WO2018158810A1 - Dispositif d'observation de cellules - Google Patents

Dispositif d'observation de cellules Download PDF

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Publication number
WO2018158810A1
WO2018158810A1 PCT/JP2017/007728 JP2017007728W WO2018158810A1 WO 2018158810 A1 WO2018158810 A1 WO 2018158810A1 JP 2017007728 W JP2017007728 W JP 2017007728W WO 2018158810 A1 WO2018158810 A1 WO 2018158810A1
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WIPO (PCT)
Prior art keywords
image
range
unit
focus
cell
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PCT/JP2017/007728
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English (en)
Japanese (ja)
Inventor
藤原　直也
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株式会社島津製作所
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Priority to JP2019502314A priority Critical patent/JP6760477B2/ja
Priority to PCT/JP2017/007728 priority patent/WO2018158810A1/fr
Publication of WO2018158810A1 publication Critical patent/WO2018158810A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
    • G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto

Definitions

  • the present invention relates to a cell observation apparatus for observing the state of a cell, and more specifically, a phase image and an intensity image of an object obtained by arithmetic processing on a hologram obtained by recording a fringe pattern of an object wave and a reference wave obtained by a digital holography microscope.
  • the present invention relates to a cell observation apparatus that creates a reconstructed image such as the above.
  • the object light reflected or transmitted from the light source by the light source and the reference light directly reaching from the same light source acquire the interference fringes (hologram) formed on the detection surface of the image sensor or the like,
  • hologram interference fringes
  • an intensity image and a phase image are created as a reconstructed image of the object.
  • the above-mentioned advantage of the holographic microscope means that it is necessary to determine an appropriate focal position at the stage of creating an image based on a hologram obtained by photographing.
  • a plurality of images for example, phase images
  • the viewer displays the plurality of images. It is necessary to find an image in the most suitable in-focus state by comparing on the screen.
  • the details are in focus. It is difficult and time consuming to determine whether or not.
  • the image of the entire area of one cell culture plate or the entire area of one or a plurality of wells is large, it takes time to create a plurality of images with different focal positions.
  • the focal position is not necessarily the same for the entire area of one cell culture plate or the entire area of one or more wells, and the height of the bottom surface is different for each well in one cell culture plate. Since the bottom surface of the culture plate is inclined, it may not be possible to determine one focal position for the entire observation target region.
  • the present invention has been made to solve the above-mentioned problems, and in a cell observation device that creates and displays a phase image or the like based on hologram data obtained by a holographic microscope, an observer can appropriately focus while confirming the image.
  • the main purpose is to facilitate visual judgment and improve the workability in determining the position.
  • the present invention which has been made to solve the above problems, performs at least one of phase, intensity, or pseudo phase by performing arithmetic processing based on hologram data obtained by measuring a sample containing cells with a holographic microscope.
  • a cell observation device that creates a reconstructed image showing any two-dimensional distribution and displays it on a display unit, a) a focal position comparison image creating unit that creates a plurality of reconstructed images having different focal positions for the entire observation target region or a partial region thereof based on the acquired hologram data; b) Displaying a screen on which an image display frame on which one of a plurality of reconstructed images having different focal positions is displayed and a slider that is an operator for changing the focal position within a predetermined range are displayed.
  • the pseudo phase is a value corresponding to a phase difference in a phase contrast microscope, that is, phase information including an intensity element.
  • the holographic microscope may be any of an inline type, an off-axis type, a phase shift type, etc., regardless of the system.
  • the sample is a cell culture plate
  • the observation target region is the entire cell culture plate or a region including one or a plurality of wells formed on the plate. It can be. That is, the cell observation device according to the present invention is a device suitable for observing living cells in culture on a cell culture plate.
  • the focal position comparison image creating unit has different focal positions for the entire observation target region, for example, based on hologram data acquired for a cell culture plate in which cells are being cultured.
  • a plurality of phase images are created as reconstructed images.
  • the focus alignment display processing unit displays a screen on which an image display frame and a slider of a predetermined size are arranged on the display unit, but the focus position comparison image display processing unit responds to the position of the slider knob.
  • the phase image at the focal position is selected from the plurality of phase images created as described above and drawn in the image display frame. Accordingly, when the user performs an operation of appropriately moving the slider knob with the pointing device, phase images of different focal positions for the same region are displayed one after another according to the operation. Thereby, the user can visually confirm the change of the phase image accompanying the change of the focal position, that is, the state of the image blur while moving the slider knob.
  • the user for example, performs a predetermined operation after moving the slider knob so that the portion of interest can be seen most clearly, that is, in a focused state.
  • the focus position determination unit determines that the focus position corresponding to the phase image displayed at that time is the focus position.
  • the same operation may be performed using an intensity image or a pseudo phase image instead of the phase image.
  • the cell observation device is preferably configured to further include a focus alignment parameter setting unit for the user to set the range of change of the focus position assigned to the slider and the pitch of the change. .
  • the user can freely set the focal position change range and the pitch of the change assigned to the slider by the focus alignment parameter setting unit.
  • the focus position change pitch can be set wider (the change range is narrow) and the focus position can be searched more finely. This makes it easier to find a more accurate in-focus position.
  • the user designates the same focal position application range in which the same focal position can be applied on the wide-range display image which is a reconstructed image of the entire observation target area displayed on the display unit or a part of the observation target area.
  • a range designating unit for The focusing position determination unit may be configured to determine a focusing position for each same focal position application range specified by the range specification unit.
  • the range designating unit is a pointing device capable of designating a range of any shape and size on the displayed image, and an operation for recognizing the designated range as the same focal position application range.
  • a reception unit for example, as described above, the focal position can be determined for each well even if the appropriate focal position is different for each well in one cell culture plate. Then, the focus position can be made common.
  • An enlarged observation range designating unit for the user to designate a partial range on the wide-range display image as an enlarged observation range;
  • An enlarged image creating unit that creates an enlarged image with a magnification higher than at least the wide-range display image for the designated enlarged observation range, and displays the enlarged image on the screen of the display unit; It is good to set it as the structure further provided.
  • the magnified observation range designating unit is similar to the above range designating unit, for example, a pointing device capable of designating a range of any shape or size on the displayed image, and a magnified observation of the range designated thereby And an operation receiving unit recognized as a range.
  • the user can check an enlarged image in a narrow range designated on the wide-range display image and determine whether or not the image is in focus.
  • the accuracy of determination as to whether or not it is the in-focus position is improved, and workability for that is also improved.
  • the enlarged observation range may not necessarily be a desired portion.
  • a focus alignment target range designation for a user to specify a focus alignment target range used for determining a focal position in one or a plurality of the same focal position application ranges preferably, a focus alignment target range designation for a user to specify a focus alignment target range used for determining a focal position in one or a plurality of the same focal position application ranges. It can be set as the structure further provided with a part.
  • the focal position comparison image creating unit can create a plurality of reconstructed images having different focal positions for the focus alignment target range.
  • the data amount of the reconstructed image such as the phase image created by the focus position comparison image creating unit can be reduced by narrowing the focus alignment target range by the user. Accordingly, it is possible to speed up the process of calculating phase information and the like and the process of creating an image using the obtained phase information and the like. As a result, it is possible to shorten the time required for the work of determining the in-focus position and efficiently perform the cell observation work.
  • the cell observation apparatus when an observer (user) performs an operation of determining an appropriate focal position while visually confirming a reconstructed image such as a phase image, it is determined whether or not the subject is in focus. It can be done easily and accurately. As a result, the efficiency of the cell observation work itself can be improved, and highly accurate cell observation using a good reconstructed image can be performed.
  • the block diagram of the principal part of the cell observation apparatus which is one Example of this invention.
  • Explanatory drawing of the image creation process in the cell observation apparatus of a present Example. The flowchart which shows operation and the process in the case of focusing in the cell observation apparatus of a present Example.
  • Explanatory drawing at the time of the focus position alignment in the cell observation apparatus of a present Example. Explanatory drawing at the time of the focus position alignment in the cell observation apparatus of a present Example.
  • Explanatory drawing at the time of the focus position alignment in the cell observation apparatus of a present Example. Explanatory drawing at the time of the focus position alignment in the cell observation apparatus of a present Example.
  • FIG. 1 is a configuration diagram of a main part of the cell observation apparatus of this embodiment.
  • the cell observation apparatus of the present embodiment includes a microscope observation unit 1, a control / processing unit 2, an input unit 3 and a display unit 4 which are user interfaces.
  • the microscopic observation unit 1 is an in-line holographic microscope (IHM), and includes a light source unit 10 including a laser diode and an image sensor unit 11, and is provided between the light source unit 10 and the image sensor unit 11.
  • a cell culture plate 12 including cells 13 to be observed is arranged.
  • the cell culture plate 12 is movable in two axial directions of the X axis and the Y axis by a moving unit 14 including a driving source such as a motor.
  • the control / processing unit 2 controls the operation of the microscopic observation unit 1 and processes data acquired by the microscopic observation unit 1, and includes an imaging control unit 20, a measurement data storage unit 21, a phase information calculation unit 22, The whole image creation unit 23, the focal position comparison image creation unit 24, the image data storage unit 25, the focusing processing unit 26, the focal position information storage unit 27, the display processing unit 28, the operation reception processing unit 29, and the like are provided as functional blocks.
  • the entity of the control / processing unit 2 is a personal computer or a higher-performance workstation, and the function of each functional block described above is operated by operating dedicated control / processing software installed on the computer. Is realized. Therefore, the input unit 3 includes a pointing device such as a keyboard and a mouse. Further, as will be described later, the function of the control / processing unit 2 may be shared by a plurality of computers connected via a communication network instead of a single computer.
  • FIG. 2 is an explanatory diagram of image creation processing in the cell observation apparatus of the present embodiment
  • FIG. 3 is a flowchart showing operations and processing at the time of focusing
  • FIG. 4 is an explanation of a display method of a focal position comparison image at the time of focusing.
  • FIGS. 5A and 5B are schematic diagrams of a focus position alignment screen
  • FIGS. 6 to 8 are explanatory diagrams at the time of focus position alignment.
  • An observer sets a cell culture plate 12 on which cells (pluripotent cells) 13 to be analyzed are cultured at a predetermined position, and inputs information such as an identification number for identifying the cell culture plate 12 and a measurement date and time. Instruct the execution of measurement after inputting from 3.
  • FIG. 2 (a) six wells 50 having a circular shape in a top view are formed on the cell culture plate 12, and cells are cultured in the respective wells 50. Therefore, the entire cell culture plate 12, that is, the entire rectangular range including the six wells 50 is the observation target region, that is, the imaging target range.
  • the imaging control unit 20 controls the microscopic observation unit 1 to acquire data on the imaging target range as follows.
  • CMOS image sensors are installed on the same XY plane of the image sensor unit 11. These four CMOS image sensors are respectively responsible for photographing four quadrant ranges 51 obtained by dividing the entire cell culture plate 12 shown in FIG. 2A into four equal parts.
  • the range in which one CMOS image sensor can be photographed at a time is a rectangular range 52 including only one well 50 in the four-divided range 51 as shown in FIGS.
  • the four CMOS image sensors have long sides with a length corresponding to 15 imaging units in the X-axis direction and four rectangular images having a short side with a length corresponding to 12 imaging units in the Y-axis direction. Four different imaging units of the cell culture plate 12 are photographed at the same time.
  • the light source unit 10 irradiates a predetermined region of the cell culture plate 12 with coherent light having a minute angle spread of about 10 °.
  • the light (object light 16) that has passed through the cell culture plate 12 and the cell 13 reaches the image sensor unit 11 while interfering with the light (reference light 15) that has passed through the area close to the cell 13 on the cell culture plate 12.
  • the object light 16 is light whose phase has changed when passing through the cell 13.
  • the reference light 15 is light that does not pass through the cell 13 and therefore does not undergo phase change caused by the cell 13.
  • the cell culture plate 12 is moved stepwise by the moving unit 14 by a distance corresponding to the size of the imaging unit 53 in the XY plane. Thereby, the irradiation area of the coherent light emitted from the light source unit 10 moves on the cell culture plate 12, and each CMOS image sensor in the image sensor unit 11 can acquire hologram data corresponding to one imaging unit 53. it can.
  • the cell culture plate 12 is moved by the moving unit 14 stepwise by 180 times corresponding to the number of imaging units 53 included in one quadrant 51, and hologram data is acquired for each movement. In this way, hologram data for the entire cell culture plate 12 can be obtained without omission.
  • the hologram data obtained by the image sensor unit 11 of the microscopic observation unit 1 is sequentially sent to the control / processing unit 2 and stored in the measurement data storage unit 21.
  • the phase information calculation unit 22 reads out the hologram data for each imaging unit 53 from the measurement data storage unit 21 and executes the back propagation calculation of light.
  • the whole image creating unit 23 performs a tiling process (see FIG. 2D) for joining phase images in a narrow range based on the phase information calculated for each imaging unit 53, thereby observing the observation target region, that is, cell culture.
  • a phase image of the entire plate 12 is created.
  • the display processing unit 28 displays the phase image of the entire observation area on the screen of the display unit 4 (step S1).
  • phase information when calculating such phase information or creating a phase image, a known algorithm disclosed in Patent Documents 1 and 2 may be used.
  • intensity information, pseudo phase information, and the like may be calculated based on hologram data, and an intensity image and pseudo phase image based on these may be generated and displayed.
  • the observer visually recognizes the phase image of the entire observation target area on the screen of the display unit 4 and inputs a range in which the same focal position can be applied on the phase image (corresponding to the same focal position application range in the present invention). It designates by operation using the part 3 (pointing device). Specifically, when the observer designates a rectangular frame of any size both vertically and horizontally on the phase image and performs a predetermined operation with a pointing device, the operation reception processing unit 29 is surrounded by the designated frame. Is recognized as the same focal position application range (step S2). By repeating such an operation, a plurality of same focal position application ranges can be designated. Of course, the same focal position may be applicable to the entire observation target region without performing such designation.
  • FIG. 6 is a schematic diagram showing an example in which the same focal position application range 210 is designated for the region corresponding to the two wells 201 on the phase image 200 of the entire cell culture plate 12.
  • the observer designates a local focus alignment target range 220 used for focus alignment for each same focus position application range 210 on the phase image 200 of the entire cell culture plate 12.
  • This designation method may be the same as that of the same focal position application range 210, and appropriately select a part that is desired to be clearly observed or a part that is likely to be focused, such as a part where a cell focused by the observer exists. You can specify.
  • the phase image 200 of the entire cell culture plate 12 it is difficult to find the cell of interest because it is difficult to see the outline of each cell.
  • step S4 when the observer designates an appropriate focus alignment target range 220 and performs a predetermined enlargement operation, the focus processing unit 26 that has received an instruction via the operation reception processing unit 29, as shown in FIG. Then, an enlarged image 240 of the phase image corresponding only to the designated range is created and displayed on the screen of the display unit 4 (step S4).
  • the processing unit 26 creates an enlarged image after movement or enlargement / reduction by the operation, and updates the displayed enlarged image 240.
  • the observer can find an appropriate focus alignment target range 220 while searching for a cell of interest and confirming whether or not the cell can be clearly observed in the enlarged image 240. If an appropriate focus alignment target range 220 is thus determined, the focus alignment target range 220 may be determined by performing a predetermined operation with the input unit 3.
  • the observer instructs execution of the focal position alignment from the input unit 3 (step S5).
  • the focusing processing unit 26 creates a phase image having different focal positions in a plurality of stages as the focal position comparison image with respect to one or a plurality of designated focal position alignment target ranges 220 (see FIG. 4). ), Image data constituting the image is stored in the image data storage unit 25 (step S6). Since the size of the focus alignment target range 220 can be freely set by an observer, the focus alignment target range 220 may be contained in one imaging unit 53 or straddle a plurality of imaging units 53. In some cases.
  • the above-described tiling process is performed when creating the phase image.
  • smooth display can be performed during continuous confirmation of a plurality of focal position comparison images as will be described later. be able to.
  • the focus position range and pitch of the focus position comparison image created in step S6 may be determined in advance by the manufacturer that provides this apparatus. These parameters may be appropriately changed by the user (observer or manager of the apparatus). However, the focus position at the time of image display, which will be described later, is changed depending on the focus position range and pitch of the created focus position comparison image. Since the range and pitch of change are restricted, it is desirable that the focal position range is not too narrow and the focal position pitch is not too wide.
  • the display processing unit 28 displays a focus alignment screen 100 as shown in FIG. 5 on the screen of the display unit 4 (step S7).
  • a focus position adjustment screen 100 On the focus position adjustment screen 100, an image display frame 101 for displaying a focus position comparison image and a focus position change slider 102 which is one of GUI components are arranged.
  • the initial value of the focus position change range and the focus position change pitch determined by default is assigned to the focus position change slider 102, for example, the change of the focus position.
  • a focus position comparison image corresponding to the maximum or minimum focus position within the range is selected from the image data storage unit 25 and displayed in the image display frame 101.
  • the observer performs an operation of appropriately moving the knob 103 of the focus position change slider 102 by the input unit 3 while viewing the focus position comparison image displayed in the image display frame 101 of the focus position alignment screen 100.
  • the focus processing unit 26 that has received the instruction through the operation reception processing unit 29 selects a focus position comparison image associated with the focus position corresponding to the position of the knob 103 from the image data storage unit 25, and The display image in the display frame 101 is updated (step S8).
  • the observer determines, for example, whether or not the focused part such as the outline or pattern of the cell looks the clearest, that is, whether or not the focused part is in focus (step S9), and the in-focus state is the most focused.
  • the position of the knob 103 of the focal position changing slider 102 is adjusted so as to find the position (step S10). If it is determined that the in-focus state is obtained when the knob 103 is moved to a certain position (Yes in step S9), the “OK” button 104 arranged at the bottom of the focus position alignment screen 100 is clicked. Thus, the determination of the focal position is instructed (step S12).
  • the focusing processing unit 26 Upon receiving this instruction, the focusing processing unit 26 has the focal position corresponding to the position of the knob 103 at that time as the focal position for the same focal position application range 210 including the focus alignment target range 220 at that time. Information indicating this is stored in the focal position information storage unit 27 (step S13).
  • the default focus position change pitch is too coarse, or the actual focus position is out of the default focus position change range.
  • the observer appropriately changes the focal position change range and pitch assigned to the focal position change slider 102 on a parameter setting dialog screen, for example, pop-up displayed in response to a predetermined operation by the input unit 3. (Step S11).
  • the display processing unit 28 assigns the change range and pitch of the changed focus position to the focus position change slider 102.
  • a focus position comparison image corresponding to the maximum or minimum focus position within the change range of the focus position is selected from the image data storage unit 25 and displayed in the image display frame 101 (step S7).
  • the observer can determine an appropriate focal position while viewing the focal position comparison image.
  • the operations and processes shown in FIG. 3 may be performed for each focal position alignment target range 220 included in the same focal position application range 210.
  • region ie, the whole cell culture plate 12
  • the focal position is determined.
  • a process of newly creating a phase image of the whole cell culture plate 12 or a part thereof corresponding to the focal position may be performed.
  • the focus position obtained for the phase image may be used for the intensity image and the pseudo phase image, or the focus position may be determined for the intensity image and the pseudo phase image by the same method as the phase image. .
  • different focal position comparison images are displayed one after another in response to the observer manually operating the slider, but a plurality of focal position comparison images having different focal positions are automatically displayed.
  • An automatic playback function that is displayed one after another may be added.
  • a button for instructing automatic reproduction of the focal position comparison image is prepared, and when the button is operated, the focal position comparison image within the focal position change range determined at that time is automatically selected. It is good to reproduce like a movie by displaying in order. At this time, the playback speed may be adjusted.
  • Such automatic reproduction is particularly convenient for the observer to determine a rough focus position.
  • control / processing unit 2 In this case, complicated processing such as calculation of phase information based on hologram data and creation of a phase image is performed on the server side, and the terminal device receives image data created thereby, and a phase image is generated based on this image data. It is preferable to perform the process of displaying the message on the terminal device side.
  • the functional blocks of the control / processing unit 2 shown in FIG. 1 are separated into the terminal device side and the server side.
  • the functions of the control / processing unit 2 may be shared by a plurality of computers.
  • an in-line type holographic microscope is used as the microscopic observation unit 1, but other types of holographic methods such as an off-axis type and a phase shift type may be used as long as the microscope can obtain a hologram. Of course, it can be replaced by a microscope.

Abstract

L'invention concerne un dispositif d'observation de cellules dans lequel un cadre d'affichage d'image (101) et un curseur (102) qui permet de sélectionner une position de mise au point sont disposés dans un écran de positionnement de mise au point (100). Un grand nombre d'images de phase ayant différentes positions de mise au point dans une pluralité de niveaux sont créées à l'avance, les images de phase correspondant à la plage désignée par un observateur sur l'image de phase entière d'une plaque de culture cellulaire à observer, et l'une des images de phase est affichée dans le cadre d'affichage d'image (101). Lorsque l'observateur déplace une languette (103) du curseur (102), une unité de traitement de mise au point sélectionne une image de phase à une position de mise au point correspondant à la position de la languette (103), et affiche l'image de phase sélectionnée dans le cadre d'affichage d'image (101). L'observateur vérifie si l'image affichée est dans l'état focalisé tout en actionnant de manière appropriée le curseur (102), et lorsque l'image est dans l'état focalisé, il clique sur un bouton "confirmation" (104) pour déterminer la position de mise au point. Par conséquent, une position de mise au point appropriée peut être déterminée de manière efficace.
PCT/JP2017/007728 2017-02-28 2017-02-28 Dispositif d'observation de cellules WO2018158810A1 (fr)

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WO2021261150A1 (fr) * 2020-06-25 2021-12-30 富士フイルム株式会社 Dispositif de traitement d'informations, procédé d'exploitation associé, et programme d'exploitation associé

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WO2013070287A1 (fr) * 2011-11-07 2013-05-16 The Regents Of The University Of California Imagerie sans masque d'échantillons denses utilisant un microscope exempt de lentille multi-hauteur
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JP2016001274A (ja) * 2014-06-12 2016-01-07 レーザーテック株式会社 レーザ顕微鏡及びスキャナー
WO2016163560A1 (fr) * 2015-04-09 2016-10-13 国立大学法人神戸大学 Microscope holographique numérique

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US10690902B2 (en) * 2017-10-25 2020-06-23 Olympus Corporation Image processing device and microscope system
WO2021261158A1 (fr) * 2020-06-25 2021-12-30 富士フイルム株式会社 Dispositif de traitement d'informations, procédé de fonctionnement et programme de fonctionnement associés
WO2021261150A1 (fr) * 2020-06-25 2021-12-30 富士フイルム株式会社 Dispositif de traitement d'informations, procédé d'exploitation associé, et programme d'exploitation associé
JP7364796B2 (ja) 2020-06-25 2023-10-18 富士フイルム株式会社 情報処理装置、その作動方法及び作動プログラム
JP7397196B2 (ja) 2020-06-25 2023-12-12 富士フイルム株式会社 情報処理装置、その作動方法及び作動プログラム

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