JP5364430B2 - Cell image acquisition apparatus and cell image acquisition method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell image acquiring device for acquiring an almost focused image even with respect to an imaging field failed in focusing in scanning of the whole of a well, and a cell image acquiring method. <P>SOLUTION: The cell image acquiring device includes an imaging device equipped with an object lens 2, a feed device 3 for feeding the well to an imaging position, a device 4 for detecting the focus matching state of the target in the well at the imaging position, and a focus matching device for adjusting the focus of the object lens on the basis of the detected focus matching state. The imaging device divides the well into a plurality of imaging fields to image the same and the feed device 3 successively feeds the well so that the imaging fields spirally move to the outside from the center of the well. The device 4 detects a focal position at each imaging field to store the focal position succeeded in detection in a memory region and extracts the focal position of the most vicinal imaging field succeeded in the detection of the focal position from the memory region toward the center of the well in a case failed in the detection of the focal position. The focus matching device matches the focus of the object lens with the extracted focal position of the imaging field. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a cell image acquisition device and a cell image acquisition method.

  In the field of biotechnology, statistical analysis is performed on a large number of living cells for the purpose of elucidating the reactions of various living cells under various conditions. For this purpose, a device called a flow cytometer has been used in the past, but in recent years, statistical analysis is performed by acquiring images of many living cells using a microscopic observation device and analyzing the acquired images. Techniques for obtaining results have been used.

  By the way, in order to actually analyze the reaction of living cells under various conditions, it is necessary to analyze a very large number of living cells cultured under controlled conditions in a vessel such as a microplate. is there. For this reason, as a microscopic observation device for acquiring a cell image, an inverted microscope type optical arrangement for observing a container such as a microplate from below is adopted, and further, such as changing the imaging field of view, focusing, and imaging, An apparatus that automates a series of operations related to image acquisition is desired.

  Conventionally, as such a microscopic observation apparatus, for example, there is a cell scanning system described in Patent Document 1 below.

JP 2002-355090 A

As shown in FIG. 4, the cell scanning system described in Patent Document 1 accommodates an inverted fluorescence microscope 51 that can switch the fluorescence wavelength to be observed by switching the excitation wavelength via the electric filter wheel 50, and a sample. The XY stage 53 on which the microwell plate 54 is mounted and movable in the XY direction and the inverted fluorescence microscope 51 are provided, and the Z-axis focus drive for focusing the objective lens of the inverted fluorescence microscope 51 is performed. A device 55, a joystick 56 capable of manually operating the movement of the XY stage 53, a high-resolution digital camera 57, a camera power supply device 58, an automatic control device 59, a central processing unit 60, and a personal computer having predetermined software A computer 61 and a display 62 are included. In FIG. 4, 52 is a light source.

In the cell scanning system described in Patent Document 1 configured as described above, cell analysis is generally performed according to the following procedure.
First, before starting an autoscan, the user specifies filter settings and channels that match the biological label to be used, and adjusts camera settings such as exposure time to match the analyte brightness. Furthermore, the well to be scanned in the microwell plate 54, the number (type) of visual fields to be analyzed in each well, and the type of cell are designated.

  Next, the user selects a focusing method from the following two methods. The first method is that the cell scanning system automatically prefocuses the area of the microwell plate 54 designated to be scanned using an autofocus procedure. In the second method, the user designates three reference points for defining an area to be scanned, and the cell scanning system prefocuses the designated reference point as a scanning area. Then, during auto-scan, a “focal plane model” is calculated from these reference points by the method of least squares, and the focus of each well is estimated by interpolating from the “focal plane model” during the scan. ing.

  When the auto scan is started, the cell scanning system determines the scan pattern of the well in the entire microwell plate 54 and the scan pattern of the imaging field in each well. The microwell plate 54 is spirally moved by automatically moving the XY stage 53 from a predetermined well to the next well and from a predetermined visual field in each well to the next visual field through software control. Scanned. Furthermore, in the cell scanning system, at each field position, the Z-axis focus driving device 55 adjusts the focus of the sample using an autofocus procedure, the electric filter wheel 50 controls the filter selection, and the CCD camera 60 images the sample. Then, the personal computer 61 collects and analyzes the sampled image. Then, the display 62 dynamically displays the number of analyzed cells, the currently analyzed well, the image of each independent wavelength, the scan state including the result of screening for each well, and the like.

  At this time, the autofocus procedure is generally executed for the first imaging field of each well, and thereafter, the user selects 4 to 5 imaging fields per time in each well. It is executed according to the frequency (number of times). The autofocus procedure also calculates the starting Z-axis point by interpolating from a pre-calculated focal plane model. Above or below this setpoint, the autofocus procedure mechanically moves the Z-axis to a number of different positions, collects images at each position, and calculates a calculated focus score that estimates the contrast of each image. Detect the maximum value. Then, the Z position of the image having the maximum focus value is determined as the focus in the imaging field.

However, in the cell scanning system described in Patent Document 1, when the entire well is scanned, the side wall of the well is within the visual field in the imaging visual field including the outer peripheral part of the well. It becomes impossible to calculate. In such a case, focusing by the autofocus procedure is impossible in this imaging field of view.
Further, in the cell scanning system described in Patent Document 1, the autofocus start point is estimated using a “focal plane model” calculated from three reference points by the least square method. In many cases, the manufacturing error of the microwell plate is not of a level that can approximate a plane.
Therefore, depending on the conventional technique such as the cell scanning system described in Patent Document 1, an image that is not focused at all in the imaging field including the outer periphery of the well is likely to adversely affect the analysis result.

  The present invention has been made in view of such a conventional problem. When scanning the entire well, an image that is substantially in focus is acquired even for an imaging field of view in which focusing fails. It is an object of the present invention to provide a cell image acquisition apparatus and a cell image acquisition method capable of performing the above.

In order to achieve the above object, a cell image acquisition device according to the present invention includes an imaging device provided with an objective lens, a transport device that transports a well containing cells to the imaging position of the imaging device, and the transported to the imaging position. A focus detection device that detects a focus state of the object in the well, and a focus device that adjusts the focus of the objective lens based on the focus state detected by the focus detection device, In a cell image acquisition device that automatically acquires an image of a cell in a well, the imaging device divides the well into a plurality of imaging fields of view, and the transfer device has an outer region from a central region of the well. Next, the wells are sequentially conveyed so that the imaging field of view moves in a spiral, and the focus detection device detects the focal position for each imaging field, and the focus of the imaging field that has succeeded in detecting the focal position. Place Stored in the storage area of the further, if it fails to detect the focus position in a predetermined imaging visual field, the focus detection device, of the focal position of the imaging field stored in the predetermined storage area, the the focal position of the imaging field of view toward the center region of the predetermined imaging visual field or al before Symbol wells fails to detect the focal position located near most, extracted from the predetermined storage area, the focusing device, The objective lens is focused on the extracted focal position of the imaging field of view.

Cell image acquiring method according to the invention, the central region of the U E le the transport device containing the cells to the outside area, so the imaging field of view moves in a spiral, sequentially conveyed the well, the focus detection The apparatus detects a focal position for each imaging field of view, and the focusing apparatus focuses the objective lens included in the imaging apparatus on the detected focal position, and the imaging apparatus uses the focal point of the focused objective lens to A cell image acquisition method for imaging an imaging field of a well, wherein the imaging device divides the well into a plurality of imaging fields, and the transfer device moves from a central region of the well to an outer region thereof. The step of sequentially transporting the wells so that the imaging field of view moves in a spiral manner, and the focus detection device detects the focal position for each imaging field of view and succeeds in detecting the focal position. Focus position and the step of storing in a predetermined storage area, if it fails to detect the focal position at a predetermined imaging field of view, focal point of the imaging field of view in which the focus detection device is stored in the predetermined storage area of a position, a focus position of the imaging field located near the most towards the central region of the focal position predetermined imaging visual field or al before Symbol wells fails to detect the, extracted from the predetermined storage area, the The focusing device has a step of focusing the objective lens on the focal position of the extracted imaging field of view.

  According to the present invention, when scanning the whole well, a cell image acquisition device and a cell image acquisition method capable of acquiring an image that is substantially in focus even for an imaging field of view that fails in focusing are obtained. It is done.

In the cell image acquisition apparatus and cell image acquisition method of this invention, it is explanatory drawing which shows an example of the scanning direction of several imaging visual fields and imaging visual fields set with respect to one well which comprises a microwell plate. It is explanatory drawing which shows schematic structure of the cell image acquisition apparatus concerning 1st Embodiment of this invention. It is a flowchart which shows the cell analysis process procedure using the cell image acquisition apparatus of 1st Embodiment. It is explanatory drawing which shows schematic structure of the cell scanning system of patent document 1. As shown in FIG.

FIG. 1 is an explanatory view showing an example of a plurality of imaging fields and scanning directions of imaging fields set for one well constituting a microwell plate in the cell image acquisition device and the cell image acquisition method of the present invention. .
In the cell image acquisition device and the cell image acquisition method of the present invention, for example, as shown in FIG. 1, scanning is started from the imaging field of view of the central region of one well 1a constituting the microwell plate, and as the scanning proceeds. The conveyance by the conveyance device (XY stage 3 shown in FIG. 2) is controlled so that the imaging field of view moves spirally to the outer region of the well 1a. In this way, the imaging field of view at the initial stage of scanning does not include well sidewalls that are obstacles to focusing, so when the focus detection auxiliary light from the focus detection device is irradiated toward the imaging field of view. Since the auxiliary light irradiates the object and the reflected light from the object is detected, autofocus (AF) can be successful. When scanning progresses and the imaging visual field reaches a region including the outer region of the well 1a, the well sidewall is included in the imaging visual field. In such a case, the reflected light from the object is not correctly detected when the focus detection auxiliary light from the focus detection device is irradiated toward the imaging field of view, so autofocus is not successful.

Here, in the cell image acquisition device and the cell image acquisition method of the present invention, the focus detection device stores the focal position of the imaging field that has been successfully detected in the focal position in a predetermined storage area, and further, the predetermined imaging field. in case of failure to detect the focus position, the focus detection device, of the focal position of the imaging field of view which is stored in a predetermined storage area, the predetermined imaging visual field or Lau E Le failed to detect the focal position focus position of the imaging field located near the most toward the center region (Z-coordinate), extracted from a predetermined storage area, focusing device focuses the objective lens to the extracted focus position of the imaging field I am doing so.
In this way, it is possible to obtain an image that is substantially in focus even with respect to an imaging field of view in which there is no target and autofocus fails.

First Embodiment FIG. 2 is an explanatory diagram showing a schematic configuration of a cell image acquisition apparatus according to a first embodiment of the present invention.
The cell image acquisition apparatus according to the first embodiment includes a microwell plate 1 that contains cells, an objective lens 2, and a microwell plate 1 in a plane perpendicular to the optical axis of the objective lens 2 on which the microwell plate 1 is placed. The XY stage 3 that positions the imaging position by transporting the image and the auxiliary light for focus detection via the objective lens 3 are projected onto the object conveyed to the imaging position, and the reflected light is received to receive the object. An autofocus means comprising a focus detection device 4 for detecting the focus position of an object, and a focus device (not shown) for adjusting the focus of the objective lens 2 to the focus position detected by the focus detection device 4, and a sample A light source 5 for illuminating, a filter 6 for selecting a wavelength of illumination light from the light source 5, a filter 7 for selecting a wavelength used for imaging among wavelengths of light from the sample, and a light source 5 A dichroic mirror 8 for separating the illumination light and the light from the sample, an imaging lens 9 for condensing the light beam from the objective lens 2, and a CCD camera 10 for converting the sample image into a digital signal, In addition to controlling the driving of the XY stage 3 and autofocus means, the personal computer 11 for processing image data from the CCD camera 10 and a monitor 12 are provided.

Furthermore, the cell image acquisition device of the first embodiment stores the personal computer 11 in a predetermined storage area (not shown) of the focus position of the imaging field of view in which the focus detection device 4 has successfully detected the focus position. When the focusing device (not shown) focuses the objective lens 2 on the detected focal position of the imaging field of view and fails to detect the focal position in the predetermined imaging field of view, it is stored in a predetermined storage area. among the focal position of the imaging field of view, focal position of the imaging visual field located near the most towards the central region of a predetermined imaging visual field or Lau E Le failed to detect the focus position (Z coordinate), a predetermined storage A control program that is extracted from an area (not shown) and causes a focusing device (not shown) to function as autofocus drive control means for focusing the objective lens 2 on the extracted focal position of the imaging field of view. It is provided.

FIG. 3 is a flowchart showing a cell analysis processing procedure using the cell image acquisition apparatus of the first embodiment.
Prior to performing cell analysis using the cell image acquisition device of the first embodiment, the user, as a first step, cultivates cells in at least one well of the microwell plate 1 under the conditions to be tested. For example, an object to be observed such as a cell nucleus or an intracellular protein is fluorescently labeled by a generally known method.
Next, as a second stage, the user places the microwell plate 1 that has been cultured and contains fluorescently labeled cells on the XY stage 3.
Next, as a third stage, the user sets the next imaging condition before starting scanning by the cell image acquisition device. First, a scanning pattern is set by designating a well to be scanned and an imaging field within the well. Here, in the cell image acquisition device of the first embodiment, as shown in FIG. 1, the central region of the well is set as the imaging field of view at the start of scanning as the scanning pattern, and the outer region extends from the central region of the well. Then, the conveying operation of the microwell plate 1 by the XY stage 3 is set so that the imaging field of view sequentially moves in a spiral manner. Further, the user sets imaging conditions (exposure conditions, illumination conditions, observation conditions), and further sets autofocus conditions (focus position search range) (step S101).
As a fourth stage, the user instructs the cell image acquisition device to start scanning by pressing a start button (not shown). (Step S102)

When the start of scanning is instructed, the cell image acquisition device drives and controls the XY stage 3 on which the microwell plate 1 is placed via the personal computer 11, and a predetermined imaging field (in a predetermined well at an imaging position). In the case of the first scan in the well, the well is transported so that the imaging field of view in the central region of the well is located and stopped at that position (step S103).
Next, the cell image acquisition device uses autofocus (focus detection by the focus detection device 4 and focusing of the objective lens 2 by the focus device (not shown)) via the autofocus drive control means provided in the personal computer 11. Is executed (step S104).
When the focus detection device 4 succeeds in focus detection, the focus position of the imaging field of view where the focus position has been successfully detected is stored in a predetermined storage area (not shown). Further, a focusing device (not shown) focuses the objective lens 2 on the detected focal position of the imaging field. Next, the CCD camera 10 takes an image and stores the acquired image (step S105).
On the other hand, when the focus detection device 4 fails in focus detection, the focus position of the imaging field stored in the predetermined storage area is changed from the current imaging field in which the focus position detection has failed to the center area of the well. headed focal position of the imaging visual field located near most of the (Z-coordinate), extracted from a predetermined storage area (not shown), a focusing device (not shown), the focal position of the extracted image capturing field, The objective lens 2 is focused as the focal position (Z coordinate) of the imaging field. Next, the CCD camera 10 takes an image and stores the acquired image (step S106).
The cell image acquisition apparatus repeats the processing of step S103 to step S106 until imaging is completed for all imaging visual fields in all wells set by the scanning pattern (step S107).
After completion of imaging for all imaging fields in all wells, the cell image acquisition device performs predetermined image analysis on the captured images, extracts data necessary for cell analysis, and displays the data on the monitor 12 (step 1). S108).

According to the cell image acquisition device and the cell image acquisition method of the first embodiment, since scanning starts from the imaging field of view of the central region of the well, a focal position where autofocus is successful is always obtained. Also, if it fails to autofocus, among the focal position of the imaging field successfully autofocus stored in a predetermined storage area, toward the center region of the imaging visual field or Lau E Le failed to autofocus top Since the focus position of the imaging field of view located in the vicinity is extracted from a predetermined storage area and used as the focus position of the imaging field of view in which autofocus has failed, the focus can be detected via the autofocus means. Even if there is an imaging field that cannot be obtained, it is possible to obtain a good image that is substantially focused on the imaging field.

  INDUSTRIAL APPLICABILITY The cell image acquisition device and cell image acquisition method of the present invention are useful in the field of biotechnology that acquires images of a large number of living cells using a microscopic observation device and obtains statistical analysis results by analyzing the acquired images. is there.

DESCRIPTION OF SYMBOLS 1 Microwell plate 1a Well 2 Objective lens 3 XY stage 4 Focus detection apparatus 5 Light source 6, 7 Filter 8 Dichroic mirror 9 Imaging lens 10 CCD camera 11 Personal computer 12 Monitor 50 Electric filter wheel 51 Inverted fluorescence microscope 52 Light source 53 XY stage 54 Microwell plate 55 Z-axis focus drive device 56 Joystick 57 High-resolution digital camera 58 Camera power supply device 59 Automatic control device 60 Central processing unit 61 Personal computer 62 Display

Claims (2)

An imaging device including an objective lens, a transport device that transports a well containing cells to an imaging position of the imaging device, and a focus that detects a focus state of an object in the well transported to the imaging position A cell image acquisition device having a detection device and a focusing device that adjusts the focus of the objective lens based on a focus state detected by the focus detection device, and automatically acquiring an image of a cell in the well In
The imaging device divides the well into a plurality of imaging fields and images,
The transport device sequentially transports the wells so that the imaging field of view moves spirally from the central region of the well to the outer region thereof,
The focus detection device detects a focal position for each imaging field, stores the focal position of the imaging field that has been successfully detected in the focal position in a predetermined storage area, and
When detection of a focal position in a predetermined imaging field of view fails, the focus detection device detects a predetermined position that has failed to detect the focal position among the focal positions of the imaging field of view stored in the predetermined storage area . the focal position of the imaging field located near the most towards the center region of the imaging visual field or al before Symbol well, extracted from the predetermined storage area, the focusing device, the focal position of the imaging field of view the extracted A cell image acquisition device characterized in that the objective lens is focused. From the central region of the U E le the transport device containing the cells to the outside area, so the imaging field of view moves in a spiral, sequentially conveyed the well, the focus detection device the focal position for each imaging field A cell image is detected and the focusing device focuses the objective lens included in the imaging device on the detected focal position, and the imaging device images the imaging field of the well with the focus of the focused objective lens. An acquisition method,
The imaging device divides the well into a plurality of imaging fields and images,
The transport device sequentially transports the well so that the imaging field of view moves spirally from the central region of the well to the outer region;
The focus detection device detects a focal position for each imaging field, and stores the focal position of the imaging field that has been successfully detected in a predetermined storage area;
When detection of a focal position in a predetermined imaging field of view fails, the focus detection device detects a predetermined position that has failed to detect the focal position among the focal positions of the imaging field of view stored in the predetermined storage area . the focal position of the imaging field located near the most towards the center region of the imaging visual field or al before Symbol well, extracted from the predetermined storage area, the focusing device, the focal position of the imaging field of view the extracted And a step of focusing the objective lens on a cell image.

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