WO2019180808A1 - Dispositif d'observation de cellules - Google Patents

Dispositif d'observation de cellules Download PDF

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Publication number
WO2019180808A1
WO2019180808A1 PCT/JP2018/011002 JP2018011002W WO2019180808A1 WO 2019180808 A1 WO2019180808 A1 WO 2019180808A1 JP 2018011002 W JP2018011002 W JP 2018011002W WO 2019180808 A1 WO2019180808 A1 WO 2019180808A1
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WIPO (PCT)
Prior art keywords
image
unit
partial
hologram
phase
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PCT/JP2018/011002
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English (en)
Japanese (ja)
Inventor
倫誉 山川
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株式会社島津製作所
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Application filed by 株式会社島津製作所 filed Critical 株式会社島津製作所
Priority to PCT/JP2018/011002 priority Critical patent/WO2019180808A1/fr
Priority to JP2020507159A priority patent/JP7036192B2/ja
Publication of WO2019180808A1 publication Critical patent/WO2019180808A1/fr

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • 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. More specifically, a hologram in which interference fringes between an object wave and a reference wave are recorded by a holographic microscope, and phase information and intensity information are obtained based on the hologram data.
  • the present invention relates to a cell observation apparatus that creates a phase image, an intensity image, and the like after calculation.
  • Digital holographic microscopes acquire interference fringes (holograms) formed on the detection surface of an image sensor or the like by the object light reflected or transmitted from the light source and the reference light directly reaching from the same light source. Then, phase information and amplitude (intensity) information are acquired by performing a light wave back-propagation calculation process based on the hologram, and an intensity image and a phase image are created as a reconstructed image.
  • a digital holographic microscope has an advantage that phase information at an arbitrary distance can be obtained at the stage of arithmetic processing after obtaining a hologram, so that it is not necessary to perform focusing one by one during photographing.
  • Digital holographic microscopes include in-line, off-axis, and phase shift types.
  • the configuration of an optical system for acquiring a hologram is mainly different.
  • the off-axis type the light emitted from the laser light source is usually divided into reference light and irradiation light that irradiates the object, and the object light and reference light that have passed through the object are incident on the irradiation light differently from each other. The light is incident on the image sensor at an angle.
  • the inline type the light emitted from the laser light source is irradiated on the object without being divided, and the object light that has passed through the object and the reference light that has passed through the vicinity of the object without passing through the object are combined. Incidently incident on the image sensor.
  • the phase shift type a hologram whose phase is different in a plurality of stages is obtained by changing the optical path length of the reference light divided using the phase shift interferometer in a plurality of stages.
  • Patent Document 1 Non-Patent Document 2
  • Patent Document 1 Non-Patent Document 2
  • Patent Document 2 has proposed a phase recovery method by iterative calculation of light wave propagation based on a hologram for a plurality of wavelength lights acquired by an inline digital holographic microscope. Is quite complex and computationally intensive.
  • a cell observation apparatus for observing living cells in culture, it is necessary to create a high-resolution image so that each cell can be observed in detail over a wide range of the entire culture plate or the entire well formed on the plate. is there.
  • holograms are acquired for each of a number of small regions obtained by finely dividing the observation target region (for example, the entire culture plate). It is necessary to obtain a two-dimensional distribution of phase information and amplitude information for each small region by performing arithmetic processing based on the above, and to reconstruct an image of the observation target region by synthesizing such two-dimensional distributions of a large number of small regions.
  • the entire surface of the culture plate is divided into about 900 small areas, and holograms for a plurality of wavelengths are obtained with high resolution (for example, 4000 ⁇ 3000 pixels / sheet) for each small area. It takes 1 hour or more from the start of measurement until the phase image reconstruction process is completed. That is, the phase image and the intensity image of the sample can be observed only after 1 hour or more has elapsed since the measurement start time.
  • the existing apparatus when a hologram image of the entire observation target area is obtained in the holographic microscope, it is immediately displayed on the computer screen for controlling the microscope, and the operator can confirm it. I am doing so. Unlike the phase image and intensity image obtained by image reconstruction, the hologram image provides almost no information about individual cells, but the outline of wells and large foreign objects in the culture plate can be sufficiently visually confirmed. . Therefore, if the operator looks at the displayed hologram image and confirms an abnormal state such as, for example, a failure in installing the culture plate in the apparatus or contamination of a large foreign object, the processing is stopped at that point and the situation is stopped. Confirm. As a result, with respect to some defects, it is possible to avoid performing a wasteful process by finding the defect before confirming the reconstructed phase image, that is, immediately after the end of imaging.
  • the hologram image is not an image in which individual cells can be clearly observed, and it is difficult to discriminate even if a small foreign substance or the like is mixed. Therefore, for example, even if the culture conditions of the cells are inappropriate and differentiation has progressed abnormally or cells that are different from the target are mixed, it is necessary to discard the sample. Therefore, it is not known until the phase image and the intensity image are confirmed, and wasteful time and manpower are consumed. For these reasons, there is a strong demand for an apparatus that can detect the above-described sample and imaging problems before the execution of a complicated and time-consuming process or even after the start of the process.
  • the present invention has been made to solve the above-described problems, and the object of the present invention is to perform phase processing on a sample containing cells by performing arithmetic processing such as phase recovery based on a hologram acquired by a holographic microscope.
  • a cell observation device that reconstructs an image or intensity image, it is possible to avoid wasting time and manpower by allowing the user to quickly and accurately grasp the occurrence of a sample failure or measurement failure. It is to provide a cell observation device.
  • the present invention made to solve the above problems is a cell observation device using a holographic microscope, a) a light source, b) a detection unit that acquires a hologram that is an interference fringe between the object wave and the reference wave when the sample including cells is irradiated with light emitted from the light source unit; c) a moving unit that moves one or both of the light source unit and the detection unit and the sample so that the measurement position on the sample moves; d) Measurement for controlling the light source unit, the detection unit, and the moving unit so as to repeat acquisition of the hologram at each measurement position in a predetermined observation target region while moving the measurement position on the sample by the moving unit.
  • a control unit e) calculating phase information and / or intensity information based on hologram data obtained at each measurement position in a predetermined observation target region under the control of the measurement control unit, and corresponding to the observation target region
  • a first reconstructed image creating unit for creating a phase image showing a two-dimensional distribution of phase information and / or an intensity image showing a two-dimensional distribution of intensity information
  • the creation department g) a display processing unit for displaying the partial phase image and / or the partial intensity image created by the second reconstructed image creating unit on the display unit; It is characterized by having.
  • the sample is a cell culture container
  • the maximum area where hologram data can be acquired by the holographic microscope is the whole culture container or a part of the culture container.
  • the culture container include a cell culture plate in which one or a plurality of wells are formed, a petri dish, and a culture flask for mass culture. Therefore, the cell observation apparatus according to the present invention is a suitable apparatus for non-invasive observation of living cells being cultured in such a culture container.
  • the moving unit moves the light source unit and the detection unit integrally with respect to the culture plate whose position is fixed, so that the light emitted from the light source unit on the culture plate is The irradiated measurement position is changed.
  • the measurement control unit irradiates light (generally coherent light) to one measurement position in the culture plate while moving the light source unit and the detection unit integrally in a stepwise manner by the moving unit, thereby detecting the detection unit.
  • the operation of acquiring data (hologram data) indicating the two-dimensional distribution of light intensity by the hologram formed on the detection surface is repeated.
  • the first reconstructed image creating unit calculates phase information and / or intensity information based on hologram data obtained at different measurement positions, and sets the observation target region based on the obtained phase information and / or intensity information. A corresponding phase image and / or intensity image is created.
  • the second reconstructed image creation unit has one measurement or one specific measurement position designated by the user prior to or in parallel with the processing by the first reconstructed image creation unit.
  • Phase information and / or intensity information is calculated based only on hologram data obtained within a position or within a predetermined range spanning a plurality of measurement positions, and a partial phase image and / or partial intensity image is calculated based on the obtained information. create.
  • the range in which this partial phase image and / or partial intensity image is created is limited to a much smaller area than the entire observation target area. That is, the partial phase image or the partial intensity image is an image corresponding to a very small part of the entire observation target region.
  • the display processing unit displays the partial phase image and / or the partial intensity image on the screen of the display unit.
  • the amount of hologram data that is the target of processing in the second reconstructed image creation unit is much smaller than that of hologram data that is the target of processing in the first reconstructed image creation unit. Therefore, the time required for the second reconstructed image creation unit to read the hologram data is much shorter than the time required for the first reconstructed image creation unit to read the hologram data, and the second reconstructed image creation The time required for the arithmetic processing and image reconstruction in the unit is much shorter than that in the first reconstructed image creating unit. Therefore, the partial phase image and the partial intensity image are displayed in a relatively short time from the time when the measurement is completed.
  • the partial phase image and the partial intensity image are images corresponding to a very small part of the entire observation target area, but are displayed when the user designates an appropriate or particularly important measurement position or range. From the partial phase image and the partial intensity image, it is possible to confirm the suitability of measurement (imaging), the presence or absence of cell abnormalities, the presence of foreign matter, and the like before the phase image of the entire observation target region is created. As a result, when the operator determines that the reconstruction process of the phase image or the like of the entire observation target region is unnecessary, the process is not performed or even if the process is started By canceling, it is possible to avoid wasting time and labor.
  • the holographic microscope system such as in-line type, off-axis type, and phase shift type is not limited.
  • the optical system has a simple configuration and the distance between the sample and the light source unit is changed in a plurality of stages.
  • An in-line configuration using holograms at a plurality of wavelengths is desirable in that the drive mechanism is simplified because it is not necessary.
  • a hologram image creating unit that creates a hologram image based on the hologram data obtained at each measurement position in the predetermined observation target region and displays the hologram image on the screen of the display unit; Instruction for the user to instruct the measurement position or range corresponding to the partial phase image and / or partial intensity image created by the second reconstructed image creation unit on the hologram image displayed by the hologram image creation unit An input section; It is good to set it as the structure further provided.
  • Hologram images can be displayed immediately after the measurement, unlike phase images. Further, even when measurement is being performed, a partial hologram image corresponding to the measurement position can be displayed every time measurement for one measurement position is completed. Since this hologram image is a two-dimensional distribution of light intensity corresponding to the hologram, the living cells are often not visualized sufficiently, but the edges of wells with large steps are sufficiently visible. . Therefore, the operator instructs the instruction input unit, for example, which partial phase image or partial intensity image of which well is to be viewed on the displayed hologram image during measurement execution or immediately after the measurement is completed. Thereby, the operator can confirm the partial phase image and partial intensity image of a desired measurement position and range in a short waiting time.
  • the partial phase image and the partial intensity image may be displayed.
  • cells are easy to see but foreign matters such as dust tend to be hard to see.
  • foreign substances tend to be visible, but cells tend not to be visible. Therefore, it is more preferable to display both the partial phase image and the partial intensity image in the same range.
  • the second reconstructed image creation unit creates a partial phase image and a partial intensity image corresponding to a specific measurement position or range
  • the display processing unit may be configured to display the partial phase image and the partial intensity image on the same screen.
  • the second reconstructed image creating unit creates a partial phase image and / or a partial intensity image at a plurality of focal positions
  • the display processing unit may include a focus instruction input unit for a user to indicate a focal position, and may display a partial phase image and / or a partial intensity image at the focal position instructed by the instruction input unit.
  • the focus instruction input unit includes a slider displayed on the screen and an operation unit that moves a knob of the slider, and the focus position changes according to the position of the knob. It can be set as the thing to do.
  • the sample is a culture plate in which a plurality of wells are formed
  • the display processing unit further includes a focus instruction input unit for a user to specify a focus position for each well
  • the first reconstructed image creating unit may be configured to create a phase image and / or an intensity image at the focal position for each well instructed by the focus instruction input unit.
  • the focal position of each well can be determined at the stage of creating the partial phase image and partial intensity image before creating the phase image and intensity image of the entire culture plate. As a result, only the phase image and intensity image at the focus position of each well need be created at the stage of creating the phase image and intensity image of the entire culture plate. Can be reduced.
  • the first reconstructed image creating unit and the second reconstructed image creating unit realize their functions with the same computer as the measurement control unit and the display processing unit or with a hardware circuit housed in the same housing.
  • each function may be realized by a separate computer or a completely separate hardware circuit.
  • the functions of the first reconstructed image creating unit and the second reconstructed image creating unit which require complicated calculations, are personal computers having the function of a measurement control unit that controls the holographic microscope main body responsible for measurement. It may be realized by a server (high performance computer) connected to the computer via a communication network.
  • the function of the display processing unit may be provided in the same personal computer as the measurement control unit, and display may be performed based on data received from the server.
  • the processes in the first reconstructed image creation unit and the second reconstructed image creation unit are basically the same, they can be substantially the same constituent elements.
  • the cell observation apparatus without waiting for a long time required to create a phase image and an intensity image of the entire observation range by image reconstruction processing based on hologram data acquired by measurement, work is performed.
  • the person can confirm the suitability of the measurement and the defect of the sample quickly and accurately. This avoids wasting time by avoiding the execution of image reconstruction processing when such troubles are confirmed, or by stopping it immediately even during execution. can do. As a result, cell observation work can be performed efficiently.
  • the whole block diagram of the cell observation apparatus which is one Example of this invention.
  • FIG. 1 is an overall configuration diagram of the cell observation apparatus of this embodiment
  • FIG. 2 is a schematic configuration diagram of a measurement terminal in the cell observation apparatus of this embodiment.
  • the cell observation apparatus of this embodiment includes a measurement terminal 1, a browsing terminal 3, and a server 5 connected via a communication network 4 such as the Internet or an intranet.
  • a communication network 4 such as the Internet or an intranet.
  • FIG. 1 one measuring terminal 1 and one browsing terminal 3 are shown, but an appropriate number of each can be provided.
  • the server 5 is a high-performance computer. As functional blocks embodied by dedicated software installed in the computer, a data transmission / reception unit 51, a hologram data storage unit 52, a first phase recovery calculation unit 53, a second A phase recovery calculation unit 54, a first image reconstruction unit 55, a second image reconstruction unit 56, an image data storage unit 57, and the like are provided. In addition, although the 1st phase recovery calculation part 53 and the 2nd phase recovery calculation part 54 are provided separately, and the 1st image reconstruction part 55 and the 2nd image reconstruction part 56 are provided separately, as mentioned later, The calculations executed by the first phase recovery calculation unit 53 and the second phase recovery calculation unit 54 are substantially the same, and are executed by the first image reconstruction unit 55 and the second image reconstruction unit 56. The processing is substantially the same with some exceptions. Thus, substantially they can be the same component.
  • the measurement terminal 1 includes a microscope observation unit 10 and a control / processing unit 20.
  • the microscopic observation unit 10 is an inline digital holographic microscope, and includes a light source unit 11 including a laser diode and the image sensor 12 as shown in FIG. Between the image sensor 12, a culture plate 13 including cells 14 that are observation objects is arranged.
  • the light source unit 11 and the image sensor 12 are integrally movable in two axial directions of the X axis and the Y axis that are orthogonal to each other by a moving unit 15 including a driving source such as a motor.
  • FIG. 2 only one light source unit 11 and one image sensor 12, i.e., one set, are shown in order to avoid complication of the drawing. Is provided with four sets sandwiching one culture plate 13 and, as will be described later, holograms for different measurement positions of one culture plate 13 by the four sets of light source units 11 and the image sensor 12. It is possible to obtain in parallel.
  • the entity of the control / processing unit 20 controls the operation of the microscopic observation unit 10 and sends the data acquired by the microscopic observation unit 10 to the server 5. Further, the control / processing unit 20 receives the data processed by the server 5 and displays it.
  • a personal computer PC
  • the control / processing unit 20 is connected to an input unit 201 that is a pointing device such as a keyboard and a mouse, and a display unit 202.
  • the browsing terminal 3 is a general PC. Then, the dedicated software installed in the PC can receive data from the server 5 and display an appropriate image formed based on the data.
  • FIG. 3 is a conceptual diagram for explaining image reconstruction processing in the cell observation apparatus of the present embodiment.
  • FIG. 3 (a) is a schematic top view of the culture plate 13 used in the cell observation apparatus of this example.
  • the culture plate 13 is formed with six wells 13a having a circular shape when viewed from above, and cells are cultured in the wells 13a.
  • the entire culture plate 13, that is, the entire rectangular range including the six wells 13 a is the observation target region.
  • the microscopic observation unit 10 includes four sets of a light source unit 11 and an image sensor 12, and each set of the light source unit 11 and the image sensor 12 has a total of 4 etc. as shown in FIG. It is responsible for collecting the hologram data of the four divided areas 81 divided. That is, the four sets of the light source unit 11 and the image sensor 12 share the collection of hologram data over the entire culture plate 13.
  • the range in which one set of the light source unit 11 and the image sensor 12 can be photographed at a time includes the one well 13a in the four-divided range 81 as shown in FIGS. 3B and 3C.
  • the four light source units 11 and the four image sensors 12 are respectively arranged in the vicinity of four vertices of a rectangle having the same size as the four-divided range 81 in the XY plane including the light source unit 11 and the image sensor 12.
  • the holograms for four different imaging units 83 on the culture plate 13 are simultaneously acquired.
  • the operator When collecting hologram data for the culture plate 13, the operator first sets the culture plate 13 on which the cells 14 to be observed are cultured at a predetermined position of the microscopic observation unit 10, and identifies the culture plate 13. Information such as a number and measurement date / time is input from the input unit 201 to instruct measurement execution. Upon receiving this measurement instruction, the imaging control unit 21 controls each part of the microscopic observation unit 10 to perform imaging.
  • one light source unit 11 irradiates a predetermined area (one imaging unit 83) of the culture plate 13 with coherent light having a small angle spread of about 10 °.
  • the coherent light (object light 17) that has passed through the culture plate 13 and the cells 14 reaches the image sensor 12 while interfering with the light (reference light 16) that has passed through the region adjacent to the cells 14 on the culture plate 13.
  • the object light 17 is light whose phase has changed when passing through the cell 14.
  • the reference light 16 is light which does not pass through the cell 14 and thus does not undergo phase change caused by the cell 14.
  • an interference image that is, a hologram
  • 2D light intensity distribution data hologram data
  • the four image sensors 12 acquire hologram data of regions corresponding to different imaging units 83 on the culture plate 13. Is done.
  • the light source unit 11 and the image sensor 12 are moved by the moving unit 15 in the X-axis direction and the Y-axis direction by a distance corresponding to one imaging unit 83 in the XY plane. It is sequentially moved in steps.
  • measurement is performed with 180 imaging units 83 included in the four-divided range 81, and measurement of the entire culture plate 13 is performed with the four sets of light source units 11 and the entire image sensor 12.
  • the hologram data obtained by the four image sensors 12 of the microscopic observation unit 10 in this way is stored in the hologram data storage unit 22 together with attribute information such as measurement date and time.
  • the data transmission / reception unit 23 uses the hologram data stored in the hologram data storage unit 22 as the measurement date and time, etc.
  • the attribute information is sequentially transferred to the server 5 via the communication network 4. It should be noted that raw, that is, unprocessed hologram data may be sent from each measurement terminal 1 to the server 5, but processing that corrects an error factor specific to each measurement terminal 1 as necessary.
  • the processed hologram data may be sent to the server 5.
  • the data transmitter / receiver 51 receives the hologram data sent from the measurement terminal 1, and identifies identification information for specifying the measurement terminal 1, identification information of the culture plate input at the time of imaging, measurement date and time, and the like.
  • Hologram data is stored in the hologram data storage unit 52 together with the attribute information.
  • the first phase recovery calculation unit 53 reads out the hologram data for each imaging unit from the hologram data storage unit 52 and restores the phase information by performing propagation calculation processing of the light wave, and intensity (amplitude) information. Ask for.
  • the first image reconstruction unit 55 performs the phase information and the intensity information. Based on the above, a phase image and an intensity image of the entire observation target region are formed.
  • the first image reconstruction unit 55 reconstructs the phase image of each imaging unit 83 based on the spatial distribution of the phase information calculated for each imaging unit 83, and connects the phase images in the narrow range.
  • a phase image of the observation target region that is, the entire culture plate 13 is formed.
  • an appropriate correction process may be performed so that the phase images at the boundaries of the imaging units 83 are smoothly connected.
  • an algorithm disclosed in known documents such as Patent Documents 1 and 2 may be used.
  • the reconstructed image obtained by normal processing is the highest resolution image obtained in principle from the acquired hologram data.
  • the resolution can be improved by binning processing based on the highest resolution phase image. You may make it produce the phase image of the resolution
  • the characteristic processing described below is executed so that the operator can quickly grasp the measurement failure or the contamination of the foreign matter after the measurement is completed.
  • FIG. 4 is an explanatory diagram of this characteristic processing operation
  • FIG. 5 is a diagram showing an example of a screen displayed on the measurement terminal after the end of photographing
  • FIG. 6 is a phase image of the designated measurement position displayed on the measurement terminal and It is a figure which shows an example of an intensity
  • the display processing unit 27 displays a captured image display screen 60 as shown in FIG.
  • an image display area 61 and a plate information display area 62 are arranged, and a “stop” button 63 is arranged at the lower right.
  • the plate information display area 62 attribute information such as the name (plate name) and identification number (plate ID) of the culture plate 13 during or after measurement is displayed.
  • the screen shown in FIG. 5 is the one after the measurement is completed, and no substantial image is displayed in the image display area 61 before the measurement is started.
  • the hologram image creation unit 24 is based on the obtained data for each imaging unit 83.
  • a hologram image showing a two-dimensional distribution of light intensity is created.
  • the hologram image created at this time is a thumbnail image with the lowest resolution.
  • the display processing unit 27 pastes and displays the created hologram thumbnail image at a position corresponding to each imaging unit in the image display area 61. That is, every time hologram data of one image pickup unit is newly obtained, a thumbnail image of the hologram based on the data is added to the image in the image display area 61 and displayed.
  • the operator monitors the hologram image displayed in the image display area 61 during the measurement, and clicks the “stop” button 63 when it can be determined that there is some problem. Then, the imaging control unit 21 receives this operation and stops measurement. As described above, when there is some problem in the measurement, it is possible to avoid spending time for the remaining useless measurement by quickly stopping the measurement.
  • the operator uses an pointing device such as a mouse that is a part of the input unit 201 to display the image display area 61 in the captured image display screen 60 shown in FIG.
  • the cursor is moved onto a desired one of a number of thumbnail images constituting the hologram image displayed on the screen.
  • the confirmation position designation accepting unit 25 accepts this operation, and displays a rectangular mark 66 indicating the selected imaging unit superimposed on the image.
  • the confirmation position designation receiving unit 25 recognizes one imaging unit selected at that time as a partial phase image creation target, and accordingly Then, the display processing unit 27 displays the designated position image display screen 70 as shown in FIG. 6 on the screen of the display unit 202.
  • An image display area 71 is provided on the designated position image display screen 70.
  • the confirmation position designation receiving unit 25 reads out the hologram data obtained for one designated imaging unit from the hologram data storage unit 22, and the data transmission / reception unit 23 sends the read data through the communication network 4 to the server 5. Forward to.
  • the hologram data transfer operation for one image pickup unit may be performed in parallel with the transfer operation of the hologram data of the entire imaging target region to the server 5 as described above, or preferentially. You may go. In the latter case, if the hologram data transfer operation for the entire imaging target area has already been started, the hologram data may be transferred for one imaging unit instructed to be temporarily interrupted.
  • the data transmission / reception unit 51 receives the hologram data for one imaging unit sent from the measurement terminal 1 as described above, and temporarily stores the data in the hologram data storage unit 52. Since the amount of data transferred at this time is much smaller than the amount of hologram data in the entire observation target area, the transfer time is also significantly shorter (see FIG. 4).
  • the second phase recovery calculation unit 54 reads out hologram data of one imaging unit stored in the hologram data storage unit 22 and performs light wave propagation calculation processing to restore phase information and obtain intensity information. Subsequently, the second image reconstruction unit 56 forms a partial phase image and a partial intensity image for one imaging unit based on the calculated phase information and intensity information. In addition, when calculating phase information and intensity information from hologram data, information on an arbitrary focal position can be calculated.
  • the focal position set at this time is a default value designated from the measurement terminal 1. (For example, the focus position set at the most recent past time point for the culture plate and well having the same identification number).
  • the image data constituting the partial phase image and the partial intensity image is stored in the image data storage unit 57 and sent from the data transmission / reception unit 51 to the measurement terminal 1. Since computation processing and image reconstruction processing need only be performed for one imaging unit, tiling processing etc. are unnecessary, so the processing time until the partial phase image and partial intensity image are formed is also the phase of the entire observation target region. This is much shorter than the time required to form the image and the intensity image (see FIG. 4).
  • the time required for transferring hologram data for one imaging unit selected and instructed by the operator and forming a partial phase image and a partial intensity image based on the data is short. Therefore, as described above in the measurement terminal 1, a part of the measurement terminal 1 is transferred from the server 5 to the measurement terminal 1 within a relatively short time from the time when the operator double-clicks the thumbnail image corresponding to one imaging unit. Image data constituting the phase image and the partial intensity image is sent. The sent image data is temporarily stored in the image data storage unit 26.
  • the display processing unit 27 creates a partial phase image and a partial intensity image based on the image data, and displays the two images side by side in the image display area 71 in the designated position image display screen 70.
  • the type of image displayed in the image display area 71 can be selected by putting a check mark in the display image selection check box 72, and only one of the partial phase image and the partial intensity image is displayed in the image display area 71. Can also be displayed.
  • the focal position of the displayed partial phase image and partial intensity image is 5450 ⁇ m.
  • the focus position set initially is not necessarily the focus position in cell observation, and it is necessary to change the focus position when observing a foreign object having a height different from that of the cell. Therefore, when observing partial phase images and partial intensity images at different focal positions, the operator sets conditions for determining a plurality of stages of focal positions in the slice condition setting area 73 in the designated position image display screen 70. That is, when the range of the focus position and the slice width (the step width of the focus position) are respectively input as numerical values, the number of slices (the number of steps of the focus position) is automatically calculated and displayed. These numerical values can also be left as default.
  • the focus designation receiving unit 28 receives this operation and instructs the server 5 on the set slice conditions.
  • the second phase recovery calculation unit 54 and the second image reconstruction unit 56 perform phase information at a plurality of focal positions according to the designated slice condition based on the hologram data for the selected one imaging unit. And intensity information are calculated, and a plurality of partial phase images and partial intensity images having different focal positions are formed.
  • the image data constituting the partial phase image and the partial intensity image at the plurality of focal positions are stored in the image data storage unit 57 and sent from the data transmission / reception unit 51 to the measurement terminal 1.
  • the sent image data is temporarily stored in the image data storage unit 26.
  • the focus designation receiving unit 28 obtains the focal position corresponding to the position of the knob on the slider, and the display processing unit 27 displays the image.
  • the partial phase image and the partial intensity image displayed in the display area 71 are updated to the image at the focal position.
  • the partial phase image and the partial intensity image at 11 different focal positions can be confirmed by operating the slider. The operator can find a focal position at which the cell to be observed can be most clearly observed by comparing partial phase images or partial intensity images at different focal positions.
  • the focus designation accepting unit 28 accepts this operation and informs the server 5 of the information on the designated focus position together with the identification number of the well in which the imaging unit such as the partial phase image currently displayed exists.
  • the instructed focal position is stored in the hologram data storage unit 52 in association with the well identification number, and the first phase recovery calculation unit 53 performs phase information and intensity of the imaging unit corresponding to the well with the identification number.
  • phase information and intensity information at the focal position associated with the identification number are calculated. That is, it is possible to determine the focal position for each well when creating the phase image and the intensity image of the entire imaging target region while checking the partial phase image and the partial intensity image on the measurement terminal 1.
  • the focal position that can be determined based on the partial phase image and the partial intensity image for one imaging unit is only for one well in which the imaging unit exists. Therefore, when it is desired to individually set the focal positions for all of the six wells, one imaging unit is selected for each well on the hologram image displayed in the image display area 61 of the captured image display screen 60. It is necessary to repeat the procedure of displaying the partial phase image and the partial intensity image for the imaging unit on the designated position image display screen 70, and further setting the slice conditions to create the slice image.
  • the focal position set for one well can be used for other wells.
  • an appropriate focal position is set for each well, and the phase image of only the focal position is set. And intensity images can be created and displayed. Thereby, it is possible to save the time required to create an image at an unnecessary focal position.
  • FIG. 7 is a diagram illustrating an example of a display screen when a hologram image and a partial phase image are displayed on the browsing terminal 3.
  • the slice image confirmation display screen 90 is provided with a hologram image display area 91 and a partial phase image display area 92.
  • a hologram image based on the hologram data stored in the hologram data storage unit 52 of the server 5 after shooting is displayed.
  • the hologram image to be displayed at this time can be appropriately selected by designating, for example, the identification number of the culture plate or the measurement date / time.
  • the operator wants to see a plurality of partial phase images having different focal positions, the operator designates one imaging unit on the hologram image, and sets conditions for determining a plurality of stages of focal positions in the slice condition setting area 93. Then, a “slice image preview” button 94 is clicked.
  • This instruction and setting contents are sent from the browsing terminal 3 to the server 5, and the second phase recovery calculation unit 54 and the second image reconstruction unit 56 of the server 5 store the hologram data of the designated imaging unit as described above.
  • the phase information at a plurality of focal positions designated based on is calculated, and a partial phase image is reconstructed for each focal position.
  • the image data which comprises the some partial phase image from which a focus position differs are transmitted to the terminal 3 for browsing.
  • the browsing terminal 3 displays the partial phase image in the partial phase image display area 92 based on the received image data. Further, the displayed partial phase image is changed to an image at a different focal position in accordance with the operation of the slider in the focal position selection operation area 95. Thereby, the operator can also confirm the partial phase image of the culture plate image
  • the partial phase image in the imaging unit is reconstructed from the hologram data of one imaging unit designated by the operator.
  • the partial phase image in the range may be reconstructed from the hologram data included in the specified range.
  • the range designated by the operator in the image may be enlarged and displayed.
  • the specified range is obtained after reconstructing the partial phase image and partial intensity image for that imaging unit. Only these images may be cut out and displayed.
  • the hologram image it is possible to specify not only one but also a plurality of imaging units on the hologram image, or to arbitrarily specify a range having a size larger than one imaging unit, and the specified plurality of imaging units or ranges.
  • the partial phase image and the partial intensity image corresponding to may be reconstructed and displayed.
  • the time is increased until a partial phase image or the like can be displayed. For this reason, it is preferable to predetermine the upper limit of the number of imaging units that can be specified and the width of the range based on the time constraint.
  • the microscopic observation unit 10 is an in-line type digital holographic microscope.
  • the microscopic observation unit 10 is not limited to the inline type as long as it acquires a hologram for each measurement position in the observation target region.
  • an off-axis type or phase shift type digital holographic microscope may be used.
  • First phase recovery calculation unit 54 Second phase recovery calculation unit 55 ... First image reconstruction unit 56 ... First Two-image reconstruction unit 57 ... Image data storage unit 60 ... Captured image display screen 61 ... Image display area 62 ... Plate information display area 63 ... "Stop” button 65 ... "Create image "Execute” button 66 ... Mark 70 ... Designated position image display screen 71 ... Image display area 72 ... Display image selection check box 73 ... Slice condition setting area 74 ... "Create slice image” button 75 ... Focus position selection operation area 76 ... "This "Set value to well focal position” button 90 ... Slice image confirmation display screen 91 ... Hologram image display area 92 ... Partial phase image display area 93 ... Slice condition setting area 94 ... "Slice image preview” button 95 ... Focus position selection operation region

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Holo Graphy (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon l'invention, une unité de traitement d'affichage (27) présente un hologramme d'une zone cible d'observation entière sur une unité d'affichage (202) après que des données d'hologramme d'une plaque de culture entière (13) sont acquises par l'intermédiaire d'une unité d'observation microscopique (10) qui est un microscope holographique. Lorsqu'un utilisateur désigne une position de mesure sur l'image pour une image de phase que l'utilisateur souhaite observer, une unité d'acceptation de désignation de position de vérification (25) transfère les données d'hologramme pour la position de mesure désignée à un serveur externe, et le serveur effectue une récupération de phase et une reconstruction d'image pour créer une image de phase partielle. Lors de la réception des données d'image constituant l'image de phase partielle, l'unité de traitement d'affichage (27) présente l'image de phase partielle sur l'unité d'affichage (202). La grande quantité de données d'hologramme pour la zone cible d'observation entière nécessite beaucoup de temps pour transférer et traiter les données, tandis que la quantité de données d'hologramme dans une position de mesure est significativement petite. Par conséquent, le dispositif selon l'invention permet à un opérateur de vérifier une image de phase partielle pour déterminer l'adéquation de la mesure et la présence ou l'absence de défauts dans un échantillon avant d'effectuer une reconstruction de l'image de phase sur la base des données d'hologramme de la zone cible d'observation entière.
PCT/JP2018/011002 2018-03-20 2018-03-20 Dispositif d'observation de cellules WO2019180808A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009521216A (ja) * 2005-12-22 2009-06-04 フェイズ ホログラフィック イメージング ペーハーイー アーベー 細胞サンプル分析のための方法と装置
WO2011089908A1 (fr) * 2010-01-20 2011-07-28 株式会社ニコン Dispositif d'observation cellulaire et procédé de culture cellulaire
JP2011170212A (ja) * 2010-02-22 2011-09-01 Nikon Corp 非線形顕微鏡
WO2012005315A1 (fr) * 2010-07-07 2012-01-12 兵庫県 Microscope holographique, procédé d'enregistrement d'une image d'hologramme d'élément microscopique, procédé de création d'un hologramme permettant la reproduction d'une image haute résolution et procédé de reproduction d'une image
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
JP2015082095A (ja) * 2013-10-24 2015-04-27 株式会社キーエンス 画像処理装置、顕微鏡システム、画像処理方法およびプログラム
WO2016163560A1 (fr) * 2015-04-09 2016-10-13 国立大学法人神戸大学 Microscope holographique numérique
WO2017203718A1 (fr) * 2016-05-27 2017-11-30 株式会社島津製作所 Procédé et dispositif d'observation holographique

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009521216A (ja) * 2005-12-22 2009-06-04 フェイズ ホログラフィック イメージング ペーハーイー アーベー 細胞サンプル分析のための方法と装置
WO2011089908A1 (fr) * 2010-01-20 2011-07-28 株式会社ニコン Dispositif d'observation cellulaire et procédé de culture cellulaire
JP2011170212A (ja) * 2010-02-22 2011-09-01 Nikon Corp 非線形顕微鏡
WO2012005315A1 (fr) * 2010-07-07 2012-01-12 兵庫県 Microscope holographique, procédé d'enregistrement d'une image d'hologramme d'élément microscopique, procédé de création d'un hologramme permettant la reproduction d'une image haute résolution et procédé de reproduction d'une image
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
JP2015082095A (ja) * 2013-10-24 2015-04-27 株式会社キーエンス 画像処理装置、顕微鏡システム、画像処理方法およびプログラム
WO2016163560A1 (fr) * 2015-04-09 2016-10-13 国立大学法人神戸大学 Microscope holographique numérique
WO2017203718A1 (fr) * 2016-05-27 2017-11-30 株式会社島津製作所 Procédé et dispositif d'observation holographique

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