WO2003100086A1 - Procede et dispositif de comptage de cellules vivantes - Google Patents

Procede et dispositif de comptage de cellules vivantes Download PDF

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Publication number
WO2003100086A1
WO2003100086A1 PCT/JP2003/006325 JP0306325W WO03100086A1 WO 2003100086 A1 WO2003100086 A1 WO 2003100086A1 JP 0306325 W JP0306325 W JP 0306325W WO 03100086 A1 WO03100086 A1 WO 03100086A1
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WO
WIPO (PCT)
Prior art keywords
image
living cells
sample
counting
cells
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Application number
PCT/JP2003/006325
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English (en)
Japanese (ja)
Inventor
Naohiro Noda
Mutsuhisa Hiraoka
Kazuhito Takahashi
Masao Nasu
Nobuyasu Yamaguchi
Original Assignee
Fuji Electric Holdings Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Fuji Electric Holdings Co., Ltd. filed Critical Fuji Electric Holdings Co., Ltd.
Priority to AU2003242354A priority Critical patent/AU2003242354A1/en
Priority to JP2004507526A priority patent/JPWO2003100086A1/ja
Publication of WO2003100086A1 publication Critical patent/WO2003100086A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/144—Imaging characterised by its optical setup
    • G01N2015/1443—Auxiliary imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

Definitions

  • 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.
  • 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.
  • the description will mainly focus on bacteria.
  • a gene using a diamidino-phenylindole (DAPI) -acridine orange (AO) that binds to DNA is used.
  • DAPI diamidino-phenylindole
  • AO acridine orange
  • 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.
  • 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. .
  • 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). "
  • 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.
  • 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
  • the number of living cells is measured by labeling living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent.
  • 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.
  • second image a fluorescence image of the sample
  • counting the bright spots in the second image counting the bright spots in the second image
  • 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.
  • 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).
  • 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 ".
  • 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).
  • live cells such as microorganisms and cell tissues in the sample are labeled with a fluorescent reagent.
  • 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.
  • a fluorescent image (second image) of the sample is obtained.
  • 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).
  • the method for labeling living cells with a fluorescent reagent instead of the above-mentioned ⁇ metabolism in living cells '' (Invention of claim 6).
  • the reagent which becomes fluorescent due to metabolism in living cells the above-mentioned FDA, CFDA, CTC and the like can be used.
  • the living cells may be bacteria. (Invention of item 7) is preferred.
  • 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). ).
  • 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).
  • the washing solution is a buffer solution having a pH suitable for expressing the fluorescence of the fluorescent reagent (the invention of claim 10).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • Example 1 Method based on difference in number of bright spots
  • bacteria contained in sample 1 are captured on filter 2 by filtration.
  • 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;
  • 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.
  • 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.
  • a specific bacterium When labeling a specific bacterium, use is made of a method that recognizes an antigen antibody reaction or a specific gene sequence.
  • 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.
  • 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.
  • a buffer solution having a pH suitable for expressing the fluorescence of the fluorescent labeling reagent is suitable.
  • 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.
  • a fluorescent image (second image) of the membrane filter 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 1 the bacteria contained in the sample solution are captured on the membrane filter by filtration.
  • a fluorescent image (first image 6 shown in the center of FIG. 2) of the membrane filter capturing the bacteria is obtained.
  • a fluorescent labeling reagent is added onto the membrane filter, and the bacteria are fluorescently labeled.
  • a fluorescent image (second image 7 shown on the left side of FIG. 2) of the re-membrane filter is obtained.
  • 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.
  • 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.
  • image processing such as binarization and edge detection.
  • the above embodiment 1 can be used.
  • a fluorescence image (first image 6) of the membrane filter capturing the bacteria is acquired.
  • 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.
  • a fluorescent labeling reagent is added onto the membrane filter, and the bacteria are fluorescently labeled.
  • 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.
  • 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.
  • 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.
  • the counting accuracy can be improved by using the actual image of the sample instead of the first image instead of the fluorescent image.
  • the sample when acquiring the first image, the sample is irradiated with excitation light instead of white light.
  • 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.
  • 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.
  • 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.
  • 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.
  • a driving means for example, a stepping motor capable of precise position control is desirable.
  • 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.
  • 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.
  • 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.
  • At least the surface of the cell flow path 20 facing the imaging means 21 is made of transparent glass or plastic.
  • FIG. 6 Based on FIG. 6, an embodiment of a method using an antigen-antibody reaction for capturing living cells will be described below.
  • 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.
  • 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.
  • 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.
  • 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.
  • first image a fluorescence image or a stereoscopic image
  • second image a fluorescence image of the sample
  • Is obtained 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.
  • 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.

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Abstract

L'invention concerne un procédé et un dispositif de comptage de cellules vivantes permettant de compter des cellules vivantes telles que des microbes dans un échantillon, ou des cellules vivantes d'un tissu cellulaire marquées par un réactif fluorescent. Avant de marquer par fluorescence les cellules vivantes, on capture une image par fluorescence (première image) d'un échantillon. Après le marquage par fluorescence des cellules vivantes, on capture une image par fluorescence (seconde image) de l'échantillon. On détermine la différence (B-A) en nombre de points brillants entre la première et la seconde images. Selon un autre procédé, une image de différence entre la première et la seconde images est créée, et on compte le nombre de points brillants dans l'image de différence, ou on compte le nombre de points brillants, sur les points brillants dans la seconde image, qui ne sont pas dans la zone non visible autour de chaque point brillant dans la première image. L'influence d'impuretés fluorescentes peut être éliminée, indépendamment des propriétés de l'échantillon, ce qui améliore la précision de mesure.
PCT/JP2003/006325 2002-05-23 2003-05-21 Procede et dispositif de comptage de cellules vivantes WO2003100086A1 (fr)

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AU2003242354A AU2003242354A1 (en) 2002-05-23 2003-05-21 Living cell counting method and device
JP2004507526A JPWO2003100086A1 (ja) 2002-05-23 2003-05-21 生細胞の計数方法および装置

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JP2007060945A (ja) * 2005-08-30 2007-03-15 Matsushita Electric Ind Co Ltd 微生物計数装置
JP2008092812A (ja) * 2006-10-06 2008-04-24 Matsushita Electric Ind Co Ltd 微生物数計測方法
WO2009110462A1 (fr) * 2008-03-04 2009-09-11 株式会社ニコン Procédé de jugement de cellules vivantes pour observation de cellules, programme de traitement d'image pour observation de cellules et dispositif de traitement d'image
WO2010010670A1 (fr) * 2008-07-23 2010-01-28 株式会社ニコン Technique d’estimation de conditions cellulaires et processeur d’image pour l’examen de cellules
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