WO2003067230A1 - Procede et dispositif de mesure d'une image fluorescente - Google Patents

Procede et dispositif de mesure d'une image fluorescente Download PDF

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Publication number
WO2003067230A1
WO2003067230A1 PCT/JP2003/001224 JP0301224W WO03067230A1 WO 2003067230 A1 WO2003067230 A1 WO 2003067230A1 JP 0301224 W JP0301224 W JP 0301224W WO 03067230 A1 WO03067230 A1 WO 03067230A1
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WO
WIPO (PCT)
Prior art keywords
light
sample
fluorescence image
autofocus
image measurement
Prior art date
Application number
PCT/JP2003/001224
Other languages
English (en)
Japanese (ja)
Inventor
Naohiro Noda
Mutsuhisa Hiraoka
Kazuhito Takahashi
Akihito NARIKUNI
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
Publication date
Application filed by Fuji Electric Holdings Co.,Ltd. filed Critical Fuji Electric Holdings Co.,Ltd.
Priority to JP2003566530A priority Critical patent/JP4106626B2/ja
Priority to AU2003207248A priority patent/AU2003207248A1/en
Publication of WO2003067230A1 publication Critical patent/WO2003067230A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G02B21/244—Devices for focusing using image analysis techniques

Definitions

  • This invention fluorescently labels (stains with a fluorescent reagent) cells such as microorganisms and tissue cells and fine particles such as mineral particles, and generates fluorescence by exciting the reagent.
  • the present invention relates to a fluorescence image measurement method and apparatus for generating fluorescence by exciting fluorescent molecules originally present in fine particles such as mineral particles and measuring the fluorescent image.
  • Fluorescence image measurement of microorganisms and tissue cells is used in many fields dealing with living organisms.
  • autofocus (AF) technology has come into general use with the development of image sensors and image processing technology in recent years.
  • the amount of high frequency components (contrast) contained in an image signal is determined, and the position where the contrast is maximized is determined as the focal point.
  • the microorganisms include prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast, fungi, lower algae, viruses, and the like.
  • the tissue cells include animal and plant-derived cultured cells and It contains pollen such as cedar and cypress. In addition, it is not limited only to living things, such as measuring dead bacteria.
  • staining can be performed with one or more color-forming substances capable of staining a detection target (for example, a microorganism).
  • the color-forming substance is not particularly limited as long as it is capable of forming a color by acting on cell components contained in a microorganism, and typical examples thereof include nucleic acids and proteins.
  • a fluorescent staining solution for staining when analyzing microorganisms in general, it can be used to analyze the structure of fluorescent nucleobase analogs, fluorescent dyes that stain nucleic acids, blue liquids that stain proteins, and proteins.
  • Environmentally friendly fluorescent probes used stains used for analysis of cell membrane and membrane potential, stains used for fluorescent antibody labeling, etc.For aerobic bacteria, stains that develop color by cell respiration are used.
  • stains that stain mitochondria stains that stain the Golgi apparatus, stains that stain the endoplasmic reticulum, a stain that reacts with intracellular esterase and its modifying compounds, etc.
  • a staining solution used for observing bone tissue a staining solution that is a nerve cell tracer, and the like can be mentioned.
  • the reagent After staining the microorganisms or the like with the staining reagent, the reagent is excited to generate fluorescence, and the fluorescence image is measured by an imaging device (for example, a CCD camera device), whereby the microorganisms and the like can be counted.
  • an imaging device for example, a CCD camera device
  • the color of the generated fluorescence (green, red, etc.) or the wavelength band of the fluorescence differs depending on the microorganism and the type of the staining reagent.
  • a total cell count detection for detecting all microorganisms an assay for staining and counting only microorganisms having respiratory activity, an assay for staining and counting only microorganisms having esterase activity, Alternatively, it can be applied to a wide range of fields, such as an assay that stains and counts microorganisms of a specific genus or species by using a multiple staining method that combines multiple chromogenic substances.
  • a focus detection device for a microscope has been proposed which solves the above problems and aims at improving the efficiency of autofocusing (for example, see Patent Document 1 described later).
  • the focus detection device for a microscope disclosed in Patent Document 1 states that “the incident light fluorescence that irradiates a sample with excitation light of a specific wavelength and collects light longer than the excitation light with an objective lens.
  • the image sensor is arranged on the short wavelength side and the long wavelength side, respectively, which are separated by the image sensor and captures the observation light image of the sample, and an image sensor for accumulating the observation light image of the sample.
  • Focus detection means for detecting the focus state of the observation light image, servo means for driving at least one of the objective lens and the sample side according to the degree of focus of the focus detection means, and a focus means and an image Capacitors, Oh in those with C P U to control the focus detection means "0.
  • the apparatus disclosed in Patent Document 1 described above is provided with a transmissive illumination means on the side facing the objective lens, and irradiates the specimen with light having a wavelength longer than the fluorescence observation wavelength by this transmissive illumination, thereby obtaining the specimen. It performs AF based on the observed image, has wavelength separation means for separating the fluorescence image and the AF image, and has two systems of imaging means for obtaining each image. In addition, when fluorescence image measurement is performed in a plurality of wavelength bands by switching the fluorescence image measurement wavelength, an in-focus position is adjusted by combining an optical path length correction unit.
  • the one disclosed in Patent Document 1 has various problems as described below. That is,
  • Patent Document 2 discloses an optical measurement method for accurately focusing an optical system. Describes the following method.
  • a plurality of excitation light sources having different wavelength regions are provided, and a light is generated from a first excitation light source among the plurality of excitation light sources.
  • a wavelength region substantially equal to the wavelength of the fluorescence is selected from the plurality of excitation light sources.
  • the optical measurement method disclosed in Patent Document 2 is mainly for reading a DNA array test piece, and the position where the amount of reflected light is maximum is defined as a focus position.
  • autofocus is not performed by detecting the focus state of the observation light image of the specimen or based on image information.
  • it is necessary to irradiate the light for autofocus on the same optical path as the excitation light, and for that purpose, it must be incident by a coaxial epi-optical system.
  • FIG. 4 of the publication and the description of the relevant part there is a problem that the device configuration is complicated and the cost is high, for example, it is necessary to rotate a plurality of types of dichroic mirrors with a motor.
  • the present invention has been made in view of the above points, and has a simple configuration having a small number of constituent elements.
  • a fluorescence image measurement method that enables AF based on image information without quenching even for specimens with low fluorescence intensity, and for specimens supplemented on the membrane filter surface or specimens with unclear contrast.
  • An object of the present invention is to provide a fluorescence image measurement device. Disclosure of the invention
  • the invention according to claim 1 provides a fluorescent image measurement method for performing autofocus based on image information obtained via an imaging unit. First, irradiate autofocusing light emitted in the fluorescence image measurement wavelength band, determine the degree of focusing based on the image information obtained as a result, and determine the degree of focusing based on the degree of focusing. Drive the at least one of them to search for the in-focus position, and after arriving at the in-focus position, stop irradiating the auto-focusing light. Then, irradiate the specimen with excitation light, and obtain a fluorescence image emitted from the specimen.
  • autofocus can be performed without irradiating the sample with excitation light, and autofocus can be performed using a simple configuration with few components, so that a decrease in light-receiving efficiency is minimized, and Thus, it is possible to measure a specimen having a low fluorescence intensity.
  • the measurement sample is a cell such as a microorganism or a tissue cell (the invention of claim 2).
  • the measurement sample includes a plurality of types of cells, and when the plurality of types of cells are measured, the measurement sample is set in advance according to the cells to be measured.
  • the autofocus light in a plurality of fluorescence image measurement wavelength bands is sequentially switched and irradiated to determine the degree of focusing, and measure a fluorescence image of each cell (claim 4).
  • the present invention can be applied to a system that performs image measurement with a plurality of excitation-fluorescence characteristics by switching light for autofocus.
  • a pattern close to the specimen may be provided, and autofocus may be performed based on image information of the pattern.
  • Invention of range 5 the contrast is evaluated.However, malfunction such as focusing on a position other than the sample holding position is prevented, and the contrast evaluation is performed. P Hiring 224
  • the method using patterns is effective for accurate and convenient flight.
  • the pattern can be provided on a surface of a slide glass holding a specimen (the invention according to claim 6). In the invention according to claim 5, the pattern can be provided on a surface of a filter that filters and supplements the sample (the invention according to claim 7). Further, in the invention of claims 5 to 7, the pattern includes a number or a figure, and by identifying these, it is possible to obtain positional information of the specimen. (Invention of paragraph 8). Further, as an apparatus for performing the fluorescence image measurement method, the inventions of the following claims 9 to 11 are preferable.
  • an autofocus light irradiation unit that irradiates a sample with light in a fluorescence image measurement wavelength band;
  • Excitation light irradiating means autofocus and fluorescence image measurement imaging means, fluorescence and excitation filters and a fluorescent filter block having a dichroic mirror, and a light receiving optical system or sample according to the degree of focus Focusing driving means for driving at least one of the following;
  • arithmetic control means wherein the arithmetic control means determines the degree of focus based on image information obtained by irradiating the autofocus light, At least one of the receiving optical system or the sample is driven according to the degree of focus to search for the in-focus position. Stopping the irradiation, then, irradiated with excitation light to the specimen, characterized in that it comprises a control function of measuring the fluorescence image emitted by the sample (the invention of Claim C.
  • the light source in the autofocus light irradiating means and / or the excitation light irradiating means is a light emitting diode or a semiconductor laser.
  • the autofocus light irradiation unit in order to perform autofocus by switching light in a plurality of fluorescence image measurement wavelength bands, includes a plurality of autofocus light irradiation units.
  • a light source, and a plurality of the fluorescent filter blocks are provided; and the arithmetic control means includes a light source of a plurality of predetermined fluorescence image measurement wavelength bands respectively corresponding to a plurality of types of cells.
  • a function of performing auto focus by sequentially switching (invention of claim 11).
  • FIG. 1 is a schematic configuration diagram of a fluorescence image measurement device according to a first embodiment of the present invention.
  • FIG. 2 is a schematic configuration diagram of a fluorescence image measurement device according to a second embodiment of the present invention.
  • FIG. 3 is an explanatory diagram illustrating an example in which a pattern for AF is provided.
  • FIG. 4 is an explanatory diagram illustrating a different example in which an AF pattern is provided.
  • FIG. 5 is an explanatory diagram illustrating still another example in which positional information is provided in an AF pattern.
  • FIG. 1 is a schematic configuration diagram of a fluorescence image measurement device according to a first embodiment of the present invention.
  • Fig. 1 is a sample
  • 2 is a light source for AF
  • 3 is a dichroic mirror
  • 4 is a filter
  • 5 is an objective lens
  • 6 is an imaging lens
  • 7 is an image sensor
  • 8 is a calculation unit
  • 9 is a stage movement.
  • 10 is a light source for excitation
  • 11 is a condenser lens
  • 13 is a fluorescence filter block.
  • the sample 1 is irradiated obliquely from above.
  • a method of performing AF with incident light and performing fluorescence image measurement using an epi-illumination optical system is not limited to this method.
  • a modification in which excitation light and AF light are irradiated from an oblique direction Includes an example in which the light for AF is transmitted light, but the structure shown in FIG. 1 is the simplest in structure and can reduce the cost.
  • a general method using contrast between pixels is used.
  • the specimen 1 is irradiated from the AF light source 2 with light containing a wavelength in the fluorescence image measurement wavelength band, so as to suppress the fading of the specimen.
  • the fluorescence image measurement wavelength band is determined by the spectral transmission characteristics of the dichroic mirror 3 and the spectral transmission characteristics of the fluorescent light receiving filter 4.
  • a light emitting diode (LED) or a semiconductor laser is suitable.
  • the reason is that the light emission spectrum can be variously selected by selecting the element, and the characteristics are not easily deteriorated even if ONZOFF is repeated. Furthermore, because these elements are small and lightweight, they can be easily assembled as light sources for AF.
  • an image for AF can be obtained in the fluorescence image measurement wavelength band
  • a wavelength separating unit and two image pickup devices as in the device disclosed in Patent Document 1 are not required, and the devices described in Patent Documents 1 and 2 are required.
  • the device configuration can be simplified. It is also possible to obtain a desired wavelength by using a white lamp as the light source for AF and combining an optical filter, and to perform irradiation and non-irradiation by opening and closing the shutter. Further, the angle of irradiation of the AF light is preferably such that the inclination angle with respect to the sample surface is about 5 to 45 °.
  • the image of the specimen at the time of irradiating the AF light is captured by the image sensor 7 via the objective lens 5, the dichroic mirror 13, the fluorescent light receiving side filter 4, and the imaging lens 6.
  • the imaging element a CCD camera element or a CMOS camera element is suitable.
  • the image obtained by the image sensor 7 is used as the arithmetic control means.
  • the contrast is evaluated by a general AF method, for example, calculating as a luminance difference between adjacent pixels, and setting a position where the contrast becomes maximum as a focal point position.
  • the processing up to the in-focus position is generally as follows.
  • stage moving mechanism 9 is not an essential requirement, as long as it can search the focal point by driving at least one of the sample 1 and the light receiving system.
  • the AF light is turned off, and then the excitation light source 10 is turned on.
  • the light from the excitation light source 10 irradiates the sample 1 via the condenser lens 11, the excitation side filter 12, the dichroic mirror 3, and the objective lens 5, thereby enabling fluorescence image measurement. .
  • a high-pressure mercury lamp is often used as the excitation light source.
  • the characteristics of the high-pressure mercury lamp deteriorate significantly when ONZNZ OFF is repeated in a short time.
  • a shutter is required. Therefore, if the wavelength characteristics and the amount of light can be satisfied, it is desirable to use a light emitting diode (LED) or a semiconductor laser as the excitation light source, similarly to the AF light source.
  • LED light emitting diode
  • the wavelength of the branch point between the reflectance and the transmittance is 505 nm (that is, the transmittance is about 50% at 505 nm, and the transmittance decreases at wavelengths shorter than 505 nm (reflectance)
  • the transmittance is about 50% at 505 nm
  • the transmittance decreases at wavelengths shorter than 505 nm (reflectance)
  • FIG. 2 a fluorescence image measurement device according to a second embodiment of the present invention shown in FIG. 2 will be described. 2, the same functional members as those shown in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted.
  • FIG. 2 shows a case where a plurality of living cells, for example, a plurality of bacteria are simultaneously measured, or a case where the same bacterium contains live and dead bacteria.
  • 1 shows an apparatus according to the invention of item 1.
  • a plurality of staining reagents are used depending on the type of bacteria, and the fluorescent color differs depending on the type of bacteria and the staining agent.
  • the three elements of the excitation side filter 12, the dichroic mirror 3, and the image measurement side filter 4 are usually combined into one unit.
  • a plurality of fluorescent filter blocks 13 are provided. By switching these, it is possible to perform image measurement with multiple excitation-fluorescence characteristics using white light emitted from a high-pressure mercury lamp.
  • An example in which the present invention is applied to a fluorescence image measurement system having such an image measurement condition switching mechanism will be described below with reference to FIG. In FIG.
  • a position recognition mechanism 14 is provided for five fluorescent filter blocks 13, and when a block is switched, its output is taken in, so that the calculation unit 8 automatically recognizes the block setting status. To do it. Further, the calculating unit 8 determines the set fluorescence image measurement wavelength of the fluorescence filter block 13 and irradiates the sample 1 with AF light suitable for the wavelength.
  • the AF light source 15 a plurality of types of light-emitting diode semiconductor lasers may be switched and used, or a switching mechanism of a white light source and an optical filter having a predetermined spectral transmittance and a shutter mechanism may be combined. May be used.
  • the excitation light source 10 is turned on, or the excitation light source is turned on in advance, and the specimen 1 is irradiated with the excitation light by opening the shirt that has blocked the excitation light, and fluorescence image measurement is performed.
  • the excitation light source may be, in addition to the white light, a plurality of LEDs that emit excitation light according to the measurement sample.
  • a pattern is drawn on the surface of the slide glass G in advance.
  • the pattern is drawn using non-fluorescent paint or non-fluorescent metal deposition.
  • the thickness and density of the pattern shall be set so that at least a part of the boundary appears in the field of view when the image is captured.
  • Specimens such as fluorescently labeled microorganisms and tissue cells are dropped on slide glass, covered with a cover glass, and used as a measurable sample. At this time, the specimen 1224
  • Irradiate light for AF Since at least a part of the pattern is shown in the AF image, autofocus is performed with that part as the target. Subsequently, the excitation light source is turned on and fluorescence image measurement is performed in the same manner as in the embodiment shown in FIGS.
  • the pattern which is the reference of the autofocus
  • the sample are close to or close to each other.
  • the distance between the pattern and the specimen should be shorter than the depth of focus of the light receiving system composed of the objective lens, lens barrel, imaging lens, and image sensor.
  • FIG. 4 When measuring microorganisms and tissue cells, the process of capturing a sample by filtration through a membrane filter and measuring it is an extremely common operation.
  • a fluorescent image measurement method for providing a pattern on the surface of the membrane filter will be described with reference to FIG.
  • the pattern is drawn on the surface of the membrane filter F by depositing a non-fluorescent paint or a non-fluorescent metal.
  • the thickness and density of the pattern should be set so that at least some of the boundaries are visible in the field of view when the image is captured.
  • Samples of fluorescently labeled microorganisms and tissue cells are filtered through a membrane filter.
  • the sample captured on the membrane filter is used as a sample for measurement. At this time, the sample is in close proximity to the surface of the membrane filter.
  • the differential interference method is used for a sample having no coloration and a refractive index close to water.
  • Image measurement using is an essential requirement.
  • the operation of supplementing the sample by filter filtration and measuring the sample is an extremely well performed operation, but in this state, it is difficult to obtain a differential interference image. That is, what is disclosed in the gazette of Patent Document 1 cannot be applied to the sample supplemented on the filter.
  • FIG. 5 When measuring specimens such as microorganisms and tissue cells, it is not uncommon to scan a sample on a plane perpendicular to the fluorescence image measurement direction and measure the image on multiple screens in order to reduce statistical variations. In such a case, if the pattern is not a simple line, but a form that can obtain positional information, for example, as shown in Fig. 5, for example, by drawing a combination of sections and serial numbers on the surface of the membrane filter F, The location of a specimen such as an object or a tissue cell can be grasped.
  • microorganisms or tissue cells change over time, this method can be used to recognize individual specimens individually and track those changes.
  • Typical examples are applications that capture the growth of microorganisms and cases where the effects of drugs on tissue cells are evaluated.
  • Such positional information may be formed on the surface of the slide glass.
  • the present invention can be used for a measurement method and an apparatus for labeling a sample containing fine particles such as cells such as microorganisms and tissue cells and mineral particles with a staining reagent and measuring a fluorescent image emitted from the sample.
  • Main measurement method and apparatus The fields of application include medical care, food production, and water and sewage.
  • autofocus can be performed without irradiating the sample with excitation light, and a reduction in light receiving efficiency can be minimized by realizing a simple mechanism with few components.
  • it can measure samples with low fluorescence intensity.
  • the present invention can be applied to a system that performs image measurement with a plurality of excitation-fluorescence characteristics while switching the fluorescence filter block. Furthermore, even for a sample whose contrast is unclear, autofocus can be performed by using a pattern provided close to the sample.

Abstract

L'invention concerne un procédé et un dispositif de mesure d'une image fluorescente, comprenant la mise en oeuvre d'une auto-focalisation (AF) à partir d'informations d'image obtenues à l'aide de moyens d'imagerie. Un échantillon utilisé comme spécimen de mesure est irradié de lumière pour l'auto-focalisation dans une bande de longueur d'onde de mesure d'image fluorescente, et l'état focalisé est évalué en fonction des informations d'image obtenues par l'irradiation. En fonction de l'état focalisé, au moins un système optique de réception de lumière ou l'échantillon est entraîné, la position de focalisation est recherchée, l'irradiation de la lumière d'auto-focalisation s'arrête lorsque la position de focalisation est atteinte, l'échantillon est irradié par une lumière d'excitation, et une image fluorescente formée par la lumière émise par le spécimen est mesurée. On peut ainsi procéder à l'auto-focalisation à partir des informations d'image et au moyen d'une structure simple comprenant un petit nombre d'éléments constitutifs sans causer l'extinction, y compris pour un échantillon à faible intensité de fluorescence.
PCT/JP2003/001224 2002-02-07 2003-02-06 Procede et dispositif de mesure d'une image fluorescente WO2003067230A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2003566530A JP4106626B2 (ja) 2002-02-07 2003-02-06 蛍光画像計測方法および装置
AU2003207248A AU2003207248A1 (en) 2002-02-07 2003-02-06 Fluorescent image measuring method and device

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002030648 2002-02-07
JP2002-30648 2002-02-07

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WO2003067230A1 true WO2003067230A1 (fr) 2003-08-14

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AU (1) AU2003207248A1 (fr)
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JP2005284136A (ja) * 2004-03-30 2005-10-13 Olympus Corp 観察装置および観察装置の焦点合わせ方法
JP2006011149A (ja) * 2004-06-28 2006-01-12 Nikon Corp 蛍光顕微鏡
JP2007093250A (ja) * 2005-09-27 2007-04-12 Yokogawa Electric Corp バイオチップ読み取り装置およびバイオチップ読み取り方法
JP2007108223A (ja) * 2005-10-11 2007-04-26 Olympus Corp 顕微鏡システム
WO2007139201A1 (fr) * 2006-05-31 2007-12-06 Olympus Corporation Procédé et dispositif d'imagerie d'un organisme échantillon
WO2008022139A2 (fr) 2006-08-14 2008-02-21 Westover Scientific, Inc. Ensemble de lumière fluorescente à l'état solide et microscope
CN101930116A (zh) * 2009-06-23 2010-12-29 索尼公司 生物样本图像获取装置、获取方法以及获取程序
JP2011133366A (ja) * 2009-12-24 2011-07-07 Ihi Corp 微生物検出方法、フィルタ及び蛍光印配置板
CN102834759A (zh) * 2010-09-14 2012-12-19 欧姆龙株式会社 观察光学系统及激光加工设备
US9000399B2 (en) 2011-05-03 2015-04-07 Samsung Electronics Co., Ltd. Fluorescence detecting optical system and multi-channel fluorescence detection apparatus including the same
EP2315005B1 (fr) * 2004-01-14 2016-01-13 Life Technologies Corporation Dispositif de fluorométrie et procédé de détection dans des échantillons biologiques
WO2016157458A1 (fr) * 2015-03-31 2016-10-06 株式会社ニコン Appareil de mesure, système de mesure, procédé de traitement de chaîne de signal, et programme
EP3466335A1 (fr) * 2011-12-21 2019-04-10 Catherine M. Shachaf Méthode pour l'imagerie de fluorecence avec autofocus
WO2022264495A1 (fr) * 2021-06-18 2022-12-22 浜松ホトニクス株式会社 Dispositif de mesure
US11815458B2 (en) * 2019-12-31 2023-11-14 Illumina, Inc. Autofocus functionality in optical sample analysis

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