JP2015099085A - Cell image processing method, cell image processing apparatus, and cell image processing program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell image processing method of acquiring a cell image with improved spatial resolution, a cell image processing apparatus, and a cell image processing program.SOLUTION: A cell image processing method includes: a first acquisition step of acquiring a quantitative phase image of a cell; a second acquisition step of acquiring a high-frequency component image of the cell; a setting step of setting luminance of the quantitative phase image and the high-frequency component image; and a subtraction step of subtracting the set high-frequency component image from the set quantitative phase image to obtain a subtraction image.

Description

  The present invention relates to a cell image processing method, a cell image processing apparatus, and a cell image processing program.

  Traditionally, as a method to determine cell area or confluence, prepare quantitative phase data on the cell and background around the cell; determine the cell boundary from the quantitative phase data; and determine the area within the boundary. Methods are known that comprise determining cell area or confluence by determining (e.g., Patent Document 1).

Special table 2007-509314

  However, the quantitative phase image is an interference image, the spatial resolution is low, and the internal structure and boundary (contour) portion of the cell in the quantitative phase image are often unclear.

  Therefore, an object of the present invention is to provide a cell image processing method, a cell image processing apparatus, and a cell image processing program that can acquire an image of a cell with excellent spatial resolution.

  The present invention that achieves the above-described object includes a first acquisition step of acquiring a quantitative phase image of a cell, a second acquisition step of acquiring a high-frequency component image of the cell, the quantitative phase image, and the high-frequency component image. A cell image processing method comprising: a setting step for setting luminance; and a subtraction step for subtracting a set high-frequency component image from a set quantitative phase image to obtain a subtracted image.

  Further, the present invention sets a first acquisition unit that acquires a quantitative phase image of a cell, a second acquisition unit that acquires a high-frequency component image of a cell, and the luminance of the quantitative phase image and the high-frequency component image. A cell image processing apparatus comprising: setting means; and subtracting means for subtracting a set high-frequency component image from a set quantitative phase image to obtain a subtracted image.

  In the present invention, the computer sets the first acquisition means for acquiring the quantitative phase image of the cell, the second acquisition means for acquiring the high-frequency component image of the cell, the luminance of the quantitative phase image and the high-frequency component image. And a subtracting unit for obtaining a subtracted image by subtracting a set high-frequency component image from a set quantitative phase image.

  According to the present invention as described above, an image of a cell having excellent spatial resolution can be acquired.

  In the present invention, “cell” means “single cell or cell population”.

  “High-frequency component image” means “an image containing a high spatial frequency component not included in the spatial frequency component of the quantitative phase image”. The high-frequency component image includes a bright-field image, a phase contrast image, and the like. It is. The “bright field image” is an “image taken with a bright field microscope”, and the “phase contrast image” is an “image taken with a phase contrast microscope”.

  The “set quantitative phase image” of the present invention means a quantitative phase image in which luminance is set. The brightness of the “set quantitative phase image” may be the same as the brightness of the “quantitative phase image” acquired in the first acquisition step.

  The “set high-frequency component image” means a high-frequency component image in which luminance is set. The luminance of the “set high-frequency component image” may be the same as the luminance of the “high-frequency component image” acquired in the second acquisition step.

  The “subtracted image” means “an image obtained by subtracting the set high-frequency component image from the set quantitative phase image”.

  The setting step of the present invention may be a step of setting the maximum luminance and the minimum luminance of the quantitative phase image and the high frequency component image to be the same. With the cell image processing method of the present invention including such steps, the spatial resolution of the subtracted image can be further improved and the contrast can be improved.

  The high frequency component image of the present invention may be a bright field image. If the high-frequency component image is a bright-field image, a subtracted image with a clear cell internal structure (for example, cell nucleus) can be acquired.

  According to the present invention, a cell image processing method, a cell image processing apparatus, and a cell image processing program capable of acquiring a cell image with excellent spatial resolution are provided.

1 is a configuration diagram of a cell observation system 1. FIG. 3 is a schematic diagram showing a hardware configuration of a cell image processing apparatus D. FIG. 3 is a schematic diagram showing a functional configuration of a cell image processing apparatus D. FIG. It is a flowchart of a cell image processing method. In order to explain that the subtraction image ((C) in the figure) obtained by subtracting the bright field image ((B) in the figure) from the quantitative phase image ((A) in the figure) includes a high spatial frequency component and is excellent in spatial resolution. FIG. 1 is a configuration diagram of a cell observation system 2. FIG. The subtraction image ((C) in the figure) obtained by subtracting the phase contrast image ((B) in the figure) from the quantitative phase image ((A) in the figure) includes a high spatial frequency component and is excellent in spatial resolution. It is a schematic diagram for. It is a quantitative phase image ((A) in the figure), a bright field image ((B) in the figure), and a subtracted image ((C) in the figure) of the human iPS cells of Example 1. Quantitative phase image of human iPS cells of Example 2 ((A) in the figure), set quantitative phase image ((A ′) in the figure), bright field image ((B) in the figure)) and subtracted image ((C) in the figure) It is. It is a quantitative phase image (in the figure (A)) and subtraction image (in the figure (C)) of the human iPS cell of Example 3. A three-dimensional plot image of the portion surrounded by the white line in the quantitative phase image of FIG. 10A ((A) in the figure) and a three-dimensional plot image of the portion surrounded by the white line in the subtraction image of FIG. ((C) in the figure). It is a quantitative phase image ((A) in the figure) and subtracted image ((C) in the figure) of the ES cells of Example 4. It is a fluorescence image of the ES cell of Example 4 immunostained with the anti-Nanog antibody. They are the quantitative phase image (in the figure (A)), bright field image (in the figure (B)), and subtraction image (in the figure (C)) of the human iPS cell of Example 5. 14A is a cross-sectional view of the quantitative phase image (A in the figure), a cross-sectional view of the phase difference contrast image in FIG. 14B (B in the figure), and a subtraction image in FIG. 14C. It is sectional drawing ((C) in a figure).

  Preferred embodiments of the cell image processing method, cell image processing apparatus and cell image processing program of the present invention will be described in detail. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

(cell)
In carrying out the present invention, first, cells are prepared. A cell means a “single cell or a population of cells” and is not particularly limited as long as it is. The cell may be not only a human but also a mouse, monkey, rabbit, dog, cat or the like. The cells may be cells (stem cells) having the ability to differentiate into cells of multiple lines (multipotency) and the ability to maintain pluripotency (self-replication ability) even after cell division (differentiated cells). Examples of the stem cells include pluripotent stem cells such as ES cells and iPS cells.

(Measurement sample)
Next, a cell is used as a measurement sample. The measurement sample is prepared by, for example, injecting a solution containing cells into the slide chamber.

(Cell observation system)
Next, the configuration of the cell observation system 1 to be used will be described. FIG. 1 is a configuration diagram of a cell observation system 1.

  As shown in FIG. 1, the cell observation system 1 is mainly configured by a quantitative phase microscope A and a cell image processing device D.

(Quantitative phase microscope)
The quantitative phase microscope A has a lens A2 facing the emission side end face B1 of the optical fiber B that guides irradiation light from a light emission unit (not shown) on the light incident side, and a reflection that reflects the light transmitted through the lens A2. Part A3. On the other hand, on the light emission side of the quantitative phase microscope A, an imaging device C1 such as a CCD camera that images interference fringes (not shown, the same applies hereinafter) generated by the optical interference unit A7 and forms a quantitative phase image; An imaging device C2 is provided that captures the light to be measured reflected by the partial reflection portion A8 and produces a bright field image.

  The quantitative phase microscope A includes a microscope main body A1 including a sample stage A4 that supports the measurement sample S, an objective lens A5, a reflection part A6, a light interference part A7, and a partial reflection part A8.

  The sample stage A4 is, for example, substantially plate-shaped having a light transmission part A41 capable of transmitting light at the center and having a placement surface A42 on which the measurement sample S can be placed. By irradiating light from above with the measurement sample S placed on the placement surface A42, the light to be measured that has passed through the measurement sample S passes through the light transmission part A41 and travels toward the objective lens A5. . The light transmitting portion A41 may be formed from a member that can transmit light, such as glass, or may be a simple hole. The objective lens A5, for example, expands incident measurement light at a predetermined magnification according to the operation and emits it as parallel light based on an operation of an operation unit (not shown). The partial reflection part A8 is, for example, a dichroic mirror (dichroic mirror), and transmits the measurement light when the irradiation light is laser light (for example, 830 nm), but is measured when the irradiation light is white light. It reflects light. The reflection part A6 is, for example, a total reflection type mirror, and allows the light to be measured H1 transmitted through the partial reflection part A8 to be totally reflected and introduced into the light interference part A7. The optical interference unit A7 splits the light to be measured H1 into two lights H1a and H1b, and the light to be measured H1 (H1a and H1b) emitted from the light separation element A71 is converged light H2 (H2a). , H2b), a converging lens A72, a spatial filter A73 disposed at the convergence position of the convergent light H2, an object fringe passing through the spatial filter A73 and the reference light H4 are superimposed to generate an interference fringe. And a lens A75. Here, the light separating element A71 is configured using a diffraction grating. Furthermore, the light separation element A71 may be a polarization separation element that separates two light beams having different polarization directions. In that case, the optical interference unit A7 separates the measured light H1 into two light beams H1a and H1b having different polarization directions, and a condensing lens A72 that converts the light beam to be converged light H2 (H2a and H2b). A spatial filter A73 disposed at the convergence position of the convergent light H2, an object light H3 and reference light H4 transmitted through the spatial filter A73, a half-wave plate A74 disposed on the emission side of the spatial filter A73, and the half-wavelength And a synthetic lens A75 that generates interference fringes by superimposing the object light H3 and the reference light H4 whose polarization directions are aligned by the plate A74. Alternatively, instead of the half-wave plate A74 disposed on the emission side of the spatial filter A73, a polarizer may be disposed to align the polarization directions of the object light H3 and the reference light H4.

  When the irradiation light is laser light, the light to be measured of the quantitative phase microscope A is transmitted through the partial reflection portion A8, reflected by A6, and introduced into the light interference portion A7. And the interference fringe produced | generated in optical interference part A7 is imaged with the imaging device C1, and becomes a quantitative phase image.

  On the other hand, when the irradiation light is white light, the light to be measured of the quantitative phase microscope A is reflected by the partial reflection portion A8, is imaged by the imaging device C2 through the imaging lens A10, and becomes a bright field image.

(Cell image processing device)
Next, the configuration of the cell image processing apparatus D will be described. FIG. 2 is a schematic diagram showing a hardware configuration of the cell image processing apparatus D, and FIG. 3 is a schematic diagram showing a functional configuration of the cell image processing apparatus D.

  As shown in FIG. 2, the cell image processing apparatus D physically includes a main storage device such as a CPU D11, a ROM D12 and a RAM D13, an input device D14 such as a keyboard and a mouse, and an output device D15 such as a display. It is configured as a normal computer including a communication module D16 such as a network card for transmitting and receiving data to and from other devices such as the imaging devices C1 and C2, an auxiliary storage device D17 such as a hard disk, and the like. Each function of the cell image processing apparatus D, which will be described later, has an input device D14 and an output device D15 under the control of the CPU D11 by reading predetermined computer software on hardware such as the CPU D11, ROM D12, and RAM D13. This is realized by operating the communication module D16 and reading and writing data in the main storage devices D12 and D13 and the auxiliary storage device D17.

  As shown in FIG. 3, the cell image processing apparatus D includes first acquisition means D1, second acquisition means D2, setting means D3, subtraction means D4, and display means D5 as functional components.

  The first acquisition unit D1 acquires a quantitative phase image of cells from the imaging device C1. The 2nd acquisition means D2 acquires the bright field image of a cell from the imaging device C2. The setting means D3 sets the luminance of the acquired quantitative phase image or bright field image. The subtracting means D4 obtains a subtracted image by subtracting the set bright field image from the set quantitative phase image. The display means D5 displays a subtraction image.

(Cell image processing program)
The cell image processing program causes the computer to function as the above-described first acquisition unit D1, second acquisition unit D2, setting unit D3, subtraction unit D4, and display unit D5. By causing the computer to read the cell image processing program, the computer operates as the cell image processing apparatus D. The cell image processing program is provided by being stored in a recording medium, for example. Examples of the recording medium include a flexible disk, a recording medium such as a CD and a DVD, a recording medium such as a ROM, or a semiconductor memory.

(Cell image processing method)
A cell image processing method performed by the cell image processing apparatus D will be described. FIG. 4 is a flowchart of the cell image processing method. With the cell image processing method performed by the cell image processing apparatus D, it is possible to acquire an image of a cell with excellent spatial resolution.

[First acquisition step S1]
The first acquisition unit D1 acquires a quantitative phase image from the imaging device C1.

[Second acquisition step S2]
The second acquisition unit D2 acquires a bright field image from the imaging device C2. The second acquisition step S2 may be performed before or after the first acquisition step S1.

[Setting step S3]
The setting means D3 sets the luminance of the acquired quantitative phase image and bright field image. The setting is usually performed so that the DC component of the subtracted image is reduced.

  The setting unit D3 may leave the acquired quantitative phase image and the bright field image without changing the brightness, or may change and set the brightness of the acquired quantitative phase image or the bright field image. .

  There is no particular limitation on the method for determining whether to change the luminance by the setting means D3. When the luminance of the quantitative phase image or the bright field image is within a predetermined luminance value or numerical range, it may be determined to change, or conversely, a predetermined luminance value or numerical value is determined. You may make it determine with changing, when not in the range.

  Alternatively, the luminance of the quantitative phase image and the bright field image may be compared to determine whether to change the luminance of the quantitative phase image or the bright field image. In this case, it is preferable to determine whether or not to change the brightness histogram.

  When changing the brightness, the setting unit D3 changes the brightness to be higher or lower.

  The brightness change may be performed once or a plurality of times. As a result of changing the luminance a plurality of times, the luminance of the set quantitative phase image may be the same as that of the quantitative phase image acquired in the first acquisition step, or the luminance of the set bright field image may be the same in the second acquisition step. It may be the same as the acquired bright field image.

  As described above, the luminance of the acquired quantitative phase image and bright field image is set by the setting means D3. In the setting step S3, the maximum luminance or the minimum luminance of the quantitative phase image and the high frequency component image are made the same. It is preferable to set. In particular, by setting the maximum luminance and the minimum luminance of the quantitative phase image and the high frequency component image to be the same, the spatial resolution of the subtraction image can be further improved and the contrast can be improved.

[Subtraction step S4]
The subtracting means D4 obtains a subtracted image by subtracting the set bright field image that is the set high-frequency component image from the set quantitative phase image. The acquired subtraction image is excellent in spatial resolution, and the internal structure and boundary (contour) portion of a cell such as an intracellular organ (organelle) are clear.

  In particular, when the cell is a cell population of pluripotent stem cells such as ES cells and iPS cells, the internal structure can be known in detail from the subtraction image without staining. Moreover, since the cell nucleus is emphasized in the subtraction image, the degree of differentiation of the cell can be determined from the subtraction image.

  Since the subtraction image includes a high spatial frequency component that is not present in the quantitative phase image, it can be said that the information of the quantitative phase image is maintained and information that is not present in the quantitative phase image is supplemented.

  Although the present invention is not limited by theory, the present inventors presume that the subtracted image is excellent in spatial resolution due to the following principle.

The quantitative phase image I _QPM is an image including only a low spatial frequency component. If the phase delay Δφ due to cells in this image is Δφ _LF ,
I _QPM = Δφ _LF
It becomes. This is schematically shown in FIG.

On the other hand, the bright field image _IBF is an image that includes a high spatial frequency component and whose boundary is emphasized. _Then, in the phase lag Δφ _HF by _{I BF} and cells,
I _BF 〜1−Δφ _HF
There is a proportional relationship. FIG. 5B schematically shows Δφ _HF .

Subtracting the bright field image from the quantitative phase image
I _QPM -I _BF = Δφ _LF + Δφ _HF −1
Thus, the subtracted image includes a high spatial frequency component.

  Therefore, the subtracted image is excellent in spatial resolution. This is schematically shown in FIG. 5A, 5B, and 5C, the horizontal axis represents the spatial frequency, and the vertical axis represents the light intensity (amplitude) at each spatial frequency.

[Display step S5]
The display means D5 displays the subtraction image acquired in the subtraction step S4. At the time of display, the subtracted image may be subjected to appropriate image processing. The subtracted image may be displayed entirely or only partially.

  Further, the display means D5 can adjust the color, brightness, contrast, enlargement, reduction, etc. of the subtraction image.

  As mentioned above, although the embodiment using the cell observation system 1 has been described in detail as the first embodiment of the present invention, the cell observation system 2 that can simultaneously acquire a quantitative phase image and a phase difference contrast image is provided. A second embodiment to be used will be described. FIG. 6 is a configuration diagram of the cell observation system 2.

  The configuration of the cell observation system 2 is substantially the same as the configuration of the cell observation system 1, but the quantitative phase microscope A of the cell observation system 2 further has irradiation light H0 from a light emitting unit (not shown) on the light incident side. Irradiation optical system A2 'facing the exit-side end face B1' of the optical fiber B 'that guides, and a part that reflects the light transmitted through the irradiation optical system A2' and transmits the light reflected by the reflection part A3 And a reflection part A9. For example, a dichroic mirror (dichroic mirror) is used as the partial reflection portion A9. The irradiation optical system A2 'includes a collector lens A21', a diaphragm plate A22 ', and a condenser lens A23'. The collector lens A21 'collimates the irradiation light H0'. The diaphragm plate A22 'selectively passes through an annular region having a certain distance from the optical axis in the light beam cross section. The condenser lens A23 'converges the light collimated by the collector lens A21'. The objective lens A5 in the cell observation system 2 has a phase film and a light reducing film.

  In such a cell observation system 2, when the irradiation light H0 is simultaneously irradiated as laser light (for example, 830 nm) and the irradiation light H0 ′ as white light, the irradiation light H0 is reflected by A3, and the partial reflection portion A9 and the measurement sample are reflected. As described for the cell observation system 1 through S, the image is finally picked up by the image pickup device C1 and becomes a quantitative phase image.

  The irradiation light H0 ′ irradiated at the same time passes through the irradiation optical system A2 ′, is reflected by the partial reflection portion A9, passes through the measurement sample S, and is finally captured by the imaging device C2 to become an image. Since the objective lens A5 has a phase film and a light reducing film, the image becomes a phase contrast image.

  Therefore, the cell observation system 2 can simultaneously acquire a quantitative phase image and a phase difference contrast image.

  In the second embodiment in which the high-frequency component image is a phase contrast image, the present inventors presume that the subtraction image is excellent in spatial resolution capability due to the following principle.

As described above, for the quantitative phase image I _QPM ,
I _QPM = Δφ _LF
(FIG. 7A).

On the other hand, the phase difference contrast image I _PhC is also an image that includes a high spatial frequency component and whose boundary is emphasized. If the phase delay due to the cell is defined as Δφ _HF , the electric field E of the image is
E = exp [i (φ + Δφ _HF )) = exp [iφ] (1 + iΔφ _HF )
Can be approximated.

As a result of the phase of the scattered light from the cell being shifted by 90 degrees by the phase film provided at the objective lens pupil position, the phase delay Δφ _HF is converted into light intensity,
I _PhC = | exp (iπ / 2) + iΔφ _HF | ² = 1-2Δφ _HF
Therefore, a proportional relationship is generated between the phase difference contrast image I _PhC and the phase delay Δφ _HF .

  Further, in the phase contrast microscope, since the optical element that attenuates the 0th-order diffracted light and its vicinity (hereinafter, substantially 0th-order diffracted light) is installed at the same position as the phase film, the approximately 0th-order diffracted light is attenuated. This is schematically shown in FIG. 7B.

When subtracting the phase contrast image from the quantitative phase image,
I _QPM -I _PhC = Δφ _LF + 2Δφ _HF −1
Thus, the subtracted image includes a high spatial frequency component.

  Therefore, also in the second embodiment, the subtraction image is excellent in spatial resolution. This is schematically shown in FIG. As in FIG. 6, in FIGS. 7A, 7 </ b> B, and 7 </ b> C, the horizontal axis is the spatial frequency, and the vertical axis is the light intensity (amplitude) at each spatial frequency.

  The first and second embodiments of the present invention have been described above, but it goes without saying that the present invention is not limited to the above-described embodiments. For example, the objective lens A5 of the cell observation system 1 may have a phase film and a dimming film, or the objective lens A5 of the cell observation system 2 may not have a phase film and a dimming film.

  The cell observation system 1 and the cell observation system 2 may be a cell observation system in which a quantitative phase microscope and a bright field microscope or a phase contrast microscope are configured as separate devices.

Example 1
Using a cell image processing device, a quantitative phase image (FIG. 8A) and a bright field image (FIG. 8 (FIG. 8 ()) of human iPS cells (253G1, Nature Biotechnology, 2008, Vol. 21, No. 1, p. 101-106). B)) shows an example of obtaining a subtracted image (FIG. 8C). In this example, the cellular image processing device compares the luminance histogram of the quantitative phase image and the bright field image, automatically changes the luminance of the quantitative phase image to the same maximum luminance and minimum luminance as the bright field image, and The bright field image was subtracted from the set quantitative phase image.

  The quantitative phase image seems to have no optical thickness of the organ (organelle) and the contrast is not so clear, but the subtraction image shows that the boundary and the organ (organelle) are clear.

(Example 2)
An example in which a subtraction image (FIG. 9C) is obtained from a quantitative phase image (FIG. 9A) and a bright field image (FIG. 9B) of a human iPS cell (253G1) by a cell image processing apparatus. Show. In this example, the cell image processing apparatus automatically changes the luminance of the quantitative phase image to the set quantitative phase image (FIG. 9A) having the same maximum luminance and minimum luminance as the bright field image, and then sets the fixed quantitative phase. The bright field image was subtracted from the image.

(Example 3)
The example which acquired the subtraction image (FIG.10 (C)) from the quantitative phase image (FIG.10 (A)) and bright field image of a human iPS cell (253G1) with the cell image processing apparatus is shown.

  The three-dimensional plot image of the portion surrounded by the white line in the quantitative phase image (FIG. 10A) is FIG. 11A, and the tertiary of the portion surrounded by the white line in the subtraction image (FIG. 10C). The original plot image is shown in FIG. In each of FIGS. 11A and 11C, the horizontal axis (X and Y directions) represents the number of pixels (pixels), and the vertical axis (Z direction) represents luminance.

  From the comparison between FIG. 11A and FIG. 11C, it can be seen that the contrast becomes clear when the subtracted image is used.

Example 4
The example which acquired the subtraction image (FIG.12 (C)) from the quantitative phase image (FIG.12 (A)) and bright field image of ES cell (khES3) with the cell image processing apparatus is shown. In the subtraction image, the cell nucleus region is clearer.

  FIG. 13 is a fluorescence image of ES cells immunostained with anti-Nanog antibody.

  The substantially circular white portion in FIG. 13 is the cell nucleus region of the ES cell. From the comparison between FIG. 12C and FIG. 13, it can be seen that the cell nucleus region in the subtraction image is clear.

(Example 5)
A subtraction image (FIG. 14C) was obtained from a quantitative phase image (FIG. 14A) and a phase contrast image (FIG. 14B) of human iPS cells (253G1) by a cell image processing apparatus. An example is shown.

  15A is a cross-sectional view of the quantitative phase image of FIG. 14A, FIG. 15B is a cross-sectional view of the phase contrast image of FIG. 14B, and FIG. It is sectional drawing of the subtraction image of FIG.14 (C). In addition, a cross section is a cross section of the black line displayed on FIG. 14 (A), (B) and (C).

  When the sectional view of the quantitative phase image is viewed, the left and right sides are gentle, which indicates that the image is unclear, that is, the spatial resolution is low. On the other hand, in the cross-sectional view of the phase contrast image, the edges at both ends are undershooted and overshooted, and the effect of differential operation in the spatial direction appears. This is because the zero-order light and the spatial frequency in the vicinity thereof are blocked by the phase film and the dimming film. In the cross-sectional view of the subtraction image, the left and right end portions have a steep intensity distribution, and the resolution of the quantitative phase image is restored.

DESCRIPTION OF SYMBOLS 1, 2 ... Cell observation system, A ... A quantitative phase microscope, A1 ... Microscope main body, B ... Optical fiber, C1, C2 ... Imaging apparatus, D ... Cell image processing apparatus, D1 ... 1st acquisition means, D2 ... 2nd Acquisition means, D3 ... setting means, D4 ... subtraction means, D5 ... display means.

Claims (5)

A first acquisition step of acquiring a quantitative phase image of the cells;
A second acquisition step of acquiring a high-frequency component image of the cell;
A setting step for setting the luminance of the quantitative phase image and the high-frequency component image;
A subtraction step of subtracting the set high-frequency component image from the set quantitative phase image to obtain a subtracted image;
A cell image processing method comprising:   The cell image processing method according to claim 1, wherein the setting step is a step of setting the highest luminance and the lowest luminance of the quantitative phase image and the high frequency component image to be the same.   The cell image processing method according to claim 1, wherein the high-frequency component image is a bright field image or a phase contrast image. First acquisition means for acquiring a quantitative phase image of a cell;
Second acquisition means for acquiring a high-frequency component image of a cell;
Setting means for setting the luminance of the quantitative phase image and the high-frequency component image;
Subtracting means for subtracting the set high-frequency component image from the set quantitative phase image to obtain a subtracted image;
A cell image processing apparatus comprising: Computer
First acquisition means for acquiring a quantitative phase image of a cell;
Second acquisition means for acquiring a high-frequency component image of a cell;
Setting means for setting brightness of the quantitative phase image and the high-frequency component image;
Subtracting means for subtracting the set high frequency component image from the set quantitative phase image to obtain a subtracted image,
Cell image processing program to function as