JP6459568B2 - Target cell detection method and target cell detection system - Google Patents

Description

  The present invention relates to a method for detecting a target cell (eg, circulating tumor cell in blood) and the like from a cell suspension such as blood, and a target cell for carrying out this method Relates to the detection system.

There are about 10 × ⁹ normal cells in 1 mL of blood, but circulating tumor cells [CTC], peripheral circulating endothelial progenitor cells [CEC], peripheral circulating endothelial progenitor cells [CEP], various stem cells, etc. These cells are called rare cells because there are only a few to a dozen or so cells. For example, CTC is useful as a determination of a therapeutic effect and a prognostic predictor in metastatic cancer cases such as breast cancer, prostate cancer, and colon cancer. In addition, CEC plays an important role in angiogenesis and maintenance of the blood vessel wall. In many pathological conditions such as cardiovascular diseases, infectious diseases, immune diseases, post-transplant examinations, and cancer, the number of CEC contained in the blood is low. Since it increases, it is useful not only for determining the therapeutic effect and predicting prognosis in cancer cases, but also for quickly measuring damage to blood vessels due to cardiovascular diseases. CEP is a bone marrow-derived cell, and the number of CEP contained in blood is useful as a biomarker for quantifying angiogenesis inhibitory effect (cancer suppressive effect). Thus, it is clear that it is clinically useful to accurately detect rare cells related to diseases (particularly metastatic cancer), but the detection is extremely difficult.

  Patent Document 1 discloses a method for detecting rare cells using a general-purpose fluorescent substance, a fluorescence microscope, and cells that are not rare as methods for detecting rare cells. Specifically, in order to detect rare cells, a positive marker that is fluorescently labeled and a negative marker that is fluorescently labeled are brought into contact with a cell group that is a sample, and this cell group is developed on a substrate, In this method, rare cells contained in a sample are detected by analyzing individual cells reflected in the cell image (eg, comparing the cell morphology of one cell with another cell).

Special table 2013-508729 gazette

  In the method of Patent Document 1, cell images are acquired for a large number of cells developed on a substrate, and target cells are detected by comparing individual cells in the cell image with other cells. If the cell image is not in focus, it is difficult to discriminate and determine rare cells. Therefore, it is necessary to acquire a focused image.

  As a method of acquiring a cell image, first, in a bright field, a cell that is developed on the substrate or a mark attached to the substrate is focused, and a cell that is developed on the substrate in the dark field with the focus. A method is conceivable in which a fluorescent image for labeling attached to each cell in the group is excited and emitted to acquire a cell image, and a target cell is searched from the cell image. However, in the bright field, since all wavelengths of light are included, the depth of focus (of the image) is higher than when focusing at a specific fluorescence wavelength (various fluorescence emitted from each cell) in the dark field. Since the focus area is sharper), when moving to the dark field and the depth of focus narrows, the cell is not in focus when the cell image is acquired, and the target cell is searched for and detected. Accuracy may be insufficient.

  On the other hand, in order to acquire an image focused on the target cell, it is also possible to excite the fluorescent dye that labels the target cell and focus directly on the target cell using the fluorescence emitted from the target cell. . However, since the target cell is rare, it is difficult to find and focus on a large number of cells.

  In particular, when the substrate is divided into several regions (fields of view) and cell images are acquired for all of them, each region is focused with the respective fluorescence that labels target and non-target cells for each imaging. In the method, not only it takes time to finish all the imaging, but also it takes time to adjust the focus in one area, and during that time, the excitation light continues to be irradiated, and in the area (field of view) to be imaged Not only does the fluorescent dye that has labeled the target cell fade, but it may also fade to the fluorescent dye of the cell in the surrounding area (to be imaged later), faint fluorescence for detecting the target cell There is a risk of losing the signal.

  The present invention has been made in view of the above problems, and provides a target cell detection method capable of easily focusing on a target cell in a short time, and a target cell detection system for carrying out the method. The purpose is to do.

  When the number of target cells to be detected is smaller than the number of non-target cells, the present inventors develop a cell group on the substrate and make it a single layer. On the other hand, by focusing the optical system (microscope, etc.) for detecting the target cell, it is easy to focus on the focal point of the target cell or a focal point very close to it before searching for the target cell. It has been found that the target cell can be easily searched and the present invention has been completed.

  According to the present invention, the following target cell detection methods and target cell detection systems [1] to [14] are provided.

[1] A cell suspension in which a target cell to be detected is contained in a smaller number than a non-target cell is spread on the surface of a cell development substrate, and an area (cell detection for detecting cells provided on the surface) Cell expansion step to form a single cell layer in the region),
A first fluorescent label that is a complex of an antibody that specifically binds to the target cell and a first fluorescent dye is used to fluorescently stain cells in the cell suspension or the expanded cells. 1 fluorescent labeling process;
Using a second fluorescent label, which is a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye, the cells in the cell suspension or the expanded cells are fluorescently stained. A second fluorescent labeling process,
And / or
Third fluorescence that is a 3A fluorescent dye that binds to both the target cell and the non-target cell, or a complex of an antibody that binds to both the target cell and the non-target cell and a 3B fluorescent dye. A third fluorescent labeling step of fluorescently staining the cells in the cell suspension or the expanded cells using the label,
A method for detecting a target cell including random numbers,
A focus position adjustment process ;
While irradiating the cell group developed in the cell detection region with the excitation light of the first fluorescent dye, the target cell emitting the first fluorescence is searched with reference to the focal position adjusted in the focal position adjusting step. A target cell search step to perform,
Including
The focal position adjustment step includes:
When the target cell detection method includes both the second fluorescent labeling step and the third fluorescent labeling step, the cell is excited by the second fluorescent dye or the excitation light of the third A or 3B fluorescent dye. Irradiating a cell group developed in a detection region, and performing a process of focusing the light receiving optical system on the non-target cell emitting the second fluorescence or the third A or the third fluorescence,
When the target cell detection method does not include the third fluorescent labeling step, the cell group developed in the cell detection region is irradiated with the excitation light of the second fluorescent dye, and the second fluorescence is emitted. Perform processing to focus the receiving optical system on the non-target cells that are emitted,
When the target cell detection method does not include the second fluorescent labeling step, the cell group developed in the cell detection region is irradiated with the excitation light of the 3A or 3B fluorescent dye, and the 3A Alternatively, a method for detecting a target cell, comprising performing a process of focusing a light-receiving optical system on a non-target cell emitting 3B fluorescence .

  [2] The method for detecting a target cell according to [1], wherein the target cell is a rare cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood.

[3] When the detection method that purpose cells comprise the second fluorescent label treatment step, the wavelength of the second fluorescent used when focusing at the focal position adjusting step, the first fluorescent Does not overlap with the wavelength of
When the target cell detection method includes the third fluorescence labeling step, the wavelength of the third A or 3B fluorescence used for focusing in the focus position adjustment step is the first fluorescence. The method for detecting a target cell according to [1] or [2], wherein the target cell does not overlap with the wavelength of .

[4] The target cell is CTC,
The non-target cell is a white blood cell;
The first fluorescent label is an anti-cytokeratin antibody labeled with the first fluorescent dye;
When the target cell detection method includes the second fluorescent labeling step, the second fluorescent label is an antibody that specifically recognizes leukocytes labeled with the second fluorescent dye,
When the target cell detection method includes the third fluorescent labeling step, the third fluorescent label is a Hoechst fluorescent dye that binds to a cell nucleus.
[1] The method for detecting a target cell according to any one of [3].

[5] The surface of the cell detection region of the cell development substrate is planar.
In the cell expansion step, the target cell according to any one of [1] to [4], wherein two or more cell layers developed on the surface of the planar substrate are monolayered using a cell scraper. Detection method.

[6] The surface of the cell detection region of the cell development substrate has a microchamber or unevenness for storing cells,
The method for detecting a target cell according to any one of [1] to [4], wherein in the cell expansion step, the cell is monolayered by a liquid flow.

[7] The method further includes a focus position correction step between the focus position adjustment step and the target cell search step ,
In the focal position correction step,
When the target cell detection method includes both the second fluorescent labeling process and the third fluorescent labeling process, the light received by the light receiving element provided in the light receiving optical system is the second fluorescent labeling process . Or a shift amount of the focal position caused by the change from the third fluorescence or the third fluorescence to the first fluorescence, and the focal position adjusted in the focal position adjustment step is corrected,
When the target cell detection method does not include the third fluorescent labeling step, the light received by the light receiving element provided in the light receiving optical system changes from the second fluorescence to the first fluorescence. Calculate the amount of deviation of the focal position caused by the change, correct the focal position adjusted in the focal position adjustment step,
When the target cell detection method does not include the second fluorescent labeling step, the light received by the light receiving element provided in the light receiving optical system is transmitted from the 3A or 3B fluorescence to the first. The method for detecting a target cell according to any one of [1] to [6] , wherein an amount of shift of a focal position caused by the change in fluorescence is calculated, and the focal position adjusted in the focal position adjustment step is corrected .

[8] The target cell search process,
A first cell image acquisition step of imaging the cell detection region with the light receiving optical system capable of receiving first fluorescence; an image forming step;
Only including,
And
When the target cell detection method includes both the second fluorescent labeling process and the third fluorescent labeling process, the light receiving optical system further receives the second fluorescence immediately after the focal position adjusting process. the second cell image acquiring step of imaging the cell detecting region and the possible states and the third cell image the light receiving optical system and the fluorescence of the 3A or the 3B ready for receiving for imaging the cell detecting region includes acquiring step, in the image forming step, was synthesized by superimposing the images captured by the second cell image acquiring step and / or said third cell image acquiring step and the first cell image acquisition step,
When the target cell detection method does not include the third fluorescent labeling step, the second cell image acquisition step is further included immediately after the focal position adjustment step. In the image formation step, the second cell image acquisition step is performed. Superimposing and synthesizing each image captured in the cell image acquisition step and the first cell image acquisition step ,
When the target cell detection method does not include the second fluorescent labeling step, the third cell image acquisition step is further included immediately after the focal position adjustment step. In the image formation step, the third cell image acquisition step is performed. The method for detecting a target cell according to any one of [1] to [7], wherein the images captured in the cell image acquisition step and the first cell image acquisition step are superimposed and synthesized .

[9] The focal position adjustment step is performed by obtaining information on the focal length of each visual field by focusing on a non-target cell in each of two or more visual fields at different positions of the cell detection region. When the distances are different, an approximate expression representing a change in focal length in other fields other than the two or more fields is obtained from the difference,
In the other field of view, a step of calculating the respective focal lengths from the approximate expression and setting the focal length of the light receiving optical system to the calculated focal length is performed as the focal position adjusting step. ] The detection method of the target cell in any one of [8].

[10] A cell deployment substrate capable of spreading on the surface a cell suspension containing a smaller number of target cells to be detected than non-target cells;
A cell expansion means for forming a single cell layer in a cell detection region of the cell expansion substrate;
A first fluorescent label that is a complex of an antibody that specifically binds to the target cell and a first fluorescent dye is used to fluorescently stain cells in the cell suspension or the expanded cells. 1 fluorescent label processing means;
Using a second fluorescent label, which is a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye, the cells in the cell suspension or the expanded cells are fluorescently stained. Second fluorescent labeling means;
And / or
Third fluorescence that is a 3A fluorescent dye that binds to both the target cell and the non-target cell, or a complex of an antibody that binds to both the target cell and the non-target cell and a 3B fluorescent dye. A third fluorescent labeling means for fluorescently staining the cells in the cell suspension or the expanded cells using a label,
An irradiation optical system capable of irradiating excitation light having a predetermined wavelength with respect to a cell detection region of the cell development substrate,
A light receiving optical system capable of receiving light from the cell detection region for each wavelength band;
Control means for controlling at least the light receiving optical system and the irradiation optical system;
A target cell detection system comprising:
The control means is at least
A focus position adjusting means;
While irradiating the cells expanded in the cell detecting region by the excitation light of the first fluorescent dye by the irradiation optical system, with reference to the position of the focus combined with the focal point position adjusting means, the second by the light receiving optical system A target cell search means for performing a process of searching for a target cell emitting fluorescence of 1 ,
The focal position adjusting means includes
When the said purpose cell detection system comprises both the third fluorescent label processing means and the second fluorescent label processing means, the excitation of the second fluorescent dye by irradiation optical system or the second 3A or the 3B fluorescent dye the cell population expanded in the cell detecting region by light irradiation, focusing of the light receiving optical system in non-target cells fluoresced the second fluorescent or said second 3A or the 3B,
When the target cell detection system does not include the third fluorescent label processing means, the irradiation optical system irradiates the cell group developed in the cell detection region with the excitation light of the second fluorescent dye, and The light receiving optical system is focused on a non-target cell emitting fluorescence of 2,
When the target cell detection system does not include the second fluorescent labeling processing means, the irradiation optical system irradiates the cell group developed in the cell detection region with the excitation light of the third A or third B fluorescent dye. A target cell detection system , wherein the light receiving optical system is focused on a non-target cell emitting the 3A or 3B fluorescence .

  [11] The target cell detection system according to [10], wherein the target cell is a rare cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood.

[12] When the said purpose cell detection system comprises a second fluorescent label processing means, the wavelength of the second fluorescent used when focusing by the focus position adjusting means, the first fluorescent Does not overlap with the wavelength ,
When the target cell detection system includes the third fluorescent labeling processing unit, the wavelength of the third A or 3B fluorescence used when focusing by the focal position adjusting unit is the first fluorescence. The target cell detection system according to [10] or [11], which does not overlap with a wavelength .

[13] The target cell is CTC,
The non-target cell is a white blood cell;
The first fluorescent label is an anti-cytokeratin antibody labeled with the first fluorescent dye;
When the target cell detection system includes the second fluorescent labeling processing means, the second fluorescent label is an antibody that specifically recognizes leukocytes labeled with the second fluorescent dye,
When the target cell detection system includes the third fluorescent labeling processing means, the third fluorescent label is a Hoechst fluorescent dye that binds to a cell nucleus.
[10] The target cell detection system according to any one of [12].

[14] The surface of the cell detection region of the cell expansion substrate is planar.
The target cell detection system according to any one of [10] to [13], wherein the cell deployment means includes a cell scraper for monolayering two or more cell layers developed on the surface portion. .

[15] The surface of the cell detection region of the cell development substrate has a microchamber or unevenness for storing cells,
The target cell detection system according to any one of [10] to [13], wherein the cell expansion means includes a liquid feeding system mechanism that generates a liquid flow for monolayering the cells.

[16] The control means further includes a focus position correction means,
The focal position correcting means includes
When the target cell detection system includes both the second fluorescent label processing unit and the third fluorescent label processing unit, the light received by the light receiving element provided in the light receiving optical system is the second fluorescent label processing unit . Calculating the shift amount of the focal position caused by the change from the fluorescence or the 3A or 3B fluorescence to the first fluorescence, and correcting the focal position adjusted by the focal position adjusting means ;
When the target cell detection system does not include the third fluorescent label processing means, the light received by the light receiving element provided in the light receiving optical system changes from the second fluorescence to the first fluorescence. Calculating the shift amount of the focal position caused by the correction, correcting the focal position adjusted by the focal position adjusting means,
When the target cell detection system does not include the second fluorescent labeling processing unit, the light received by the light receiving element provided in the light receiving optical system is transmitted from the 3A or 3B fluorescence to the first The target cell detection system according to any one of [10] to [15] , wherein an amount of shift of a focal position generated by changing to fluorescence is calculated, and the focal position adjusted by the focal position adjusting unit is corrected .

[17] The target cell searching section,
First cell image acquisition means for imaging the cell detection region with the light receiving optical system in a state capable of receiving first fluorescence ; and image forming means;
With
And
When the target cell detection system includes both the second fluorescent label processing means and the third fluorescent label processing means, the light receiving optical system is immediately after the focus of the light receiving optical system is adjusted by the focus position adjusting means. the second cell image acquiring means for imaging the cell detecting region the system and the second fluorescent ready to light, and the said light-receiving optical system in the fluorescence can receive a state of the first 3A or the 3B The image forming unit further includes a third cell image acquiring unit that images the cell detection region, and the first cell image acquiring unit, the second cell image acquiring unit, and / or the third cell image acquiring unit captures the image in the image forming unit. There line processing to synthesize by superimposing the images,
If the target cell detection system does not include the third fluorescent labeling processing means, the light receiving optical system receives the second fluorescence immediately after the focus position adjusting means focuses the light receiving optical system. A second cell image acquiring unit configured to capture the cell detection region in a possible state; and in the image forming unit, the images captured by the first cell image acquiring unit and the second cell image acquiring unit are overlapped. Perform the process of combining them together ,
When the target cell detection system does not include the second fluorescent labeling processing means, the light receiving optical system is moved to the third A or 3B immediately after the focus position adjusting means focuses the light receiving optical system. And further comprising a third cell image acquiring means for capturing the cell detection region in a state where fluorescence can be received, and in the image forming means, each of the images captured by the first cell image acquiring means and the third cell image acquiring means. Perform the process of overlaying and compositing images.
[10] The target cell detection system according to any one of [16].

[18] After obtaining the focal length information by focusing on the non-target cell in each of two or more visual fields at different positions of the cell detection region,
When these focal lengths are different, an approximate expression representing a change in focal length in a visual field other than the two or more visual fields is obtained from the difference,
In the other visual field, each focal length is calculated from the approximate expression, and means for setting the focal length of the light receiving optical system to the calculated focal length is included as the focal position adjusting means. ] The target cell detection system according to any one of [17] to [17].

  ADVANTAGE OF THE INVENTION According to this invention, the target cell detection method which can focus on a target cell easily in a short time and the target cell detection system for implementing this method are provided. Thereby, the discoloration of the fluorescent dye labeled with the target cell can be suppressed as much as possible, the detection accuracy of the target cell can be improved, and the time required for the entire detection process can be shortened.

FIG. 1 is a diagram showing an example of a target cell detection system according to the present invention. FIG. 2 is a diagram illustrating a state in which a cell expansion process is performed by the cell expansion means. (A) is the figure which showed the state which is pouring cell suspension into a flow path. (B) is the figure which showed the state which made the cell layer monolayer, storing the cell in a microchamber by reciprocating liquid feeding. (C) is the figure which showed the state of the cell collection device after monolayering. In FIG. 2, “●” indicates a target cell (eg, CTC cell) to be detected. Further, the first to third fluorescent labels are not shown (the same applies to other drawings). FIG. 3 is a partial cross-sectional perspective view showing a cell development substrate having irregularities on the surface. FIG. 4 is a diagram showing a state in which cells are monolayered by a cell scraper with respect to a cell group developed on a cell development substrate of a cell recovery device. FIG. 5 is a schematic diagram of the irradiation optical system and the light receiving optical system of the target cell detection system. In this example, the cell deployment device is held by a cell collection device holder, and the cell collection device holder is driven to adjust the position in the X, Y, and Z directions with respect to the optical axis (vertical one-dot chain line). It is provided as possible. FIG. 6 shows that when the surface of the cell development substrate on which the microchamber is formed is inclined due to manufacturing errors, tolerances, etc., the inclination is uniform. Create a linear approximation formula that expresses the focal length from the multiple focal lengths obtained by focusing, calculate the focal length of the non-target cells in the other non-focused microchambers from the approximation formula, and set the focal position. FIG. 5 is an explanatory diagram when a target cell is detected based on the focal position. FIG. 7 is a view showing a main flowchart of the target cell search method according to the present invention. Here, it is shown that the focal position correcting step (broken line portion) is arbitrarily executed. FIG. 8A is a diagram showing an example of the flow of the cell expansion process. When a cell expansion substrate having a microchamber or irregularities formed on the surface is used, the cells stacked in the microchamber are shown. A process for monolayering is shown. FIG. 8 (B) is a diagram showing another example of the flow of the cell expansion process, and cells stacked using a cell scraper when a planar cell expansion substrate is used on the surface. A process for making the layers monolayer is shown. FIG. 9 is a diagram showing an example of the fluorescent labeling process. The flow is divided before and after the cell suspension is developed on the cell development substrate. The flow before deployment is shown on the right side of FIG. 9, and the flow after deployment is shown on the left side of FIG. FIG. 10 is a plan view of the cell development substrate from above, in which a plurality of fields of view that are virtually set are shown in a grid shape, and one rectangle (□) represents one field of view. Alphabets A to I indicate positions where non-target cells are focused in the focus position adjustment step. When the surface of the substrate for cell deployment is inclined due to manufacturing error / tolerance, linear approximation is performed using the focal length combined at each point (for example, points A, B, and C), for example, the correlation coefficient is R ^{When 2} > 0.90 or more, the focal length of the visual field between A and B or the visual field between B and C is calculated from the linear approximation formula. FIG. 10 shows that the focus is examined so as to cover the entire cell detection region. FIG. 11 is a diagram illustrating an example of a flow of a focal position adjustment process performed by the target cell detection system. Confirm whether non-target cells can be detected in a plurality of predetermined fields on the substrate with the focal position of the light receiving optical system for the non-target cells obtained in a certain field of view (whether the cell deployment substrate is distorted or not). Confirm) and if it can be detected (when there is no distortion), an approximate expression is not created. On the other hand, if it cannot be detected (when the cell deployment substrate is distorted), the focal length is obtained by focusing on the non-target cell in three or more fields of view and approximated based on this focal length information. Formulas are created and the focal length is set for other fields between the fields examined. FIG. 12 is a diagram illustrating an example of a flow of a target cell search process. FIG. 13 is a diagram for explaining the operation and effect of the present invention. Since the height position of the bottom surface of each microchamber is substantially the same, and the height position of the single cell layer existing on the bottom surface of each microchamber is also substantially the same, The focal position of the light receiving optical system for the target cell is substantially the same. Therefore, by focusing in advance on the non-target cell (◯) in the visual field 1 and maintaining this state, the target cell (●) can also be detected in the visual field 2. FIG. 14 is a diagram for explaining the operation and effect of the present invention. The cell deployment substrate is flat and horizontal, and the focal positions of the light receiving optical system for the non-target cells and the target cells in the visual fields 1 and 2 are substantially the same. Therefore, the target cell (●) can be detected also in the visual field 2 by previously focusing the light receiving optical system on the non-target cell (◯) in the visual field 1 and maintaining this state.

  Hereinafter, the target cell detection system according to the present invention will be described with reference to the drawings, and then the target cell detection method according to the present invention will be described by using the system as an example.

<Target cell detection system>
The target cell detection system according to the present invention includes a cell expansion substrate having a cell detection region on the surface of which a cell suspension containing a smaller number of target cells to be detected than non-target cells, and the cell expansion. A cell development means for forming a single cell layer in a cell detection region of the substrate for use, and a first fluorescent label that is a complex of an antibody that specifically binds to the target cell and a first fluorescent dye, A first fluorescent labeling treatment means for fluorescently staining cells in the cell suspension or the expanded cell group, and a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye. (2) a second fluorescent labeling means for fluorescently staining cells in the cell suspension or the expanded cell group using a fluorescent label, and / or binding to both the target cell and the non-target cell If it is a 3A fluorescent dye Is a complex of an antibody that binds to both the target cell and the non-target cell and a 3B fluorescent dye, and a cell in the cell suspension or a cell after the expansion using a third fluorescent label A third fluorescent labeling processing means for fluorescently staining the group, an irradiation optical system capable of irradiating excitation light of a predetermined wavelength to the cell detection region of the cell development substrate, and a wavelength band of light from the cell detection region A light receiving optical system capable of separately receiving light, and control means for controlling at least the light receiving optical system and the irradiation optical system, wherein the control means at least uses the second fluorescent dye or the third A or the third B by the irradiation optical system. A focal position for irradiating a cell group developed on the cell detection region with excitation light of a fluorescent dye and focusing the non-target cell emitting second fluorescence or 3A or 3B fluorescence on the light receiving optical system Adjustment And irradiating the cells developed in the cell detection area with the excitation light of the first fluorescent dye by the irradiation optical system, and using the light receiving optical system as a reference with the focus position adjusted by the focus position adjusting means as a reference And target cell searching means for performing a process of searching for target cells emitting the first fluorescence.

  FIG. 1 shows an example of a target cell detection system. The target cell detection system 100 includes a cell deployment substrate 11, cell deployment means, a liquid feeding system mechanism 9 as first to third fluorescent labeling processing means, an irradiation optical system 6, a light receiving optical system 7, a control means 8 and the like. I have. In FIG. 1, reference numeral 13 denotes a reagent container, which stores the cell suspension CL, the first fluorescent label FL1, the second fluorescent label FL2, the 3A fluorescent label FL3A, and the thirdB fluorescent label FL3B. doing. The reagent container 13 is covered with a light shielding member to be shielded from light (not shown).

(Cell suspension)
A cell suspension is one in which there are fewer target cells to be detected than non-target cells that are not to be detected, and blood, urine, or other cells that may contain rare cells or other target cells. It can be prepared by diluting specimens such as lymph, tissue fluid and body cavity fluid, or pretreatments such as cell fractions and purified products obtained from these specimens with an appropriate solvent such as PBS. The cell suspension may be, for example, peripheral blood itself, or a leukocyte fraction separated from peripheral blood and diluted with an appropriate buffer (for example, phosphate buffered saline (PBS)). It may be. When this separation is performed, density gradient centrifugation using a medium for separating human lymphocytes (for example, Ficoll-paque (GE)) is preferable, and a blood cell layer containing lymphocytes separated under the plasma layer by centrifugation. From the above, the cell suspension can be prepared. The “rare cell” is a cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood.

  The cell suspension is prepared by dispersing rare cells or other cell lines of target cells or cell populations containing the target cells cultured for testing, research, etc. in PBS or the like. Also good. As a patient's blood cell model, a cell suspension obtained by adding a rare cell line such as CTC to a suspension of blood cells collected from a healthy person may be used.

  Cell suspensions that have undergone cell immobilization and protein removal treatment, that is, cells that contain target cells such as CTC that have been treated with a predetermined immobilizing agent, and that give a blocking effect are removed. Cell suspensions are suitable. The cell suspension includes those containing any of a first fluorescent label, a second fluorescent label, a 3A fluorescent dye, and a 3B fluorescent label described later.

<Cell development substrate>
The substrate for cell deployment may be a substrate capable of developing a cell suspension, and typically corresponds to the microchamber chip 11 of the cell recovery device 10 in the example shown in FIG. In the target cell detection system 100 illustrated in FIG. 1, the cell collection device 10 is provided by combining the microchamber chip 11 and the flow path forming member 12 with the seal member 12a maintaining airtightness, The liquid delivery system mechanism 9 introduces the cell suspension CL from the inlet 2 of the device 10 into the flow path 1 in the internal space, thereby expanding the cells in the cell suspension CL on the surface of the microchamber chip 11. It is configured to be able to.

  However, the cell development substrate that can be used in the present invention is not limited to such an embodiment, and a cell suspension can be developed on the surface to adsorb cells contained therein. Anything is acceptable. For example, the substrate for cell development may be the bottom surface of a slide glass or a petri dish for cell culture. In addition, the cell deployment substrate may not necessarily be used in a state in which both the flow path forming member 12 and the seal member 12a are combined and the flow path is formed. For example, only the seal member 12a is combined with the cell expansion substrate and opened. Alternatively, the cell suspension CL or the like may be dropped from the upper part to expand the cells.

(Material)
As will be described later, the cell group developed on the cell development substrate is collected on a predetermined surface area on the cell development substrate by a liquid flow or the like, and is used for cell observation. The detection region is preferably formed of an optically transparent member so that excitation light or fluorescence for observation can pass through the substrate from any direction of the substrate. In FIG. 1, the cell detection region of the cell development substrate 11 is indicated by the symbol CDR.

  Examples of the material for the cell development substrate include transparent polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane [PDMS], polymethyl methacrylate [PMMA], and cyclic olefin copolymer [COC]. The cell expansion substrate may be a combination of a plurality of materials obtained by bonding a molded polymer to a substrate made of metal, glass, quartz glass, or the like.

  The dimensions (vertical width, horizontal width) of the cell detection region are not particularly limited, but can be set based on the amount of cell suspension to be developed or the ease of deployment of the cell suspension. As a preferred example, there is an example in which the vertical width (length in the direction of the flow path 1 in FIG. 1) is set to 10 mm or more and the horizontal width is set to 50 mm or less.

(shape)
In the cell development substrate, the surface portion of the cell development substrate corresponding to the cell detection region may have a microchamber or unevenness. Examples of the cell deployment substrate include a cell deployment substrate 11 having a microchamber 5 as shown in FIG. 1, and a cell deployment substrate 11a having irregularities as shown in FIG.

(Micro chamber)
A microchamber refers to a concave minute hole that can “accommodate” and “hold” one or more cells, and preferably has a bottom. The shape of the microchamber is preferably an inverted conical shape with a flat bottom, but is not limited thereto. In the example shown in FIGS. 1 and 2, inverted frustoconical microchambers 5... Are provided. Here, the above-mentioned “holding” means that the cells contained in the microchamber are difficult to get out of the microchamber due to the passage of staining liquid or washing liquid through the flow path on the cell development substrate.

  The diameter of the upper opening of the microchamber is preferably 20 to 500 μm. When the diameter is in the range of 20 to 500 μm, cells can be suitably accommodated and held in the microchamber. The depth of the microchamber is preferably about 10 to 15 cells per microchamber. Typically, the depth of the microchamber is 10 to 250 μm. Although the number of microchambers is not particularly limited, an example in which 1000 to 30000 or more microchambers are formed in the cell detection region can be given.

(Unevenness)
When unevenness is provided on the cell development substrate, the interval between the concave and convex portions is not particularly limited, but from the viewpoint of storing and holding more cells, as illustrated in FIG. The substrate 11a is preferable. When the substrate for cell development is provided with irregularities, the depth of the concave portion (the height of the convex portion) is set so that the expanded cell layer is not more than two layers as much as possible and the target cell can be stored and held. It is preferable to do. Specifically, a depth of about 90% of the maximum particle size to the maximum particle size of the target cell is preferable.

The diameter (average diameter) of rare cells such as CTC is described in, for example, American Journal of Pathology, Vol. 156, no. 1, 57-63, January 2000, it is reported as follows. Optical microscope photographs are taken of multiple types of circulating cancer cells, and the average projected area of various circulating cancer cells (CTC) is measured to be 396 μm ² to 796 μm ² . Therefore, assuming that the shape of various circulating cancer cells is a sphere, the diameter of the circulating cancer cells is calculated to be 22 to 32 μm. The diameters of blood cells are shown in Table 1 below. Therefore, the depth of the concave portion or the height of the convex portion of the cell development substrate is preferably about 20 to 30 μm.

<Cell expansion means>
The cell expansion means is a means for forming a single cell layer in the cell detection region of the cell expansion substrate.
As illustrated in FIGS. 1 to 3, when the cell deployment substrate 11 has the microchamber 5 or the unevenness, the cell deployment means uses a liquid flow for monolayering two or more cell layers. It is desirable to have a function of feeding liquid to be generated. This is because if the cell suspension CL is spread on the microchamber 5 or the cell development substrate 11 (or 11a) having irregularities, the cells are stacked in the microchamber 5 or the recess. This is because it is difficult for the cell scraper 14 (see FIG. 4) to form a single cell layer.

  In the example of FIG. 1, the liquid feeding system mechanism 9 corresponds to the cell deployment means, and as shown in FIGS. 2 (A) to (C), the liquid feeding system 9 makes the liquid flow for monolayering the cells. It is desirable to have a liquid-feeding function that can generate cells and make cells monolayered.

  The liquid feeding system mechanism 9 may be controlled by a control program stored in the control means 8 to generate a liquid flow for making the cells monolayer, and separately, for making the cells monolayer. It may be mechanically configured (pipette or the like) so as to be able to generate a liquid flow, and may be operated by a user. Furthermore, the liquid feeding system mechanism 9 includes a liquid feeding mechanism and a supply tank as disclosed in Japanese Patent Application Laid-Open No. 2012-000065 as long as it can generate a liquid flow for monolayering the cells. It may be.

(Cell monolayer by liquid flow)
The liquid flow for making the cells into a single layer is a liquid flow that moves at least the expanded cells, and specifically, the flux (the amount of fluid that flows per unit area of the channel cross section per unit time) is It is desirable to be (1 mm / min) to (10000 mm / min). The liquid flow may be a liquid flow of the cell suspension CL itself or an isotonic liquid (physiological saline) different from the cell suspension CL.

(Single layer by cell scraper)
On the other hand, as illustrated in FIG. 4, even when the cell expansion substrate is flat, the cells are adhered and stacked, so that the cell expansion scraper is used as a cell expansion means. The layer can be monolayered. The method of making a single layer in this way is called a gap coat method, and as described in the example of FIG. 4, after the cell suspension CL is dropped onto the cell deployment substrate 11, the upper surface of the cell deployment substrate 11 is then used. On the other hand, a bar-shaped cell scraper 14 (blocked) having a certain gap (coating gap) is slid (slid in the direction of the white arrow in FIG. 4).

  This coating gap is preferably about 1 to 3 times the maximum diameter (22 to 32 μm in the CTC example) of the target cell (eg CTC) (about 20 to 100 μm in the CTC example). Further, the coating gap may be set to the average particle diameter of the target cell to 90% thereof. The cell scraper 14 is configured to be reciprocally movable along the surface of the cell deployment substrate 11 (typically in the horizontal direction).

(Single layer by wire bar)
Another method for forming a single cell layer includes a wire bar method. For example, this method uses a wire having a thickness of about twice the maximum particle size to the maximum particle size of the target cell (eg, when the target cell is CTC, 1 to 2 times the maximum particle size of 22 to 32 μm of CTC). Specifically, a 32-65 μm wire (e.g., 0.05 or 0.08 nylon fiber) was wound around a cell scraper having a round bar portion made of glass, for example, and subjected to blocking treatment. Thereafter, the stacked cells that have been slid and developed in contact with the upper surface of the cell development substrate 11b are made into a single layer.
In addition to the method described above, the stacked cells may be monolayered using a known method capable of monolayering of cells.

<First fluorescent labeling means>
The first fluorescent labeling means includes an antibody that specifically binds to a target cell, a first fluorescent dye, and a cell group contained in a cell suspension or a cell group developed on the surface of a cell development substrate. It is a means to perform the process which makes the 1st fluorescence label | marker body which is a composite_body | complex contact.

  In the example of FIG. 1, the first fluorescent label processing means corresponds to the liquid feeding system mechanism 9 that performs operation control by the user or the control means 8 and performs a process of bringing the first fluorescent label into contact with the cell group. In the example of FIG. 1, the liquid feeding system mechanism 9 as the first fluorescent labeling processing means sucks the solution containing the first fluorescent label FL1 stored in the reagent container 13 and similarly puts it in the reagent container 13. The first fluorescent label FL1 is brought into contact with a cell group contained in the cell suspension CL before deployment, or the cell group already deployed on the cell deployment substrate 11 1 Solution containing fluorescent label FL1 is fed.

[First fluorescent label]
The first fluorescent label is a complex of an antibody that specifically binds to a target cell such as a rare cell to be detected and a first fluorescent dye. This antibody is an antibody that specifically recognizes an antigen that is specifically expressed in the target cell, and the target specific antigen varies depending on the target cell (see Table 2 below). The first labeled phosphor is preferably dissolved in an isotonic solution (PBS or the like).

  For example, when the target cell is CTC, an anti-cytokeratin antibody is preferably used as the antibody. When the target cell is CEC, an anti-CD34 antibody is suitably used as the antibody. When the target cell is CEP, an anti-CD34 antibody, an anti-CD133 antibody, or an anti-VEGFR-2 antibody is preferably used as the antibody (see Table 2). As long as the antibody specifically binds to the target cell, the antibody is not limited to those exemplified in Table 2.

(First fluorescent dye)
The first fluorescent dye is a fluorescent dye used for labeling a target cell as a detection target, and the fluorescence wavelength thereof is a second fluorescent dye described later used for labeling a cell other than the detection target (non-target cell), or The 3A and 3B fluorescent dyes used for labeling both target cells and non-target cells are those in which the fluorescence wavelengths do not overlap.

  What can be used as a 1st fluorescent dye is not specifically limited, An organic fluorescent dye or an inorganic fluorescent dye can be used. Organic fluorescent dyes include, for example, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 546 , Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750.

<Second fluorescent labeling means>
The second fluorescent labeling treatment means includes an antibody that specifically binds to a non-target cell and a second fluorescent dye for the cell group contained in the cell suspension or the cell group developed on the surface of the cell development substrate. It is a means to perform the process which makes the 2nd fluorescent label which is a composite_body | complex contact.

  In the example of FIG. 1, the second fluorescent label processing means corresponds to the liquid feeding system mechanism 9 that is controlled in operation by the user or the control means 8 and performs a process of bringing the second fluorescent label into contact with the cell group. In the example of FIG. 1, the liquid feeding system mechanism 9 as the second fluorescent labeling processing means sucks the solution containing the second fluorescent label FL2 contained in the reagent container 13 and similarly puts it in the reagent container 13. It is mixed with a cell suspension CL before development and the second fluorescent label FL2 is brought into contact with the cell group contained therein, or the cell group already developed on the cell development substrate 11 2 A solution containing the fluorescent label FL2 is fed.

[Second fluorescent label]
The second fluorescent label is a complex of an antibody that specifically binds to a non-target cell such as a white blood cell that is not detected and a second fluorescent dye. The first labeled phosphor is preferably dissolved in an isotonic solution (PBS or the like).

  Antibodies that can be used as the above-mentioned antibodies are those that specifically recognize antigens that are specifically expressed in non-target cells, and the specific antigens differ depending on the type of non-target cells. Different (see Table 3 below). When the non-target cells are leukocytes, an antibody that specifically recognizes leukocytes such as an anti-CD45 antibody is preferably used as the antibody. When the non-target cell is a red blood cell, an anti-D antibody is preferably used as the antibody. In addition to this, for example, according to the Beckman Coulter CD classification chart, an antigen can be appropriately selected according to the purpose (reference URL http://www.bc-cytometry.com/CD_Chart.html).

(Second fluorescent dye)
The second fluorescent dye is a fluorescent dye used for labeling a non-target cell that is not a detection target, and the fluorescence wavelength thereof is the first fluorescent dye used for labeling a detection target cell (non-target cell), or The 3A fluorescent dye and the 3B fluorescent dye used for labeling both target cells and non-target cells are preferably those that do not have overlapping fluorescence wavelengths. As the second fluorescent dye, a fluorescent dye whose fluorescent wavelength partially overlaps with the first fluorescent dye, the third A fluorescent dye, or the 3B fluorescent dye may be used. In that case, a fluorescent filter is used in the light receiving optical system. It is preferable to receive the light by cutting the overlapping fluorescent wavelength band.

  What can be used as a 2nd fluorescent dye is not specifically limited, For example, it can select and use from the organic fluorescent dyes enumerated as a 1st fluorescent dye. In addition, a common fluorescent dye may be used as a non-target cell.

<Third fluorescent labeling means>
The third fluorescent labeling means is a fluorescent dye (third 3A) that binds to both non-target cells and target cells with respect to the cell groups contained in the cell suspension or the cell groups developed on the surface of the cell expansion substrate. Fluorescent dye) or a means for performing a treatment of contacting a third fluorescent label, which is a complex of an antibody that binds to both non-target cells and target cells, and a fluorescent dye (third B fluorescent dye).

  In the example of FIG. 1, the third fluorescent label processing means is controlled by the user or the control means 8, and a liquid feeding system mechanism 9 that performs a process of bringing the 3A fluorescent dye or the 3B fluorescent label into contact with the cell group. Is applicable. In the example of FIG. 1, the liquid feeding system mechanism 9 as the third fluorescent labeling processing unit sucks the solution containing the third A fluorescent dye FL3A or the thirdB fluorescent label FL3B stored in the reagent container 13, Similarly, the 3A fluorescent dye FL3A or the 3B fluorescent label FL3 is brought into contact with a cell group contained in the cell suspension CL before expansion in the reagent container 13 or already expanded. A solution containing the 3A fluorescent dye FL3A or the 3B fluorescent label FL3 is sent to the cell group developed on the substrate 11.

[3A fluorescent dye]
The 3A fluorescent dye is a fluorescent dye that binds to a cell organ that exists in common in both non-target cells and target cells.

  For example, when the target cell is a rare cell such as CTC and the non-target cell is a cell other than the rare cell (eg, white blood cell), the 3A fluorescent dye is a cell in which the stained object has a nucleus ( A Hoechst fluorescent dye or DAPI that binds to the cell nucleus of the non-target cell and the target cell, which is used for discriminating that it is not a cell fragment or the like having no nucleus (see Table 4 below). ).

[3B Fluorescent Label]
The third fluorescent label is a complex of an antibody that binds to an antigen that is commonly expressed in both target cells and non-target cells and a third B fluorescent dye. The 3B fluorescent label is preferably dissolved in an isotonic solution (PBS or the like). For example, an antibody that binds to an antigen that is commonly expressed in both non-target cells and target cells is fluorescently stained as a standard for standardizing the intensity of various measured fluorescence. An antibody against a housekeeping gene product (cytochrome c, actin, etc.) that is expressed in a typical manner is preferably used (see Table 4 below).

(3A, 3B fluorescent dye)
When Hoechst fluorescent dye is used as the 3A fluorescent dye, for example, Hoechst 33258 and Hoechst 33342 can be used. The 3B fluorescent dye is a fluorescent dye used for labeling both target cells and non-target cells, and the fluorescence wavelength thereof is the first fluorescent dye used for labeling cells to be detected (non-target cells). Alternatively, the third fluorescent dye used for labeling both the target cell and the non-target cell preferably has a fluorescence wavelength that does not overlap. As the 3B fluorescent dye, a fluorescent dye whose fluorescent wavelength partially overlaps with the first fluorescent dye, the second fluorescent dye, or the 3A fluorescent dye may be used. In that case, a fluorescent filter is used in the light receiving optical system. It is preferable to receive the light by cutting the overlapping fluorescent wavelength band.

<Irradiation optics>
The irradiation optical system can irradiate the cell detection region of the cell development substrate with light in a wavelength band that excites the aforementioned fluorescent dyes. Here, an optical filter for cutting light other than the excitation wavelength may be provided on the optical axis of the irradiation optical system. In the example of FIG. 1, an irradiation optical system 6 and a light receiving optical system 7 are provided above the cell collection device 10 (see FIGS. 1 and 5). As shown in FIG. 5, the irradiation lens group 20, the dichroic mirror 17, and the objective lens 18 are arranged on the optical path of the excitation light from the light irradiation device (light source) 15 to the cell deployment device 10. The excitation light emitted from the light irradiation device (light source) 15 is directed toward a single-layer cell developed in the cell deployment device 10 through the irradiation lens group 20, the dichroic mirror 17, and the objective lens 18. Focused. On the other hand, the objective lens 18, the dichroic mirror 17, the fluorescent filter 19, and the lens 16 are arranged on the optical path of fluorescence from the cell deployment device 10 to the imaging unit 21 (the one-dot chain line in the vertical direction in FIG. 5). They are arranged in this order from the 10 side. Fluorescence (at least one of the first to third B fluorescence) emitted from the cell deployment device 10 passes through the objective lens 18, the dichroic mirror 17, and the fluorescence filter 19, and then is imaged as a light detection unit by the lens 16. An image is formed on a light receiving surface (light receiving element such as a CCD) of the unit 21. As the fluorescent filter 19, for example, an excitation light cut filter, a neutral density filter, or the like is used. Among these, the excitation light cut filter improves the S / N ratio by blocking excitation light and external light. On the other hand, the neutral density filter adjusts the fluorescence intensity according to the light detection unit. Note that unnecessary wavelengths may be removed by the dichroic mirror 17.

  The cell deployment device 10 can be moved and adjusted in the X-axis direction and the Y-axis direction by moving the cell collection device holder 26 installed. When the position is adjusted by moving in the Z-axis direction, the cell deployment device 10 is moved (moved up and down in FIG. 5), or the objective lens 18A is moved on the optical axis by an actuator (not shown). Move back and forth to adjust.

<Control means>
The control means includes a focus position adjusting means for focusing the light receiving optical system, and a target cell searching means for performing a process of searching for a target cell with reference to the adjusted focus, and optionally includes a focus position correcting means described later. Have. Here, the control means may be executed automatically by the program itself or by the program.

  In the example shown in FIG. 1, a program for adjusting the focal position of the light receiving optical system is stored as a part of the control means 8, and this program (executable body) 23 corresponds to the focal position adjustment means. Similarly, a control program for searching for a target cell is stored as a part of the control means 8, and the second program (executable body) 24 corresponds to the target cell search means.

(Focus position adjustment means)
The focal position adjusting means irradiates the cell group developed in the cell detection region with the wavelength light of the second fluorescent dye or the 3A or 3B fluorescent dye by the irradiation optical system, so that the second fluorescence or the 3A or 3A This is a means for performing processing for focusing on non-target cells emitting 3B fluorescence.

  In the example shown in FIGS. 1 and 5, the program 23 serving as the focal position adjusting means receives the second fluorescence by the light receiving optical system 7 by replacing the fluorescent filter 19 in the light receiving optical system 7. After that, the above-described position adjustment is performed via an actuator (not shown), and as a result, the second fluorescence is developed into the cell detection region CDR on the cell deployment substrate 11 (or 11a) and is made into a single layer. Processes to focus on the non-target cells.

  Further, after the focal position adjusting means obtains focal length information by focusing on the non-target cell in each of the following means, that is, two or more visual fields at different positions of the cell detection region, these focal lengths are obtained. Are different from each other, an approximate expression representing a change in focal length in another field other than the two or more fields is obtained from the difference, and in each of the other fields, the focal length is calculated from the approximate expression. Thus, a means (focal length information complementing means) for setting the focal length of the light receiving optical system to the calculated focal length may be provided.

  In the example of FIG. 1, a program for complementing focal length information for a predetermined visual field is stored as a part of the control unit 8, and this program 25 corresponds to the focal length information complementing unit. The details will be described in the focus position adjustment step described later. To explain with the example shown in FIG. 6, first, after the focus position adjustment means focuses on the non-target cell in the visual field 1 and obtains information on the focus A. Similarly, a process for examining whether or not a non-target cell can be detected in a visual field other than the visual field 1 (for example, an adjacent visual field 2 in order to reduce operation loss) is performed. When the cell detection region (including the portion shown in FIG. 6) of the cell deployment substrate 11 is inclined with respect to the horizontal plane due to a dimensional error or tolerance at the manufacturing stage, the focal length differs between the visual fields 1 to 4. In the fields other than the field 1 (fields 2 to 4 in FIG. 6), the non-target cell is not detected at the focal length A at all. The same applies to the other visual fields 2 to 4 other than the visual field 1. In this case, the focal position adjusting means obtains focal lengths B and D in the visual fields 2 and 4, for example, and an approximate expression indicating the focal length from the focal information (focal lengths A, B and D) in the visual fields 1, 2 and 4 Based on this approximate expression, information on the focal length C of the non-target cell in the remaining visual field (the visual field 3 between the visual fields 2 and 4 in the example of FIG. 6) is calculated, and an appropriate value in each visual field is calculated. Set the focus position. In this case, the target cell search means to be described later refers to the information on the proper focal position calculated and set for each visual field, and the target cell search means selects the target cell for each visual field based on the proper focal position of each visual field. It is desirable to search.

  Note that the order of visual fields for confirming the presence / absence of detection of non-target cells is not limited, and the order of visual fields to be focused by the user so as to cover the entire cell deployment substrate is set (for example, in FIG. As shown, the point of view A → B → C and C → I → G are set so as to cover the entire cell detection region of the cell deployment substrate), and focus information for creating an approximate expression is obtained. It may be obtained. If the order of visual fields to be confirmed is set so as to cover the entire cell deployment substrate, even if the cell deployment substrate is partially distorted, the distortion is easily detected. Further, when the cell development substrate is distorted or the like, the focus information for creating the approximate expression may be two or more, and is not limited to the case based on the three focus information. Moreover, as long as the visual field for obtaining the focus information is not the same visual field position, a part of the visual field may overlap each other so that there is no omission of imaging.

  It is desirable that the visual field for setting the focus information calculated from the approximate expression exists between the visual fields obtained by examining the focal length, and the number of visual fields to be set may be two or more. In addition to this, for example, when the cell deployment substrate is made of resin and the cell detection region is distorted in a curved surface, it can be approximated by another approximate line such as a multi-order curve. Based on the above, the focal length may be calculated and set as described above.

(Target cell search means)
The target cell search means irradiates the cells developed on the cell detection region with the excitation light of the first fluorescent dye by the irradiation optical system, and uses the light receiving optical system based on the focus position adjusted by the focus position adjustment means. This is means for performing a process for searching for a target cell emitting the first fluorescence in the system. Here, “to be used as a reference” may be used while keeping the focal position obtained by the focal position adjusting means as it is, or may be the focal position after being corrected by the focal position correcting means. This means that at least the focus position set by focusing (re-focusing) on the target cell is not used.

  As an example of “processing to search for target cells”, information obtained by a light receiving optical system (eg, microscope) may be displayed on a monitor for the user, or the user is encouraged to perform visual observation with a microscope. Alternatively, the image photographing process may be performed for the entire visual field (continuously or integrally). Further, for each pixel in the captured image, processing may be performed so as to detect the target cell by performing a comparison operation with reference pixel information (luminance, color) obtained when the target cell is captured. The “processing for searching for target cells” is preferably performed continuously for each visual field when processing a plurality of visual fields.

  The program 24 being executed as the target cell search means scans the cell development substrate by irradiating the excitation light of the first fluorescent dye with the light receiving optical system 7 focused on the non-target cell. And a process for searching for a target cell emitting the first fluorescence. Here, for example, when the target cell is detected, the target cell search means may perform a process of displaying the fact on the display unit.

  The target cell search means further takes an image of the cell detection region by setting the light receiving optical system 7 to receive the second fluorescence immediately after the light receiving optical system 7 is focused by the focus position adjusting means. Two-cell image acquisition means, and / or third cell image acquisition means for imaging the cell detection region with the light-receiving optical system 7 in a state capable of receiving the 3A or 3B fluorescence, and the light-receiving optical system 7 A first cell image acquisition unit that captures the cell detection region in a state in which the fluorescence of 1 can be received, and the images captured by the first cell image acquisition unit and the second cell image acquisition unit are superimposed and synthesized. Image forming means for performing the processing.

  In the case of the example shown in FIG. 1, the program 24 as the target cell search means replaces the fluorescence filter 19 to make the light receiving optical system 7 capable of receiving the second fluorescence, and then the cell deployment substrate. 11 is irradiated with the excitation light of the second fluorescent dye and the second image acquisition means for imaging the cell detection region CDR on the cell development substrate 11 as described above. First cell image acquisition means for imaging the cell detection area with the excitation light of the first fluorescent dye in the state in which the optical system 7 can receive the first fluorescence, and images obtained by each imaging Image forming means for synthesizing the image. The synthesis here is a process of synthesizing a cell image so that, for example, fluorescent bright spots derived from a target cell and a non-target cell are simultaneously displayed in one image. The same applies when the 3A fluorescent dye and the 3B fluorescent dye are used instead of the second fluorescent dye.

(Focus position correction means)
The focal position correction means is generated when the light after passing through the optical filter provided in the light receiving optical system changes from the second fluorescence or the third A or the third fluorescence to the first fluorescence. It is means for calculating the amount of deviation of the focal position and correcting the focal distance adjusted by the focal position adjusting means.

  Even in the same light receiving optical system, since the refractive index changes when the wavelength of the received fluorescence changes, chromatic aberration that changes the focal position for each wavelength occurs. Therefore, the light received by the light receiving optical system is a first fluorescence emitted from the first fluorescent dye used for labeling the target cell, a second fluorescence emitted from the second fluorescent dye used for labeling the non-target cell, When the wavelength of the fluorescence received by changing between the 3A fluorescence or the 3B fluorescence emitted from the third fluorescent dye used for labeling the target cell and the non-target cell is changed, the light receiving optics adapted to the non-target cell The focus position of the system is shifted. Therefore, the focal position correction means calculates the focal position deviation amount based on a known focal length calculation formula, and adjusts the focal position so as to eliminate the focal position deviation quantity in advance according to the wavelength of fluorescence to be used next. Perform the process of adjusting the position. This adjustment process includes a process of moving the object together with the cell deployment substrate to a focal position after shifting, or a lens position of the light receiving optical system is moved to adjust the focus position of the light receiving optical system to the non-target cell. Process.

<< Detection method of target cells >>
The target cell detection method according to the present invention comprises the following cell expansion step, first fluorescent labeling step, and fluorescent labeling step (second fluorescent labeling step and / or third fluorescent labeling step). A target cell detection method including random order, which includes a focus position adjustment step and a target cell search step (see FIG. 7). Further, it may optionally include a focus position correction step described later.

  Hereinafter, the method for detecting a target cell according to the present invention will be described step by step including an example in which the target cell detection system 100 is used.

[Cell expansion process]
In the cell expansion step, a cell suspension containing a smaller number of target cells to be detected than non-target cells is developed on at least the cell detection region on the surface of the cell expansion substrate, and a single layer is formed on the cell detection region. It is a step of forming a cell layer, and is performed in any order in the order of the first fluorescent labeling step, the second fluorescent labeling step, or the third fluorescent labeling step of the fluorescent labeling step (see FIG. 7).

Two specific examples of the cell expansion step shown in FIGS. 8A and 8B are shown.
(Transfer and development of cell suspension: Step 1A-1)
In Step 1A-1, the liquid suspension system 9 (see FIG. 1) is operated by an operation command from the control means 8 or a user operation, and the cell suspension stored in one container of the reagent container 13 After the CL is sucked up, the cell suspension CL is fed to the cell development substrate 11 (or 11a) and the cell suspension CL is developed in the cell detection region CDR. Here, when the cell group does not fit in the cell detection region CDR, an isotonic solution (PBS or the like) is arbitrarily transferred to the cell detection region CDR by a predetermined liquid feeding described later. In this process, when the surface of the cell detection region CDR of the cell development substrate has a microchamber 5 for storing cells or unevenness, the cell is moved into the microchamber 5 or the recess. It is good.

  As the above-mentioned predetermined liquid feeding method, an isotonic solution is intermittently fed to the cell development substrate 11 to move the cells into the cell detection region (for example, after feeding 1 to 50 μL, 5 to 15 seconds) Liquid feeding method in which the liquid feeding stop is repeated as one set), the liquid feeding direction of the liquid feeding system mechanism is reversed at a predetermined interval, and the cell is moved into the cell detection region, and the cell deployment substrate 11 is physically used. (In FIG. 1), a method of moving the cell into the cell detection region by tilting it to the right or left downward, a part of the cell suspension CL on the cell deployment substrate 11, for example, from downstream to upstream (or downstream) The cell is moved to the cell detection region by recirculation (upstream from the upstream) (e.g., the cell-free supernatant accumulated in the reservoir 4 from the outlet 3 to the inlet 2 is partially refluxed to the inlet 2). For cell deployment) Method of moving the cells by applying a centrifugal force to the plate 11 to the cell detection area, the method further combination of these, and the like. Note that various liquid supply conditions such as a liquid supply amount and a stop time in a predetermined liquid supply can be arbitrarily changed in consideration of a cell position, a movement amount, and the like.

(Liquid feeding / development of cell suspension: Step 1A-1 (FIG. 8B))
In Step 1B-1, the liquid suspension system 9 (see FIG. 1) is operated by an operation command from the control means 8 or a user operation, and the cell suspension stored in one container of the reagent container 13 After the CL is sucked up, the cell suspension CL is dropped from above the cell development substrate 11b to develop the cell suspension CL in the cell detection region CDR.

(Cell monolayer formation by liquid flow; Step 1A-2 (FIG. 8A))
In step 1A-2, as shown in FIGS. 2A to 2C, the cell deployment means or the user controls the liquid feeding of the liquid feeding mechanism to generate a liquid flow for making the cells monolayered. To make the cells monolayer. When the user himself / herself delivers the fluid, the fluid delivery mechanism (in this case, a pipette, etc.) is mechanically controlled so that a fluid flow is generated to make the expanded cell layer monolayer. It may be configured. When the surface of the cell detection region of the cell deployment substrate 11 has a microchamber 5 for storing cells or unevenness, the cell scraper 14 or the like is used to make the monolayer by liquid flow. Alternatively, it is difficult to monolayer the cells stacked in the microchamber 5.

(Monolayerization with a cell scraper; Step 1B-2 (FIG. 8B))
In Step 1B-2, the cells are monolayered by the cell scraper. The cell deployment means controls the operation of the cell scraper 14 as shown in FIG. 4 and translates the cell scraper 14 along the surface of the cell deployment substrate 11, whereby the surface of the cell deployment substrate 11. The process which makes 2 or more cell layers expand | deployed in (1) into a single layer is performed.

(Confirmation of cell monolayer; Steps 1A-3 and 1B-3 (FIGS. 8A and 8B))
Thereafter, in Steps 1A-3 and 1B-3, it is confirmed whether or not the cells are monolayered. If the cell is monolayered and Yes, the process proceeds to the next step. If the cell is not monolayered, the process returns to Step 1A-2 and Step 1B-2, and the cell monolayer is again formed. Processing is performed. Confirm that the cells are monolayered, for example, by confirming that there is virtually no non-target cell overlap (remains at an acceptable level of overlap) in bright field observation or fluorescence observation. Can do. This confirmation may be performed by automatically determining whether or not the non-target cell overlap is substantially seen in the real-time image obtained by the above-mentioned observation by the program.

(Confirmation of fluorescent labeling process; Steps 1A-4 and 1B-4 (FIGS. 8A and 8B))
In Steps 1A-4 and 1B-4, it is confirmed whether or not the phosphor labeling process is performed. If the phosphor labeling process is performed and the answer is Yes, it is stored in the storage means that the cell expansion process has been performed. After recording, etc., the process shifts to the focal position adjustment process, and when the phosphor labeling process is not performed, the process proceeds to the fluorescence labeling process.

[Fluorescent labeling process]
The fluorescent labeling step includes (1) using the first fluorescent label, which is a complex of an antibody that specifically binds to the target cell and the first fluorescent dye, to develop the cells in the cell suspension CL or the development. A first fluorescent labeling step of fluorescently staining a subsequent cell group, and (2) a second fluorescent label that is a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye, A second fluorescent labeling step of fluorescently staining cells in the cell suspension CL or the expanded cell group, and (3) a 3A fluorescent dye that binds to both the target cells and the non-target cells. Or a cell in the cell suspension CL or after the expansion using a third fluorescent label, which is a complex of an antibody that binds to both the target cell and the non-target cell and a third B fluorescent dye. A third fluorescent labeling process for fluorescently staining the cell population of Constructed. In addition, since it is as having mentioned above about the detail of the 1st-3rd B fluorescent label, the description is abbreviate | omitted.

FIG. 9 shows an example of the fluorescent labeling process.
(Confirmation of cell suspension development; Step 2A-1 (FIG. 9))
In the example of FIG. 9, first, in step 2A-1, it is determined whether or not the cell suspension CL has already been developed. If the cell suspension CL is expanded and Yes, the process proceeds to Step 2A-2, and if the cell suspension CL is not expanded and No, the process proceeds to Step 2A-5.

(Light-shielding treatment; Step 2A-2)
In step 2A-2, a light shielding process is performed to shield the entire cell collection device 10 including the cell development substrate 11 (or 11a, 11b) on which the fluorescent labeling process is performed. For example, the control means 8 performs an operation of closing the opening of the light shielding box that covers the entire cell deployment substrate 11.

(Liquid feeding process of fluorescent label mixed solution; Step 2A-3)
In Step 2A-3, the first fluorescent dye label (FL1) that specifically binds to the target cell, the second fluorescent label (FL2) that specifically binds only to the non-target cell, and / or the target cell and the non-target A liquid mixture containing a 3A fluorescent dye (FL3A) or a 3B fluorescent label (FL3B) exemplified by a nuclear staining reagent that binds to both cells is fed to the cell development substrate 11 protected from light. Here, each fluorescent labeling body or the like may be individually sent in random order (the first to third labeling process steps may be performed individually in random order), but the fluorescent dyes are discolored. From the viewpoint of reducing as much as possible, it is preferable to send the mixture simultaneously as a mixed solution.

(Binding reaction; Step 2A-4)
Finally, a binding reaction is performed in step 2A-4, and the first fluorescent label is bound to the target cell by this binding reaction, and the second fluorescent label, the third A fluorescent dye, or the third B fluorescent dye label is not used. Combine with target cells. Examples of the binding reaction include an example in which the reaction is performed for about 20 to 40 minutes in a dark room temperature. Then, the process proceeds to the focus position adjustment process.

(Fluorescent-labeled body feeding process; Step 2A-5)
On the other hand, when the cell suspension CL is not spread on the cell development substrate 11, in step 2A-5, the first fluorescent dye label, the second fluorescent label, the third A dye, and / or the first The 3B fluorescent label is mixed with the cell suspension CL that is shielded from light, and the process moves to the next step.

(Binding reaction; Step 2A-6)
In Step 2A-6, the binding reaction (eg, about 20 minutes to 40 minutes at room temperature in a dark place) is performed at a predetermined temperature and time with the mixing in Step 2A-5, and then the process proceeds to the cell expansion process. To do.

[Focus position adjustment process]
The focal position adjusting step irradiates the cell group developed on the cell detection region with the excitation light of the second fluorescent dye or the 3A or 3B fluorescent dye, and emits the second fluorescence or the 3A or 3B fluorescence. In this step, the light receiving optical system is focused on the non-target cells.

  The focal position adjustment step is performed by obtaining information on the focal length of each visual field by focusing on a non-target cell in each of the two or more visual fields at different positions in the cell detection region, When these focal lengths are different from each other, an approximate expression representing a change in focal length in a field other than the two or more fields is obtained from the difference. And the step of setting the focal length of the light receiving optical system to the calculated focal length may be included.

  For example, as shown in FIG. 10, when there are visual fields 1 to 100 obtained by partitioning the cell detection region and the focal information of the spots A to C in the visual field is 50300 μm, 50600 μm, and 51200 μm, linearity is obtained from these focal information. An example is provided in which an approximate expression is created, and focus information is calculated for the visual field between the visual fields A to C (the grid between AB and the grid between BC) from the approximate expression. Similarly, a linear approximation formula can be created from the focus information of the visual fields C to E, E to G, A to G, etc., and the focus information can be calculated and acquired in the same manner. Since the distortion of the cell deployment substrate 11 may not be uniform, the entire cell detection region shown in FIG. 10 is covered (for example, spots A to C, C to E, E to G, and G to C in the visual field). , H to D, respectively, create an approximate expression, calculate the focal length from the approximate expression for the visual field existing between the spots, and set the focal distance), and set the focal distance for each visual field in advance. It is desirable. Here, it may include a step of confirming whether or not non-target cells are detected for the visual field calculated from the approximate expression and set with the focal length.

FIG. 11 shows an example of the focal position adjustment process. Hereinafter, a flow when the substrate for cell deployment 11 without distortion and in which non-target cells can be confirmed in the entire cell detection region is subjected to the focal position adjustment step will be described with reference to FIG.
The total number of fields to be examined for the presence / absence of detection of non-target cells is “5”. The list array includes fields A to C as the first array, fields C to G as the second array, and coordinates (x , Y) are included (see FIG. 10), and the counter (m) is “0” in the initialized state.

(Reading information of list array; Step 3A-1)
First, in step 3A-1, the target cell detection system 100 reads information on a visual field for checking whether or not it is possible to focus on a non-target cell as a list (see Table 5 below).

Note that the processing order is performed from the young elements of the array.

(Focus processing; Step 3A-2)
Next, in step 3A-2, the first visual field to be scanned by the irradiation optical system and the light receiving optical system is selected from the read information, and after the scanning has moved to the visual field, the second fluorescent dye and / or the third A fluorescent dye Alternatively, a process of focusing on non-target cells (focusing process) is performed in a state where the cell group is irradiated with the excitation light of the 3B fluorescent dye. In the specific example of FIG. 11, the visual field A (0, 0) is selected, and the focusing process is performed with the visual field A (0, 0).

(Acquisition of focus information and information of field of view n; Step 3A-3)
Next, in step 3A-3, focus information and visual field information in the visual field are acquired. In a specific example, the focal length of the non-target cell in the visual field A (focal length A) = 50000 μm is acquired.

(Setting of focal length to be used; Step 3A-4)
Next, in step 3A-4, the focal length obtained in step 3A-3 is set as the focal length used in another field of view. In a specific example, the focal length to be used = focal length A is set. Further, the temporary counter (m) is incremented by one.

(Judgment of necessity of approximate expression creation; Step 3A-5)
Next, it is confirmed whether the counter (m) is equal to or greater than the number of array elements, and it is confirmed whether an approximate expression has been created. In the specific example, since the number of array elements of the visual field array [0] [] is 3 and the counter (m) = 1, the condition is not satisfied and the result is No, and the process proceeds to the next step.

(Move to the next predetermined field of view; Step 3A-6)
In step 3A-6, the scanning of each optical system is moved by obtaining information on the next field of view to be measured. In the specific example, the visual field B (0, 2) is selected, and the scanning of the irradiation optical system and the light receiving optical system is moved to the visual field B (0, 2).

(Detection of non-target cells in another visual field; Step 3A-7)
Next, in Step 3A-7, it is determined whether or not a non-target cell can be detected based on the “focal length to be used”. In the specific example, in the visual field (0, 2), it is determined whether or not non-target cells are detected at the focal length A set in the visual field (0, 0). Here, since the cell development substrate 11 is not distorted, a non-target cell is detected at the focal length A, and the determination is Yes, and the process proceeds to the next step.

(Confirmation of scanning status; Step 3A-8)
In step 3A-8, it is determined whether all fields in the array have been scanned. In the specific example, only two fields (field A (0, 0) and field B (0, 2)) of the fields (fields A to C) included in the field array [0] [] are examined. It will be judged No and will return to previous step 3A-6.

(Move to next predetermined field of view-detection of non-target cell in another field of view; Steps 3A-6 to 7)
And it moves to the visual field C (0, 4) which is not investigated, and it is confirmed whether a non-target cell is detected in the visual field C (0, 4). As described above, since the cell development substrate 11 is not distorted, non-target cells are confirmed even in the field of view C (0, 4).

(Confirmation of scanning status; Step 3A-8)
In step 3A-8, it is determined whether all the arrays in the list (list in the above table) have been examined. If not, the original array is returned to and the next array is processed in the same manner. In the specific example, since all the elements (fields A to C) of the visual field array [0] [] have been confirmed to be focused on the non-target cells, the process proceeds to the next target cell search step. Therefore, when there is no distortion in the cell deployment substrate, an approximate expression is not created and the process proceeds to the next target cell search step.

  Hereinafter, with reference to FIG. 11, a flow when a substrate having a distortion in the cell deployment substrate 11 unlike the above example is subjected to the focal position adjustment step will be described (FIG. 11). It is assumed that the initial conditions such as the counter (m) are the same as those described above.

(Reading array list information; Step 3A-1)
First, in step 3A-1, a visual field array (see Table 5 above) including information on the visual field for checking whether or not the target cell can be focused is read.

(Focus processing; Step 3A-2)
Next, in step 3A-2, the first visual field to be scanned by the irradiation optical system and the light receiving optical system is selected from the read information, and after the scanning has moved to the visual field, the second fluorescent dye and / or the third A fluorescent dye Alternatively, a process of focusing on non-target cells (focusing process) is performed in a state where the cell group is irradiated with the excitation light of the 3B fluorescent dye. In the specific example of FIG. 11, the visual field A (0, 0) is selected, and the focusing process is performed with the visual field A (0, 0).

(Acquisition of focus information and information of field of view n; Step 3A-3)
Next, focus information and visual field information in the visual field A are acquired. In a specific example, the focal length of the non-target cell in the visual field A is acquired, and the focal length (focal length A) in the visual field A is acquired at 50000 μm.

(Setting of focal length to be used; Step 3A-4)
Next, in step 3A-4, the focal length obtained in step 3A-3 is set as the focal length used in another field of view. In a specific example, the focal length to be used = focal length A is set. Further, the temporary counter (m) is incremented by one.

(Judgment of necessity of approximate expression creation; Step 3A-5)
Next, it is determined whether m is greater than or equal to the number of array elements, and it is confirmed whether an approximate expression has been created. Here, since the number of array elements of the visual field array [0] [] is 3, and m = 1, the condition is not satisfied and the result is No, and the process proceeds to the next step.

(Move to the next predetermined field of view; Step 3A-6)
In step 3A-6, the scanning of each optical system is moved by obtaining information on the next field of view to be measured. In the specific example, the visual field B (0, 2) is selected, and the scanning of the irradiation optical system and the light receiving optical system is moved to the visual field B (0, 2).

(Detection of non-target cells in another visual field; Step 3A-7)
Next, it is determined whether the non-target cell can be detected at the “focal length to be used”. In the specific example, it is determined whether or not a non-target cell is detected at the focal length A set in the visual field A (0, 0) in the visual field B (0, 2). In the specific example, since the cell development substrate 11 is distorted, the non-target cell is not detected at the focal length A, and it is determined as No, and the process returns to the focusing process in step 3A-2.

(Focus processing; Step 3A-2)
Next, in step 3A-2, a process of focusing on the non-target cell in the visual field B (0, 2) in which the non-target cell is not detected in step 3A-7 is performed.

(Acquisition of focus information and information of field of view n; Step 3A-3)
Next, in step 3A-3, focus information and visual field information in the visual field are acquired. In the specific example, the focal length of the non-target cell in the visual field B (focal length B) = 50400 μm is acquired.

(Setting of focal length to be used; Step 3A-4)
Next, in step 3A-4, the focal length B obtained in step 3A-3 is set as the focal length used in another field of view. In a specific example, the focal length to be used = focal length B is set. Further, the temporary counter (m) is incremented by 1, so that m = 2.

(Judgment of necessity of approximate expression creation; Step 3A-5)
Next, it is determined whether m is greater than or equal to the number of array elements, and it is confirmed whether an approximate expression has been created. Here, since the number of array elements of the visual field array [0] [] is 3, and m = 2, the condition is not satisfied and the result is No, and the process proceeds to the next step.

(Move to the next predetermined field of view; Step 3A-6)
In step 3A-6, the scanning of each optical system is moved by obtaining information on the next field of view to be measured. In the specific example, the field C (0, 4) is selected, and the scanning of the irradiation optical system and the light receiving optical system is moved to the field C (0, 4).

(Detection of non-target cells in another visual field; Step 3A-7)
Next, it is determined whether the non-target cell can be detected at the “focal length to be used”. In a specific example, in the visual field C (0, 4), it is determined whether or not non-target cells are detected at the focal length B set in the visual field B (0, 2). In the specific example, since the cell development substrate 11 is distorted, the non-target cell is not detected at the focal length B, and it is determined as No, and the process returns to the focusing process in step 3A-2.

(Focus processing; Step 3A-2)
Next, in step 3A-2, a process of focusing on the non-target cell in the visual field C (0, 4) in which the non-target cell is not detected in step 3A-7 is performed.

(Acquisition of focus information and information of field of view n; Step 3A-3)
Next, in step 3A-3, focus information and visual field information in the visual field are acquired. In the specific example, the focal length of the non-target cell in the visual field C (focal length C) = 50800 μm is acquired.

(Setting of focal length to be used; Step 3A-4)
Next, in step 3A-4, the focal length obtained in step 3A-3 is set as the focal length used in another field of view. In a specific example, the focal length to be used = focal length C is set. Further, the temporary counter (m) is incremented by 1, so that m = 3.

(Judgment of necessity of approximate expression creation; Step 3A-5)
Next, in step 3A-5, it is determined whether m is an array element or more, and it is confirmed whether an approximate expression has been created. Here, since the array element of the visual field array [0] [] is 3, and m = 3, the condition is judged Yes and the process moves to the next step 3A-10.

(Approximation formula creation, calculation and setting of focal length; steps 3A-10 to 11)
In step 3A-10, a linear approximation formula is created from the collected focal length information (not shown) of a plurality of points, and the focal lengths for different visual fields between the visual fields whose focal lengths are examined are calculated. In a specific example, a linear approximation formula: focal length f (y) = 200y + 50000 is created from the collected focal length information (50000 μm, 50400 μm, 50800 μm), and the focal length is between the visual fields examined. Focal lengths (50200 μm, 50600 μm) for different fields of view (field of view (0, 1), field of view (0, 3)) are set. In step 3A-11, the calculated focal lengths (50200 μm, 50600 μm) are set in the respective fields (0, 1) and fields (0, 3), respectively.

  Next, it is determined whether all the arrays in the list of Table 5 have been scanned. Since the visual field array [1] [] is not examined, each visual field of the array is examined in the same manner (an approximate expression is created in some cases), and then the process proceeds to a target cell search step.

[Focus position correction process]
The focal position correction step is caused when the light after passing through the fluorescence filter provided in the light receiving optical system changes from the second fluorescence or the 3A or 3B fluorescence to the first fluorescence. This is a step of calculating the amount of shift of the focal position and correcting the focal position adjusted in the focal position adjustment step, and is performed as necessary between the focal position adjustment step and the target cell search step described later.

In the case of an optical system with one lens, the focal length is expressed by the equation 1 / f = (n _c −1) (1 / r ₁ −1 / r ₂ ). Here, r ₁ is the curvature of one surface of the lens, r ₂ is the radius of curvature of the other surface of the lens, the n _c is the refractive index of the lens. Further, the composite focal length is expressed by 1 / f composite = 1 / f ₁ + 1 / f ₂ −d / f ₁ f ₂ . Here, d is the distance between the second principal point of the first lens and the first principal point of the second lens.

  Since the curvature of each lens of the eyepiece lens and objective lens of the light receiving optical system is constant, only the wavelength of the fluorescence passing through the light receiving optical system is changed without changing the distance between the principal points of the eyepiece lens and the objective lens The focal position of the light receiving optical system changes. For example, when the light passing through the lens changes from a red fluorescent dye to a blue fluorescent dye, the blue fluorescent light refracts more strongly, so the refractive index of the lens changes and the focal length of the light receiving optical system becomes shorter. Become. Similarly, chromatic aberration occurs when there are a plurality of lenses. Therefore, to adjust the focus, the position of the observation target on the optical axis is changed and moved to the focus position of the light receiving optical system after the change, or the change amount of the focus position is canceled. It is necessary to change the distance between the principal points of the lenses of the light receiving optical system.

  In the present invention, the light received by the light receiving optical system at the time of transition from the focal length adjustment step to the target cell search step is changed from the second fluorescence (or 3A fluorescence or 3B fluorescence) to the first fluorescence. Therefore, as described above, the focal length of the light receiving optical system changes (increases or decreases), and the focal position shifts. Therefore, in order to focus with the first fluorescence, the position of the observation target (cell group) on the optical axis is changed and moved to the changed focus position of the light receiving optical system, or the focal length is changed. Therefore, it is necessary to change the distance between the principal points of the lenses of the light receiving optical system so as to cancel out the change (increment and decrement). Therefore, by performing either of these processes between the two steps, even if the fluorescence received by the light receiving optical system is switched as described above, the target cell can be detected (the target cell). Is in focus).

[Target cell search process]
The target cell search step emits the first fluorescence with reference to the focal position adjusted in the focal position adjustment step while irradiating the cell group developed in the cell detection region with the excitation light of the first fluorescent dye. This is a step of searching for a target cell.

  In the target cell search step, for example, the light receiving optical system 7 is set in a state capable of receiving the first fluorescence, and the focus position set in the focus position adjustment step is used as a reference (a state in which the target cell can be detected (the focus on the target cell is In the combined state)), the target cell is searched for each field of view of the cell detection region and the bright spot of the target cell is detected to count the number of target cells, or the entire cell detection region or each field of view is described later. Thus, by acquiring each cell image (first cell image or the like) and determining whether each pixel of the image has the first fluorescent color, the presence or absence of the target cell is determined, and the target cell An example of measuring the number of

(Cell image acquisition process)
In the latter case of acquiring a cell image, the target cell search step further includes the following cell image acquisition step (at least a first cell image acquisition step), an image formation step, and the like immediately after the focal position adjustment step.

  FIG. 12 shows an example of the cell image acquisition process. This cell image acquisition step includes the following first cell image acquisition step and / or second cell image acquisition step or third cell image acquisition step.

  The second cell image acquisition step (step 4A-1) is a step of imaging the cell detection region with the light receiving optical system 7 in a state capable of receiving the second fluorescence, and the second fluorescence labeling step is performed. It is executed when Here, when the focal length is different for each visual field due to circumstances such as distortion in the cell deployment substrate 11 (or 11a), and the focal distance is set for each visual field in the focal position adjustment step described above. A second cell image is acquired for each field of view and focal length.

  The third cell image acquisition step (step 4A-2) is a step of imaging the cell detection region with the light receiving optical system 7 in a state capable of receiving the fluorescence of the third A and / or the third B, and the third fluorescent label. It is executed when a processing step is performed. Here, when the focal length is different for each visual field due to circumstances such as distortion in the cell deployment substrate 11 and the focal distance is set for each visual field in the above-described focal position adjustment step, similarly, A third cell image is acquired for each field of view and focal length.

  The first cell image acquisition step is a step of imaging the cell detection region with the light receiving optical system 7 in a state capable of receiving the first fluorescence. In this step, as described above, the cell image is acquired at the focal position adjusted to the non-target cell in the focal position adjusting step. Here, due to circumstances such as distortion in the cell deployment substrate 11, the focal length is different for each visual field, and is set when the focal distance is set for each visual field in the above-described focal position adjustment step. A first cell image is acquired at each focal length.

  The image forming step is a step of performing processing for superimposing and synthesizing the images captured in the first cell image acquiring step, the second cell image acquiring step, and / or the third cell image acquiring step.

  The image composition includes a reference point (for example, a mark provided in a cell detection region of the cell development substrate 11 and a first to third B fluorescent dyes that may be used) that serves as a mark when images are superimposed. After matching a specific shape (for example, a mark that emits light in a star shape) when excited with excitation light, the first cell image and the second and third cell images are compared in pixel units, For example, when the compared pixels are pixels having the same brightness (eg, a black portion common as the background), the luminance average of both pixels is taken to form one pixel at the compared position. In addition, in the case where one pixel is clearly brighter than the other pixel among the compared pixels, an example is considered in which processing for forming the one pixel portion is performed with a bright pixel.

[Analysis process]
The analysis step is a step of detecting a target cell in a cell image obtained by image synthesis, a first cell image, or a real-time image received by the first fluorescence. Specifically, for example, the first fluorescence for each pixel is obtained using pixel information that is a criterion for detecting the target cell, such as the color and brightness of the target cell obtained when the first fluorescence is imaged. There is an example in which the target cell is detected by checking whether or not the pixel exists. Here, when the detected pixels are adjacent and continuous, one island constituted by the continuous pixels is detected as one target cell.

Hereinafter, the operation and effect of the target cell detection method and target cell detection system according to the present invention will be described with reference to the drawings.
(1) A method for detecting a target cell comprising the above-described cell development step, first fluorescent labeling step, and second (or third) fluorescent labeling step in any order, wherein the second fluorescent dye or 3A or 3B A process of irradiating a cell group developed on the cell detection region with excitation light of a fluorescent dye and focusing the light receiving optical system 7 on a non-target cell emitting second fluorescence or 3A or 3B fluorescence. A focus position adjusting step to be performed; and irradiating a cell group developed in the cell detection region with the excitation light of the first fluorescent dye, and emitting the first fluorescence with reference to the focus position adjusted in the focus position adjusting step. And a target cell search step for searching for a target cell. Since the target cell and the non-target cell are almost at the same height on the cell development substrate 11, the target cell can be detected without focusing on the target cell by focusing on the non-target cell in advance. The state of the light receiving optical system at a possible focal position can be obtained. Therefore, the trouble of exciting the first fluorescent dye that labels the target cell and focusing on the target cell with the first fluorescence can be omitted. Therefore, the fading of the first fluorescent dye due to the work of focusing on the target cell can be suppressed. As a result, it is possible to eliminate the trouble of focusing on the target cell and suppress the fading of the first fluorescent dye. As a result, the detection rate of the target cell can be improved and the time until detection can be shortened.

  (2) If the target cell is a rare cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood, the rare cell that is hardly present in the blood is quickly detected. Can do. In addition, provision of data that contributes to early detection of diseases and the like related to rare cells can be achieved.

  (3) If the wavelength of the second fluorescence or the 3A or 3B fluorescence used for focusing in the focus position adjusting step does not overlap with the wavelength of the first fluorescence, only the target cell is the first. Therefore, the target cell can be detected without using a fluorescent filter for cutting the overlapping band, and the original amount of fluorescence emitted from the first fluorescent dye that labels the target cell can be obtained. Can be obtained. As a result, the target cell can be easily detected.

  (4) The target cell is CTC, the non-target cell is leukocyte, the first fluorescent label is an anti-cytokeratin antibody labeled with the first fluorescent dye, and the second fluorescent label Is an antibody that specifically recognizes leukocytes labeled with the second fluorescent dye, and / or the third fluorescent label is a Hoechst fluorescent dye that binds to cell nuclei. When CTC is contained in a sample such as blood, it is possible to detect CTC as soon as possible and to provide data that contributes to early detection of metastatic cancer associated with CTC.

  (5) The surface of the cell detection region of the cell expansion substrate is planar, and in the cell expansion step, two or more cell layers expanded on the planar surface are made into a single layer using a cell scraper. If the target cell and the non-target cell are arranged at the same height, the focus position where the target cell can be detected by focusing on the non-target cell. The state of the light receiving optical system is obtained (see FIGS. 4 and 14).

  (6) The surface of the cell detection region of the cell expansion substrate has a microchamber or irregularity for storing cells, and the cells stored by liquid flow in the cell expansion process are made into a single layer. For example, the cells stacked locally in the microchamber or in the recess can be removed to make the cell monolayer, and the removed cells can be placed in the microchamber or recess that does not store the cells. And a single cell layer can be formed uniformly over the entire area of the cell development substrate. As a result, by focusing on the non-target cell in the microchamber or in the recess, the state of the light receiving optical system at the focal position where the target cell can be detected is obtained (see FIGS. 2 and 13).

  (7) Between the focal position adjustment step and the target cell search step, the light after passing through the fluorescence filter provided in the light receiving optical system is the second fluorescence or the third A or the third B The light receiving optical system further includes a focus position correcting step of calculating a shift amount of the focus position caused by the change from the fluorescence to the first fluorescence and correcting the focus position adjusted in the focus position adjusting step. Even if the fluorescence passing through the lens is switched and the focal position of the light receiving optical system changes due to chromatic aberration, the correction is made and the focused state on the non-target cell (the focal position where the target cell can be detected) is maintained.

  (8) Immediately after the focal position adjustment step, the second cell image acquisition step of imaging the cell detection region with the light receiving optical system in a state capable of receiving the second fluorescence, and / or the light receiving optical system 7 A third cell image acquisition step of imaging the cell detection region in a state where the 3A or 3B fluorescence can be received, and the light receiving optical system can receive the first fluorescence as the target cell search step The first cell image acquisition step, the second cell image acquisition step, and / or the third cell image acquisition step and the first cell image acquisition step for imaging the cell detection region in a simple state are superimposed and synthesized. If the processing to be performed is included, the non-target cell and the target cell are displayed in the same image. Therefore, in the situation where only the target cells are picked up from the cell expansion substrate, the position of the target cells on the cell expansion substrate is referred to with reference to the synthesized image while the fluorescent dye that labels the non-target cells is excited. Can be easily grasped. As a result, the target cell can be picked up in a short time by shortening the time for exciting the first fluorescent dye labeling the target cell as much as possible.

(9) The focal position adjustment step is performed by obtaining information on the focal length of each visual field by focusing on a non-target cell in each of two or more visual fields at different positions of the cell detection region. When the distances are different, an approximate expression representing a change in focal length in another field other than the two or more fields is obtained from the difference, and in each of the other fields, the focal length is calculated from the approximate expression. Then, if the step of setting the focal length of the light receiving optical system to the calculated focal length is performed as the focal position adjusting step, the surface of the cell expansion substrate is inclined due to a manufacturing error or the like. Even if the focal length shifts for each field of view, it is not necessary to focus on non-target cells for all fields of view, reducing the effort to focus on non-target cells as much as possible. It can be. As a result, the fading of the second (or 3A or 3B) fluorescent dye can be suppressed.
Since the target cell detection system according to the present invention is a system that executes the above-described methods (1) to (9) as means, the effects described in (1) to (9) are achieved.

  Hereinafter, examples and comparative examples of the target cell detection method will be described with reference to the drawings.

<Preparation of cell suspension>
White blood cells obtained by isolation from peripheral blood by density gradient centrifugation were suspended in PBS, and a suspension A containing white blood cells at a predetermined concentration (> 10 ⁶ cells / mL) was prepared. Next, to this suspension A, CTC cells as cancer cells were added in a rare amount (white blood cell count ratio: 1/10000) (hereinafter referred to as suspension B). Next, the suspension B was subjected to cell tissue fixing treatment and first to third fluorescent labeling treatment steps described later. The white blood cell concentration of the suspension obtained through this step was adjusted to 10 ⁶ cells / mL (including 10 rare cells), and this was used as a cell suspension in the Examples and the like described later.

(Cell tissue fixation)
To the suspension B (1 mL), paraformaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) as a cell / tissue immobilizing agent is added to 4% and gently mixed, and the reaction is performed in the dark at room temperature for 15 minutes. I let you. To this reaction solution, a sufficient amount of phosphate buffered saline (PBS) is added and mixed. After centrifugation ((300) × g, (1) minutes), the supernatant is discarded and only the cells are recovered. A sufficient amount of PBS was added again. The washing process from the centrifugation process to the addition of PBS was repeated three times.

(First to third fluorescent labeling process)
1 mL of PBS containing 0.1% Tween for cell membrane permeabilization treatment, and anti-cytokeratin antibody (anti-CK antibody) labeled with Alexa Fluor 647 (first fluorescent dye) to label cancer cells (target cells) ( Micromet) (first fluorescent label) 10 μL solution and anti-CD45 antibody (Santa Cruz Biotechnology) labeled with Alexa Fluor 488 (Invitrogen) to label leukocytes (non-target cells) (second) 10 μL of the fluorescent label) solution was added to the suspension of the cells that had been washed by fixing the cell tissue and allowed to react at room temperature for 30 minutes with gentle mixing. For this purpose, 10 μL of a Hoechist (3A fluorescent dye) (manufactured by Invitrogen) solution was added and reacted.

  After the reaction, the above centrifugation treatment was performed to remove the first and second fluorescent labels and the 3A fluorescent dye not bound to the cells, and PBS was newly added and suspended again. Thereafter, the antibody reagent not bound to the cells was removed by centrifugation, and phosphate buffered saline (PBS) was newly added and resuspended.

[Example 1] (Detection of cells on microchamber 5)
The cell collection device 10 having the flow path 1 was produced by combining the flow path top plate (flow path forming member) 12 and the microchamber chip (substrate for cell deployment) 11. The microchamber chip 11 has 20000 microchambers 5 (chamber depth 50 μm, chamber diameter 100 μφ) formed in a 20 mm × 50 mm cell detection region (CDR). The width was 25 mm, the length was 70 mm, and the height was 10 μm.

  Next, a blocking solution (3% BSA-containing PBS) was fed from the inlet 2 of the cell recovery device 10 to the channel 1 at a high flow rate (16 mL / min). Thereafter, the blocking agent present in the flow channel 1 was removed from the flow channel 1 by feeding PBS to the flow channel 1. Thereafter, the cell suspension CL subjected to the first to third fluorescent labeling treatments is fed into the 100 μL channel 1 at a flow rate of 0.1 mL / min, and then intermittently fed (0.5 sec. (Liquid stop) was repeated 100 times to store the cells in the microchamber 5 and to form a single layer. Thereafter, the cells stored in the microchamber 5 are irradiated with excitation light (light having a wavelength of 488 nm) of the second fluorescent dye bound to the CD45 antibody bound to leukocytes at nine locations in the microchamber chip 11. However, the light receiving optical system 7 was focused on the non-target cell.

Thereafter, the excitation light was switched to CTC detection light (light with a wavelength of 635 nm), and the emission wavelength was made receivable, and the entire surface of the cell detection region CDR of the microchamber chip 11 was photographed (first cell image was acquired). ).
Next, the excitation light was changed to CD45 detection light (light with a wavelength of 488 nm), and the entire CDR surface was photographed (second cell image was acquired).
Next, the excitation light was changed to cell nucleus detection light (light having a wavelength of 350 to 352 nm), and the entire CDR surface was imaged (a third cell image was acquired).
Finally, the entire CDR surface was photographed with light (full wavelength) for bright field observation.

  Note that when the excitation light was switched, the focal position of the light receiving optical system 7 was adjusted so as to eliminate the amount of dye aberration (focal position shift amount) caused by the difference in each fluorescence wavelength. As a result of the determination, the time from the start of detection to the completion of detection was 54 minutes, and the detection rate of cancer cells was 90%. Note that the time from the start of detection to the completion of detection includes the time for focus adjustment.

[Example 2] (Detection of cells developed on a plane)
The above-described cells in which the first to third fluorescent labeling treatments are performed on the surface of the planar cell development substrate using a 25 mm × 50 mm planar substrate (cell development substrate) 11b instead of the microchamber chip 11 of Example 1. 100 μL of the suspension was dropped, and the cells were monolayered with a cell scraper 14. Then, while irradiating the cell nucleus of the Hoechst-stained cell group present on the cell development substrate with Hoechst (3A fluorescent dye) excitation light (light with a wavelength of 400 nm), for each imaging area (field of the light receiving optical system) The light receiving optical system 7 was focused on the cell nucleus of one cell included in the cell group.

  Next, the excitation light is switched to CTC detection light (light having a wavelength of 635 nm), the light receiving optical system is set to receive light having a light emission wavelength, and the focus position of the light receiving optical system is adjusted to the excitation wavelength of 400 nm. The entire surface of the cell detection region CDR of the cell development substrate was photographed by adjusting the focal position to a position approaching 20 μm on the optical axis (the first cell image was acquired).

Next, the excitation light is switched to the excitation light for detecting leukocytes (light having a wavelength of 488 nm) so that the light receiving optical system can receive light having the emission wavelength, and the focal position of the light receiving optical system is set to the excitation wavelength of 400 nm. The entire focus detection position was adjusted to a position approaching 20 μm on the optical axis, and the entire cell detection area of the cell development substrate was photographed (second cell image was acquired).
Then, the first and second cell images were instantaneously synthesized by arithmetic processing and analyzed. As a result of the determination, the time from the start of detection to the completion of detection was about 67 minutes, and the detection rate of cancer cells was 80%.

[Comparative Example 1]
As in Example 1, blocking treatment and washing with PBS are performed, and the cell suspension subjected to the first to third fluorescent labeling treatments is sent to the flow path 1, and the cells are transferred intermittently as in Example 1. Was made into a single layer.

  First, a non-target cell on a cell development substrate is focused in a bright field, and then a Hoechst-stained cell (= Hoechst33242 (3A)) while irradiating a cell group developed with excitation light (light having a wavelength of 400 nm). The third cell image was obtained by focusing on one cell (non-target cell)) of the cell group stained with the dye) and imaging the entire cell detection region.

  Next, the excitation light is switched to excitation light for CTC detection (light having a wavelength of 635 nm) and the light receiving optical system is set so as to be able to receive light of the emission wavelength, and the target cell is focused, and the cell detection region CDR is The entire cell was imaged to capture the first cell image.

  Finally, the excitation light is switched to excitation light for detecting leukocytes (light with a wavelength of 488 nm), and the light receiving optical system is set to receive light of the emission wavelength, and the cells are focused on the white blood cells (non-target cells). The entire cell of the detection region CDR was imaged to capture the second cell image. As a result of instantaneously synthesizing and determining the first to third cell images by arithmetic processing, the CTC detection rate was 30%.

[Comparative Example 2]
As in Example 1, blocking treatment and washing with PBS are performed, and the cell suspension subjected to the first to third fluorescent labeling treatments is sent to the flow path 1 to form a cell monolayer as in Example 1. Went. Thereafter, for each imaging area (field of view of the light receiving optical system) set on the cell deployment substrate, imaging was performed as follows.

  First, irradiate a cell group developed with excitation light (light with a wavelength of 400 nm), and focus on a cell stained with Hoechst staining (= one cell (non-target cell) stained with Hoechst33342 (third A dye)). In addition, the light receiving optical system was set to be capable of receiving light of the emission wavelength, and a third cell image was acquired.

  Next, the excitation light is switched to excitation light for CTC detection (light having a wavelength of 635 nm), the light receiving optical system is set to receive light of the emission wavelength, and the target cell is focused to focus on the first cell image. Imaging was performed.

  Finally, the excitation light is switched to the excitation light for detecting leukocytes (light having a wavelength of 488 nm), and the light receiving optical system is set to receive light of the emission wavelength, and is focused on the white blood cells (non-target cells). Two-cell images were taken. As a result of instantaneously synthesizing and determining the first to third cell images by arithmetic processing, the time from the start of detection to the completion of detection was about 106 minutes, and the detection rate of cancer cells was 90%.

  As above, the target cell detection method and target cell detection system according to the present invention have been described with reference to the embodiments and examples, but the present invention is not limited to these embodiments and examples. Changes in the design are permitted without departing from the spirit of the invention as set forth in the appended claims.

DESCRIPTION OF SYMBOLS 1 Flow path 2 Inlet 3 Outlet 4 Reservoir 5 Microchamber 6 Irradiation optical system 7 Light reception optical system 8 Control means 9 Liquid supply system mechanism (cell expansion means, the 1st-3rd fluorescence label processing means)
10 Cell recovery device 11 Substrate for cell deployment (including microchamber chip)
11a, 11b Cell development substrate 12 Flow path forming member 12a Seal member 13 Reagent container 14 Cell scraper 15 Light source 16 Lens 17 Dichroic mirror 18 Objective lens 19 Optical filter 20 Irradiation lens group 21 Imaging unit 22 Analysis unit 23 Program ( (Focus position adjustment means)
24 Program (Target cell search means)
25 Program (Focal distance information supplement means)
26 Cell Recovery Device Holder 100 Target Cell Detection System CL Cell Suspension CDR Cell Detection Area

Claims (18)

A cell suspension containing a smaller number of target cells to be detected than non-target cells is spread on the surface of the cell development substrate, and is provided in a region (cell detection region) for detecting cells provided on the surface. A cell expansion step to form a single cell layer;
A first fluorescent label that is a complex of an antibody that specifically binds to the target cell and a first fluorescent dye is used to fluorescently stain cells in the cell suspension or the expanded cells. 1 fluorescent labeling process;
Using a second fluorescent label, which is a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye, the cells in the cell suspension or the expanded cells are fluorescently stained. A second fluorescent labeling process,
And / or
Third fluorescence that is a 3A fluorescent dye that binds to both the target cell and the non-target cell, or a complex of an antibody that binds to both the target cell and the non-target cell and a 3B fluorescent dye. A third fluorescent labeling step of fluorescently staining the cells in the cell suspension or the expanded cells using the label,
A method for detecting a target cell including random numbers,
A focus position adjustment process ;
While irradiating the cell group developed in the cell detection region with the excitation light of the first fluorescent dye, the target cell emitting the first fluorescence is searched with reference to the focal position adjusted in the focal position adjusting step. A target cell search step to perform,
Including
The focal position adjustment step includes:
When the target cell detection method includes both the second fluorescent labeling step and the third fluorescent labeling step, the cell is excited by the second fluorescent dye or the excitation light of the third A or 3B fluorescent dye. Irradiating the cell group developed in the detection region, and performing a process of focusing the light receiving optical system on the non-target cell emitting the second fluorescence or the third A or the third fluorescence,
When the target cell detection method does not include the third fluorescent labeling step, the cell group developed in the cell detection region is irradiated with the excitation light of the second fluorescent dye, and the second fluorescence is emitted. Perform processing to focus the receiving optical system on the non-target cells that are emitted,
When the target cell detection method does not include the second fluorescent labeling step, the cell group developed in the cell detection region is irradiated with the excitation light of the 3A or 3B fluorescent dye, and the 3A Alternatively, a method for detecting a target cell, comprising performing a process of focusing a light-receiving optical system on a non-target cell emitting 3B fluorescence .   The method for detecting a target cell according to claim 1, wherein the target cell is a rare cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood. When the target cell detection method includes the second fluorescent labeling step, the wavelength of the second fluorescence used for focusing in the focus position adjusting step is the wavelength of the first fluorescence. Do not overlap,
When the target cell detection method includes the third fluorescence labeling step, the wavelength of the third A or 3B fluorescence used for focusing in the focus position adjustment step is the first fluorescence. The method for detecting a target cell according to claim 1 or 2, wherein the method does not overlap with the wavelength of the target cell. The target cell is CTC;
The non-target cell is a white blood cell;
The first fluorescent label is an anti-cytokeratin antibody labeled with the first fluorescent dye;
When the target cell detection method includes the second fluorescent labeling step, the second fluorescent label is an antibody that specifically recognizes leukocytes labeled with the second fluorescent dye,
When the target cell detection method includes the third fluorescent labeling step, the third fluorescent label is a Hoechst fluorescent dye that binds to a cell nucleus.
The method for detecting a target cell according to any one of claims 1 to 3. The surface of the cell detection region of the cell expansion substrate is planar,
The target cell according to any one of claims 1 to 4, wherein in the cell spreading step, two or more cell layers developed on the surface of the planar substrate are monolayered using a cell scraper. Detection method. The surface of the cell detection region of the cell deployment substrate has a microchamber or unevenness for storing cells,
The method for detecting a target cell according to any one of claims 1 to 4, wherein in the cell expansion step, the cell is monolayered by a liquid flow. A focal position correcting step between the focal position adjusting step and the target cell searching step ;
In the focal position correction step,
When the target cell detection method includes both the second fluorescent labeling process and the third fluorescent labeling process, the light received by the light receiving element provided in the light receiving optical system is the second fluorescent labeling process . Or a shift amount of the focal position caused by the change from the third fluorescence or the third fluorescence to the first fluorescence, and the focal position adjusted in the focal position adjustment step is corrected,
When the target cell detection method does not include the third fluorescent labeling step, the light received by the light receiving element provided in the light receiving optical system changes from the second fluorescence to the first fluorescence. Calculate the amount of deviation of the focal position caused by the change, correct the focal position adjusted in the focal position adjustment step,
When the target cell detection method does not include the second fluorescent labeling step, the light received by the light receiving element provided in the light receiving optical system is transmitted from the 3A or 3B fluorescence to the first. The method of detecting a target cell according to any one of claims 1 to 6 , wherein an amount of shift of a focal position caused by changing to fluorescence of the target is calculated, and the focal position adjusted in the focal position adjustment step is corrected . The target cell search step includes
A first cell image acquisition step of imaging the cell detection region with the light receiving optical system capable of receiving first fluorescence; an image forming step;
Only including,
And
When the target cell detection method includes both the second fluorescent labeling process and the third fluorescent labeling process, the light receiving optical system further receives the second fluorescence immediately after the focal position adjusting process. the second cell image acquiring step of imaging the cell detecting region and the possible states and the third cell image the light receiving optical system and the fluorescence of the 3A or the 3B ready for receiving for imaging the cell detecting region includes acquiring step, in the image forming step, was synthesized by superimposing the images captured by the second cell image acquiring step and / or said third cell image acquiring step and the first cell image acquisition step,
When the target cell detection method does not include the third fluorescent labeling step, the second cell image acquisition step is further included immediately after the focal position adjustment step. In the image formation step, the second cell image acquisition step is performed. Superimposing and synthesizing each image captured in the cell image acquisition step and the first cell image acquisition step ,
When the target cell detection method does not include the second fluorescent labeling step, the third cell image acquisition step is further included immediately after the focal position adjustment step. In the image formation step, the third cell image acquisition step is performed. Superimposing and synthesizing each image captured in the cell image acquisition step and the first cell image acquisition step ,
The method for detecting a target cell according to any one of claims 1 to 7. The focal position adjustment step is performed by obtaining information on the focal length of each visual field by focusing on a non-target cell in each of two or more visual fields at different positions in the cell detection region. If so, an approximate expression representing a change in focal length in a field other than the two or more fields is obtained from the difference,
In the other visual field, the step of calculating each focal length from the approximate expression and setting the focal length of the light receiving optical system to the calculated focal length is performed as the focal position adjusting step. The method for detecting a target cell according to any one of 1 to 8. A cell spreading substrate capable of spreading on the surface a cell suspension containing a smaller number of target cells to be detected than non-target cells;
A cell expansion means for forming a single cell layer in a cell detection region of the cell expansion substrate;
A first fluorescent label that is a complex of an antibody that specifically binds to the target cell and a first fluorescent dye is used to fluorescently stain cells in the cell suspension or the expanded cells. 1 fluorescent label processing means;
Using a second fluorescent label, which is a complex of an antibody that specifically binds to the non-target cell and a second fluorescent dye, the cells in the cell suspension or the expanded cells are fluorescently stained. Second fluorescent labeling means;
And / or
Third fluorescence that is a 3A fluorescent dye that binds to both the target cell and the non-target cell, or a complex of an antibody that binds to both the target cell and the non-target cell and a 3B fluorescent dye. A third fluorescent labeling means for fluorescently staining the cells in the cell suspension or the expanded cells using a label,
An irradiation optical system capable of irradiating excitation light having a predetermined wavelength with respect to a cell detection region of the cell development substrate,
A light receiving optical system capable of receiving light from the cell detection region for each wavelength band;
Control means for controlling at least the light receiving optical system and the irradiation optical system;
A target cell detection system comprising:
The control means is at least
A focus position adjusting means;
While irradiating the cells expanded in the cell detecting region by the excitation light of the first fluorescent dye by the irradiation optical system, with reference to the position of the focus combined with the focal point position adjusting means, first by the light receiving optical system 1 and a target cell search means for performing a process of searching for the target cells that emit fluorescence,
The focal position adjusting means includes
When the said purpose cell detection system comprises both the third fluorescent label processing means and the second fluorescent label processing means, the excitation of the second fluorescent dye by irradiation optical system or the second 3A or the 3B fluorescent dye Irradiating a cell group developed on the cell detection region with light, and focusing the light receiving optical system on a non-target cell emitting second fluorescence or fluorescence of 3A or 3B ,
When the target cell detection system does not include the third fluorescent label processing means, the irradiation optical system irradiates the cell group developed in the cell detection region with the excitation light of the second fluorescent dye, and The light receiving optical system is focused on a non-target cell emitting fluorescence of 2,
When the target cell detection system does not include the second fluorescent labeling processing means, the irradiation optical system irradiates the cell group developed in the cell detection region with the excitation light of the third A or third B fluorescent dye. A target cell detection system , wherein the light receiving optical system is focused on a non-target cell emitting the 3A or 3B fluorescence .   The target cell detection system according to claim 10, wherein the target cell is a rare cell contained in the blood at a content rate of less than 0.01% with respect to the total number of cells in the blood. When the said purpose cell detection system comprises a second fluorescent label processing means, the wavelength of the second fluorescent used when focusing by the focus position adjusting means, overlap the first wavelength of the fluorescent Without
When the target cell detection system includes the third fluorescent labeling processing unit, the wavelength of the third A or 3B fluorescence used when focusing by the focal position adjusting unit is the first fluorescence. The target cell detection system according to claim 10 or 11, which does not overlap with a wavelength . The target cell is CTC;
The non-target cell is a white blood cell;
The first fluorescent label is an anti-cytokeratin antibody labeled with the first fluorescent dye;
When the target cell detection system includes the second fluorescent labeling processing means, the second fluorescent label is an antibody that specifically recognizes leukocytes labeled with the second fluorescent dye,
When the target cell detection system includes the third fluorescent labeling processing means, the third fluorescent label is a Hoechst fluorescent dye that binds to a cell nucleus.
The target cell detection system according to any one of claims 10 to 12. The surface of the cell detection region of the cell expansion substrate is planar,
The target cell detection system according to any one of claims 10 to 13, wherein the cell deployment means includes a cell scraper for monolayering two or more cell layers developed on the surface portion. . The surface of the cell detection region of the cell deployment substrate has a microchamber or unevenness for storing cells,
The target cell detection system according to any one of claims 10 to 13, wherein the cell expanding means includes a liquid feeding system mechanism for generating a liquid flow for monolayering the cells. The control means further includes a focus position correction means,
The focal position correcting means includes
When the target cell detection system includes both the second fluorescent label processing unit and the third fluorescent label processing unit, the light received by the light receiving element provided in the light receiving optical system is the second fluorescent label processing unit . Calculating the shift amount of the focal position caused by the change from the fluorescence or the 3A or 3B fluorescence to the first fluorescence, and correcting the focal position adjusted by the focal position adjusting means ;
When the target cell detection system does not include the third fluorescent label processing means, the light received by the light receiving element provided in the light receiving optical system changes from the second fluorescence to the first fluorescence. Calculating the shift amount of the focal position caused by the correction, correcting the focal position adjusted by the focal position adjusting means,
When the target cell detection system does not include the second fluorescent labeling processing unit, the light received by the light receiving element provided in the light receiving optical system is transmitted from the 3A or 3B fluorescence to the first Calculating the amount of shift of the focal position caused by changing to fluorescence, and correcting the focal position adjusted by the focal position adjusting means;
The target cell detection system according to any one of claims 10 to 15. The target cell search means includes :
First cell image acquisition means for imaging the cell detection region with the light receiving optical system in a state capable of receiving first fluorescence ; and image forming means;
With
And
When the target cell detection system includes both the second fluorescent label processing means and the third fluorescent label processing means, the light receiving optical system is immediately after the focus of the light receiving optical system is adjusted by the focus position adjusting means. the second cell image acquiring means for imaging the cell detecting region the system and the second fluorescent ready to light, and the said light-receiving optical system in the fluorescence can receive a state of the first 3A or the 3B The image forming unit further includes a third cell image acquiring unit that images the cell detection region, and the first cell image acquiring unit, the second cell image acquiring unit, and / or the third cell image acquiring unit captures the image in the image forming unit. There line processing to synthesize by superimposing the images,
If the target cell detection system does not include the third fluorescent labeling processing means, the light receiving optical system receives the second fluorescence immediately after the focus position adjusting means focuses the light receiving optical system. A second cell image acquiring unit configured to capture the cell detection region in a possible state; and in the image forming unit, the images captured by the first cell image acquiring unit and the second cell image acquiring unit are overlapped. Perform the process of combining them together ,
When the target cell detection system does not include the second fluorescent labeling processing means, the light receiving optical system is moved to the third A or 3B immediately after the focus position adjusting means focuses the light receiving optical system. And further comprising a third cell image acquiring means for capturing the cell detection region in a state where fluorescence can be received, and in the image forming means, each of the images captured by the first cell image acquiring means and the third cell image acquiring means. Perform the process of overlaying and compositing images.
The target cell detection system according to any one of claims 10 to 16. In each of two or more fields of view in different positions of the cell detection region, focusing on non-target cells to obtain focal length information,
When these focal lengths are different, an approximate expression representing a change in focal length in a visual field other than the two or more visual fields is obtained from the difference,
In the other field, each focal length is calculated from the approximation equation, the means for setting the focal length of the light receiving optical system on the calculated focal length, including as the focal position adjusting means, wherein Item 18. The target cell detection system according to any one of Items 10 to 17.