based on the refractive index distribution, the phase distribution, or the complex amplitude distribution at one wavelength.

[0021] One aspect of the present invention is "the evaluation method according to any one of [9] to [8], wherein the evaluation of the distribution of the labeled substance and / or the surrounding environment is performed based on the ratio and / or difference between the refractive index information at corresponding coordinates in the first refractive index information distribution and the second refractive index information distribution." According to the evaluation method of this aspect, the distribution of the labeled substance and / or the surrounding environment can be evaluated for one wavelength based on the ratio or difference that is an index of the change in refractive index at corresponding coordinates.

[0022] One aspect of the present invention is "the evaluation method according to any one of [1] to [9], wherein the cell sample is a cell, and at least one protein, at least one organelle, cytoplasm, cell membrane, or the entire cell of the cell is labeled with the labeling substance."

[10] According to the evaluation method of this aspect, information on the distribution of a predetermined protein and the environment of a predetermined location can be obtained at the single-cell level by evaluating the distribution of the labeling substance and / or the surrounding environment.

[0023] One aspect of the present invention is "(11) the evaluation method according to any one of (1) to (10) above, wherein the cell sample is a cell aggregate, and at least one cell contained in the cell aggregate is partially or entirely labeled with the labeling substance." According to the evaluation method of this aspect, by evaluating the distribution of the labeling substance and / or the surrounding environment, it is possible to obtain information about the distribution of a specific cell and the environment in which the specific cell is located, for cells contained in a cell aggregate.

[0024] One aspect of the present invention is a cell sample evaluation device comprising: "

[12] a refractive index information distribution acquisition unit that acquires a refractive index information distribution of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously; and an analysis unit that compares a first refractive index information distribution, which is the refractive index information distribution at the predetermined wavelength of the cell sample before the refractive index change of the labeling substance, with a second refractive index information distribution, which is the refractive index information distribution at the predetermined wavelength of the cell sample after the refractive index change of the labeling substance, to evaluate the distribution of the labeling substance and / or the surrounding environment." The cell sample evaluation device of this aspect can evaluate the distribution of the labeling substance and / or the surrounding environment based on the refractive index information distribution at a single wavelength. This makes it possible to avoid problems during evaluation that arise from acquiring refractive index information distributions at multiple wavelengths.

[0025] One aspect of the present invention is a program for causing a computer to execute the following steps: (13) for a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to a stimulus, by acquiring a refractive index information distribution at the predetermined wavelength of the cell sample before the refractive index of the labeling substance changes to obtain a first refractive index information distribution; (2) for acquiring a refractive index information distribution at the predetermined wavelength of the cell sample after the refractive index of the labeling substance changes to obtain a second refractive index information distribution; and (3) comparing the first refractive index information distribution with the second refractive index information distribution to evaluate the distribution of the labeling substance and / or the surrounding environment. The program of this aspect allows the distribution of the labeling substance and / or the surrounding environment to be evaluated based on the refractive index information distribution at a single wavelength. Therefore, problems associated with acquiring refractive index information distributions at multiple wavelengths can be avoided during evaluation. [Effects of the Invention]

[0026] The present disclosure provides a method, an apparatus, and a program for evaluating a cell sample based on the refractive index information distribution of the cell sample labeled with a labeling substance, acquired using light of a single wavelength. Such a method, an apparatus, and a program enable evaluation of the cell sample while avoiding the problems of artifacts and the need for a light source that arise from using multiple wavelengths. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the absorption spectrum and refractive index spectrum of pigment protein A before and after light irradiation in Evaluation Example 1. [Figure 2] FIG. 1 is a diagram showing cells having intracellular site X in Evaluation Example 1. [Figure 3] FIG. 10 shows the absorption spectrum and refractive index spectrum of chromoprotein B before and after the passage of time in Evaluation Example 2. [Figure 4] FIG. 10 is a diagram showing spheroids produced from cells P and Q in Evaluation Example 2. [Figure 5]10 is an example of a tomographic image showing whether or not there is a significant increase in the second phase distribution relative to the first phase distribution in Evaluation Example 2. Black cells indicate cells present in a location where the phase in the second phase distribution is significantly increased relative to the first phase distribution, and white cells indicate cells present in a location where the phase in the second phase distribution is not significantly increased relative to the first phase distribution. [Figure 6] FIG. 1 is a diagram showing an example of the configuration of a cell sample evaluation device used in an evaluation method of one embodiment. [Figure 7] FIG. 1 is a diagram showing the configuration of an observation device 1A. [Figure 8] 2 is a diagram schematically illustrating incidence of first light and second light on an observation object S, and incidence of the first light and second light on an imaging unit 50 after passing through the observation object S. FIG. [Figure 9] FIG. 2 is a diagram showing the configuration of a processing unit 60 of the observation device. [Figure 10] 1 is a flowchart of an observation method. DETAILED DESCRIPTION OF THE INVENTION

[0028] An embodiment for carrying out the present disclosure will be described below, but the present disclosure is not limited to the following embodiment.

[0029] A method for evaluating a cell sample according to one embodiment includes, in this order, a step of labeling a cell sample with a labeling substance that changes its refractive index at a predetermined wavelength in response to a stimulus and / or spontaneously (labeling step), a step of acquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a first refractive index information distribution (first refractive index information distribution acquisition step), a step of causing a refractive index change at the predetermined wavelength of the labeling substance that labeled the cell sample (refractive index change step), a step of reacquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a second refractive index information distribution (second refractive index information distribution acquisition step), and a step of comparing the first refractive index information distribution with the second refractive index information distribution to evaluate the distribution of the labeling substance and / or the surrounding environment (analysis step).

[0030] <Cell samples> A cell sample according to the present disclosure is an object containing cells and having optical transparency. The cell sample is not limited to a single cell or an aggregate of cells. In addition to cells, the cell sample may further contain components that may be contained in a cell sample, such as extracellular matrix and / or neutral fat. The cell sample may be a cell or a cell aggregate. In the present disclosure, a cell aggregate refers to an object containing multiple cells aggregated in three dimensions and having optical transparency, such as a spheroid, organoid, or biological tissue. The cell sample may be an artificially prepared sample, or may be a sample collected from a human or non-human animal or a processed product thereof. The cell sample may be, for example, a cell, a plate culture of cells, a spheroid, an organoid, or biological tissue. The cell sample may contain one or more types of cells. The cells contained in the cell sample may be, for example, human-derived cells. The cells contained in the cell sample may be live cells, dead cells, and / or a mixture thereof, preferably including live cells, e.g., 50% or more of the cells contained therein being live cells. The thickness of the cell sample may be, for example, 1 μm or more, 5 μm or more, 10 μm or more, or 50 μm or more, or 200 μm or less, 100 μm or less, or 50 μm or less, and these may be freely combined. The cell sample evaluation method according to one embodiment uses the refractive index information distribution as an index, and, like fluorescence imaging, can be used when the sample is thick or when evaluating a deep portion of the sample.

[0031] <Labeling substance> The labeled substance according to the present disclosure is a substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to a stimulus. In the present disclosure, the labeled substance labels a portion or all of the cells contained in a cell sample, and the presence or absence and / or degree of change in refractive index at the predetermined wavelength serves as an indicator of the distribution and / or surrounding environment of the cells. The refractive index of the labeled substance may increase or decrease spontaneously and / or in response to a stimulus, and in one embodiment, may increase. The mechanism of the change in refractive index of the reference substance may be any mechanism that produces a detectable change in refractive index, such as a change in molecular structure, three-dimensional structure, oxidation state, electronic state, coordination state, motion state, or folding.

[0032] The labeling substance may take any form as long as it can label cells contained in a cell sample, and may be, for example, a protein, an organic molecule, a metal complex, a quantum dot, a metal oxide, or a complex of any of these with an antibody, a peptide, a nucleic acid, an organic molecule, or a polymer. In one embodiment, the labeling substance may take the form of a protein, in which case labeling with the labeling substance can be carried out simply and with a high degree of freedom using bioengineering techniques.

[0033] The maximum absorption wavelength of the labeling substance may be, for example, 350 nm or more, 400 nm or more, 450 nm or more, 480 nm or more, 500 nm or more, 515 nm or more, or 525 nm or more, or 1500 nm or less, 1000 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, or 625 nm or less, and these may be freely combined. Light sources and detectors for light in these wavelength ranges are widely installed in optical systems commonly used to acquire refractive index information distributions. Therefore, when the maximum absorption wavelength of the labeling substance is within the above range, commonly used optical systems can be used, providing high convenience. Furthermore, light in these wavelength ranges is less absorbed by cells than light with shorter wavelengths. Therefore, when the maximum absorption wavelength of the labeling substance is within the above range, cell samples can be evaluated while suppressing the reduction in signal intensity due to light absorption by cells and the scattering effects caused by refractive index changes caused by substances internal to the cells.

[0034] The molar extinction coefficient ε [L / mol cm] at the maximum absorption wavelength of the labeling substance may be, for example, 10,000 or more, 30,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, or 100,000 or more, or 300,000 or less, 200,000 or less, or 150,000 or less, and these may be freely combined. When the molar extinction coefficient at the maximum absorption wavelength of the labeling substance is equal to or greater than the above-mentioned lower limit, refractive index changes due to the labeling substance can be easily discriminated from refractive index changes due to substances with small molar extinction coefficients derived from cells, thereby enabling suitable evaluation of cell samples.

[0035] The fluorescence quantum yield of the labeling substance may be, for example, 30% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.3% or less, 0.1% or less, 0.03% or less, 0.01% or less, or 0.001% or less. The phosphorescence quantum yield of the labeling substance may be, for example, 30% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.3% or less, 0.1% or less, 0.03% or less, 0.01% or less, or 0.001% or less. When the fluorescence quantum yield and / or phosphorescence quantum yield of the labeling substance are equal to or less than the upper limit, artifacts in the refractive index information distribution resulting from the fluorescence and / or phosphorescence of the labeling substance can be suppressed. When the fluorescence quantum yield and / or phosphorescence quantum yield of the labeling substance are equal to or less than the upper limit, photobleaching of the labeling substance due to the fluorescence and / or phosphorescence of the labeling substance can be suppressed.

[0036] In one embodiment, the labeling substance may be a chromoprotein. A chromoprotein is a protein that has high absorbance in the visible light region. The chromoprotein may be, for example, a protein having a molar extinction coefficient ε [L / mol cm] of 100,000 or more and a fluorescence quantum yield and a phosphorescence quantum yield of 1% or less, or may be a protein having a maximum absorption wavelength of 400 nm or more and 700 nm or less, a molar extinction coefficient ε [L / mol cm] of 100,000 or more, and a fluorescence quantum yield and a phosphorescence quantum yield of 1% or less, 0.1% or less, or 0.01% or less. The chromoprotein may be, for example, a chromoprotein described in Non-Patent Document 1. The chromoprotein may be a natural or artificial chromoprotein. Examples of natural chromoproteins include the blue protein (cjBlue) derived from the sea anemone (Cnidopus japonicus), the blue protein (rpulFKz1) derived from the jellyfish (Rhizostoma pulmo), the purple protein (gfasCP) derived from the coral (Galaxea fascicularis), the pink protein (asFP595) derived from the sea anemone (Anemonia sulcata), the blue protein (Rtms5) derived from the coral (Montipora efflorescens), the blue protein (aeCP597) derived from the sea anemone (Actinia equina), and phytochrome. Artificial proteins may be modified versions of the above natural chromoproteins.

[0037] In this embodiment, the predetermined wavelength is the wavelength used to acquire the refractive index information distribution. The predetermined wavelength may be a wavelength at which a refractive index change occurs in the labeled substance, and the refractive index change can be detected based on the refractive index information distribution acquired from the cell sample. For example, the predetermined wavelength may be a wavelength that is 70 nm or less, 50 nm or less, 30 nm or less, or 15 nm or less different from the maximum absorption wavelength of the labeled substance. Furthermore, for example, the predetermined wavelength may be a wavelength that is 70 nm or less, 50 nm or less, 30 nm or less, or 15 nm or less different from the maximum absorption wavelength of the labeled substance in the wavelength range of 400 to 1000 nm. It is generally known that dyes exhibit large fluctuations in refractive index values ​​near the wavelength at which the absorbance is maximum or locally maximum. Therefore, if the predetermined wavelength satisfies the above condition, the refractive index change of the standard substance is likely to be large, making it easier to detect the refractive index change based on the refractive index information distribution acquired from the cell sample.

[0038] The rate of change in refractive index (rate of increase or decrease) of the labeled substance before and after the change in refractive index at a predetermined wavelength may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more. When the rate of change in refractive index of the labeled substance before and after the change in refractive index at a predetermined wavelength is equal to or greater than the above-mentioned lower limit, the change in refractive index can be easily detected based on the refractive index information distribution.

[0039] The rate of change (increase or decrease) in absorbance of the labeled substance before and after a change in refractive index at a predetermined wavelength may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more. Generally, a sudden change in refractive index with respect to wavelength in a region of high absorbance is caused by a change in the electronic state due to the labeled substance absorbing light, and therefore the magnitude of the change in refractive index in such a region is also determined by the magnitude of the absorbance. Therefore, if the rate of change in absorbance of the labeled substance before and after a change in refractive index at a predetermined wavelength is equal to or greater than the above-mentioned lower limit, the change in refractive index can be easily detected based on the refractive index information distribution.

[0040] The difference between the maximum absorption wavelength or the maximum absorption wavelength in the wavelength range of 400 to 1000 nm before and after a change in refractive index at a predetermined wavelength of the labeling substance may be 5 nm or more, 10 nm or more, 20 nm or more, 30 nm or more, or 50 nm or more. It is generally known that dyes exhibit large fluctuations in refractive index values ​​near the wavelength at which the absorbance is maximum or maximum. Therefore, if the difference between the maximum absorption wavelength or the maximum absorption wavelength in the wavelength range of 400 to 1000 nm before and after a change in refractive index is equal to or greater than the lower limit, the shift in the refractive index spectrum before and after the change is large, which tends to increase the change in refractive index before and after the change, making it easier to detect a change in refractive index based on the refractive index information distribution.

[0041] The labeling substance may be a substance that reversibly changes the refractive index at a predetermined wavelength. In one embodiment, if the labeling substance is a substance that reversibly changes the refractive index at a predetermined wavelength, the same cell sample can be evaluated at multiple time points by evaluating the cell sample once and then returning the refractive index at the predetermined wavelength to the value before the change. This makes it possible to, for example, evaluate time-dependent changes in the cell sample (time-lapse observation) or compare the evaluation results of the cell sample before and after exposing the cell sample to a drug or the like.

[0042] The labeled substance may be a substance whose refractive index at a predetermined wavelength changes instantaneously or time-dependently. In one embodiment, if the labeled substance is a substance whose refractive index at a predetermined wavelength changes instantaneously, the refractive index state distribution before and after the change in refractive index can be obtained in a short time, and the distribution of the labeled substance (intracellular transport or cell migration) and / or the surrounding environment at a specific time point can be obtained in real time. On the other hand, in another embodiment, if the labeled substance is a substance whose refractive index at a predetermined wavelength changes time-dependently, for example, if the rate of change over time varies depending on the surrounding environment, the distribution of the labeled substance and / or the surrounding environment can be evaluated based on the time dependency of the change in refractive index information distribution.

[0043] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to light irradiation. Such a substance's refractive index at a predetermined wavelength changes due to structural or conformational changes that occur when the substance, excited by light irradiation, returns to its ground state. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to light irradiation is irradiated with light as a stimulus, causing a change in the refractive index of the labeling substance. The substance whose refractive index at a predetermined wavelength changes in response to light irradiation may be a substance whose refractive index at a predetermined wavelength changes instantaneously or over time under light irradiation, or may be a substance whose refractive index at a predetermined wavelength changes instantaneously after light irradiation. Light irradiation can induce a change in the refractive index of the labeling substance without physical contact with the cell sample, making it simple and capable of suppressing artifacts caused by stimuli.

[0044] In one embodiment, the wavelength of light that causes a refractive index change in a substance whose refractive index at a predetermined wavelength changes in response to light irradiation may differ from the predetermined wavelength by 25 nm or more, 50 nm or more, 100 nm or more, or 150 nm or more. If the difference between the predetermined wavelength and the wavelength of light that causes a refractive index change is equal to or greater than the above-mentioned lower limit, the refractive index change of the labeled substance due to irradiation with light of the predetermined wavelength can be suppressed when acquiring refractive index information distribution at the predetermined wavelength.

[0045] A substance whose refractive index at a predetermined wavelength changes in response to light irradiation may be, for example, a photochromic substance. A photochromic substance is a substance whose absorption spectrum changes when irradiated with light; typically, the color of the substance itself changes with a change in the maximum absorption wavelength. A photochromic substance may be in the form of, for example, a protein or an organic molecule. Examples of protein-based photochromic substances include the chromoproteins asFP595, Dronpa, Kaede, rhodopsin, and phytochrome. Examples of organic molecule-based photochromic substances include azobenzene and spiropyran. Another example of a photochromic substance is a substance (photocaged substance) that has a leaving group (caged group) that is released in response to light irradiation, and whose absorption spectrum changes due to a change in conjugated structure caused by the release of the caged group upon light irradiation. Among these, the chromoprotein asFP595 is a suitable example. AsFP595 is a chromoprotein with a molar extinction coefficient of approximately 120,000 under steady-state conditions. However, upon irradiation with green light, it converts into a fluorescent protein, resulting in a decrease in the absorption peak at 568 nm and a decrease in the molar extinction coefficient (Dmitriy M. Chudakov et al., "Chromophore Environment Provides Clue to 'Kindling Fluorescent Protein' Riddle", THE JOURNAL OF BIOLOGICAL CHEMISTRY 278, 9(28), 7215-7219 (2003); Dmitriy M. Chudakov et al., "Photoswitchable cyan fluorescent protein for protein tracking", Nature Biotechnology 22, 1435-1439 (2004)). Therefore, the resulting change in refractive index can be used as an index for evaluating cell samples in this embodiment.

[0046] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to a change in pH. Such substances change their refractive index at a predetermined wavelength due to protonation / deprotonation or elimination of a partial structure caused by a change in pH. The substance whose refractive index at a predetermined wavelength changes in response to a change in pH may be a substance whose refractive index at a predetermined wavelength changes reversibly or irreversibly depending on the pH. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to a change in pH can undergo a change in the refractive index of the labeling substance by increasing (basifying) or decreasing (acidifying) the pH of the solution surrounding the cell sample through the replacement of the solution surrounding the cell sample or the addition of an acid or base to the solution. In these cases, the pH range in which a change in refractive index occurs may overlap with 4.0 to 11.0, 5.0 to 10.0, 5.5 to 9.5, or 6.0 to 9.0. If the pH range in which the refractive index change occurs overlaps with the above range, the refractive index change can be induced at a pH close to physiological conditions, thereby minimizing damage to cells contained in the cell sample and suppressing artifacts caused by refractive index changes of substances other than the labeling substance.

[0047] A substance whose refractive index at a predetermined wavelength changes in response to a change in pH may be, for example, a halochromic substance. A halochromic substance is a substance whose absorption spectrum shape changes reversibly with pH, ​​and typically, the color of the substance itself changes with a change in the maximum absorption wavelength. Halochromic substances are also widely used as pH indicators for various samples, regardless of whether or not they contain cells, and such commonly used pH indicators can also be used as the halochromic substance of the present disclosure. Examples of halochromic substances include phenolphthalein, bromophenol blue, bromothymol blue, and phenol red.

[0048] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to changes in the substance concentration. Such a substance changes its refractive index in response to changes in the concentration of a predetermined substance present in the surrounding environment, typically by forming a complex with the predetermined substance, resulting in a change in refractive index. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to changes in substance concentration can undergo a change in the refractive index of the labeling substance by increasing or decreasing the concentration of the predetermined substance in the solution surrounding the cell sample through stimulation by replacing the solution surrounding the cell sample or adding a corresponding predetermined substance to the solution. The substance whose refractive index at a predetermined wavelength changes in response to changes in substance concentration may be, for example, an ionochromic substance. An ionochromic substance is a substance whose absorption spectrum shape reversibly changes in response to changes in the concentration of a predetermined ion present in the surrounding environment, and typically changes in the shape of the absorption spectrum by forming a coordinate bond with the predetermined ion. An ionochromic substance can be, for example, a metal ion detection probe that is sometimes used by those skilled in the art as an ion sensor in a cell system, and the metal ion to be detected is Ca. 2+ (which can be detected by probes such as Fluo3), Mg 2+ , Cu 2+ , Fe 2+ , Fe 3+ and Zn 2+ Another example of a substance whose refractive index at a predetermined wavelength changes depending on the concentration of the substance is hemoglobin, a substance whose absorption spectrum shape changes reversibly with the addition and detachment of oxygen molecules. Hemoglobin is a tetramer consisting of two α chains and two non-α chains (β chains, γ chains, or δ chains), and each polypeptide chain contains porphyrin and Fe. 2+ Therefore, not only does it bind to oxygen molecules, but it also binds to Fe 2+ It can be said that the shape of the absorption spectrum of hemoglobin reversibly changes depending on the presence or absence of .

[0049] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes with temperature. Such a substance's refractive index changes due to changes in the three-dimensional structure or motion state of the compounds constituting the substance with temperature. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes with temperature is stimulated by heating to increase the temperature or cooling by heat dissipation to cause a change in the refractive index of the labeling substance. The substance whose refractive index at a predetermined wavelength changes with temperature may be, for example, a thermochromic substance. A thermochromic substance is a substance whose absorption spectrum shape changes reversibly with temperature. The shape of the absorption spectrum usually changes due to changes in the three-dimensional structure or motion state of the compounds constituting the substance with temperature. An example of a thermochromic substance is a copper-N,N-diethylethylenediamine complex.

[0050] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to a change in solvent. The refractive index of such a substance changes due to changes in the electronic or kinetic state, which depend on the dielectric constant, polarity, viscosity, etc., of the surrounding environment. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to a change in solvent can undergo a change in the refractive index of the labeling substance by stimulating the replacement of the solution surrounding the cell sample or the addition of a different solvent to the solution. The substance whose refractive index at a predetermined wavelength changes in response to a change in solvent may be, for example, a solvatochromic substance. A solvatochromic substance is a substance whose refractive index at a predetermined wavelength changes depending on the dielectric constant, polarity, viscosity, etc., of the surrounding solvent. Examples of solvatochromic substances include POLARIC and pyridinium N-phenoxide betaine.

[0051] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to the application of a voltage. Such a substance changes its refractive index when exposed to an electric field, either permanently or transiently changing its charge state. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to the application of a voltage is stimulated by applying a voltage in a conventional manner, causing a change in the refractive index of the labeling substance. The substance whose refractive index at a predetermined wavelength changes in response to the application of a voltage may be, for example, an electrochromic substance. An electrochromic substance is a substance whose absorption spectrum shape changes reversibly as the charge state of the substance changes in response to exposure to an electric field. Examples of electrochromic substances include tungsten oxide and viologen.

[0052] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes in response to a mechanical stimulus. The refractive index of such a substance changes due to a change in chemical structure or kinetic state caused by a mechanical stimulus. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes in response to a mechanical stimulus is subjected to a mechanical stimulus such as pressure or traction, which causes a change in the refractive index of the labeling substance. The substance whose refractive index at a predetermined wavelength changes in response to a mechanical stimulus may be, for example, a mechanochromic substance. A mechanochromic substance is a substance whose absorption spectrum shape changes in response to a mechanical stimulus. There are no particular limitations on the mechanochromic substance, as long as it is a known substance.

[0053] The labeling substance may be a substance whose refractive index at a predetermined wavelength changes upon exposure to a gas. Such a substance changes its refractive index at a predetermined wavelength when exposed to a gas, causing gas molecules to be incorporated into the substance, resulting in a change in its three-dimensional structure. A cell sample labeled with a substance whose refractive index at a predetermined wavelength changes upon exposure to a gas is exposed to a gas as a stimulus in a conventional manner, thereby causing a change in the refractive index of the labeling substance. The substance whose refractive index at a predetermined wavelength changes upon exposure to a gas may be, for example, a vapochromic substance. A vapochromic substance is a substance whose absorption spectrum changes upon exposure to a gas. Vapochromic substances are not particularly limited as long as they are well known.

[0054] The labeling substance may be a substance that spontaneously changes its refractive index at a predetermined wavelength. The refractive index of such a substance changes due to spontaneous maturation or degradation of the substance, independent of external stimuli. A cell sample labeled with a substance that spontaneously changes its refractive index at a predetermined wavelength causes a change in the refractive index of the labeling substance when the cell sample is left standing. The substance that spontaneously changes its refractive index at a predetermined wavelength may be in the form of a protein, for example. A protein-like substance that spontaneously changes its refractive index at a predetermined wavelength changes its refractive index due to gradual time-dependent folding or gradual degradation within the cell. A preferred substance that spontaneously changes its refractive index at a predetermined wavelength is the chromoprotein aeCP597. aeCP597 is known to change color over time (Non-Patent Document 1, Maria A. Shkrob et al., "Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina," Biochem J 15;392(Pt 3):649-54 (2005)). If the labeling substance is a substance that spontaneously changes its refractive index at a predetermined wavelength, the change in the refractive index of the labeling substance can be induced without stimulating the cell sample, which is simple and eliminates artifacts caused by stimuli.

[0055] The labeled substance may be a substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to at least one stimulus selected from the group consisting of light irradiation, pH change, substance concentration change, temperature change, solvent change, voltage application, mechanical stimulus, and gas exposure. From the viewpoint of simplicity and artifact suppression, the labeled substance is preferably a substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to light irradiation, more preferably a substance whose refractive index at a predetermined wavelength changes spontaneously or in response to light irradiation, and even more preferably a substance whose refractive index at a predetermined wavelength changes instantaneously in response to light irradiation or a substance whose refractive index at a predetermined wavelength changes spontaneously and time-dependently.

[0056] <Refractive index information distribution> The refractive index information distribution is a distribution of parameters (refractive index information) including information on the magnitude of the refractive index, acquired for a space containing a cell sample. The refractive index information distribution may be a spatial distribution (three-dimensional distribution) or a planar distribution (two-dimensional distribution), preferably a spatial distribution. The refractive index information may be the refractive index itself, or a parameter that includes the refractive index as a direct or indirect variable and is capable of detecting changes in the refractive index as changes in value. For example, the refractive index information is a polynomial function, exponential function, logarithmic function, power function, trigonometric function, inverse trigonometric function, hyperbolic function, or inverse hyperbolic function of the refractive index, preferably a polynomial function or exponential function of the refractive index, and more preferably a linear function of the refractive index, or a natural exponential function with the refractive index multiplied by i (where i represents the imaginary unit) or a function of a real constant multiple thereof. Furthermore, the refractive index information may be a polynomial function, exponential function, logarithmic function, power function, trigonometric function, inverse trigonometric function, hyperbolic function, or inverse hyperbolic function of the wavelength and the state of the labeled substance. Preferably, it may be a polynomial function or exponential function of the wavelength and the state of the labeled substance. More preferably, it may be a linear function of the wavelength and the state of the labeled substance, or a natural exponential function with an exponent equal to i times the refractive index (i represents an imaginary unit) or a function multiplied by a real constant. When the refractive index information is one of these functions, the refractive index information distribution can be easily obtained, thereby enabling the cell sample to be suitably evaluated. Note that the polynomial function of the refractive index according to the present disclosure is understood as a concept that includes both the refractive index itself and a function expressed by a monomial. Furthermore, the exponential function of the refractive index according to the present disclosure is understood as a concept that includes both a function with a real number and a function with a complex number as the exponent. In one aspect, the refractive index information may be a parameter that does not include information on the magnitude of absorption (e.g., absorbance).

[0057] In one embodiment, the refractive index information may be a refractive index, a phase, or a complex amplitude. In other words, in one embodiment, the refractive index information distribution may be a refractive index distribution, a phase distribution, or a complex amplitude distribution. The phase distribution φ(r) with respect to the coordinate r is expressed by the following relational expression (1A) using the refractive index distribution n(r). In the following relational expression (1A), d represents the thickness of the sample, λ represents the wavelength of the light source (predetermined wavelength), π represents the ratio of the circumference to the diameter of the circle, and r represents the coordinate. The phase is a linear function of the refractive index. For example, by integrating the following equation (1A) in which d is replaced with a minute thickness Δd, the phase change after light along the thickness direction passes through the cell sample can be obtained.

number

number

[0058] <Labeling process> Next, each step of this embodiment will be described. In the labeling step, a cell sample is labeled with a labeling substance. In the labeling step, at least some or all of the cells contained in the cell sample are partially or entirely labeled with the labeling substance. As a method for labeling a cell sample with a labeling substance, a person skilled in the art can appropriately select and use a method typically used for labeling cell samples, depending on the form of the labeling substance, the cells in the cell sample that are to be labeled, and the portion of the cells that are to be labeled. Labeling of a cell sample may be performed by subjecting the cell sample to a process for labeling the cells contained therein with a labeling substance, or may be performed by first subjecting the cells to a process for labeling with a labeling substance and then preparing a cell sample using the labeled cells. The former method is simple and avoids artifacts that may occur due to labeling because it involves labeling a prepared cell sample, and is suitable for use in evaluation methods using cell samples, such as biological samples and organoids, where analysis of interactions between multiple types of cells and other components is effective. The latter method uses cells labeled with a labeling substance to prepare a cell sample, and therefore is highly efficient in labeling cells with the labeling substance and can selectively label only the target cells among the cells contained in the cell sample. The latter method is suitable for use when it is desired to label only a portion of the cells contained in a cell sample such as a cell aggregate.

[0059] When a labeling substance, a complex or fusion protein containing the same, or a nucleic acid that expresses the same does not have cell membrane permeability, the labeling substance or a complex containing the same may be introduced into a cell according to a method that may be used by those skilled in the art for introducing a substance that cannot permeate the cell membrane into a cell, for example, by lipofection or electroporation.

[0060] The cells contained in the cell sample are labeled with a labeling substance for at least one selected from the group consisting of, for example, at least one protein, at least one organelle, cytoplasm, cell membrane, and the whole cell, and preferably, at least one protein, at least one organelle, cytoplasm, cell membrane, or the whole cell is labeled with a labeling substance.

[0061] The protein to be labeled with a labeling substance may be endogenous to the cell or may be genetically introduced into the cell. Those skilled in the art can select an appropriate method for labeling a protein with a labeling substance depending on the form of the labeling substance. For example, when the labeling substance is in the form of a protein, a fusion protein containing the protein to be labeled with the labeling substance and the protein serving as the labeling substance is expressed in the cell by introducing a nucleic acid that expresses the fusion protein or by genome editing. Examples of the form of the nucleic acid that expresses the fusion protein include plasmid DNA, vectors, and mRNA and its precursors. Examples of genome editing technologies include CRISPR-Cas9, TALEN, and ZFN. Furthermore, for example, when the labeling substance is in a form other than a protein, a cell sample can be labeled with the labeling substance by introducing into the cell a complex of the labeling substance and a ligand that binds to the protein to be labeled, or by expressing in advance in the cell a fusion protein of the protein to be labeled with a peptide tag (e.g., FLAG tag) or protein tag (e.g., HALO protein or SNAP) that binds to a specific ligand, and then introducing into the cell sample or cells a complex of the labeling substance and a specific ligand that binds to the tag. Labeling a protein whose intracellular location is known can indirectly label, for example, the organelle in which the protein is localized. For example, because H2B protein is a nuclear-localized protein, introducing a complex of a labeling substance and H2B protein can label the nuclei of cells in a cell sample with the labeling substance. Furthermore, labeling a protein can provide information about, for example, the intracellular location of the protein and its surrounding environment.

[0062] Organelles labeled with a labeling substance include, for example, the cell nucleus, mitochondria, lysosomes, Golgi apparatus, peroxisomes, and endoplasmic reticulum. Organelles, cytoplasm, and cell membranes can be indirectly labeled by labeling proteins expressed in these organelles with a labeling substance, as described above. Alternatively, they can be labeled by expressing or introducing into cells a labeling substance that has been modified to facilitate transport or localization within the cell. A modification that facilitates transport to organelles or cytoplasm within the cell is, for example, a localization signal. A localization signal is a peptide sequence that facilitates transport and localization of a substance to which it is attached within the cell. Examples of localization signals include nuclear localization signals (NLS), nuclear export signals (NES), and mitochondrial localization signals (MTS). A modification that facilitates localization to the cell membrane is, for example, modification with a lipid such as a phospholipid.

[0063] When labeling the whole cell with a labeling substance, for example, a labeling substance (eg, a protein) that has not been modified for binding to and localizing with other proteins may be introduced into the cell.

[0064] When the cell sample is a cell aggregate, all cells contained in the cell sample may be labeled, or only a portion of the cells may be labeled. Labeling of only a portion of the cells contained in the cell sample can be achieved, for example, by preparing a cell aggregate using labeled cells and unlabeled cells, or by introducing a labeling substance into only a portion of the cells, for example, by encapsulating the labeling substance in liposomes whose surface has been modified with a ligand for a cell surface marker expressed only in a portion of the cells and contacting the liposomes with the cell sample. Thus, when the cell sample is a cell aggregate, at least one cell contained in the cell aggregate may be partially or entirely labeled with the labeling substance.

[0065] <First refractive index information distribution acquisition step> In the first refractive index information distribution acquisition step, a refractive index information distribution at a predetermined wavelength of the cell sample labeled in the labeling step is acquired to obtain the first refractive index information distribution. The refractive index information distribution is acquired using a method capable of acquiring a refractive index information distribution at a predetermined wavelength of the cell sample labeled with a labeling substance. A method capable of acquiring a refractive index information distribution at a predetermined wavelength of the cell sample labeled with a labeling substance is, for example, a method capable of acquiring the optical path length of light at a predetermined wavelength in the cell sample labeled with a labeling substance. The optical path length can be expressed as the value obtained by integrating the product of the refractive index and an infinitesimal distance element at coordinates on the optical path with respect to the spatial length of the optical path. Therefore, a method capable of acquiring the optical path length can obtain a refractive index information distribution at a predetermined wavelength of the cell sample labeled with a labeling substance. In particular, if the method capable of acquiring the optical path length is an acquisition method capable of eliminating the influence of spatial length (thickness), or if the method capable of acquiring the optical path length is combined with an analysis method capable of eliminating the influence of spatial length (thickness), a three-dimensional refractive index information distribution can be acquired, such as a three-dimensional refractive index distribution, phase distribution, or complex amplitude distribution. A technique capable of acquiring the optical path length is, for example, to acquire the optical path length based on an evaluation of the optical interference between light that reaches a detector due to reflection, transmission, refraction, diffraction, or scattering of light irradiated onto a cell sample and light that reaches the detector directly or after reflection from a reference mirror without irradiating the cell sample. The refractive index information distribution may be acquired using, for example, quantitative phase imaging, optical diffraction tomography, or optical coherence tomography. Among these, quantitative phase imaging and optical diffraction tomography are preferred due to their excellent spatial resolution and ease of signal interpretation, and optical diffraction tomography is even more preferred due to the ability to obtain three-dimensional information.

[0066] Quantitative phase imaging (QPI) is a technique for quantitatively imaging the amount of phase delay (interference intensity image) based on the optical interference between light transmitted through a sample and a reference light (i.e., optical interferometry). Here, the amount of phase delay at a coordinate on a plane is proportional to the optical path length, which is calculated by integrating the product of the refractive index and the small distance element corresponding to the height at that coordinate with respect to the thickness. Therefore, images obtained by QPI can provide information about the refractive index and thickness of a sample. In particular, comparing images of the same sample taken at multiple times can provide information about changes in the refractive index, since the thickness at corresponding coordinates is assumed to be the same. Quantitative phase imaging can be performed using, for example, a quantitative phase microscope, an optical microscope equipped with a quantitative phase imaging unit, a phase-contrast microscope, or a differential interference microscope. These microscopes and units are commercially available. A detailed example of quantitative phase imaging includes the method and apparatus described in the literature (Osamu Yasuhiko, Kozo Takeuchi, et al., "Single-shot quantitative phase imaging as an extension of differential interference contrast microscopy", Genes Cells 26:596-610 (2021).).

[0067] Optical Diffraction Tomography (ODT) is a technique that develops QPI into a three-dimensional imaging technology. It is a method that can acquire spatial phase distribution by acquiring images in multiple light irradiation directions and then performing three-dimensional reconstruction. Detailed ODT techniques and devices used therein are described, for example, in International Publication No. 2022 / 054305, International Publication No. 2023 / 095441, International Publication No. 2023 / 095440, or in literature (Yasuhiko O, Takeuchi K. In-silico clearing approach for deeprefractive index tomography by partial reconstruction and wave-backpropagation. Light Sci Appl. 12:101 (2023).), and the following can also be used.

[0068] 7 is a diagram showing the configuration of an observation device 1A. This observation device 1A includes a light source 10, an irradiation unit 31, an imaging unit 50, and a processing unit 60. The light source 10 outputs spatially coherent light. The light output from the light source 10 may or may not be temporally coherent.

[0069] The light source 10 may be a laser light source, or may be a light source such as an SLD (Super Luminescent Diode), an SC (Super Continuum) light source, an optical frequency comb light source, etc. Alternatively, the light source 10 may be configured to increase spatial coherence by passing spatially incoherent light output from an LED (Light Emitting Diode), a mercury lamp, or the like through a pinhole or the like.

[0070] The lens 21 is optically connected to the light source 10, and focuses the light output from the light source 10 onto a light input end 22 of an optical fiber 23, causing the light to be incident on the light input end 22. The optical fiber 23 guides the light that has entered the light input end 22 to a light output end 24. The light guided by the optical fiber 23 is output from the light output end 24 as divergent light. The lens 25 is optically connected to the light output end 24, and inputs the light that has been output from the light output end 24 as divergent light, collimates it, and outputs the collimated light to the irradiation unit 31.

[0071] The illumination unit 31 receives light output from the light source 10 and transmitted through the lens 21, the optical fiber 23, and the lens 25, and generates first light and second light from the received light. The illumination unit 31 also superimposes the first light and second light on each other and illuminates the observation object S. The illumination unit 31 illuminates the observation object S with the first light along a fixed light illumination direction, and illuminates the observation object S with the second light along each of a plurality of light illumination directions.

[0072] The irradiation unit 31 includes a beam splitter 311 , a phase-modulating spatial light modulator 313 , a polarizer 314 , a half-wave plate 315 , a polarizer 316 , a lens 318 and an objective lens 319 .

[0073] Beam splitter 311 reflects light that has reached it via polarizer 314 and half-wave plate 315, which are provided between it and lens 25, to spatial light modulator 313. Beam splitter 311 also receives light that has reached it from spatial light modulator 313 and outputs this light to polarizer 316.

[0074] Of the linearly polarized light beams incident on the modulation surface in a first and second orthogonal directions, the spatial light modulator 313 selectively phase-modulates the linearly polarized light beam in the second direction without phase-modulating the linearly polarized light beam in the first direction. The polarizer 314 and the half-wave plate 315 set the polarization state of the light so that the light incident on the modulation surface of the spatial light modulator 313 from the beam splitter 311 contains linearly polarized components in the first and second directions to the same extent.

[0075] Polarizer 316 receives the light that has arrived from spatial light modulator 313 via beam splitter 311 and allows the linearly polarized light in the first and second directions contained in the light to interfere with each other. Polarizer 316 has an optical axis whose orientation differs by 45 degrees from the polarization orientation of the light (linearly polarized light in the first and second directions) that has arrived from spatial light modulator 313 via beam splitter 311, and selectively transmits the polarized component of the input light in the orientation of the optical axis. Lens 318 and objective lens 319 irradiate the first light and second light output from polarizer 316 onto observation object S as plane waves.

[0076] The irradiation unit 31 having such a configuration can treat linearly polarized light in a first direction that has not been phase-modulated by the spatial light modulator 313 as first light, and can irradiate this first light along a fixed light irradiation direction onto the observation object S. The irradiation unit 31 can treat linearly polarized light in a second direction that has been phase-modulated by the spatial light modulator 313 as second light, and can irradiate this second light along each of a plurality of light irradiation directions onto the observation object S.

[0077] The direction of irradiation of the second light onto the observation object S can be set by the orientation and spacing of the phase modulation pattern on the modulation surface of the spatial light modulator 313. In addition, the phase difference between the first light and the second light can be set by shifting the phase modulation pattern on the modulation surface of the spatial light modulator 313.

[0078] The objective lens 41 receives light (first light and second light) that has been irradiated onto the observation object S by the irradiation unit 31 and passed through the observation object S, and outputs the light to the mirror 42. The lens 43 receives light that has been output from the objective lens 41 and reflected by the mirror 42, and causes the light to be incident on the imaging surface of the imaging unit 50.

[0079] The imaging unit 50 receives both the first light and the second light that have reached the imaging surface from the lens 43, and captures an interference intensity image resulting from interference between the first light and the second light. The imaging unit 50 captures interference intensity images when the phase difference between the first light and the second light is set to each of a plurality of phase differences for each of a plurality of light irradiation directions of the second light.

[0080] The processing unit 60 is electrically connected to the imaging unit 50, and performs required processing based on the interference intensity image captured by the imaging unit 50 to generate a complex amplitude image, etc. The processing content of the processing unit 60 will be described later.

[0081] Next, an observation method using the observation device 1A (FIG. 7) will be described.

[0082] 8 is a diagram schematically illustrating the incidence of the first light and the second light on the observation object S, and the incidence of the first light and the second light on the imaging unit 50 after passing through the observation object S. The wavefront of the first light incident on the observation object S is denoted by u. 0,in The wavefront of the second light incident on the observation object S along the n-th light irradiation direction (n=1 to N) among the multiple (N) light irradiation directions of the second light is represented as u (r). n,in It is expressed as (r)exp(iφ).

[0083] r is a variable representing the position. φ is the phase difference between the first light and the second light. i is the imaginary unit. The wavefront of the first light on the imaging plane or focal plane (a plane optically conjugate to the imaging plane) of the imaging unit 50 is represented as u0(r), and the wavefront of the second light is represented as u n It is expressed as (r)exp(iφ).

[0084] Interference intensity image I acquired by imaging by the imaging unit 50 n (r, φ) is expressed by the following equation (1): Interference intensity image I n(r, φ) is an interference intensity image acquired by imaging with the imaging unit 50 when the first light is incident on the observation object S along a certain light irradiation direction and the second light is incident on the observation object along the nth light irradiation direction, with φ being the phase difference between the first light and the second light. The focal plane (a plane optically conjugate to the imaging plane) may be on the observation object S, may be on the imaging unit 50 side of the observation object S, or may be on the irradiation unit 31 side of the observation object S.

number

[0085] 9 is a diagram showing the configuration of a processing unit 60 of the observation device. The processing unit 60 includes an interference term calculation unit 61, a first complex amplitude image generation unit 62, a second complex amplitude image generation unit 63, a complex differential interference image generation unit 64, a differential phase image generation unit 65, and a refractive index distribution image generation unit 66. The processing unit 60 may be, for example, a computer.

[0086] 10 is a flowchart of the observation method. In an irradiation step S1, the irradiation unit 31 irradiates the observation object S with a first light and a second light in an overlapping manner. At this time, the light irradiation direction of the first light with respect to the observation object S is constant, the light irradiation direction of the second light with respect to the observation object S is set to each of a plurality of light irradiation directions, and the phase difference φ between the first light and the second light is set to each value.

[0087] In the imaging step S2, the imaging unit 50 captures an interference intensity image (Equation (1)) when the phase difference φ between the first light and the second light is set to each of the multiple phase differences for each of the multiple light irradiation directions of the second light.

[0088] The processing steps performed by the processing unit 60 include steps S3 to S8. In the interference term calculation step S3, the interference term calculation unit 61 of the processing unit 60 calculates the interference term by a phase shift method based on the interference intensity image (Equation (1)) acquired by the imaging unit 50 when each of the plurality of phase differences φ is set for each of the plurality of light irradiation directions of the second light.

[0089] For example, when the three-point phase shift method is used, the interference term calculation unit 61 calculates the interference term C by the following equation (2): n (r)=u0 * (r)·u n (r) is calculated. The interference term u0(r) u n * (r) can be calculated. The phase shift method with four or more points can also be used. n (r) is found for each of the plurality of light irradiation directions of the second light (that is, for each n (=1 to N)).

number

[0090] In the first complex amplitude image generating step S4, the first complex amplitude image generating unit 62 of the processing unit 60 generates the interference term C n A complex amplitude image of the first light is generated based on (r). The phase of the complex amplitude u0(r) of the first light can be estimated as follows.

[0091] After correcting the phase gradient between the first and second light (difference in the light incident direction), the coherent sum of the corrected interference terms, C sum (r) is calculated by the following formula (3). Here, the light incident direction of the first light is parallel to the z axis, and the wave vector representing the n-th light incident direction of the second light is k n Let's say.

number

[0092] The first factor (u0 * The phase of (r)) is the component of the first light incident on the observation object S along a fixed light irradiation direction, and is greatly affected by scattering because there is no optical sectioning effect. On the other hand, the second factor (u n (r)exp(ik nThe phase of the signal (sum of r) is the sum of the incident light from multiple directions, and has a relatively flat distribution because the information near the focal plane is selectively extracted by the optical sectioning effect of the coherent sum.

[0093] From this, the phase φ0(r) of the complex amplitude u0(r) of the first light is expressed by the coherent sum C of the interference term after phase gradient correction, as expressed by the following equation (4): sum It can be approximately expressed by the phase of (r).

number

[0094] The amplitude of the complex amplitude u0(r) of the first light is the intensity image |u0(r)| captured by the imaging unit 50 when the observation object S is irradiated with only the first light without irradiating the observation object S with the second light. 2 It can be found from

[0095] For example, in the observation device 1A (FIG. 7), by adjusting the orientation of the optical axis of each of the polarizer 314 and the half-wave plate 315 in the irradiation unit 31, only the first light can be irradiated onto the observation object S.

[0096] The amplitude of the complex amplitude u0(r) of the first light is the interference term C n Based on (r), the interference term C can be estimated as follows: n Intensity sum of (r) I sum (r) is calculated using the following formula (5).

number

[0097] The first factor on the right side of equation (5) (|u0(r)| 2 ) is the component of the first light incident on the observation object S along a fixed light irradiation direction, and is significantly affected by scattering because there is no optical sectioning effect. On the other hand, the second factor (|u n (r)| 2The sum of the scattered light rays (sum of the scattered light rays) is the sum of the scattered light rays for multiple incident directions of light, and the optical sectioning effect of the coherent sum averages the scattered light to a relatively uniform distribution.

[0098] From this, the amplitude A0(r) of the complex amplitude u0(r) of the first light is calculated by the interference term C as shown in the following equation (6): n Intensity sum of (r) I sum It can be approximately expressed as the square root of (r).

number

[0099] In the first complex amplitude image generation step S4, the first complex amplitude image generation unit 62 can generate a complex amplitude image u0(r) of the first light using the following equation (7) based on the phase φ0(r) and amplitude A0(r) of the complex amplitude u0(r) of the first light obtained as described above.

number

[0100] The optical sectioning effect is the ability to selectively capture an image at the focal plane of an objective lens in, for example, a confocal microscope or a differential interference microscope. In these microscopes, the optical sectioning effect can be achieved by increasing the numerical aperture on both the entrance and detection sides, thereby achieving selective illumination and detection.

[0101] In the second complex amplitude image generating step S5, the second complex amplitude image generating unit 63 of the processing unit 60 generates a complex amplitude image u0(r) of the first light and an interference term C n Based on (r), the complex amplitude image u of the second light in each of the plurality of light irradiation directions is calculated by the following equation (8). n However, in this equation (8), when the denominator on the right side is 0, u n Since (r) is indefinite, the complex amplitude image u of the second light in each of the multiple light irradiation directions is calculated by the following equation (9) instead of equation (8).n It is preferable to generate (r), where ε is a small positive value. When ε=0, equation (9) is equal to equation (8).

number

number

[0102] In the complex differential interference contrast image generating step S6, the complex differential interference contrast image generating unit 64 of the processing unit 60 generates a complex amplitude image u of the second light in each of the plurality of light irradiation directions. n Based on (r), a complex differential interference image W(r) can be generated using equation (10) below. δr represents shear. At least one of the x-component δx and y-component δy of δr is non-zero. If δx ≠ 0 and δy = 0, a complex differential interference image with the x-direction as the shear direction is obtained. If δx = 0 and δy ≠ 0, a complex differential interference image with the y-direction as the shear direction is obtained. If δx ≠ 0 and δy ≠ 0, a complex differential interference image with the direction according to the ratio of δx to δy as the shear direction is obtained.

number

[0103] In the phase differential image generating step S7, the phase differential image generating section 65 of the processing section 60 can generate a phase differential image represented by the phase of the complex differential interference image W(r). Furthermore, in the refractive index distribution image generating step S8, the refractive index distribution image generating section 66 of the processing section 60 can obtain a phase differential image (i.e., a three-dimensional phase differential image) at each position in the z-axis direction using the equation for free propagation of a wavefront, and can generate a refractive index distribution image of the observation object S by deconvolution based on this.

[0104] Optical coherence tomography (OCT) is a technique for obtaining information about the optical path length by evaluating the interference between light from the same light source that is scattered by a sample and reaches a detector, and light that passes through a fixed optical path inside the OCT device and reaches a detector. For example, information about the optical path length of light that is scattered by a sample and reaches a detector can be obtained from the change in optical interference that occurs when the optical path length of the fixed optical path is changed. OCT can be performed using commercially available equipment according to standard procedures.

[0105] The light source used to acquire the refractive index information distribution may be any light source capable of emitting light of a predetermined wavelength at an intensity and in a manner that allows the signal resulting from the labeling substance that labeled the cell sample to be acquired as refractive index information. For example, a lamp or laser that emits light over a wide wavelength range, such as a mercury light source, halogen light source, xenon light source, or LED light source, may be used as the light source. In this case, it is preferable that the light emitted from the light source has passed through a filter such as a bandpass filter and the component of the wavelength range around the predetermined wavelength is irradiated onto the cell sample. Alternatively, for example, a laser light source that emits laser light over a narrow wavelength range, such as a helium-neon laser, argon ion laser, or diode laser, may be used as the light source.

[0106] The wavelength range of the light irradiated onto the cell sample in acquiring the refractive index information distribution has an upper and lower limit wavelength, and the difference between the upper and lower limit wavelengths and the predetermined wavelength is, for example, 35 nm or less, 30 nm or less, or 26 nm or less, respectively. Examples of the wavelength range of the light irradiated onto the cell sample include 433±12 nm, 530±21.5 nm, 620±26 nm, and 694±22 nm.

[0107] The refractive index information distribution may be acquired for a part or the whole of the cell sample, for example, for a specific site within a cell, a specific cell within a cell population, or a specific region within a cell aggregate. Furthermore, light irradiation in acquiring the refractive index distribution may be performed for a part or the whole of the cell sample, and the refractive index information distribution may be acquired for a part or the whole of the region irradiated with light.

[0108] <Refractive index changing process> In the refractive index changing step, after the first refractive index information distribution acquisition step, a change in the refractive index at a predetermined wavelength of the labeling substance that labels the cell sample is caused. The change in refractive index can be caused by applying a stimulus depending on the type of labeling substance or by leaving the labeling substance undisturbed. The detailed method for causing a change in refractive index depending on the labeling substance has already been explained in the section "Labeling substance."

[0109] <Second refractive index information distribution acquisition process> In the second refractive index information distribution acquisition step, the refractive index information distribution at a predetermined wavelength of the cell sample after the refractive index of the labeling substance has been changed in the refractive index change step is acquired again to obtain the second refractive index information distribution. The second refractive index information distribution acquisition step is performed under the same or substantially the same conditions as the first refractive index distribution acquisition step, except for the conditions for maintaining the changed state of the refractive index of the labeling substance. For example, the refractive index information distribution in the second refractive index information distribution acquisition step is acquired using the same device and settings, in the same or substantially the same method and environment as the refractive index information distribution in the first refractive index information distribution acquisition step, except for the conditions for maintaining the changed state of the refractive index of the labeling substance. This allows refractive index information distributions under the same conditions before and after the refractive index change to be acquired for the same planar or spatial coordinates of the cell sample, and allows the first refractive index information distribution and the second refractive index information distribution to be directly compared in the subsequent analysis step. In the above, "substantially the same" means that although the two are not exactly the same with respect to conditions that may vary depending on the time of acquisition, such as temperature, humidity, atmospheric pressure, and the placement of the cell sample, they can be considered to be the same in the subsequent analysis process, the influence of which can be ignored, or they can be corrected to be the same using methods commonly used by those skilled in the art.

[0110] <Analysis process> In the analysis step, the first refractive index information distribution and the second refractive index information distribution are compared to evaluate the distribution of the labeled substance and / or the surrounding environment. More specifically, in the analysis step, the distribution of the labeled substance and / or the surrounding environment is evaluated based on the presence or absence and / or degree of change in the refractive index information distribution at a predetermined wavelength between the first refractive index information distribution and the second refractive index information distribution. The presence or absence and / or degree of change in the refractive index information distribution at a predetermined wavelength can be evaluated based on, for example, the ratio and / or difference between the first refractive index information distribution and the second refractive index information distribution. When the first refractive index information distribution and the second refractive index information distribution are spatial distributions of refractive index information, the comparison between the two may be performed on the spatial distributions or on the planar distributions of the two on the same tomographic plane.

[0111] The distribution of the labeled substance in the analysis step can be evaluated based on the presence or absence and degree of change in refractive index information at a predetermined wavelength at a specific location in the cell sample. For example, when a refractive index information distribution is acquired for the cell sample, it can be evaluated that a labeled substance is present at a location where a change in refractive index information occurs between the first refractive index information distribution and the second refractive index information distribution, and that a labeled substance is not present at a location where no change in refractive index information occurs. Furthermore, for example, when a refractive index information distribution for the cell sample is acquired, it can be evaluated that a labeled substance is present at a high concentration at a location where a change in refractive index information between the first refractive index information distribution and the second refractive index information distribution is large, and that a labeled substance is present at a low concentration at a location where a change in refractive index information is small.

[0112] The evaluation of the environment surrounding the labeled substance in the analysis step can be based on the presence or absence and degree of an environment-dependent change in refractive index information at a predetermined wavelength of the labeled substance at a specific location in the cell sample. For example, if the presence or absence and / or degree of change in refractive index at a predetermined wavelength when a stimulus is applied to the labeled substance depends on pH, substance concentration, temperature, and / or solvent, the pH, substance concentration, temperature, and / or solvent can be evaluated as the environment surrounding the labeled substance present at that location based on the presence or absence and / or degree of change in refractive index information between a first refractive index information distribution and a second refractive index information distribution at that location. For example, if the degree of change in refractive index information in response to light irradiation of the labeled substance depends on calcium ion concentration, the calcium ion concentration in the environment surrounding the labeled substance can be evaluated based on the magnitude of the difference in refractive index information between the first refractive index information distribution and the second refractive index information distribution. In this case, for example, if the presence or absence and / or degree of refractive index change at a predetermined wavelength when a stimulus is applied to a labeled substance varies depending on multiple parameters of the surrounding environment, a parameter other than one of the multiple parameters (parameter A) may be determined from information other than refractive index information (for example, fluorescent imaging using a fluorescent dye), and the refractive index change caused by those parameters may be subtracted from the actual refractive index change to obtain a corrected refractive index change, which may be used as an index to evaluate the value of parameter A in the vicinity of the labeled substance.

[0113] In the analysis step, it is preferable to compare the first refractive index information distribution and the second refractive index information distribution at the same location in the cell sample, from the viewpoint of reducing the influence of differences in labeling substance concentration and shape. For example, it is preferable to compare corresponding coordinates in the cell sample, the same organelle, or the same cell. It is more preferable to compare corresponding coordinates in the cell sample, from the viewpoint of reducing the influence of differences in labeling substance concentration and shape. Here, because the physical shape and arrangement of the cell sample may change between the first refractive index information distribution acquisition step and the second refractive index information distribution acquisition step, the corresponding coordinates to be compared do not have to be exactly the same coordinates. Rather, it is preferable that the coordinates in the first refractive index information distribution and the second refractive index information distribution correspond to the same location in the cell sample, determined by correction based on the physical shape and arrangement of the cell sample obtained from the refractive index information distribution. Thus, in a preferred embodiment, the distribution of the labeling substance and / or the surrounding environment may be evaluated based on the ratio and / or difference of refractive index information at corresponding coordinates in the first refractive index information distribution and the second refractive index information distribution. The refractive index information at a certain coordinate may be the refractive index information at that coordinate, or the average value of the refractive index information at coordinates within a predetermined distance from that coordinate may be used as the refractive index information at that coordinate. Furthermore, when comparing the first refractive index information distribution and the second refractive index information distribution for the same organelle or the same cell, the comparison may be performed using numerical values ​​obtained by statistical processing of the refractive index information distribution, such as the average value, median, or variance of the refractive index information. In the analysis step, the degree of change in the refractive index information distribution between a portion of the cell sample labeled with a labeling substance and an unlabeled portion may be compared, although typically, no change in the refractive index information distribution occurs in a portion not labeled with a labeling substance.

[0114] <Evaluation example 1> Next, the cell sample evaluation method according to one embodiment described above will be described in more detail using evaluation examples. In evaluation example 1, chromoprotein A is used as the labeling substance. Chromoprotein A is a chromoprotein whose absorbance and refractive index instantaneously decrease in response to green light irradiation, as shown in the absorption and refractive index spectra before and after light irradiation in FIG. 1 , and has a maximum absorption wavelength of 620 nm and a wavelength at which the refractive index is maximized in the visible light region of 650 nm.

[0115] 2 shows a cell having an intracellular site X. Evaluation example 1 aims to identify the distribution of intracellular site X, whose distribution within the cell is unknown and difficult to identify from a bright-field image.

[0116] First, in the labeling step, a fusion protein of chromoprotein A and a protein known to be localized at intracellular site X is expressed in cells using a viral vector. The fusion protein expressed in the cells localizes to intracellular site X. As a result, intracellular site X of the cells (cell sample) is labeled with chromoprotein A (labeling substance).

[0117] Next, as the first refractive index information acquisition process, a helium-neon laser with a wavelength of 632.8 nm is used as the light source, and the phase distribution (refractive index information distribution) of the entire cell at 632.8 nm (predetermined wavelength) is acquired by ODT to obtain the first phase distribution (first refractive index information distribution).

[0118] Next, in the refractive index change step, the entire cell sample is irradiated with light from an argon ion laser (green laser) with a wavelength of 514.5 nm, which causes a decrease (change) in the refractive index at 632.8 nm (predetermined wavelength) of the chromoprotein A (labeling substance) that labels the intracellular site X.

[0119] Next, as a second refractive index information acquisition process, the phase distribution (refractive index information distribution) of the entire cell at 632.8 nm (predetermined wavelength) is acquired again by ODT under the same conditions using the same light source as in the first refractive index information acquisition process to obtain a second phase distribution (second refractive index information distribution).

[0120] Finally, in the analysis step, the first phase distribution and the second phase distribution are compared. In a cell, a difference in phase occurs between the first phase distribution and the second phase distribution due to a change in the refractive index of the chromoprotein A at a location where the chromoprotein A is present, whereas no difference in phase occurs between the first phase distribution and the second phase distribution at a location where the chromoprotein A is not present. Based on this, a location where a significant decrease (change) in phase between the first phase distribution and the second phase distribution is observed can be evaluated as a location where the chromoprotein A is present. Furthermore, since the chromoprotein A is localized to intracellular site X in a cell as described above, a location where a significant decrease (change) in phase between the first phase distribution and the second phase distribution is observed can be identified as the intracellular site X.

[0121] An example of a labeling substance that can be used in the same manner as chromoprotein A used in Evaluation Example 1 is asFP595.

[0122] <Evaluation example 2> In Evaluation Example 2, chromoprotein B is used as the labeling substance. Chromoprotein B is a chromoprotein whose absorbance and refractive index increase spontaneously and time-dependently, as shown in the absorption and refractive index spectra before and after the passage of time in Figure 3. It has a maximum absorption wavelength of 620 nm and a wavelength at which the refractive index is maximum in the visible light region of 650 nm.

[0123] In Evaluation Example 2, when spheroids are produced using two types of cells, cell P and cell Q, the objective is to identify the spatial position of cell P in the cell aggregate. Figure 4 shows a spheroid produced from cell P and cell Q.

[0124] First, in the labeling step, a spheroid is created in which only cell P is labeled. The detailed procedure is as follows: first, mRNA encoding chromoprotein B is introduced into free cell P by lipofection, and then cell P is cultured to express chromoprotein B. Next, cell P expressing chromoprotein B and unlabeled cell Q are seeded into a microwell plate for spheroid creation and cultured to create spheroids. This results in a spheroid (cell sample) in which cell P is labeled with chromoprotein B (labeling substance).

[0125] Next, as the first refractive index information acquisition process, a helium-neon laser with a wavelength of 632.8 nm is used as the light source, and a three-dimensional phase distribution (refractive index information distribution) of the entire spheroid at 632.8 nm (predetermined wavelength) is acquired by ODT according to the description in International Publication No. 2022 / 054305 to obtain the first phase distribution (first refractive index information distribution).

[0126] Next, as a refractive index change step, the spheroids are statically cultured for a time sufficient for the chromoprotein B expressed in the cells P to spontaneously change the refractive index. This causes the refractive index at 632.8 nm (predetermined wavelength) of the chromoprotein B (labeling substance) that labeled the cells P to spontaneously increase (change).

[0127] Next, as a second refractive index information acquisition process, ODT is performed under the same conditions using the same light source as in the first refractive index information acquisition process to again acquire a three-dimensional phase distribution (refractive index information distribution) of the entire spheroid at 632.8 nm (predetermined wavelength), thereby obtaining a second phase distribution (second refractive index information distribution).

[0128] Finally, in the analysis step, the first phase distribution and the second phase distribution are compared. In the spheroid, a difference in phase occurs between the first and second phase distributions due to a change in the refractive index of chromoprotein B in areas where chromoprotein B is present, whereas no difference in phase occurs between the first and second phase distributions in areas where chromoprotein B is absent. Based on this, areas where a significant increase (change) in phase between the first and second phase distributions is observed can be evaluated as areas where chromoprotein B is present. Furthermore, since, as described above, only cell P among the cells contained in the spheroid is labeled with chromoprotein B, the presence of cell P can be identified in areas where a significant increase (change) in phase between the first and second phase distributions is observed. Figure 5 is an example of a tomographic image showing the presence or absence of a significant increase in the second phase distribution relative to the first phase distribution in Evaluation Example 2. In Figure 5, black cells indicate cells present in areas where the phase in the second phase distribution is significantly increased relative to the first phase distribution, and white cells indicate cells present in areas where the phase in the second phase distribution is not significantly increased relative to the first phase distribution. From the results in Figure 5, the black cells can be identified as cell P and the white cells as cell Q, and it can be determined that cell P is located near the center of the spheroid.

[0129] <Action and effect> In the evaluation method of this embodiment, a cell sample is labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously, and the distribution of the labeling substance and / or the surrounding environment is evaluated by comparing a first refractive index information distribution and a second refractive index information distribution, which are refractive index information distributions acquired at the same predetermined wavelength before and after the change in refractive index. This allows the distribution of the labeling substance and / or the surrounding environment to be evaluated based on the refractive index information distribution at a single wavelength, thereby avoiding the problems of artifacts caused by acquiring refractive index information distributions at multiple wavelengths and the need to prepare multiple light sources.

[0130] <Cell sample evaluation device> Fig. 6 shows an example of the configuration of a cell sample evaluation device used in the evaluation method of this embodiment. The cell sample evaluation device shown in Fig. 6 comprises a refractive index information distribution acquisition section, a stimulation section, and an analysis section.

[0131] The refractive index information distribution acquisition unit acquires the refractive index information distribution of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to a stimulus. The labeling substance, the cell sample, and the refractive index information distribution are the same as those described in the cell sample evaluation method. The refractive index information distribution acquisition unit may have the same configuration as the device described in the section on refractive index information distribution in the cell sample evaluation method, and more specifically, the same configuration as the device described as being usable in, for example, quantitative phase imaging, optical diffraction tomography, or optical coherence tomography. The refractive index information distribution acquisition unit also includes a calculation unit that performs various calculations, including a step of acquiring a refractive index information distribution at a predetermined wavelength of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to a stimulus, before the refractive index of the labeling substance changes, thereby obtaining a first refractive index information distribution, and a step of acquiring a refractive index information distribution at a predetermined wavelength of the cell sample after the refractive index of the labeling substance changes, thereby obtaining a second refractive index information distribution.

[0132] The stimulation unit supplies a stimulus to the cell sample that causes a change in refractive index when the cell sample is labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to the stimulus. The stimulation unit has the same configuration as a device commonly used to apply a stimulus that causes a change in refractive index to a labeling substance. For example, if the stimulus is light irradiation, the stimulation unit may have the same configuration as a device commonly used as a light source. For example, if the stimulus is a temperature change, the stimulation unit may have the same configuration as a device commonly used as a heating device that raises the temperature of a cell sample or a heat dissipation device that lowers the temperature. For example, if the stimulus is a pH change, a substance concentration change, or a solvent change, the stimulation unit may have the same configuration as a device commonly used as a perfusion device that replaces a liquid present around a cell sample with another liquid.

[0133] The analysis unit compares a first refractive index information distribution, which is a refractive index information distribution at a predetermined wavelength of the cell sample before a change in the refractive index of the labeling substance occurs, with a second refractive index information distribution, which is a refractive index information distribution at a predetermined wavelength of the cell sample after a change in the refractive index of the labeling substance occurs, to evaluate the distribution of the labeling substance and / or the surrounding environment. That is, the analysis unit is a unit that performs the analysis step described in the cell sample evaluation method. In other words, the analysis unit is a unit that compares the first refractive index information distribution with the second refractive index information distribution, and executes the step of evaluating the distribution of the labeling substance and / or the surrounding environment.

[0134] The calculation unit and analysis unit of the refractive index information distribution acquisition unit described above include a processing unit including a CPU that performs various calculation processes, a memory unit including a hard disk drive, RAM, ROM, etc. that stores data and programs, a display unit including a liquid crystal display that displays processing results, etc., and an input unit including a keyboard, mouse, etc. that accepts input of various conditions for acquiring and displaying interference images. The calculation unit and analysis unit may be configured as a smart device such as a tablet terminal that has a touch panel, etc. as an input unit. Furthermore, the processing unit and memory unit of the calculation unit and analysis unit may be configured as an FPGA (field-programmable gate array) or a microcomputer.

[0135] The cell sample evaluation device described above comprises a refractive index information distribution acquisition unit that acquires a refractive index information distribution of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously, and an analysis unit that compares a first refractive index information distribution, which is the refractive index information distribution at the predetermined wavelength of the cell sample before the refractive index change of the labeling substance, with a second refractive index information distribution, which is the refractive index information distribution at the predetermined wavelength of the cell sample after the refractive index change of the labeling substance, to evaluate the distribution of the labeling substance and / or the surrounding environment. This makes it possible to evaluate the distribution of the labeling substance and / or the surrounding environment based on the refractive index information distribution at a single wavelength, thereby avoiding the problems of artifacts and the need for multiple light sources when evaluating a cell sample. [Explanation of symbols]

[0136] 1A...observation device, 10...light source, 21...lens, 22...light input end, 23...optical fiber, 24...light output end, 25...lens, 31...irradiation unit, 41...objective lens, 42...mirror, 43...lens, 50...imaging unit, 60...processing unit, 61...interference term calculation unit, 62...first complex amplitude image generation unit, 63...second complex amplitude image generation unit, 64...complex differential interference image generation unit, 65...phase differential image generation unit, 66...refractive index distribution image generation unit, 311...beam splitter, 313...spatial light modulator, 314...polarizer, 315...half-wave plate, 316...polarizer, 318...lens, 319...objective lens.

Claims

1. labeling a cell sample with a labeling substance that changes its refractive index at a predetermined wavelength in response to a stimulus and / or spontaneously; acquiring a refractive index information distribution of the cell sample at the predetermined wavelength to obtain a first refractive index information distribution; causing a change in refractive index at the predetermined wavelength of the labeling substance that labels the cell sample; a step of acquiring a second refractive index information distribution by again acquiring refractive index information distribution of the cell sample at the predetermined wavelength; comparing the first refractive index information distribution with the second refractive index information distribution to evaluate the distribution of the labeled substance and / or the surrounding environment; A method for evaluating a cell sample, comprising, in this order:

2. 2. The evaluation method according to claim 1, wherein the labeled substance is a substance whose refractive index at a predetermined wavelength changes spontaneously and / or in response to at least one stimulus selected from the group consisting of light irradiation, a pH change, a substance concentration change, a temperature change, a solvent change, voltage application, a mechanical stimulus, and gas exposure.

3. The evaluation method according to claim 1 , wherein the labeling substance is a substance whose refractive index at a predetermined wavelength changes in response to light irradiation or spontaneously.

4. The evaluation method according to claim 1 , wherein the change in refractive index of the labeling substance is time-dependent.

5. The evaluation method according to claim 1 , wherein the marker substance is a substance whose refractive index at a predetermined wavelength increases in response to a stimulus and / or spontaneously.

6. The evaluation method according to claim 1 , wherein the labeling substance is a chromoprotein.

7. The evaluation method according to claim 1 , wherein the refractive index information distribution is acquired using quantitative phase imaging, optical diffraction tomography, or optical coherence tomography.

8. The evaluation method according to claim 1 , wherein the refractive index information distribution is a refractive index distribution, a phase distribution, or a complex amplitude distribution.

9. The evaluation method according to claim 1, wherein the evaluation of the distribution of the labeled substance and / or the surrounding environment is performed based on the ratio and / or difference of refractive index information at corresponding coordinates in the first refractive index information distribution and the second refractive index information distribution.

10. 2. The evaluation method according to claim 1, wherein the cell sample is a cell, and at least one type of protein, at least one type of organelle, cytoplasm, cell membrane, or the entire cell of the cell is labeled with the labeling substance.

11. The evaluation method according to claim 1 , wherein the cell sample is a cell aggregate, and at least one cell contained in the cell aggregate is partially or entirely labeled with the labeling substance.

12. a refractive index information distribution acquisition unit that acquires a refractive index information distribution of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously; an analysis unit that compares a first refractive index information distribution, which is a refractive index information distribution at the predetermined wavelength of the cell sample before a change in the refractive index of the labeling substance occurs, with a second refractive index information distribution, which is a refractive index information distribution at the predetermined wavelength of the cell sample after a change in the refractive index of the labeling substance occurs, and evaluates the distribution of the labeling substance and / or the surrounding environment; A cell sample evaluation device comprising:

13. a step of obtaining a first refractive index information distribution by acquiring a refractive index information distribution at a predetermined wavelength of a cell sample labeled with a labeling substance whose refractive index at a predetermined wavelength changes in response to a stimulus and / or spontaneously, before the refractive index of the labeling substance changes; obtaining a second refractive index information distribution by acquiring a refractive index information distribution at the predetermined wavelength of the cell sample after a change in the refractive index of the labeling substance has occurred; comparing the first refractive index information distribution with the second refractive index information distribution to evaluate the distribution of the labeled substance and / or the surrounding environment; A program that causes a computer to execute the following.

Images