WO2003100086A1

WO2003100086A1 - Living cell counting method and device

Info

Publication number: WO2003100086A1
Application number: PCT/JP2003/006325
Authority: WO
Inventors: Naohiro Noda; Mutsuhisa Hiraoka; Kazuhito Takahashi; Masao Nasu; Nobuyasu Yamaguchi
Original assignee: Fuji Electric Holdings Co., Ltd.
Priority date: 2002-05-23
Filing date: 2003-05-21
Publication date: 2003-12-04
Also published as: AU2003242354A1; JPWO2003100086A1

Abstract

A living cell counting method and device for counting living cells such as microbe in a sample or living cells of a cell tissue is labeled by a fluorescent reagent. Before living cells are fluorescence-labeled, a fluorescence image (first image) of a sample is captured. After the living cells are fluorescence labeled, a fluorescence image (second image) of the sample is captured. The difference (B-A) in number of bright points between the first and second images is determined. In another method, a difference image between the first and second images is created, and the number of bright points in the difference image is counted, or the number of bring points, out of the bright points in the second image, not in the blind area around each bright point in the first image is counted. The influence of fluorescent impurities can be eliminated irrespective of the properties of the sample, and thus the measurement accuracy is improved.

Description

Description ^: Method and apparatus for counting live cells

The present invention provides a method for fluorescently labeling (staining with a fluorescent reagent) living cells such as microorganisms and tissue cells and generating fluorescence by exciting the reagent, or the fluorescent property of living cells such as microorganisms and tissue cells. The present invention relates to a method and an apparatus for counting living cells, which generate fluorescence by exciting molecules and count the number of living cells using the fluorescence image. Background art

The detection of living cells such as microorganisms and tissue cells such as animals and plants in a sample is an extremely important industrial technique for, for example, confirming the sterilization state and detecting abnormalities in the survival state of the cells. In the following description, for convenience of explanation, the description will mainly focus on bacteria.

There is a high percentage of bacteria in the natural environment that are alive but difficult to culture by conventional methods. These bacteria do not form colonies on common agar plates and often do not grow in liquid media. For this reason, there is a possibility that it cannot be detected by the conventional culture-based method. As a method for solving such a problem, in addition to the method of labeling the living cells with a fluorescent reagent, for example, a gene using a diamidino-phenylindole (DAPI) -acridine orange (AO) that binds to DNA is used.方法 Methods of detecting bacteria by labeling, fluorescein diacetate (FDA), carboxy fluorescein diacetate (CFDA), which is metabolized in bacteria and becomes fluorescent, 5-cyano-2,3-dito Lil Tetrazori A measurement method has been proposed for detecting bacteria maintaining physiological activity using, for example, microclide (CTC).

The FDA and CFDA are hydrolyzed by the action of an enzyme (esterase) in living cells such as bacteria, and become fluorescent. In addition, CTC becomes fluorescent when reduced with the respiration of living cells. Each of the above reagents comes into contact with living cells in a sample to be measured as a solution, reacts by being taken into the living cells, and becomes a state of emitting fluorescence. The fluorescence detects living cells such as bacteria. .

However, the method of detecting bacteria by fluorescence has a problem that if fluorescent contaminants coexist in a sample, they are mistaken for bacteria to be detected, resulting in counting errors.

Patent Document 1 described later discloses the following method as a method for detecting bacteria invented to solve this problem. That is, “(a) the medium is stained with a fluorescent enzyme substrate, and the fluorescence image is recorded. (B) The stained medium is irradiated with light to cause photobleaching, and then the fluorescence image is recorded. A) a live cell detection method, which comprises taking a difference image between the fluorescence image obtained in (a) and the fluorescence image obtained in (b). "

In the invention described in Patent Document 1, in short, focusing on the fact that bacteria labeled with a fluorescent reagent are more likely to undergo photobleaching than fluorescent contaminants originally contained in a sample, the procedure described above was used. To remove the influence of fluorescent impurities.

However, the invention described in Patent Document 1 also has the following problems.

Live cells labeled with a fluorescent reagent are more susceptible to photobleaching than fluorescent contaminants contained in the sample, but the fluorescent properties of the contaminants cannot be controlled, so that only stained bacteria are necessarily fading. And all the fluorescence of the impurities does not fade. Is not always maintained.

If the fluorescence of the contaminant fades in the same way as the fluorescence-labeled bacteria, a measurement error will result. Therefore, it is not always possible to count viable cells with high accuracy, and it is difficult to ensure the accuracy of the measured values when the sample properties change.

(Patent Document 1)

Japanese Patent Application Laid-Open No. H10-10-215

The present invention has been made in view of the above points, and provides a method and apparatus for counting living cells which eliminates the influence of fluorescent contaminants regardless of the properties of a sample and improves the measurement accuracy. Aim. Disclosure of the invention

In order to solve the above-mentioned problems, in the invention set forth in claim 1, the number of living cells is measured by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent. In the method for counting living cells, a fluorescence image (first image) of a sample is obtained before the living cells are fluorescently labeled, bright spots in the first image are counted, and the living cells are fluorescently labeled. Later, by obtaining a fluorescence image (second image) of the sample, counting the bright spots in the second image, and calculating the difference between the number of bright spots in the first image and the number of bright spots in the second image The method is characterized in that the number of living cells is measured. According to the above counting method, the influence of fluorescent contaminants can be eliminated regardless of the properties of the sample, and viable cells can be counted accurately.

In order to achieve the same object, the method of the invention described in claims 2 to 3 below may be adopted. That is, in a live cell counting method for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the method comprises the steps of: Obtaining a fluorescent image (first image) of the sample, fluorescently labeling the living cells, obtaining a fluorescent image (second image) of the sample, obtaining a difference image between the first image and the second image, By calculating the number of bright spots in the difference image, the number of the living cells is measured (the invention of claim 2).

In addition, in the method for counting the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the method for counting the number of living cells may be performed by: Acquiring an image (first image), acquiring position information of a bright point in the first image, and further setting in advance an insensitive area such as a radius and a width and width for the bright point, After recognizing a dead area associated with each bright spot in the first image and fluorescently labeling the living cells, a fluorescent image (second image) of the sample is obtained, and the bright spot in the second image is obtained. The live cells are obtained by acquiring position information and calculating the number of luminescent spots, of the luminescent spots in the second image, which are not included in a dead area associated with each luminescent spot in the first image. Is measured (the invention of claim 3).

The setting of the dead area varies depending on the measurement conditions and the state of the sample.For example, for example, "a region with a radius of 10 m relative to the position of the bright spot obtained in the first image" Area ". By properly setting the dead area, the number of bacteria can be measured appropriately.

Further, from the viewpoint of more reliably eliminating the influence of fluorescent contaminants and further improving the counting accuracy of living cells, the inventions of claims 4 to 5 below are preferable. That is, in the method for counting living cells according to any one of claims 1 to 3, the amount of excitation light at the time of obtaining the first image, the excitation light amount at the time of obtaining the second image. It is larger than the amount of light (the invention of claim 4).

In addition, live cells such as microorganisms and cell tissues in the sample are labeled with a fluorescent reagent. In the method for counting the number of living cells, the live cell is labeled with a fluorescent substance, instead of the first image according to claim 3, Acquire an image and use this as the first image, and acquire the position information of the bright points of all the particles in the stereoscopic image, and further set in advance a dead area such as a radius and a vertical and horizontal width for the bright points Then, after recognizing a dead area associated with each bright spot in the stereoscopic image and fluorescently labeling the living cells, a fluorescent image (second image) of the sample is obtained. By obtaining the position information of the luminescent spots, of the luminescent points in the second image, the position of the luminescent spots is obtained by calculating the number of luminescent spots that are not included in a dead area associated with each luminescent spot in the real image. The number of living cells is measured (the invention of claim 5). Further, in the method for counting living cells according to any one of claims 1 to 5, the method for labeling living cells with a fluorescent reagent instead of the above-mentioned `` metabolism in living cells '' (Invention of claim 6). As the reagent which becomes fluorescent due to metabolism in living cells, the above-mentioned FDA, CFDA, CTC and the like can be used.

Further, from the viewpoint of application advantages, in the method for counting living cells according to any one of claims 1 to 6, the living cells may be bacteria. (Invention of item 7) is preferred.

Furthermore, as the embodiments of the invention set forth in claims 1 to 5, the inventions set forth in claims 8 to 10 described below are preferable from the viewpoint of improving measurement accuracy. That is, the sample is filtered to capture live cells on the filter, a first image of the sample containing the live cells captured on the filter is obtained, and a fluorescent reagent is added to the sample on the filter to fluoresce the live cells. After the fluorescent reagent that has been labeled and not taken up by the living cells is removed by filtration, a fluorescence image (second image) of the sample containing the living cells captured on the filter is obtained (Claim 8). ). Further, in the method for counting living cells according to claim 8, after removing the fluorescent reagent not taken up by the living cells by filtration, the washing solution is filtered by the filter so as to wash away the remaining fluorescent reagent even after the filtration. The washing solution is added to the above sample and filtered (the invention of claim 9).

Further, in the method for counting live cells according to claim 9, the washing solution is a buffer solution having a pH suitable for expressing the fluorescence of the fluorescent reagent (the invention of claim 10).

Further, as the invention relating to a method different from the invention of claim 8 when acquiring the first image and the second image, the invention of the following claims 11 to 14 is described. Each can be suitably adopted according to the needs.

First, the invention according to claim 11 is a method using electrostatic force for capturing living cells, that is, the method according to any one of claims 1 to 5 In the method for counting living cells, the sample is introduced into a cell flow path provided with an imaging means for counting live cells, and a part of the cell flow path is positively charged, thereby being negatively charged in a natural state. The captured live cells in the sample are captured by the positive charging section, the first image of the sample containing the captured live cells is obtained, and then a fluorescent reagent is introduced into the cell flow path to obtain the captured live cells. After fluorescently labeling the cells and removing the fluorescent reagent not taken up by the living cells with a washing solution, a fluorescence image (second image) of the sample containing the captured living cells is obtained.

Next, the invention according to claim 12 is a method for utilizing an antigen-antibody reaction for capturing living cells, that is, the method according to any one of claims 1 to 5 described above. In the method for counting live cells according to the above, the sample is introduced into a cell flow path provided with an imaging means for live cell counting, and a part of the cell flow path is specified in advance as a live cell to be captured. To fix antibodies that bind By capturing live cells in a sample by binding to the antibody, obtaining the first image of the sample containing the live cells captured by the antibody, and then introducing a fluorescent reagent into the cell flow path, Capturing the captured living cells with a fluorescent label, removing the fluorescent reagent that has not been incorporated into the living cells with a washing solution, and acquiring a fluorescence image (second image) of the sample containing the captured living cells. You.

Next, the invention according to claim 13 is a method of using a filter for capturing living cells, and holding a live cell capturing surface with a transparent plate for preventing adhesion of dust and facilitating handling, That is, in the method of counting live cells according to any one of claims 1 to 5, the sample is filtered, and the live cells are captured on a filter. The capture surface is covered with a transparent plate such as glass or plastic, and the first image of the sample containing the captured live cells is obtained from the transparent plate side. The fluorescent image of the sample containing the captured living cells from the transparent plate side after the captured living cells are fluorescently labeled by adding from the side and the fluorescent reagent not taken up by the living cells is removed by filtration.And obtaining a second image). The transparent plate is preferably a thin film.

Next, the invention according to claim 14 uses the electrostatic force or the antigen-antibody reaction for capturing live cells, and uses the reaction time of the fluorescent reagent for the acquisition timing of the first image and the second image. A live cell counting method for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent assay, wherein an imaging means for counting live cells is provided. The sample is introduced together with a fluorescent reagent into a cell flow path provided with a positively charged portion or an antibody fixing portion for capturing live cells in the sample, and the live cells react with the fluorescent reagent to fluoresce. Before being labeled, the captured live cells Acquiring a first image of a sample containing the living cells, and then obtaining a fluorescent image (second image) of the sample containing the captured living cells after the living cells are fluorescently labeled in response to the fluorescent reagent. And measuring the number of the living cells.

In the case of the invention set forth in claim 14, there is an advantage that the counting procedure is simplified.However, if necessary, the reaction time is delayed by lowering the concentration of the fluorescent reagent, or By lowering the temperature at the time and slowing the reaction time, the interval between the acquisition timings of the first image and the second image can be increased.

Further, as an apparatus for performing the counting method, the inventions of the following claims 15 to 17 are suitable. That is, an optical means for irradiating the sample with excitation light in a live cell counting device for measuring the number of the living cells by labeling living cells such as microorganisms and cell tissues in the sample with a fluorescent reagent. Optical means for capturing the fluorescence emitted by the sample, an image sensor for capturing the captured fluorescence as an image and converting it into an electrical signal, Image processing means for calculating the area of the luminescent spot; and calculating means for counting the number of luminescent spots having an area of a preset range and calculating the difference in the number of luminescent spots between the first image and the second image. (Invention of Claim 15).

Further, in a live cell counting device for measuring the number of live cells by labeling live cells such as microorganisms and cell tissues in the sample with a fluorescent reagent, optical means for irradiating the sample with excitation light, An optical means for collecting the fluorescence emitted from the sample, an imaging element for capturing the collected fluorescence as an image and converting it into an electric signal; and obtaining a difference image between the first image and the second image. Image processing means for recognizing bright spots in the difference image and calculating the area of each recognized bright spot, and calculating means for counting bright spots having an area within a preset range are provided. (Invention of claim 16).

Further, an optical means for irradiating the sample with excitation light in a live cell counting device for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in the sample with a fluorescent reagent. Optical means for collecting the fluorescence emitted from the sample, an image sensor that captures the captured fluorescence as an image and converts it into an electric signal, and recognizes the bright spots in the image obtained by the image sensor and recognizes each individual. Image processing means for calculating the area of the luminescent spots of each of the luminescent spots and recording the positions of the luminescent spots having the area of a preset range; Computing means for recognizing and counting the bright spots in the second image that are not included in the dead areas recognized in the first image among the bright spots in the second image. The invention of claim 17). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a procedure of a method for counting living cells according to an embodiment of the present invention. FIG. 2 is a schematic explanatory view of a method using a difference image according to the embodiment of the present invention. '

FIG. 3 is a schematic explanatory view of a method using the position information of a bright spot according to the embodiment of the present invention.

FIG. 4 is a schematic configuration diagram of an apparatus for counting live cells according to an embodiment of the present invention. FIG. 5 is an explanatory diagram of an embodiment using electrostatic force according to the present invention.

FIG. 6 is an explanatory view of an embodiment utilizing the antigen-antibody reaction of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail with reference to FIGS. 1 to 6. (Example 1: Method based on difference in number of bright spots)

Based on FIG. 1, an embodiment of a method for counting the number of bacteria using the determination of the difference in the number of bright spots between the first image and the second image will be described below. The following description assumes the counting of bacteria in liquid samples. However, bacteria adhering to the solid sample can be spread in sterile water by wiping and post-macing, and can be handled in the same manner as liquid samples thereafter.

First, bacteria contained in sample 1 are captured on filter 2 by filtration. As the filter, a membrane filter having a high uniformity in pore size is preferable. The pore size of the membrane filter used must be selected according to the size of the bacteria to be measured. When the number of bacteria is counted, it is usually 0.2 to 0.6;

Next, the fluorescence image (first image) of the membrane filter capturing the bacteria is acquired using the fluorescence image acquisition means 3. The acquired image is subjected to image processing by the image processing unit 4, and the number of fluorescent bright spots A present in the image is obtained. Bright spots that are already present before fluorescent labeling are foreign substances.

The following processing is specifically performed as the image processing.

① Capture multiple screens in the same field of view and average them to reduce random noise

② Shading (shade) correction

③ Extract bright spots by edge detection

認識 Recognize the distance between individual bright spots by labeling

輝 Select bright spots whose area falls within a preset range

輝 Count bright spots selected in ⑤

The area set in advance in ⑤ is a numerical value determined by the size of the bacteria to be counted and the characteristics of the device used for detection. By conducting experimental studies in advance, it is possible to set, for example, “area equivalent to 0.2 to 10 m in diameter”. Subsequently, the fluorescent labeling reagent 5 is added onto the membrane filter. Fluorescent marker All bacteria can be labeled by using DAPI or AO, which has gene affinity, as the recognition reagent 5. PI is suitable for labeling dead bacteria. By using CFDA or CTC, which expresses fluorescence by the physiological activity of bacteria such as enzymatic reaction and respiration, only viable bacteria can be selectively labeled. When labeling a specific bacterium, use is made of a method that recognizes an antigen antibody reaction or a specific gene sequence. In the former case, an antibody that specifically binds to the antigen of the target bacterium is fluorescently labeled in advance, and this fluorescently labeled antibody is reacted with a sample to label only a specific kind of bacterium. In the latter case, the target bacterium can be labeled using a specific gene as a target by using techniques such as FISH and in situ PCR.

Fluorescent labeling reagents that do not react with bacteria may cause measurement noise. In this case, it is effective to remove the reagent not taken up by the bacteria by filtration. In order to further enhance the removal effect, it is effective to remove the reagent that has not been taken up by bacteria by filtration and then wash away the reagent with a washing solution.

As the washing solution, a buffer solution having a pH suitable for expressing the fluorescence of the fluorescent labeling reagent is suitable. For example, AO and CFDA as fluorescent labeling reagents are highly viable and have good fluorescence expression efficiency, so using a highly viable pH buffer solution as a washing solution can provide a more contrasting fluorescent image. Can be done.

After fluorescent labeling, obtain a fluorescent image (second image) of the membrane filter again. The same image processing as the first image is performed on the acquired second image, and the number B of fluorescent bright spots existing in the image is obtained. Then, the difference (B−A) in the number of bright spots between the first image and the second image, that is, the number of bright spots appearing by the fluorescent label is obtained, and this is determined as the bacterial count.

(Example 2: Method using difference image)

Based on Fig. 2, it is assumed that a difference image is obtained between the first image and the second image. An example of a method for measuring the number of bacteria used will be described below.

First, as in Example 1, the bacteria contained in the sample solution are captured on the membrane filter by filtration. Next, a fluorescent image (first image 6 shown in the center of FIG. 2) of the membrane filter capturing the bacteria is obtained. Subsequently, a fluorescent labeling reagent is added onto the membrane filter, and the bacteria are fluorescently labeled. After that, a fluorescent image (second image 7 shown on the left side of FIG. 2) of the re-membrane filter is obtained. Then, a difference image 8 shown on the right side of FIG. 2 is obtained from the second image 7 and the first image 6.

The bright spots present in the difference image 8 are the bright spots appearing by the fluorescent labels, which are counted as the number of bacteria.

When actually performing a difference between images, there are cases in which an ideal result cannot be obtained by the above processing. Specifically, in the following cases, it is difficult to obtain an appropriate number of bright spots with a simple difference image.

① When the image acquisition position is shifted between the first image and the second image

② When the brightness of the entire image (background) differs between the first and second images due to the influence of the fluorescent labeling operation and the difference in the optical conditions at the time of image acquisition.

③ The shape and size of the images derived from the corresponding bright spots differed between the first image and the second image due to differences in focusing when acquiring images and setting conditions during image processing. If

場合 When the bright spot itself has moved between the first and second images

In the case of the above (1), a correct result can be obtained by recognizing the positional relationship between the two images by image processing called pattern matching and obtaining a difference image at the corresponding position.

The above case (2) can be dealt with by a method of clarifying a distinction between a signal derived from a bright spot and a signal derived from a background by image processing such as binarization and edge detection. In the case of (3), it is difficult to deal with this embodiment, but According to this, it is possible to respond effectively. In the case of ④, the above embodiment 1 can be used.

In addition, in this example and Example 3 described later, if the position of bacteria moves during fluorescent labeling or washing, an error occurs in the analysis of the bright spot between the first image and the second image. In order to prevent this, it is effective to perform fluorescent labeling and washing while performing suction filtration.

(Example 3: Method using location information)

Based on Fig. 3, an embodiment of a method for counting the number of bacteria using the position information of fluorescent bright spots is described below.

As a procedure, as in Examples 1 and 2, first, bacteria contained in the sample solution are captured on a membrane filter by filtration. Next, a fluorescence image (first image 6) of the membrane filter capturing the bacteria is acquired. At the time of image acquisition, the reference point 9 (indicated by a white arrow in the figure) is used, the position of the membrane filter is grasped based on this, and the position information 10 of the bright spot in the first image is recognized. I do. Subsequently, a fluorescent labeling reagent is added onto the membrane filter, and the bacteria are fluorescently labeled. After that, a fluorescent image (second image 7) of the membrane filter is acquired. Similar to the first image, the reference point 9 (arrow in the figure) is used when acquiring the image, the position of the membrane filter is grasped based on this, and the position information of the bright spot in the second image 1 1 Recognize.

The first image 6 and the second image 7 shown in FIG. 3 show an example in which the translation and the rotation occur, though they are minute. Thus, even when the image acquisition position is shifted, the position of the bright spot can be correctly recognized as a relative position from the reference point. Then, the position information of the bright spot is compared between the second image 7 and the first image 6, and the bright spot information 12 that first appears after the second image 7 is obtained. The number of bright spots obtained in the bright spot information 12 is the bacterial count.

In practice, it is difficult to find a match between points that do not have an area. _λ

14 Therefore, with respect to the location of the bright spot obtained in the first image 6, a predetermined area set in advance is recognized as an insensitive area associated with each bright spot. As described above, the dead area setting may be, for example, "a region having a radius of 10 m with respect to the position of the bright spot obtained in the first image" or "a region of ± 5 pixels both vertically and horizontally as the image" I do. By appropriately setting the dead area, it is possible to appropriately measure the number of bacteria even in the case of (3), which has been a problem in the second embodiment described above.

In the embodiment shown in FIG. 3, the amount of excitation light at the time of acquiring the first image is set to be larger than the amount of excitation light at the time of acquiring the second image, as in the invention of claim 4. As a result, the influence of the fluorescent contaminants can be more reliably eliminated, and the counting accuracy of living cells can be further improved.

Further, as in the invention of claim 5, the counting accuracy can be improved by using the actual image of the sample instead of the first image instead of the fluorescent image. In this case, when acquiring the first image, the sample is irradiated with excitation light instead of white light.

(Example 4: Example of apparatus)

An embodiment of the viable cell counting apparatus according to the present invention will be described with reference to FIG.

The excitation light emitted from the light source 13 is reflected by the dichroic mirror 14, condensed by the objective lens 15, and irradiates the sample 16 on the membrane filter 2. As the light source, a light emitting diode, a semiconductor laser, a mercury lamp, or the like is suitable. The fluorescence emitted from the sample is collected by the objective lens, passes through the dichroic mirror, and forms an image on the image sensor 17. As the imaging device, a CCD device or a CMOS device can be used. The image sensor captures the fluorescence as an image and converts it into electrical signals. The image obtained by the image sensor is transmitted to the image / arithmetic processing unit 4, and performs various image processing and arithmetic processing as described above. Further, it is preferable that the membrane filter 2 on which the sample is placed is sequentially scanned in the horizontal direction 18 by a driving means (not shown), and a plurality of screens are acquired and analyzed to enlarge the visual field area. This is effective, for example, in reducing the statistical variability of the counting results when the distribution of bacteria varies. Further, driving in the vertical direction 19 is a necessary function when performing autofocus for image observation. As the driving means, for example, a stepping motor capable of precise position control is desirable.

In the case where the first image is replaced with a fluorescence image and a real image of the sample is used, when the first image is acquired, the sample is irradiated with excitation light instead of white light. A switching unit (not shown) for switching between the excitation light and the white light is provided.

(Embodiment 5: Method of using electrostatic force)

Based on FIG. 5, an embodiment of a method of using electrostatic force for capturing living cells will be described below.

In FIG. 5, a sample is introduced into a cell channel 20 provided with an imaging means 21 for counting live cells and an electrode 22 for generating electrostatic force, and a part of the cell channel 20 is corrected. The positively charged portion captures the living cells 30 in the sample negatively charged in the natural state by the positively charged portion, and acquires the first image of the sample containing the captured living cells by the imaging means 21. Thereafter, a fluorescent reagent is introduced into the cell flow path 20 to label the captured living cells 30 with fluorescence. After removing the fluorescent reagent not taken up by the living cells with a washing solution, a sample containing the captured living cells is obtained. The fluorescence image (second image) is obtained by the imaging means 21.

In FIG. 5, at least the surface of the cell flow path 20 facing the imaging means 21 is made of transparent glass or plastic.

(Example 6: Method using antigen-antibody reaction)

Based on FIG. 6, an embodiment of a method using an antigen-antibody reaction for capturing living cells will be described below. In FIG. 6, a sample is introduced into a cell flow path 20 provided with an imaging means 21 for counting living cells, and a part of the cell flow path 20 is specified in advance with a living cell to be captured. By immobilizing the antibody 23 that binds specifically, the live cells 30 in the sample are captured by binding with the antibody 23, and the first sample containing the live cells 30 captured by the antibody 23 is removed. An image is obtained by the imaging means 21, and thereafter, a fluorescent reagent is introduced into the cell flow path 20 to fluorescently label the captured living cells 30, and the fluorescent reagent not taken up by the living cells is washed with a washing liquid. After the removal, a fluorescence image (second image) of the sample containing the captured living cells is obtained by the imaging means 21. Industrial applicability

According to the present invention, as described above, living cells such as microorganisms and tissue cells are stained with a fluorescent reagent and fluorescence is generated by exciting the reagent, or living cells such as microorganisms and tissue cells originally have The present invention can be applied to a method and an apparatus for counting living cells, in which fluorescence is generated by exciting fluorescent molecules, and the number of living cells is counted using the fluorescence image. The microorganisms include bacteria, fungi, viruses, yeasts, molds, and the like, and the application fields of the present counting method and apparatus include medical treatment, food production, and water and sewage.

According to the present invention, a fluorescence image or a stereoscopic image (first image) of a sample is obtained before the living cells are fluorescently labeled, and a fluorescence image of the sample (second image) is obtained after the living cells are fluorescently labeled. ) Is obtained, and the difference between the number of bright spots in the first image and the second image is calculated, or the difference image between the first image and the second image is calculated, and the number of bright spots in the difference image is calculated. Or the number of luminescent points in the second image where the position is not included in the dead area associated with each luminescent point in the first image.

Improves measurement accuracy by eliminating the effects of fluorescent contaminants regardless of sample properties Thus, a method and apparatus for counting living cells can be provided.

Claims

The scope of the claims

1. In a live cell counting method for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the fluorescence of the sample is measured before the living cells are fluorescently labeled. Obtaining an image (first image), counting bright spots in the first image,

Obtaining a fluorescence image (second image) of the sample after fluorescent labeling the living cells, counting the bright spots in the second image,

A viable cell counting method, wherein the number of viable cells is measured by calculating a difference between the number of bright points in the first image and the number of bright points in the second image.

2. In a live cell counting method of measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the fluorescence of the sample is measured before the living cells are fluorescently labeled. After obtaining an image (first image) and fluorescently labeling the living cells, obtaining a fluorescent image (second image) of the sample,

A method for counting living cells, comprising: obtaining a difference image between the first image and the second image; and measuring the number of the living cells by calculating the number of bright spots in the difference image.

3. In a live cell counting method of measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the fluorescence of the sample may be measured before the live cells are fluorescently labeled. Acquiring an image (first image), acquiring position information of a luminescent spot in the first image, and further setting in advance an insensitive area such as a radius and a vertical and horizontal width with respect to the luminescent point, Recognize the dead area associated with each bright spot in one image, Obtaining a fluorescent image (second image) of the sample after fluorescent labeling the living cells; and obtaining position information of a bright spot in the second image;

Among the bright spots in the second image, by determining the number of bright spots whose position is not included in the dead area associated with each bright spot in the first image, the number of living cells can be measured. Characteristic method for counting live cells.

4. The method according to any one of claims 1 to 3, wherein the excitation light quantity at the time of acquiring the first image is larger than the excitation light quantity at the time of acquiring the second image. The method for counting live cells as described above.

5. In a live cell counting method for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, the method according to claim 1, In place of the first image described in the third paragraph of the range, a real image of the sample is obtained and used as the first image, and the position information of the bright spots of all the particles in the real image is obtained. In advance, a dead area such as a radius and a vertical and horizontal width is set in advance for the bright point, and a dead area associated with each bright point in the real image is recognized.

Obtaining a fluorescent image (second image) of the sample after fluorescent labeling the living cells; and obtaining position information of a bright spot in the second image;

Among the bright spots in the second image, the position is determined by determining the number of bright spots that are not included in a dead area associated with each bright spot in the real image, thereby measuring the number of living cells. Characteristic method for counting live cells.

6. The method according to any one of claims 1 to 5, characterized in that instead of "labeling living cells with a fluorescent reagent", "fluorescence is caused by metabolism in living cells". 4. The method for counting living cells according to the above item.

7. The method for counting living cells according to any one of claims 1 to 6, wherein the living cells are bacteria.

8. Filter the sample to capture live cells on the filter, obtain a first image of the sample containing the live cells captured on the filter, and add a fluorescent reagent to the sample on the filter to remove the live cells. Claims: A fluorescent image (second image) of a sample containing live cells captured on a filter is obtained after removing the fluorescent reagent that has been fluorescently labeled and not taken up by the live cells by filtration. 6. The method for counting living cells according to any one of Items 1 to 5.

9. After removing the fluorescent reagent not taken up by the living cells by filtration, a washing solution is added to the sample on the filter so as to wash away the remaining fluorescent reagent even after the filtration, and the washing solution is filtered. 9. The method for counting living cells according to claim 8, wherein

10. The method for counting live cells according to claim 9, wherein the washing solution is a buffer solution having a pH suitable for expressing the fluorescence of the fluorescent reagent.

1 1. The sample is introduced into a cell flow path provided with an imaging means for viable cell counting, and a part of the cell flow path is positively charged. The live cells are captured by the positively charged portion, the first image of the sample containing the captured live cells is obtained, and then a fluorescent reagent is introduced into the cell flow path to label the captured live cells with fluorescence. After removing the fluorescent reagent that has not been taken up by the living cells with a washing solution, it contains the captured living cells. The method for counting living cells according to any one of claims 1 to 5, wherein a fluorescence image (second image) of the sample is obtained.

12. Introduce the sample into a cell flow path provided with an imaging means for viable cell counting, and place an antibody that specifically binds to live cells to be captured in advance in a part of the cell flow path By immobilizing, the live cells in the sample are captured by binding to the antibody, the first image of the sample containing the live cells captured by the antibody is obtained, and then the fluorescent reagent is passed through the cell flow path. After capturing the living cells and labeling the captured living cells with a fluorescent reagent, removing the fluorescent reagent not taken up by the living cells with a washing solution, a fluorescent image (second image) of the sample containing the captured living cells is obtained. 6. The method for counting living cells according to any one of claims 1 to 5, wherein the method includes counting live cells.

13. Filter the sample to capture live cells on a filter, cover the live cell capture surface on the filter with a transparent plate such as glass or plastic, and from the transparent plate side, Obtaining the first image of the sample containing cells, and thereafter adding a fluorescent reagent from the side opposite to the live cell capturing surface of the filter, thereby labeling the captured live cells with fluorescence, and 6. The method according to claim 1, wherein after removing the fluorescent reagent by filtration, a fluorescence image (second image) of the sample containing the captured living cells is obtained from the transparent plate side. The method for counting living cells according to any one of the above items.

14. A live cell counting method for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, comprising: an imaging means for counting live cells; and To capture live cells in a sample The sample is introduced together with a fluorescent reagent into a cell flow path provided with a positively charged portion or an antibody fixing portion, and the captured live cells are contained before the live cells are fluorescently labeled in response to the fluorescent reagent. Obtaining a first image of the sample, and thereafter obtaining a fluorescence image (second image) of the sample containing the captured living cells after the living cells are fluorescently labeled in response to the fluorescent reagent. A method for counting living cells, comprising measuring the number of living cells.

15. A live cell counting apparatus for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, wherein an optical means for irradiating the sample with excitation light. Optical means for capturing the fluorescence emitted from the sample, an image sensor that captures the captured fluorescence as an image and converts it into electrical signals, and recognizes the bright spots in the image obtained by the image sensor and Image processing means for calculating the area of the luminescent spot; and calculating means for counting the number of luminescent spots having an area of a preset range and calculating the difference in the number of luminescent spots between the first image and the second image. A viable cell counting device, characterized in that:

16. A live cell counting device for measuring the number of living cells by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent. Means, optical means for collecting the fluorescence emitted by the sample, an image sensor for capturing the collected fluorescence as an image and converting it into an electric signal, and obtaining a difference image between the first image and the second image, Image processing means for recognizing bright spots in the difference image and calculating the area of each recognized bright spot, and arithmetic means for counting bright spots having an area of a preset range are provided. Live cell counting device.

17 7. Label living cells such as microorganisms and cell tissues in the sample with a fluorescent reagent. Thus, in the living cell counting device for measuring the number of living cells, an optical unit for irradiating the sample with excitation light, an optical unit for collecting fluorescence emitted from the sample, and an image of the collected fluorescence Recognizes the image sensor and converts it to an electrical signal.Recognizes the bright spots in the image obtained by the image sensor, calculates the area of each recognized bright spot, and records the position of the bright spot having an area within a preset range. Image processing means, and recognizes a preset area as a dead area associated with each bright point with respect to the position of each recorded bright point, and, among the bright points in the second image, A counting means for determining and counting a position whose position is not included in the insensitive area recognized in the first image.