Cell evaluation method

Abstract

To provide a method that can evaluate a cell easily even if the cell is not unknown.SOLUTION: A cell evaluation method comprises: a label step S1; a refractive index distribution acquisition step S2; and an analysis step S3. In the label step S1, the cell evaluation method is configured to label a specific part of a cell to be evaluated with a label substance mutually different in a refractive index in each of a first wavelength and second wavelength. In the refractive index distribution acquisition step S2, the cell evaluation method is configured to acquire a refractive index distribution of the cell having the specific part labelled in the label step S1 in each of the first wavelength and second wavelength. In the analysis step S3, the cell evaluation method is configured to compare the refractive index distribution in each of the first wavelength and second wavelength, and thereby evaluate a distribution of the specific part in the cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and an apparatus for evaluating the distribution of a specific site in a cell. [Background technology]

[0002] For example, since it is difficult to observe a transparent object such as a cell using a normal microscope, an observation device capable of acquiring a phase image or a refractive index distribution image of the object is generally used. Several methods for realizing such an observation device are known.

[0003] One of the conventional techniques uses a Michelson interferometer or a Mach-Zehnder interferometer that splits and combines two beams, and places the object to be observed on the optical path of one of the two beams.Then, by moving a mirror or the like on the optical path, the optical path length difference (phase difference) between the two beams is set to each value in sequence, multiple interference images of the object to be observed are obtained, and a phase image of the object to be observed can be created based on these multiple interference images.

[0004] The technology described in Non-Patent Document 1 uses a configuration in which a differential interference microscope is combined with a quarter-wave plate and a polarization camera. Then, a phase image of the observed object can be created based on the interference images for each of the multiple polarization components acquired by the polarization camera.

[0005] If the thickness of the object being observed is d, the wavelength of light is λ, and the refractive index is n, the phase φ is expressed by the relational formula φ = 2π n d / λ. Therefore, if the thickness d and wavelength λ are known, a refractive index distribution image can be created from the phase image.

[0006] The Optical Diffraction Tomography (ODT) technology described in Non-Patent Document 2 uses a Mach-Zehnder interferometer that splits and combines two light beams, and places an observation object on the optical path of one of the two light beams. Then, by setting various directions of light incidence on the observation object, multiple interference images of the observation object are obtained, and a refractive index distribution image of the observation object can be created based on these multiple interference images.

[0007] Patent Document 1 discloses an invention for evaluating stem cells based on phase images. The invention disclosed in this document compares the phase of the cell nucleus and the phase of the cytoplasm in a phase image of a stem cell, and evaluates the quality of the stem cell based on the comparison result. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5745919 [Non-patent literature]

[0009] [Non-Patent Document 1] Yasuhiko O, Takeuchi K, Yamada H,Ueda Y. Single-shot quantitative phase imaging as an extension of differentialinterference contrast microscopy. Genes Cells. 2021. [Non-Patent Document 2] Yasuhiko O, Takeuchi K, Yamada H,Ueda Y. Multiple-scattering suppressive refractive index tomography for the label-free quantitative assessment of multicellular spheroids. Biomed OptExpress. 2022. [Non-Patent Document 3] Fu et al. Quantitative dispersion microscopy. Biomed Opt Express. 2010. [Non-Patent Document 4] Sai et al. Designing refractiveindex fluids using the Kramers-Kronig relations. Faraday Discuss. 2020. Summary of the Invention [Problem to be solved by the invention]

[0010] When the type of cell is known, the cell can be evaluated (e.g., evaluated for quality) based on the phase image or refractive index distribution image of the cell acquired by the above-mentioned conventional technology. However, when the type of cell is unknown, it may not be clear what information about the cell is represented by the phase image and refractive index distribution image of the cell acquired by the above-mentioned conventional technology, and it is difficult to evaluate the cell based on these images.

[0011] The present invention has been made to solve the above problems, and has an object to provide a method and apparatus that can easily evaluate even unknown cells. [Means for solving the problem]

[0012] The cell evaluation method of the present invention includes a labeling step of labeling a specific portion 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 portion has been labeled in the labeling step, and an analysis step of evaluating the distribution of the specific portion of the cell by comparing the refractive index distributions at the first wavelength and the second wavelength.

[0013] In the labeling step, the specific site of the cell may be labeled with a labeling substance having a higher refractive index at the longer wavelength of the first wavelength and the second wavelength than at the shorter wavelength, or the specific site of the cell may be labeled with a labeling substance having a lower refractive index at the longer wavelength of the first wavelength and the second wavelength than at the shorter wavelength.

[0014] In the labeling step, it is preferable to label a specific site of a cell with a light-absorbing labeling substance having an absorption peak at a specific wavelength, and in this case, it is preferable to acquire the refractive index distribution of the cell whose specific site is labeled in the labeling step at each of a first wavelength and a second wavelength on the short wavelength side or long wavelength side of the specific wavelength in the refractive index distribution acquisition step. In addition, it is preferable to label a specific site of a cell with a light-absorbing labeling substance having an absorption peak at a specific wavelength in the labeling step, and acquire the refractive index distribution of the cell whose specific site is labeled in the labeling step at each of a first wavelength and a second wavelength, with one of the first wavelength and the second wavelength being on the short wavelength side of the specific wavelength and the other being on the long wavelength side of the specific wavelength. Furthermore, in the labeling step, it is also preferable to introduce into a cell an intracellular expression construct in which a DNA sequence that commands a chromoprotein is connected to the N-terminus or C-terminus of a DNA sequence that commands an arbitrary protein, and to label the specific site using this intracellular expression construct as a labeling substance.

[0015] In the analysis step, it is preferable to evaluate the distribution of the specific part of the cell based on the ratio or difference between the refractive index distributions at the first wavelength and the second wavelength, and it is also preferable to perform an analysis of the specific part of the cell by superimposing the result of the comparison of the refractive index distributions at the first wavelength and the second wavelength on an intensity image or a fluorescent image of the cell.

[0016] In the labeling step, it is preferable to label another specific site of the cell with another labeling substance having a different refractive index at the third wavelength and the fourth wavelength, in the refractive index distribution acquisition step, the refractive index distribution of the cell labeled in the labeling step is acquired at the third wavelength and the fourth wavelength, and in the analysis step, the refractive index distribution at the third wavelength and the fourth wavelength are compared to evaluate the distribution of the other specific site of the cell. In this case, the first wavelength or the second wavelength may be equal to the third wavelength or the fourth wavelength.

[0017] The cell evaluation device of the present invention includes a refractive index distribution acquisition unit that acquires, at each of the first wavelength and the second wavelength, the refractive index distribution of a cell whose specific site is labeled with a labeling substance having a different refractive index at each of the first wavelength and the second wavelength, and an analysis unit that evaluates the distribution of the specific site in the cell by comparing the refractive index distribution at each of the first wavelength and the second wavelength.

[0018] The program of the present invention is a program for causing a computer to execute a refractive index distribution acquisition step of acquiring, at each of the first and second wavelengths, the refractive index distribution of a cell whose specific site is labeled with a labeling substance having a different refractive index at each of the first and second wavelengths, and an analysis step of evaluating the distribution of the specific site in the cell by comparing the refractive index distribution at each of the first and second wavelengths. Effect of the Invention

[0019] According to the present invention, even unknown cells can be easily evaluated. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a flow chart of a cell evaluation method. [Diagram 2] FIG. 2 is a diagram showing the configuration of the cell evaluation apparatus 1. As shown in FIG. [Diagram 3] FIG. 3 is a diagram showing the configuration of the cell evaluation apparatus 2A. [Figure 4]FIG. 4 is a diagram showing the configuration of the cell evaluation device 2B. [Diagram 5] 5 is a diagram showing the absorption spectrum and the refractive index spectrum of a light-absorbing labeling substance, in which (a) shows the absorption spectrum and (b) shows the refractive index spectrum. [Figure 6] 6(a) and (b) are diagrams showing schematic diagrams of refractive index spectra of light-absorbing labeling substances. [Figure 7] Fig. 7 is a diagram for explaining the refractive index distribution of a cell whose nucleus as a specific site is labeled with a light-absorbing labeling substance. Fig. 7(a) shows the refractive index distribution of the cell at a first wavelength λ1. Fig. 7(b) shows the refractive index distribution of the cell at a second wavelength λ2. [Figure 8] Fig. 8 is a diagram for explaining a first analysis example in the analysis step S3. Fig. 8(a) is a diagram that shows a refractive index distribution image. Fig. 8(b) is a diagram that shows a refractive index fluctuation image. Fig. 8(c) is a diagram in which the refractive index distribution image (Fig. 8(a)) and the refractive index fluctuation image (Fig. 8(b)) are superimposed. [Figure 9] Fig. 9 is a diagram for explaining a second analysis example in the analysis step S3. Fig. 9(a) is a diagram showing a typical refractive index fluctuation image acquired by labeling a cell nucleus with a labeling substance. Fig. 9(b) is a diagram showing a typical refractive index fluctuation image acquired by labeling an entire cell with a labeling substance. Fig. 9(c) is a diagram in which the refractive index fluctuation image (Fig. 9(a)) and the refractive index fluctuation image (Fig. 9(b)) are superimposed. [Figure 10] FIG. 10 is a diagram for explaining a third analysis example in the analysis step S3, and is a diagram that diagrammatically illustrates a plurality of refractive index fluctuation images acquired at regular time intervals. [Figure 11] FIG. 11 is a diagram for explaining a third analysis example in the analysis step S3, and is a diagram in which a plurality of refractive index fluctuation images (FIG. 10) acquired at regular time intervals are superimposed. [Figure 12] FIG. 12 is a diagram for explaining a third analysis example in the analysis step S3, and is a diagram in which a plurality of refractive index fluctuation images (FIG. 10) acquired at regular time intervals are superimposed on a refractive index distribution image. [Figure 13]Fig. 13 is a diagram for explaining a fourth analysis example in analysis step S3. Fig. 13(a) is a diagram that shows a schematic of a fluorescent image or intensity image of an entire cell. Fig. 13(b) is a diagram that shows a schematic of a refractive index fluctuation image acquired by labeling a cell nucleus with a labeling substance. Fig. 13(c) is a diagram in which the fluorescent image or intensity image of the entire cell (Fig. 13(a)) is superimposed on the refractive index fluctuation image (Fig. 13(b)). [Figure 14] Fig. 14 is a diagram for explaining a fifth analysis example in the analysis step S3. Fig. 14(a) is a diagram showing a schematic diagram of a fluorescent image or intensity image of a cell nucleus. Fig. 14(b) is a diagram showing a schematic diagram of a refractive index fluctuation image acquired by labeling with a labeling substance whose labeling target is unknown. Fig. 14(c) is a diagram in which the fluorescent image or intensity image of the cell nucleus (Fig. 14(a)) is superimposed on the refractive index fluctuation image (Fig. 14(b)). [Figure 15] FIG. 15 is a diagram for explaining a sixth analysis example in the analysis step S3. FIG. 15(a) is a diagram showing a typical refractive index fluctuation image obtained by labeling a specific vesicle with a labeling substance α. FIG. 15(b) is a diagram showing a typical refractive index fluctuation image obtained by labeling another specific vesicle with a labeling substance β. FIG. 15(c) is a diagram showing a typical refractive index distribution image. FIG. 15(d) is a diagram in which the refractive index fluctuation image (FIG. 15(a)), the refractive index fluctuation image (FIG. 15(b)), and the refractive index distribution image (FIG. 15(c)) are superimposed. [Figure 16] FIG. 16 is a diagram showing a schematic diagram of a cell cluster. [Figure 17] Fig. 17 is a diagram for explaining a seventh analysis example in the analysis step S3. Fig. 17(a) is a diagram showing a typical refractive index fluctuation image acquired by labeling a specific portion of each cell constituting a cell cluster with a labeling substance α. Fig. 17(b) is a diagram showing a typical refractive index distribution image. Fig. 17(c) is a diagram in which the refractive index fluctuation image (Fig. 17(a)) and the refractive index distribution image (Fig. 17(b)) are superimposed. [Figure 18]FIG. 18 is a diagram for explaining an eighth analysis example in the analysis step S3. FIG. 18(a) is a diagram showing a typical refractive index fluctuation image obtained by labeling a specific portion of each cell constituting a cell cluster with a labeling substance α. FIG. 18(b) is a diagram showing a typical refractive index fluctuation image obtained by labeling another specific portion of each cell constituting a cell cluster with another labeling substance β. FIG. 18(c) is a diagram showing a typical refractive index distribution image. FIG. 18(c) is a diagram in which the refractive index fluctuation image (FIG. 18(a)), the refractive index fluctuation image (FIG. 18(b)), and the refractive index distribution image (FIG. 18(cb)) are superimposed. [Figure 19] FIG. 19 is a table summarizing the light-absorbing labeling substances, central wavelengths, and cells used in each example. [Figure 20] FIG. 20 is a graph showing the measured absorbance spectrum and estimated refractive index spectrum of a 0.002% brilliant cresyl blue solution. [Figure 21] FIG. 21 is a graph showing the measured absorbance spectrum and estimated refractive index spectrum of a 0.003% new methylene blue solution. [Figure 22] FIG. 22 is a graph showing the measured absorbance spectrum and estimated refractive index spectrum of a 0.066% neutral red solution. [Figure 23] FIG. 23 is a graph showing the measured absorbance spectrum and estimated refractive index spectrum of a 0.004% Janus Green B solution. [Figure 24] Figure 24 shows images of Example 1A. Figure 24(a) is a phase image at a central wavelength of 433 nm. Figure 24(b) is a phase image at a central wavelength of 694 nm. [Diagram 25] Fig. 25 shows images of Example 1A. Fig. 25(a) is a refractive index distribution image at a central wavelength of 433 nm. Fig. 25(b) is a refractive index distribution image at a central wavelength of 694 nm. [Figure 26] Fig. 26 shows images of Example 1A. Fig. 26(a) is an enlarged view of the area shown by the dashed rectangle in Fig. 25(a). Fig. 26(b) is an enlarged view of the area shown by the dashed rectangle in Fig. 25(b). [Figure 27] Figure 27 shows images of Example 1A. Figure 27(a) is an intensity image at a central wavelength of 530 nm. Figure 27(b) is a refractive index fluctuation image. [Figure 28] Figure 28 shows images of Example 1B. Figure 28(a) is a phase image at a central wavelength of 433 nm. Figure 28(b) is a phase image at a central wavelength of 694 nm. [Figure 29] Figure 29 shows images of Example 1B. Figure 29(a) is an intensity image at a central wavelength of 530 nm. Figure 29(b) is a refractive index fluctuation image. [Diagram 30] Figure 30 shows images of Example 2A. Figure 30(a) is a phase image at a central wavelength of 530 nm. Figure 30(b) is a phase image at a central wavelength of 694 nm. [Diagram 31] Figure 31 shows images of Example 2A. Figure 31(a) is an intensity image at a central wavelength of 530 nm. Figure 31(b) is a refractive index fluctuation image. [Diagram 32] Figure 32 shows images of Example 2B. Figure 32(a) is a phase image with a central wavelength of 530 nm. Figure 32(b) is a phase image with a central wavelength of 694 nm. [Diagram 33] Figure 33 shows images of Example 2B. Figure 33(a) is an intensity image at a central wavelength of 530 nm. Figure 33(b) is a refractive index fluctuation image. [Diagram 34] Fig. 34 shows images of Example 3. Fig. 34(a) is a phase image at a central wavelength of 433 nm. Fig. 34(b) is a phase image at a central wavelength of 620 nm. [Diagram 35] Figure 35 shows images from Example 3. Figure 35(a) is an intensity image at a central wavelength of 530 nm. Figure 35(b) is a refractive index fluctuation image. [Diagram 36] Fig. 36 shows images of Comparative Example 3. Fig. 36(a) is a phase image at a central wavelength of 433 nm. Fig. 36(b) is a phase image at a central wavelength of 620 nm. [Figure 37]Fig. 37 shows images of Comparative Example 3. Fig. 37(a) is an intensity image at a central wavelength of 530 nm. Fig. 37(b) is a refractive index fluctuation image. [Figure 38] Fig. 38 shows images of Example 4. Fig. 38(a) is a phase image at a central wavelength of 530 nm. Fig. 38(b) is a phase image at a central wavelength of 694 nm. [Figure 39] Figure 39 shows images from Example 4. Figure 39(a) is an intensity image at a central wavelength of 530 nm. Figure 39(b) is a refractive index fluctuation image. [Diagram 40] Fig. 40 shows images of Comparative Example 4. Fig. 40(a) is a phase image at a central wavelength of 530 nm. Fig. 40(b) is a phase image at a central wavelength of 694 nm. [Diagram 41] Fig. 41 shows images of Comparative Example 4. Fig. 41(a) is an intensity image at a central wavelength of 530 nm. Fig. 41(b) is a refractive index fluctuation image. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are given the same reference numerals, and duplicated descriptions are omitted. The present invention is not limited to these examples, but is indicated by the claims, and is intended to include all modifications within the meaning and scope equivalent to the claims.

[0022] FIG. 1 is a flow chart of a cell evaluation method. The cell evaluation method includes a labeling step S1, a refractive index distribution acquisition step S2, and an analysis step S3. In the labeling step S1, a specific site of a cell to be evaluated is labeled with a labeling substance having different refractive indices at a first wavelength and a second wavelength. In the refractive index distribution acquisition step S2, the refractive index distribution of the cell whose specific site has been labeled in the labeling step S1 is acquired at the first wavelength and the second wavelength. In the analysis step S3, the refractive index distribution at the first wavelength and the second wavelength are compared to evaluate the distribution at the specific site of the cell.

[0023] A cell evaluation device capable of performing the processes of refractive index distribution acquisition step S2 and analysis step S3 in this cell evaluation method can be constructed by employing the techniques described in Non-Patent Document 1 or Non-Patent Document 2, for example.

[0024] 2 is a diagram showing the configuration of the cell evaluation device 1. The cell evaluation device 1 includes a refractive index distribution acquisition unit 10 and an analysis unit 40. The refractive index distribution acquisition unit 10 performs the process of the refractive index distribution acquisition step S2, and is based on the technology described in Non-Patent Document 1. The analysis unit 40 performs the process of the analysis step S3.

[0025] The refractive index distribution acquisition unit 10 includes a light source 11, a lens 12, a polarizer 13, a first prism 14, a condenser lens 15, an objective lens 21, a second prism 22, a lens 24, a mirror 27, an optical module 30, and a calculation unit 39. The optical module 30 includes a lens 31, a quarter-wave plate 33, a lens 34, a polarization camera 35, and mirrors 36 to 38. The configuration of the refractive index distribution acquisition unit 10, except for the optical module 30 and the calculation unit 39, can be the same as that of a differential interference microscope. That is, the refractive index distribution acquisition unit 10 can be configured by mounting the optical module 30 to a camera port of a differential interference microscope and providing the calculation unit 39.

[0026] A lens 12, a polarizer 13, a first prism 14, and a condenser lens 15 are provided, in this order, on the optical path of an irradiation optical system that condenses and irradiates the light output from the light source 11 onto the evaluation object S. Furthermore, an objective lens 21, a second prism 22, a lens 24, a lens 31, a quarter-wave plate 33, and a lens 34 are provided, in this order, on the optical path of an imaging optical system that inputs light generated on the evaluation object S in response to the irradiation of the evaluation object S with light by the irradiation optical system, and forms an image on the imaging surface of a polarization camera 35.

[0027] The light source 11 outputs spatially incoherent light. The light output from the light source 11 may be either temporally coherent or temporally incoherent. The light output from the light source 11 may be either linearly polarized or non-polarized. The light source 11 may include, for example, a halogen lamp, a light-emitting diode, or a laser diode, and may further include a diffusion plate in the downstream thereof.

[0028] The light source 11 can output light having different central wavelengths. For example, the wavelength of the output light may be made variable by selecting a laser diode to be used from a plurality of laser diodes having different output wavelengths. Alternatively, the wavelength of the output light may be made variable by using a light emitting diode that outputs broadband light and a plurality of filters having different transmission bands, and switching the filter to be used.

[0029] The lens 12 is optically connected to the light source 11. The lens 12 collimates the light output from the light source 11 and outputs the collimated light to the polarizer 13.

[0030] The polarizer 13 is optically connected to the lens 12. The polarizer 13 receives the light collimated and output by the lens 12, and outputs linearly polarized light having a polarization plane (vibration plane) according to the orientation of the optical axis of the polarizer 13 to the first prism 14.

[0031] The first prism 14 is optically connected to the polarizer 13. The first prism 14 inputs the linearly polarized light output from the polarizer 13, and outputs two linearly polarized light beams that are orthogonal to each other in different directions. The polarization planes of the two linearly polarized light beams that are orthogonal to each other and output from the first prism 14 are inclined by 45° with respect to the polarization plane of the linearly polarized light input from the polarizer 13 to the first prism 14.

[0032] The condenser lens 15 is optically connected to the first prism 14. The condenser lens 15 condenses and irradiates the two mutually orthogonal linearly polarized light beams output from the first prism 14 onto the evaluation object S. Since the two mutually orthogonal linearly polarized light beams output from the first prism 14 travel in different directions after being output from the first prism 14, the positions at which the light beams are condensed by the condenser lens 15 on the evaluation object S are slightly different.

[0033] The objective lens 21 inputs two mutually orthogonal linearly polarized light beams output from the evaluation object S in response to the irradiation of the evaluation object S with light by the irradiation optical system, collimates these light beams, and outputs them to the second prism 22.

[0034] The second prism 22 is optically connected to the objective lens 21. The second prism 22 inputs two mutually orthogonal linearly polarized light beams collimated and output by the objective lens 21, combines these light beams in the optical paths shifted by the first prism 14, and outputs the combined light to the lens 24.

[0035] The first prism 14 and the second prism 22 may be a Wollaston prism used in a typical differential interference microscope, or may be a Nomarski prism.

[0036] Lenses 24, 31, and 34 on the optical path between second prism 22 and polarized camera 35 input the light output from second prism 22 and form an image on the imaging surface of polarized camera 35. Lens 24 inputs the light output from second prism 22 and converges this light. Lens 31 inputs the light output from lens 24 and reflected by mirror 27 and collimates this light. Lens 34 inputs the light output from lens 31 and reflected by mirrors 36 and 37 and converges this light. Lenses 31 and 34 constitute a relay optical system.

[0037] A quarter-wave plate 33, which is provided on the optical path between the lens 31 and the lens 34, inputs the collimated light output from the second prism 22 and passed through the lenses 24 and 31, and outputs two circularly polarized lights having different rotation directions.

[0038] The polarization camera 35 is optically connected to the lens 34. The polarization camera 35 has an imaging surface disposed at a position where an image is formed by the imaging optical system. The polarization camera 35 inputs two lights that have been circularly polarized in different rotation directions by the quarter-wave plate 33, and obtains an interference image on the imaging surface for each of the three or more polarization components. As the polarization camera 35, for example, an image sensor (Polarsens (registered trademark)) commercialized by Sony Corporation, or an image sensor (Area Scan Polarization Sensor) or an image sensor (Line Scan Polarization Sensor) commercialized by Teledyne DALSA, Inc. may be used.

[0039] The calculation unit 39 is electrically connected to the polarization camera 35. The calculation unit 39 creates a complex amplitude image (amplitude image and phase image) of the evaluation object S based on the interference image for each of the three or more polarization components acquired by the polarization camera 35. The analysis unit 40 evaluates the evaluation object S based on the phase image acquired by the calculation unit 39.

[0040] The calculation unit 39 and the analysis unit 40 execute each calculation process of the refractive index distribution acquisition step S2 and the analysis step S3 according to a program, and are, for example, a computer. The two may be integrated. The calculation unit 39 and the analysis unit 40 include a processing unit including a CPU that performs various calculation processes, a storage unit including a hard disk drive, RAM, and ROM, etc. that stores data and programs, a display unit including a liquid crystal display that displays the processing results, etc., and an input unit including a keyboard, mouse, etc. that accepts input of various conditions when acquiring an interference image and displaying the image. The calculation unit 39 and the analysis unit 40 may be configured by a smart device such as a tablet terminal that has a touch panel or the like as an input unit. The calculation unit and the storage unit of the calculation unit 39 and the analysis unit 40 may be configured by an FPGA (field-programmable gate array) or a microcomputer.

[0041] 3 is a diagram showing the configuration of the cell evaluation apparatus 2A. The cell evaluation apparatus 2A includes a refractive index distribution acquisition section 50A and an analysis section 90. The refractive index distribution acquisition section 50A performs the process of the refractive index distribution acquisition step S2, and is based on the technology described in Non-Patent Document 2. The analysis section 90 performs the process of the analysis step S3.

[0042] The refractive index distribution acquisition unit 50A includes laser light sources 51, 52, a mirror 55, a dichroic mirror 56, a beam splitter 57, lenses 61, 62, optical fibers 63, 64, a lens 71, a mirror 72, a lens 73, a condenser lens 74, an objective lens 75, a beam splitter 81, a lens 82, a camera 83A, and a calculation unit 84.

[0043] The laser light sources 51 and 52 output laser light of different center wavelengths. The mirror 55 is optically connected to the laser light source 51. The mirror 55 reflects the laser light output from the laser light source 51 to the dichroic mirror 56. The dichroic mirror 56 is optically connected to the laser light source 52 and the mirror 55. The dichroic mirror 56 inputs the laser light output from the laser light source 52 and the laser light arriving from the mirror, combines these input laser lights, and outputs the combined laser light to the beam splitter 57.

[0044] The beam splitter 57 is optically connected to the dichroic mirror 56. The beam splitter 57 receives the light arriving from the dichroic mirror 56, branches the light into two, and outputs one of the branched light beams to a lens 61 and the other branched light beam to a lens 62.

[0045] The lens 61 is optically connected to the beam splitter 57. The lens 61 inputs one of the split lights arriving from the beam splitter 57, and collects the light on a light incident end 63a of the optical fiber 63, causing the light to enter the light incident end 63a. The optical fiber 63 guides the light incident on the light incident end 63a, and emits it as divergent light from a light emitting end 63b.

[0046] The lens 62 is optically connected to the beam splitter 57. The lens 62 inputs the other branched light arriving from the beam splitter 57, and collects the light on a light incident end 64a of the optical fiber 64, causing the light to enter the light incident end 64a. The optical fiber 64 guides the light incident on the light incident end 64a, and emits it as diverging light from a light emitting end 64b.

[0047] The lens 71 is optically connected to the light emitting end 63b of the optical fiber 63, and collimates the light outputted as divergent light from the light emitting end 63b. The mirror 72 is optically connected to the lens 71, and reflects the light arriving from the lens 71 to the lens 73. The orientation of the reflecting surface of the mirror 72 is variable. The lens 73 is optically connected to the mirror 72. The condenser lens 74 is optically connected to the lens 73. The lens 73 and the condenser lens 74 preferably constitute a 4f optical system. The lens 73 and the condenser lens 74 irradiate light onto the evaluation object S from a light irradiation direction according to the orientation of the reflecting surface of the mirror 72.

[0048] The objective lens 75 is optically connected to the condenser lens 74. The evaluation object S is disposed between the objective lens 75 and the condenser lens 74. The objective lens 75 inputs light (object light) that is output from the condenser lens 74 and passes through the evaluation object S, and outputs the light to the beam splitter 81.

[0049] The beam splitter 81 is optically connected to the objective lens 75, and is also optically connected to the light emitting end 64b of the optical fiber 64. The beam splitter 81 inputs light (object light) output from the objective lens 75 and reaching it, and inputs light (reference light) output from the light emitting end 64b and reaching it, and outputs both light beams to the lens 82. The lens 82 is optically connected to the beam splitter 81, and collimates each of the object light and reference light arriving from the beam splitter 81, and outputs them to the camera 83A.

[0050] Camera 83A is a color camera and is optically connected to lens 82. Camera 83A captures an interference fringe image (interference intensity image) caused by interference between object light and reference light arriving from lens 82. The incident direction of the reference light is inclined with respect to the incident direction of the object light on the imaging surface of camera 83A. The position where the object light and the reference light are combined by beam splitter 81 may be after the imaging lens, but considering the influence of aberration, it is preferably between objective lens 75 and lens 82 as shown in the figure.

[0051] The calculation unit 84 is electrically connected to the camera 83A. The calculation unit 84 analyzes the interference image (color image) acquired by the camera 83A to create an interference image at the output wavelength of each of the laser light sources 51 and 52. Furthermore, the calculation unit 84 calculates a three-dimensional refractive index distribution of the evaluation object S at each wavelength by processing the interference image obtained by setting various light incidence directions to the evaluation object S by changing the orientation of the reflecting surface of the lens 71. The analysis unit 90 evaluates the evaluation object S based on the refractive index distribution image acquired by the calculation unit 84. The calculation unit 84 and the analysis unit 90 also execute each calculation process of the refractive index distribution acquisition step S2 and the analysis step S3, and may be, for example, a computer, or may be integrated into one.

[0052] Fig. 4 is a diagram showing the configuration of the cell evaluation device 2B. The cell evaluation device 2B includes a refractive index distribution acquisition unit 50B and an analysis unit 90. Compared to the refractive index distribution acquisition unit 50A (Fig. 3), the refractive index distribution acquisition unit 50B (Fig. 4) differs in that it further includes shutters 53, 54, and in that it includes a monochrome camera 83B instead of the color camera 83A.

[0053] The shutter 53 is provided on the optical path between the laser light source 51 and the mirror 55, and allows or prohibits the propagation of the laser light from the laser light source 51 to the mirror 55. The shutter 54 is provided on the optical path between the laser light source 52 and the dichroic mirror 56, and allows or prohibits the propagation of the laser light from the laser light source 52 to the dichroic mirror 56. The shutters 53 and 54 do not allow the propagation of the laser light at the same time. The camera 83B captures an interference image at the output wavelength of the laser light source 51 and an interference image at the output wavelength of the laser light source 52 separately.

[0054] The processing of the refractive index distribution acquisition step S2 of the cell evaluation method of this embodiment can be performed using the refractive index distribution acquisition units 10, 50A, and 50B of these cell evaluation devices, but is not limited to these configurations and may be performed using other configurations that can acquire a phase image or a refractive index distribution image of the evaluation object S.

[0055] Next, the labeling step S1, the refractive index distribution acquisition step S2, and the analysis step S3 of the cell evaluation method will each be described in more detail.

[0056] It is generally known that the longer the wavelength of light, the lower the refractive index of the substance. For example, Non-Patent Document 3 shows that the refractive index of proteins and DNA at a wavelength of 400 nm is lower than that at a wavelength of 310 nm. In addition, in a non-labeled site, the longer the wavelength of light, the lower the refractive index of the entire non-labeled site. Therefore, it is not easy to observe a specific site in a cell unless labeling (labeling that shows a larger change in refractive index than the change in refractive index of the non-labeled site) is performed.

[0057] In the labeling step S1, a labeling substance having different refractive indices at a first wavelength and a second wavelength, which are wavelengths of light used in the subsequent refractive index distribution acquisition step S2, is used as a labeling substance for labeling a specific site of a cell to be evaluated. The specific site may be a specific molecule or a specific structure, such as a vesicular structure, a nucleolus, a mitochondria, a specific protein, or the entire cell.

[0058] The labeling substance that labels the specific site in the labeling step S1 may be a labeling substance whose refractive index at the longer wavelength of the first wavelength and the second wavelength is higher than that at the shorter wavelength, or conversely, may be a labeling substance whose refractive index at the longer wavelength is lower than that at the shorter wavelength. In addition, the labeling substance that labels the specific site in the labeling step S1 is preferably a light-absorbing labeling substance having an absorption peak at a specific wavelength λ0, and in this case, in the refractive index distribution acquisition step S2, it is preferable to acquire the refractive index distribution of the cell at each of the first wavelength λ1 and the second wavelength λ2 that are on the short wavelength side or the long wavelength side of the specific wavelength λ0. In addition, in the refractive index distribution acquisition step S2, it is also preferable to acquire the refractive index distribution of the cell at each of the first wavelength λ1 and the second wavelength λ2, with one of the first wavelength λ1 and the second wavelength λ2 being on the short wavelength side of the specific wavelength λ0 and the other being on the long wavelength side of the specific wavelength λ0.

[0059] FIG. 5 is a diagram showing the absorption spectrum and the refractive index spectrum of a light-absorbing labeling substance. FIG. 5(a) shows the absorption spectrum. FIG. 5(b) shows the refractive index spectrum. As shown in this figure, the light-absorbing labeling substance has an absorption peak at a specific wavelength λ0, and the refractive index is minimal on the short wavelength side of the specific wavelength λ0, and is maximal on the long wavelength side of the specific wavelength λ0. In general, the relationship between the absorption spectrum and the refractive index spectrum is expressed by the Kramers-Kronig equation (see Non-Patent Document 4).

[0060] Therefore, when using a light-absorbing labeling substance having an absorption peak at a specific wavelength λ0, as shown in FIG. 5(b), a first wavelength λ1 can be set near the wavelength at which the refractive index is minimum on the short wavelength side of the specific wavelength λ0, and a second wavelength λ2 can be set near the wavelength at which the refractive index is maximum on the long wavelength side of the specific wavelength λ0.

[0061] As shown in the refractive index spectrum of the light-absorbing labeling substance in Fig. 6(a), there is a wavelength range in which the refractive index is highly dependent on the wavelength from the vicinity of the wavelength where the refractive index is minimal to the short wavelength side, so the first wavelength λ1 and the second wavelength λ2 may be set in this wavelength range. Also, as shown in the refractive index spectrum of the light-absorbing labeling substance in Fig. 6(b), there is a wavelength range in which the refractive index is highly dependent on the wavelength from the vicinity of the wavelength where the refractive index is maximized to the long wavelength side, so the first wavelength λ1 and the second wavelength λ2 may be set in this wavelength range.

[0062] By setting the first wavelength λ1 and the second wavelength λ2 in this manner, in the refractive index distribution acquisition step S2, the refractive index distribution of cells whose specific sites have been labeled with a light-absorbing labeling substance is acquired at each of the first wavelength λ1 and the second wavelength λ2.

[0063] Examples of light-absorbing markers capable of labeling specific sites of living cells include brilliant cresyl blue, new methylene blue, neutral red, Janus green B, Nile blue, Bismarck brown, Nile red, oil red, etc. Examples of light-absorbing markers capable of labeling specific sites of dead cells include trypan blue, etc. Examples of light-absorbing markers capable of labeling specific sites of cells include hematoxylin, eosin, picric acid, orange G, azocarmine G, fuchsin, aniline blue, azure blue, azure II, Sudan III, Oizudan black, resorcin, Kern Echtrot, etc., although they require cell fixation treatment.

[0064] Natural chromoproteins can be used as examples of light-absorbing labeling substances capable of labeling specific sites of cells. Examples of such natural chromoproteins include blue protein (cjBlue) derived from sea anemone (Cnidopus japonicus), blue protein (rpulFKz1) derived from jellyfish (Rhizostoma pulmo), purple protein (gfasCP) derived from coral (Galaxea fascicularis), pink protein (asFP595) derived from sea anemone (Anemonia sulcata), blue protein (Rtms5) derived from coral (Montipora efflorescens), and blue protein (aeCP597) derived from sea anemone (Actinia equina). Artificial chromoproteins obtained by modifying natural chromoproteins may also be used.

[0065] The labeling substance for labeling a specific site of a cell may be an intracellular expression construct in which a DNA sequence encoding a chromoprotein V (light-absorbing polypeptide) is connected to the N-terminus or C-terminus of a DNA sequence encoding an arbitrary protein W. The intracellular expression construct may be designed so as to be expressed under the control of a CMV (Cytomegalovirus) promoter or the like. In the labeling step S1, the intracellular expression construct is introduced into a cell by lipofection, electroporation, or the like to label a specific site of the cell. This introduction results in the expression of an artificial fusion protein.

[0066] A plurality of kinds of labeling substances may be used simultaneously. That is, in the labeling step S1, other specific parts of the cell may be labeled with other labeling substances having different refractive indices at the third wavelength and the fourth wavelength. In this case, in the refractive index distribution acquisition step S2, the refractive index distribution of the cell labeled in the labeling step S1 is acquired at the first wavelength and the second wavelength, and also at the third wavelength and the fourth wavelength. In the analysis step S3, the refractive index distribution at the third wavelength and the fourth wavelength are compared to evaluate the distribution of other specific parts of the cell. The first wavelength or the second wavelength may be equal to the third wavelength or the fourth wavelength. When two kinds of labeling substances are used simultaneously, the number of wavelengths of light used in the refractive index distribution acquisition step S2 may be four, three, or two.

[0067] The first wavelength and the second wavelength can be determined, for example, as follows. When the absorption spectrum of the light-absorbing labeling substance is known, the refractive index spectrum is estimated from the absorption spectrum based on the Kramers-Kronig relation, and the first wavelength and the second wavelength are set near the minimum and maximum values ​​in this refractive index profile. When the absorption spectrum of the light-absorbing labeling substance is unknown, the absorption spectrum is measured, and then the refractive index spectrum is estimated from the absorption spectrum based on the Kramers-Kronig relation, and the first wavelength and the second wavelength are set near the minimum and maximum values ​​in this refractive index profile.

[0068] After the first wavelength and the second wavelength are set, the refractive index distribution acquisition step S2 may be performed for the set first wavelength and second wavelength. Alternatively, after the refractive index distributions for several wavelengths are acquired in the refractive index distribution acquisition step S2, the first wavelength and the second wavelength for which a difference in the refractive index distribution is recognized may be set from among those wavelengths.

[0069] When the first wavelength and the second wavelength can be freely set, the first wavelength and the second wavelength may be set to the minimum and maximum values ​​in the refractive index profile. When the first wavelength and the second wavelength are selected by selecting from among several fixed wavelengths, the two wavelengths having the largest refractive index difference among the fixed wavelengths may be set as the first wavelength and the second wavelength. In addition, a refractive index distribution may always be obtained for one of the first wavelength and the second wavelength, and a refractive index distribution may be obtained for the other wavelength at a wavelength set based on the color of the light-absorbing marker substance.

[0070] In the analysis step S3, the distribution of the specific site in the cell is evaluated by comparing the refractive index distribution at the first wavelength λ1 and the second wavelength λ2. FIG. 7 is a diagram for explaining the refractive index distribution of a cell in which the cell nucleus as the specific site is labeled with a labeling substance. FIG. 7(a) shows the refractive index distribution of the cell at the first wavelength λ1. FIG. 7(b) shows the refractive index distribution of the cell at the second wavelength λ2. Compared with the refractive index distribution of the cell at the first wavelength λ1 (FIG. 7(a)), the refractive index of the region of the cell nucleus labeled with the labeling substance (hatched region in FIG. 7(b)) can be made higher in the refractive index distribution of the cell at the second wavelength λ2 (FIG. 7(b)). By comparing the refractive index distribution of the cell at the first wavelength λ1 (FIG. 7(a)) with the refractive index distribution of the cell at the second wavelength λ2 (FIG. 7(b)), the position, shape, size, etc. of the cell nucleus in the cell can be evaluated. Thus, in the analysis step S3, the distribution of the specific site in the cell can be evaluated by comparing the refractive index distribution at the first wavelength λ1 and the refractive index distribution at the second wavelength λ2.

[0071] In the analysis step S3, the distribution of a specific part of the cell may be evaluated based on the difference in the refractive index distribution of the cell at the first wavelength λ1 and the second wavelength λ2. The distribution of a specific part of the cell may also be evaluated based on the ratio of these two refractive index distributions. When a refractive index distribution image is created after a phase image of the cell is acquired, the difference or ratio of the two refractive index distributions may be obtained after correction based on the difference in wavelength, or the ratio of the two refractive index distributions may be obtained without correction based on the difference in wavelength. It is also preferable to perform an analysis of a specific part of the cell by superimposing the result of the comparison of the refractive index distribution at the first wavelength λ1 and the second wavelength λ2 on the intensity image or fluorescence image of the cell. Hereinafter, an image showing the difference or ratio of the refractive index distribution images of the cell at the first wavelength λ1 and the second wavelength λ2 is referred to as a "refractive index fluctuation image".

[0072] In the analysis step S3, various analyses can be performed on a specific portion of the cell using the refractive index distribution image (and also using other images).

[0073] FIG. 8 is a diagram for explaining a first analysis example in the analysis step S3. FIG. 8(a) is a diagram that shows a typical refractive index distribution image. FIG. 8(b) is a diagram that shows a typical refractive index fluctuation image. FIG. 8(c) is a diagram in which the refractive index distribution image (FIG. 8(a)) and the refractive index fluctuation image (FIG. 8(b)) are superimposed. These diagrams show examples in which a specific vesicular structure among intracellular vesicular structures or cellular structures is labeled with a labeling substance.

[0074] The refractive index distribution image ( FIG. 8(a) ) does not allow for distinction between a specific labeled vesicle structure in a cell and an unlabeled vesicle structure or other cellular structure. On the other hand, the refractive index fluctuation image ( FIG. 8(b) ) allows for distinction between a specific labeled vesicle structure in a cell and an unlabeled vesicle structure or other cellular structure. Therefore, by comparing the refractive index distribution image ( FIG. 8(a) ) with the refractive index fluctuation image ( FIG. 8(b) ), or by displaying the refractive index distribution image ( FIG. 8(a) ) and the refractive index fluctuation image ( FIG. 8(b) ) superimposed on each other, it is possible to determine the distribution of vesicle structures and other cellular structures in a cell, and further, the distribution of a specific labeled vesicle structure among them.

[0075] In the first analysis example, the average refractive index of the labeled specific vesicle structure can be analyzed, and the specific vesicle structure can be evaluated based on this value. For example, the substance contained in the vesicle can be estimated, and the vacuole or secretory vesicle can be identified. If the localization of the labeled substance is unknown and the intracellular structure can be roughly identified from the refractive index distribution image, it is possible to evaluate where the labeled substance is localized, and for example, the intracellular localization of the specific vesicle structure (tendency to be localized around the cell nucleus, etc.) can be known. If the localization of the labeled substance is known and it is difficult to identify the intracellular structure from the refractive index distribution image, it is possible to perform segmentation of the refractive index distribution image, and it is possible to evaluate, for example, what refractive index characteristics can be identified as a specific vesicle structure.

[0076] The wavelength of light used to obtain the refractive index distribution image may be equal to or different from any of the wavelengths of light used to obtain the refractive index variation image. For example, when it is desired to compare the refractive index between a labeled site and a non-labeled site, the wavelength of light used to obtain the refractive index distribution image may be one that is not in a wavelength range in which the refractive index spectrum of the labeling substance changes significantly.

[0077] FIG. 9 is a diagram for explaining a second analysis example in the analysis step S3. FIG. 9(a) is a diagram showing a refractive index fluctuation image obtained by labeling a cell nucleus with a labeling substance. FIG. 9(b) is a diagram showing a refractive index fluctuation image obtained by labeling an entire cell with a labeling substance. FIG. 9(c) is a diagram showing a refractive index fluctuation image (FIG. 9(a)) superimposed on a refractive index fluctuation image (FIG. 9(b)). In the second analysis example, the area of ​​the cell nucleus is calculated based on the refractive index fluctuation image (FIG. 9(a)), and the area of ​​the entire cell is calculated based on the refractive index fluctuation image (FIG. 9(b)), and the ratio of the two areas is calculated, thereby analyzing the ratio of the cell nucleus / cytoplasm. In addition, it is also possible to identify the area other than the cell nucleus as the cytoplasm in the entire cell, and to identify the position of the cell nucleus in the entire cell. It is also possible to estimate the number of cells contained in a cell mass by labeling cells only with a labeling substance for the cell nucleus and counting the number of cell nuclei.

[0078] 10 to 12 are diagrams for explaining a third analysis example in the analysis step S3. FIG. 10 is a diagram showing a plurality of refractive index fluctuation images acquired at a fixed time interval. FIG. 11 is a diagram showing a plurality of refractive index fluctuation images (FIG. 10) acquired at a fixed time interval superimposed on a refractive index distribution image. In the third analysis example, it is possible to analyze the state in which the position of a specific site labeled with a labeling substance changes over time in a cell. When acquiring a fluorescent image, it is necessary to irradiate high-power excitation light, which causes problems of phototoxicity and photobleaching. However, in this embodiment, it is not necessary to irradiate high-power light, so there are fewer problems of phototoxicity and photobleaching, and time-lapse observation of cells over a long period of time is possible.

[0079] FIG. 13 is a diagram for explaining a fourth analysis example in the analysis step S3. FIG. 13(a) is a diagram showing a fluorescent image or intensity image of the whole cell. FIG. 13(b) is a diagram showing a refractive index fluctuation image obtained by labeling the cell nucleus with a labeling substance. FIG. 13(c) is a diagram showing a fluorescent image or intensity image of the whole cell (FIG. 13(a)) superimposed on a refractive index fluctuation image (FIG. 13(b)). In the fourth analysis example, the area of ​​the whole cell is calculated based on the fluorescent image or intensity image (FIG. 13(a)) of the whole cell, and the area of ​​the cell nucleus is calculated based on the refractive index fluctuation image (FIG. 13(b)), and the ratio of the two areas is calculated, thereby analyzing the ratio of the cell nucleus / cytoplasm. In addition, the area other than the cell nucleus in the whole cell can be identified as the cytoplasm, and the position of the cell nucleus in the whole cell can be identified.

[0080] FIG. 14 is a diagram for explaining a fifth analysis example in the analysis step S3. FIG. 14(a) is a diagram showing a fluorescence image or intensity image of a cell nucleus. FIG. 14(b) is a diagram showing a refractive index fluctuation image obtained by labeling with a labeling substance whose labeling target is unknown. FIG. 14(c) is a diagram showing a fluorescence image or intensity image (FIG. 14(a)) of a cell nucleus superimposed on a refractive index fluctuation image (FIG. 14(b)). In the fifth analysis example, if the fluorescence image or intensity image (FIG. 14(a)) of the cell nucleus and the refractive index fluctuation image (FIG. 14(b)) match each other, it can be evaluated that the labeling substance whose labeling target is unknown can label the cell nucleus. In addition, by comparing the fluorescence image or intensity image (FIG. 14(a)) of the cell nucleus with the refractive index fluctuation image (FIG. 14(b)), correction can be performed when creating a refractive index fluctuation image based on a refractive index distribution image.

[0081] The wavelength of light used when acquiring a fluorescence image or an intensity image may be equal to or different from any of the wavelengths of light used when acquiring a refractive index fluctuation image.

[0082] When two types of labeling substances are used, in the analysis step S3, various further analyses can be performed on two types of specific sites of the cell using two refractive index distribution images (and other images as well). FIG. 15 is a diagram for explaining a sixth analysis example in the analysis step S3. FIG. 15(a) is a diagram showing a refractive index fluctuation image obtained by labeling a specific vesicle with a labeling substance α. FIG. 15(b) is a diagram showing a refractive index fluctuation image obtained by labeling another specific vesicle with a labeling substance β. FIG. 15(c) is a diagram showing a refractive index distribution image. FIG. 15(d) is a diagram in which the refractive index fluctuation image (FIG. 15(a)), the refractive index fluctuation image (FIG. 15(b)), and the refractive index distribution image (FIG. 15(c)) are superimposed. In the sixth analysis example, it is possible to know that the region in which a particular vesicle labeled with labeling substance α exists is different from the region in which another particular vesicle labeled with labeling substance β exists; for example, it is possible to know that the former vesicles exist in a region close to the cell nucleus and the latter vesicles exist in a region far from the cell nucleus.

[0083] The distribution of specific sites can be evaluated not only for a single cell but also for a cell cluster in which multiple cells are assembled in a three-dimensional shape. FIG. 16 is a diagram showing a cell cluster as a model. A phase image or a refractive index distribution image can be obtained in any cross section of the cell cluster (cross section shown by a broken line in the diagram), and a refractive index fluctuation image in the cross section can be obtained from the image. Not only a cross section of the cell cluster but also a tomographic or three-dimensional refractive index distribution image can be obtained. FIG. 17 is a diagram explaining a seventh analysis example in the analysis step S3. FIG. 17(a) is a diagram showing a refractive index fluctuation image obtained by labeling a specific site of each cell constituting the cell cluster with a labeling substance α. FIG. 17(b) is a diagram showing a refractive index distribution image as a model. FIG. 17(c) is a diagram showing the refractive index fluctuation image (FIG. 17(a)) and the refractive index distribution image (FIG. 17(b)) superimposed on each other. In the seventh analysis example, it is possible to know the region in the cell cluster where a cell having a specific site labeled with the labeling substance α exists, for example, to know that the cell exists in the peripheral region.

[0084] Using two types of labeling substances, the distribution of two types of specific sites in the cell cluster can also be evaluated. FIG. 18 is a diagram for explaining an eighth analysis example in the analysis step S3. FIG. 18(a) is a diagram that shows a typical refractive index fluctuation image obtained by labeling a specific site of each cell constituting the cell cluster with labeling substance α. FIG. 18(b) is a diagram that shows a typical refractive index fluctuation image obtained by labeling another specific site of each cell constituting the cell cluster with another labeling substance β. FIG. 18(c) is a diagram that shows a typical refractive index distribution image. FIG. 18(c) is a diagram in which the refractive index fluctuation image (FIG. 18(a)), the refractive index fluctuation image (FIG. 18(b)), and the refractive index distribution image (FIG. 18(cb)) are superimposed. In the eighth analysis example, it is possible to know the region in the cell cluster where cells having specific sites labeled with the labeling substances α and β are present, and for example, it is possible to know that cells having specific sites labeled with the labeling substance α are present in the peripheral region, and that cells having specific sites labeled with the labeling substance β are present in the central region.

[0085] Next, an example will be described. An optical module 30 was attached to the camera port of a differential interference microscope (ECLIPSETi-2) manufactured by Nikon Corporation to configure a cell evaluation device 1 (FIG. 2), which was used to obtain a phase image of a cell whose specific site was labeled with a labeling substance, and further to obtain a refractive index distribution image and a refractive index fluctuation image. Depending on the type of labeling substance, a phase image was obtained using light of any two wavelengths of 433±12 nm, 530±21.5 nm, 620±26 nm, and 694±22 nm. By combining a light-emitting diode with a bandpass filter manufactured by Semrock, light of each wavelength was selected and output.

[0086] The labeling substances used were brilliant cresyl blue, new methylene blue, Janus green B (the above three labeling substances were purchased from Muto Chemical Co., Ltd.), and neutral red (purchased from Sigma-Aldrich Co., Ltd.) The cells used for evaluation were the human lung cancer-derived A549 cell line and the human liver cancer-derived HepG2 cell line.

[0087] 19 is a table summarizing the labeling substance, central wavelength, and cells used in each Example. The table also shows the central wavelength and cells for Comparative Examples 3 and 4, in which no labeling substance was used.

[0088] Brilliant cresyl blue used in Examples 1A and 1B is a vital dye (basic dye) that is known to be taken up into cells by endocytosis (pinocytosis) and is also known as a dye for staining nucleoli. New methylene blue used in Examples 2A and 2B is known as a reagent with properties similar to those of brilliant cresyl blue.

[0089] Neutral red used in Example 3 is one of the vital dyes and is used in the cellular uptake assay. The uptake of neutral red is related to the maintenance of an ATP-dependent pH gradient. Janus Green B used in Example 4 is one of the vital dyes and is known to stain mitochondria blue, as well as to stain various other proteins. Janus Green B is also known to stain the cell nuclei and cytoplasm in cultured cells.

[0090] The absorption spectrum of the labeled substance was measured using a spectrophotometer U-4100 manufactured by Hitachi, Ltd. The refractive index spectrum of the labeled substance was estimated from the absorption spectrum based on the Kramers-Kronig relation. Figure 20 is a graph showing the measured absorption spectrum and estimated refractive index spectrum of a 0.002% brilliant cresyl blue solution. Figure 21 is a graph showing the measured absorption spectrum and estimated refractive index spectrum of a 0.003% new methylene blue solution. Figure 22 is a graph showing the measured absorption spectrum and estimated refractive index spectrum of a 0.066% neutral red solution. Figure 23 is a graph showing the measured absorption spectrum and estimated refractive index spectrum of a 0.004% Janus Green B solution. Based on these refractive index profiles, the first and second wavelengths of light used in measuring the refractive index distribution were set as shown in Figure 19.

[0091] 24 to 27 are images of Example 1 A. In Example 1 A, brilliant cresyl blue was used as the labeling substance, light with central wavelengths of 433 nm and 694 nm was used when measuring the refractive index distribution, and the A549 cell line was used as the cells to be evaluated.

[0092] FIG. 24(a) is a phase image at a central wavelength of 433 nm. FIG. 24(b) is a phase image at a central wavelength of 694 nm. The position indicated by the arrow (a) in the figure is a site labeled with a labeling substance, where the phase is 1.51 and the refractive index is 1.354 at a central wavelength of 433 nm, and the phase is 2.05 and the refractive index is 1.378 at a central wavelength of 694 nm, and the refractive index is high at long wavelengths. The refractive index values ​​were estimated assuming a cell thickness of 5 μm and a refractive index of the background medium of 1.333. The position indicated by the arrow (b) in the figure is a site not labeled with a labeling substance, where the phase is 1.52 and the refractive index is 1.354 at a central wavelength of 433 nm, and the phase is 0.72 and the refractive index is 1.349 at a central wavelength of 694 nm, and the refractive index is low at long wavelengths.

[0093] FIG. 25(a) is a refractive index distribution image with a central wavelength of 433 nm. FIG. 25(b) is a refractive index distribution image with a central wavelength of 694 nm. These refractive index distribution images were created by correcting each pixel value of the phase image based on the wavelength. The refractive index distribution image of FIG. 25(a) is obtained by multiplying the phase image of FIG. 24(a) by a coefficient of 4.33. The refractive index distribution image of FIG. 25(b) is obtained by multiplying the phase image of FIG. 24(b) by a coefficient of 6.94. The value of the coefficient by which each phase image is multiplied may be a constant multiple of the wavelength of the light used when the phase image was acquired.

[0094] FIG. 26(a) is an enlarged view of the range indicated by the dashed rectangle in FIG. 25(a). FIG. 26(b) is an enlarged view of the range indicated by the dashed rectangle in FIG. 25(b). FIG. 27(a) is an intensity image at a central wavelength of 530 nm. FIG. 27(b) is a refractive index fluctuation image created from FIG. 26(a) and FIG. 26(b). The refractive index fluctuation image (FIG. 27(b)) is obtained by subtracting the refractive index distribution image at a central wavelength of 433 nm (FIG. 26(a)) from the refractive index distribution image at a central wavelength of 694 nm (FIG. 26(b)) and extracting an area with a phase larger than the background (phase 0) (area with a positive phase value). The refractive index fluctuation image may be created based on the ratio of the refractive index distribution images at two wavelengths, or may be created based on a comparison between the difference or ratio of the refractive index distribution images at two wavelengths and a threshold value. Comparing the intensity image (FIG. 27(a)) with the refractive index fluctuation image (FIG. 27(b)), it can be seen that the area labeled with the labeling substance is detected as a change in refractive index.

[0095] 28 and 29 are images of Example 1B. In Example 1B, brilliant cresyl blue was used as the labeling substance, light with central wavelengths of 433 nm and 694 nm was used when measuring the refractive index distribution, and the HepG2 cell line was used as the cells to be evaluated.

[0096] Fig. 28(a) is a phase image at a central wavelength of 433 nm. Fig. 28(b) is a phase image at a central wavelength of 694 nm. The position indicated by the arrow in the figure is the part labeled with a labeling substance, where the phase is 0.14 and the refractive index is 1.335 at a central wavelength of 433 nm, and the phase is 3.59 and the refractive index is 1.412 at a central wavelength of 694 nm, with the refractive index increasing at longer wavelengths.

[0097] Fig. 29(a) is an intensity image at a central wavelength of 530 nm. Fig. 29(b) is a refractive index fluctuation image. The refractive index fluctuation image (Fig. 29(b)) was created in the same manner as in Example 1A. Comparing the intensity image (Fig. 29(a)) and the refractive index fluctuation image (Fig. 29(b)), it can be seen that in Example 1B, as in Example 1A, the site labeled with the labeling substance was detected as a change in refractive index.

[0098] 30 and 31 are images of Example 2A. In Example 2A, new methylene blue was used as the labeling substance, light with central wavelengths of 530 nm and 694 nm was used when measuring the refractive index distribution, and A549 cell line was used as the cell to be evaluated. FIG. 30(a) is a phase image at a central wavelength of 530 nm. FIG. 30(b) is a phase image at a central wavelength of 694 nm. FIG. 31(a) is an intensity image at a central wavelength of 530 nm. FIG. 31(b) is a refractive index fluctuation image. The refractive index fluctuation image (FIG. 31(b)) was created in the same manner as in Example 1A. Comparing the intensity image (FIG. 31(a)) and the refractive index fluctuation image (FIG. 31(b)), it can be seen that the site labeled with the labeling substance is also detected as a change in refractive index in Example 2A.

[0099] 32 and 33 are images of Example 2B. In Example 2B, new methylene blue was used as the labeling substance, light with central wavelengths of 530 nm and 694 nm was used when measuring the refractive index distribution, and HepG2 cell line was used as the cell to be evaluated. FIG. 32(a) is a phase image at a central wavelength of 530 nm. FIG. 32(b) is a phase image at a central wavelength of 694 nm. FIG. 33(a) is an intensity image at a central wavelength of 530 nm. FIG. 33(b) is a refractive index fluctuation image. The refractive index fluctuation image (FIG. 33(b)) was created in the same manner as in Example 1A. Comparing the intensity image (FIG. 33(a)) and the refractive index fluctuation image (FIG. 33(b)), it can be seen that the site labeled with the labeling substance is also detected as a change in refractive index in Example 2B.

[0100] 34 and 35 are images of Example 3. In Example 3, neutral red was used as the labeling substance, light with central wavelengths of 433 nm and 620 nm was used when measuring the refractive index distribution, and HepG2 cell line was used as the cell to be evaluated. FIG. 34(a) is a phase image at a central wavelength of 433 nm. FIG. 34(b) is a phase image at a central wavelength of 620 nm. FIG. 35(a) is an intensity image at a central wavelength of 530 nm. FIG. 35(b) is a refractive index fluctuation image. The refractive index fluctuation image (FIG. 35(b)) was created in the same manner as in Example 1A. Comparing the intensity image (FIG. 35(a)) and the refractive index fluctuation image (FIG. 35(b)), it can be seen that the site labeled with the labeling substance is also detected as a change in refractive index in Example 3.

[0101] Figures 36 and 37 are images of Comparative Example 3. Comparative Example 3 differs from Example 3 in that no labeling substance was used. Figure 36(a) is a phase image at a central wavelength of 433 nm. Figure 36(b) is a phase image at a central wavelength of 620 nm. Figure 37(a) is an intensity image at a central wavelength of 530 nm. Figure 37(b) is a refractive index fluctuation image. The refractive index fluctuation image (Figure 37(b)) was created in the same manner as Example 1A. In Comparative Example 3, in which no labeling substance was used, it was almost impossible to detect specific sites within the cells.

[0102] 38 and 39 are images of Example 4. In Example 4, Janus Green B was used as the labeling substance, light with central wavelengths of 530 nm and 694 nm was used when measuring the refractive index distribution, and the A549 cell line was used as the cell to be evaluated. FIG. 38(a) is a phase image at a central wavelength of 530 nm. FIG. 38(b) is a phase image at a central wavelength of 694 nm. FIG. 39(a) is an intensity image at a central wavelength of 530 nm. FIG. 39(b) is a refractive index fluctuation image. The refractive index fluctuation image (FIG. 39(b)) was created in the same manner as in Example 1A. Comparing the intensity image (FIG. 39(a)) and the refractive index fluctuation image (FIG. 39(b)), it can be seen that the site labeled with the labeling substance is also detected as a change in refractive index in Example 4.

[0103] Figures 40 and 41 are images of Comparative Example 4. Comparative Example 4 differs from Example 4 in that no labeling substance was used. Figure 40(a) is a phase image at a central wavelength of 530 nm. Figure 40(b) is a phase image at a central wavelength of 694 nm. Figure 41(a) is an intensity image at a central wavelength of 530 nm. Figure 41(b) is a refractive index fluctuation image. The refractive index fluctuation image (Figure 41(b)) was created in the same manner as Example 1A. In Comparative Example 4, in which no labeling substance was used, it was almost impossible to detect specific sites within the cells.

[0104] As described above, in this embodiment, a specific portion of a cell is labeled with a labeling substance having a different refractive index at a first wavelength and a second wavelength, the refractive index distribution of the cell is obtained at the first wavelength and the second wavelength, and the refractive index distribution at the first wavelength and the second wavelength are compared to evaluate the distribution of the specific portion of the cell. According to this embodiment, even an unknown cell can be easily evaluated.

[0105] Intensity images (such as Figure 27(a)) also show the distribution of specific sites labeled with a labeling substance. However, intensity images show the effects of light scattering and the like, in addition to light absorption by the labeling substance. Intensity images may be observed to have a reduced light intensity due to light refraction and scattering at the boundaries of structures with different refractive indices, in addition to the effects of absorption (this may also be observed in unlabeled cells). The effects of light scattering and the like can cause noise. This is a problem with intensity images at one wavelength, but it can also be a problem when comparing intensity images at multiple wavelengths. On the other hand, refractive index distribution images make it easy to avoid this problem because the refractive index can be quantified. Therefore, refractive index fluctuation images can have less noise caused by light scattering and the like.

[0106] When using a light-absorbing labeling substance, light is less likely to penetrate in the wavelength region where there is light absorption (region near the specific wavelength λ0 in Figure 5(a)), which may affect the acquisition of refractive index distribution images. This effect is expected to be greater when observing cell clusters using light from multiple directions with ODT, making deep observation difficult. However, by setting the first and second wavelengths near the wavelength where the refractive index is minimal on the short wavelength side of the specific wavelength λ0 in the refractive index spectrum of the light-absorbing labeling substance, or near the wavelength where the refractive index is maximal on the long wavelength side of the specific wavelength λ0 (Figures 5(b) and 6), it is possible to acquire phase images or refractive index distribution images using light with wavelengths where light absorption is small.

[0107] In addition, when a light-absorbing labeling substance is used, the first and second wavelengths can be set to be relatively close to each other (Fig. 5(b), Fig. 6). By comparing the refractive index distribution images at the first and second wavelengths that are relatively close to each other, it becomes easier to extract the refractive index fluctuation image, and data with a high signal-to-noise ratio can be obtained.

[0108] The following can be said when comparing a case where a wavelength intensity image is used to identify a target site, assuming that a refractive index distribution image is obtained using light of multiple wavelengths (hereinafter referred to as "Case 1"), with a case where a refractive index distribution image of multiple wavelengths is used to identify a target site (hereinafter referred to as "Case 2"). In Case 2, when a low refractive index peak (first wavelength) and a high refractive index peak (second wavelength) are relatively close to each other, by comparing the refractive index distribution images at the first and second wavelengths, it becomes easy to extract the refractive index fluctuation region, and data with a high S / N ratio can be easily obtained. This is because Case 2 can capture a large change in the refractive index distribution of a labeled site when the change in the refractive index distribution of a non-labeled site is small, and is an advantage over Case 1. On the other hand, even if the low refractive index peak (first wavelength) and the high refractive index peak (second wavelength) are not relatively close to each other due to the relationship between the absorption spectrum and the refractive index spectrum of the labeling substance, it is assumed that the low refractive index peak is often clear in the refractive index spectrum in Case 2, so by comparing the refractive index distribution images at the first and second wavelengths, it becomes easy to extract the refractive index fluctuation region and to obtain data with a high S / N ratio. The advantage of Case 2 over Case 1 is due to the fact that, while it is necessary to focus on the high absorbance peak of the optical absorption spectrum in Case 1, attention is paid to the low refractive index peak as well as the high refractive index peak of the refractive index spectrum in Case 2.

[0109] When acquiring a fluorescent image, it is necessary to irradiate high-power excitation light, which can cause problems of phototoxicity and photobleaching. In contrast, in this embodiment, it is not necessary to irradiate high-power light, so there are fewer problems of phototoxicity and photobleaching, and time-lapse observation of cells over a long period of time is possible. In addition, since there is less of a phototoxicity problem, it is possible to observe the cells while maintaining their original functions, and since there is less of a photobleaching problem, quantitative observation is possible.

[0110] Moreover, a single device can acquire refractive index distribution images and refractive index fluctuation images, as well as intensity images (Figure 27(a) and the like). Biologically meaningful observations become possible by comparing the refractive index fluctuation image and intensity image of cells. [Explanation of symbols]

[0111] Reference Signs List 1, 2A, 2B... cell evaluation device, 10... refractive index distribution acquisition unit, 11... light source, 12... lens, 13... polarizer, 14... first prism, 15... condenser lens, 21... objective lens, 22... second prism, 24... lens, 27... mirror, 30... optical module, 31... lens, 33... quarter-wave plate, 34... lens, 35... polarization camera, 36-38... mirror, 39... calculation unit, 40... analysis unit, 50A, 50 B...refractive index distribution acquisition unit, 51, 52...laser light source, 53, 54...shutter, 55...mirror, 56...dichroic mirror, 57...beam splitter, 61, 62...lens, 63, 64...optical fiber, 71...lens, 72...mirror, 73...lens, 74...condenser lens, 75...objective lens, 81...beam splitter, 82...lens, 83A, 83B...camera, 84...calculation unit, 90...analysis unit.

Claims

1. a refractive index distribution acquiring step of acquiring a refractive index distribution of a cell at each of the first wavelength and the second wavelength, the specific site being labeled with a labeling substance having refractive indexes different from each other at a first wavelength and a second wavelength, the refractive index distribution of the cell being caused to have a detectable refractive index difference between a labeled site and an unlabeled site at both or either one of the first wavelength and the second wavelength; an analysis step of evaluating a distribution of the specific site in the cell by comparing a refractive index distribution at the first wavelength and a refractive index distribution at the second wavelength; A cell evaluation method comprising:

2. The cell evaluation method according to claim 1 , wherein the labeling substance has a refractive index at a longer wavelength of the first wavelength and the second wavelength that is higher than a refractive index at a shorter wavelength.

3. The cell evaluation method according to claim 1 , wherein the labeling substance has a lower refractive index at a longer wavelength of the first wavelength and the second wavelength than at a shorter wavelength.

4. The labeling substance is a light-absorbing labeling substance having an absorption peak at a specific wavelength, A cell evaluation method according to any one of claims 1 to 3, wherein in the refractive index distribution acquisition step, the refractive index distribution of the cell is acquired at each of the first wavelength and the second wavelength which are on the shorter wavelength side and the longer wavelength side of the specific wavelength.

5. The labeling substance is a light-absorbing labeling substance having an absorption peak at a specific wavelength, 4. The cell evaluation method according to claim 1, wherein in the refractive index distribution acquisition step, the refractive index distribution of the cell is acquired at each of the first wavelength and the second wavelength, with one of the first wavelength and the second wavelength being on the shorter wavelength side with respect to the specific wavelength, and the other being on the longer wavelength side with respect to the specific wavelength.

6. The cell evaluation method according to any one of claims 1 to 3, wherein the labeling substance is a chromoprotein.

7. The cell evaluation method according to any one of claims 1 to 3, wherein the cell is a cell into which an intracellular expression construct has been introduced, in which a DNA sequence encoding a chromoprotein is connected to the N-terminus or C-terminus of a DNA sequence encoding an arbitrary protein, and the labeling substance is this intracellular expression construct.

8. The cell evaluation method according to any one of claims 1 to 7, wherein in the analysis step, the distribution of the specific site in the cell is evaluated based on a ratio or difference between refractive index distributions at the first wavelength and the second wavelength.

9. The cell evaluation method according to any one of claims 1 to 8, wherein in the analysis step, an analysis of the specific portion of the cell is performed by overlaying a result of a comparison of refractive index distributions at the first wavelength and the second wavelength on an intensity image or a fluorescence image of the cell.

10. The cell further labels another specific site with another labeling substance having a refractive index different from that at a third wavelength and a fourth wavelength, respectively, and generates a detectable refractive index difference between a labeled site and an unlabeled site at both or either one of the third wavelength and the fourth wavelength, In the refractive index distribution acquisition step, a refractive index distribution of the cell is acquired at each of the third wavelength and the fourth wavelength; The cell evaluation method according to any one of claims 1 to 9, wherein in the analysis step, the distribution of the other specific site in the cell is evaluated by comparing the refractive index distribution at the third wavelength and the fourth wavelength, respectively.

11. The cell evaluation method according to claim 10 , wherein the first wavelength or the second wavelength is equal to the third wavelength or the fourth wavelength.

Images