JP2007205973A - Droplet observation device and droplet observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a droplet observation device that has improved operability and can acquire improved images, and to provide a droplet observation method. <P>SOLUTION: In the observation of a drop 15, containing a protein solution existing in a well 8a, provided in a lattice array, on a plate for detecting protein crystal, a unit cell in which the image of the drop 15 that has not been acquired appropriately, due to positional misalignment is selected by a cursor 11a out of unit cells 42 in a list display layout 41, where the reduced image of a drop image obtained by imaging the drop 15 with a high imaging magnification is displayed corresponding to a well array in the plate for displaying a well image 43 on the screen overlappingly. A cross cursor 44 is made to coincide with the center of the drop 15, and proper position is instructed and inputted, thus correcting droplet position information corresponding to the well image. Accordingly, improved image of the drop 15 is acquired, and droplet observation having improved operability is brought into realization. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a droplet observation apparatus and a droplet observation method for observing a droplet containing a protein solution.

  In recent years, efforts to make effective use of genetic information in fields such as medicine have become active, and efforts have been made to analyze the structure of proteins, which are gene products, as the basic technology. This protein structure analysis specifies the three-dimensional structure of the protein, and is performed by a method such as X-ray crystal structure analysis. In order to perform such protein structural analysis, it is first required to crystallize the protein to be analyzed, and various apparatuses for crystallization have been proposed (for example, see Patent Document 1).

In this example of patent literature, a robot handles a large number of microplates each provided with a plurality of wells for storing protein solutions for crystal formation, thereby imaging the inside of the wells with a camera and observing the crystallization state. It can be done automatically. Thereby, the protein crystal observation work for a huge number of samples can be efficiently performed without requiring manual work or visual observation by the person in charge of the observation work.
JP 2005-516201 gazette

  By the way, various methods such as a vapor diffusion method and a microbatch method are used for protein crystallization. In any of these methods, the state of a droplet containing a protein solution is observed with a camera microscopically during the crystallization process. Thus, the presence or absence of crystallization is determined. In order to correctly determine the presence or absence of crystallization, it is necessary to obtain a good image of a droplet to be observed. However, in many cases, the droplets are present in a random state in the well, and in micro-imaging, the image is taken in an abnormal state where the droplet is out of the imaging field of view, and a correct image may not be obtained. is there.

  In such a case, there is no clear handling method in the related art including the above-described patent document examples, and the person in charge of observation confirms the images individually and repeatedly performs imaging by manual operation for abnormal images. I had to be treated. For this reason, it is difficult to improve the work efficiency of observation work when targeting a large number of samples, it is possible to obtain a good image that satisfies the target image accuracy requirements, and the droplets have excellent operability. There was a need for an observation device.

  Therefore, an object of the present invention is to provide a droplet observation apparatus and a droplet observation method that can acquire a good image with excellent operability.

The droplet observation apparatus according to the present invention is a droplet observation apparatus that observes a droplet including a protein solution existing in the crystal generation space on a plate having a plurality of crystal generation spaces in a lattice arrangement for generating protein crystals. A mounting unit for mounting the plate, an imaging unit for imaging the crystal generation space at a desired imaging magnification, and positioning for positioning the plate on the mounting unit relative to the imaging unit. An image storage unit for storing an image acquired by the imaging unit and the liquid are connected to the mechanism, an input device for an operator to operate the droplet observation device, and a display unit for displaying predetermined display contents. A control unit having a droplet position storage unit that stores a droplet position for each crystal space, the control unit sets an imaging magnification of the imaging unit to a low magnification, and a crystal in the plate By controlling the positioning mechanism based on the arrangement information of the formation space, the imaging unit and the plate are aligned, the crystal generation space is imaged by the imaging unit, and a low-magnification image for each crystal generation space is obtained. A first imaging processing unit that acquires the first image as a single image and stores the first image in a first image storage area of the image storage unit; detects the position of the droplet from the first image; A droplet detection processing unit that stores in the droplet position storage unit as droplet position information for each crystal generation space; and an imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information. By controlling, the imaging unit and the plate are aligned, the droplet is captured by the imaging unit, an enlarged image of the droplet is acquired as a second image, and the second image is stored in the image storage unit. Second image storage area A second imaging processing unit that stores the image, a reduced image creation processing unit that creates a reduced image obtained by reducing the second image and stores the reduced image in a reduced image storage area of the image storage unit, and a plurality of reduced image storage units In response to the operation of selecting a list display unit that reads out the reduced image and displaying the list on the display unit, and selecting one of a plurality of reduced images displayed on the display unit by the input device. A first image selection display unit that reads a first image of the crystal generation space corresponding to the selected reduced image from the first image storage area and displays the first image on the display unit; and a first image displayed on the display unit. A droplet position information correcting unit that corrects the droplet position information corresponding to the first image in response to the operation of instructing the correct position of the droplet being performed by the input device;

  Further, the droplet observation apparatus of the present invention is a droplet observation device for observing a droplet containing a protein solution existing in the crystal generation space on a plate having a plurality of crystal generation spaces for generating a protein crystal in a lattice arrangement. The mounting unit for mounting the plate, the imaging unit for imaging the crystal generation space at a desired imaging magnification, and the plate on the mounting unit relative to the imaging unit are positioned relative to each other. A positioning mechanism, an input device for an operator to operate the droplet observation device, and a display unit for displaying predetermined display contents are connected, and an image storage unit for storing an image acquired by the imaging unit and the image storage unit A control unit having a droplet position storage unit that stores a droplet position for each crystal space, the control unit sets an imaging magnification of the imaging unit to a low magnification, The imaging unit and the plate are aligned by controlling the positioning mechanism based on the arrangement information of the crystal generation space, and the crystal generation space is imaged by the imaging unit to obtain a low-magnification image for each crystal generation space. A first imaging processing unit that acquires the first image and stores the first image in a first image storage area of the image storage unit; and detects the position of the droplet from the first image and stores the information. A droplet detection processing unit that stores, as droplet position information, for each crystal generation space in the droplet position storage unit, and a cut-out obtained by cutting a predetermined range based on the droplet position information of the first image from the first image An image cut processing unit that creates an image and stores the cut image in a cut image storage area of the image storage unit, and a list display unit that reads a plurality of cut images from the cut image storage area and displays a list on the display unit When, In response to the operation of selecting one of the plurality of cut images displayed on the display unit being performed by the input device, the first image of the crystal generation space corresponding to the selected cut image is selected as the first image. A first image selection display unit that reads from the first image storage unit and displays it on the display unit, and an operation for instructing the correct position of the droplet in the first image displayed on the display unit is performed by the input device. In response to this, a droplet position information correction unit that corrects the droplet position information corresponding to the first image and an imaging magnification of the imaging unit are set to a high magnification, and based on the droplet position information The positioning mechanism is controlled to align the imaging unit and the plate, and an image of the droplet is captured by the imaging unit to obtain an enlarged image of the droplet as a second image. Second of the image storage unit And a second imaging processing unit for storing in the image storage area.

The droplet observation method of the present invention includes a mounting unit for mounting a plate having a plurality of crystal generation spaces in a lattice arrangement for generating protein crystals, and an imaging unit for imaging the crystal generation space at a desired imaging magnification. A positioning mechanism for positioning the plate on the mounting unit relative to the imaging unit, an input device for an operator to operate the droplet observation device, and a display for displaying predetermined display contents An image storage unit that stores an image acquired by the imaging unit, and a droplet position storage unit that stores a position of a droplet including a protein solution existing in the crystal generation space for each crystal space; A droplet observation method comprising: a droplet observation device comprising a control unit comprising: a droplet observation device that observes the droplet on the plate, wherein the imaging magnification of the imaging unit is set to a low magnification, and The imaging mechanism and the plate are aligned by controlling the positioning mechanism based on the arrangement information of the generation space, and the crystal generation space is imaged by the imaging unit to obtain a low-magnification image for each crystal space. A first imaging process for acquiring the first image as an image and storing the first image in a first image storage area of the image storage unit; detecting the position of the droplet from the first image; As the droplet detection process stored in the droplet position storage unit, the imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information, thereby the imaging unit and the plate The image is picked up by the image pickup unit, an enlarged image of the liquid drop is acquired as a second image, and the second image is stored in the second image storage area of the image storage unit. 2 imaging process Reduced image creation processing for creating a reduced image obtained by reducing the second image and storing the reduced image in the reduced image storage area of the image storage unit, and reading a plurality of reduced images from the reduced image storage area and listing them on the display unit In response to the reduced image list display processing to be displayed and the operation of selecting one of the plurality of reduced images displayed on the display unit being performed by the input device, the selected reduced image is handled. A first image selection display process for reading the first image of the crystal generation space from the first image storage area and displaying it on the display unit, and indicating the correct position of the droplet in the first image displayed on the display unit And a droplet position information correction process for correcting the droplet position information corresponding to the first image in response to the operation performed by the input device.

  Further, the droplet observation method of the present invention includes a mounting unit for mounting a plate having a plurality of crystal generation spaces for generating protein crystals in a lattice arrangement, and imaging for imaging the crystal generation space at a desired imaging magnification. A positioning mechanism for positioning the plate on the mounting unit relative to the imaging unit, an input device for an operator to operate the droplet observation device, and predetermined display contents An image storage unit that is connected to a display unit and stores an image acquired by the imaging unit, and a droplet position storage unit that stores a position of a droplet including a protein solution existing in the crystal generation space for each crystal space A droplet observation apparatus comprising a control unit having a control unit, and observing the droplets on the plate, wherein an imaging magnification of the imaging unit is set to a low magnification, The imaging unit and the plate are aligned by controlling the positioning mechanism based on the arrangement information of the crystal generation space, and the crystal generation space is imaged by the imaging unit to obtain a low-magnification image for each crystal space. A first imaging process for acquiring the first image and storing the first image in the first image storage area of the image storage unit; detecting the position of the droplet from the first image; Droplet detection processing stored as information in a droplet position storage unit, and a cutout image obtained by cutting out a predetermined range based on the droplet position information of the first image from the first image is created. Image cut-out processing stored in the cut-out image storage area of the image storage unit, cut-out image list display processing for reading out a plurality of cut-out images from the cut-out image storage region and displaying the list on the display unit, and display on the display unit In response to the operation of selecting one of a plurality of cut images performed by the input device, the first image of the crystal generation space corresponding to the selected cut image is read from the first image storage unit. In response to the first image selection display process displayed on the display unit and the operation of instructing the correct position of the droplet in the first image displayed on the display unit being performed by the input device, Droplet position information correction processing for correcting the droplet position information corresponding to the first image, setting the imaging magnification of the imaging unit to a high magnification, and controlling the positioning mechanism based on the droplet position information The imaging unit and the plate are aligned with each other, a droplet is imaged by the imaging unit, an enlarged image of the droplet is acquired as a second image, and the second image is stored in the second image of the image storage unit. Store in storage area Second imaging processing.

According to the present invention, in observing droplets existing in the crystal generation space, a liquid from a reduced image of the first image obtained by imaging the crystal generation space at a low imaging magnification or a second image obtained by imaging the crystal generation space at a high imaging magnification. A list of cut images obtained by cutting out the corresponding portions of the drop is displayed, and a first image of the crystal generation space corresponding to the selected image is displayed by an operation of selecting one of these displayed images. By correcting the droplet position information corresponding to the first image by an operation of instructing and inputting the correct position of the droplet, a good image with no droplet displacement can be obtained, and the operability is excellent Droplet observation is realized.

(Embodiment 1)
FIG. 1 is a perspective view of a droplet observation apparatus according to Embodiment 1 of the present invention, FIG. 2 is a cross-sectional view of a target plate of the droplet observation apparatus according to one embodiment of the present invention, and FIG. FIG. 4 is a block diagram showing the configuration of a control system of the droplet observation apparatus according to the first embodiment of the present invention, and FIGS. 5 and 6 are embodiments of the present invention. FIG. 7, FIG. 8, FIG. 9 are diagrams showing display screens in the droplet observation apparatus according to Embodiment 1 of the present invention.

  First, the overall configuration of the droplet observation apparatus 1 will be described with reference to FIG. The droplet observation apparatus 1 includes a plurality of crystallization plates used for generating protein crystals by a method such as an oil batch method or a hanging drop method, that is, a plurality of wells, which are crystal generation spaces for generating protein crystals, in a lattice arrangement. The provided plate has a function of observing a droplet containing the protein solution present in the well.

  In FIG. 1, the droplet observation apparatus 1 has a configuration in which an observation unit 3 and an operation unit 4 are arranged on the upper surface of a base 2, and each unit described below is controlled by a control unit 13 built in the base 2. Yes. The observation unit 3 includes an imaging unit 5 supported by a frame 3a, and an observation stage 6 is disposed below the imaging unit 5. The observation stage 6 includes a mounting portion 7 that can be moved in the X, Y, and Z directions by an XYZ table mechanism 6b incorporated in a rectangular table frame 6a.

  The mounting unit 7 mounts a plate 8 provided with a plurality of wells 8a, which are crystal generation spaces for generating protein crystals, in a lattice arrangement. The plate 8 placed on the placement unit 7 is positioned relative to the imaging unit 5 by driving the XYZ table mechanism 6 b and moving the placement unit 7. That is, the observation stage 6 is a positioning mechanism that positions the plate 8 on the mounting unit 7 relative to the imaging unit 5.

  The operation unit 4 includes a keyboard 10, an input device including a mouse 11, an identification code reading unit 12, and a display unit 9, which are connected to a control unit 13. (See FIG. 4). The input device is used by an operator to operate the droplet observation device, and the identification code reading unit 12 identifies the barcode attached to the plate 8 when the plate 8 is set on the mounting unit 7. Used to read and identify codes. On the display unit 9, a predetermined screen such as a screen in which each well 8 a of the plate 8 is imaged by the imaging unit 5 is displayed in addition to an operation screen when performing operation input and data input by the keyboard 10 and the mouse 11. .

  In the present embodiment, as described above, a crystallization plate produced when protein crystallization is performed by two different methods (oil batch method and hunting drop method) can be used as an observation target. Yes. When the oil batch method is used, as shown in FIG. 2A, a drop 15 containing a protein solution to be observed is held in oil 14 dispensed in a predetermined amount into the well 8a of the plate 8. . In this case, the well 8a itself becomes a crystal generation space.

  When the hanging drop method is used, as shown in FIG. 2B, a predetermined amount of the crystallization solution 17 is dispensed into the well 8a, and then the cover plate 16 with the drop 15 attached to the lower surface side is used. The drop 15 is held in a gap space 18 that covers the well 8 a and is surrounded by the liquid surface of the crystallization solution 17 and the cover plate 16. That is, in this case, the gap space 18 becomes a crystal generation space.

  FIG. 3 shows a state in which the plate 8 holding the observation target drop 15 in the well 8 a is placed on the placement unit 7 and imaged by the imaging unit 5. The imaging unit 5 includes a zoom optical system 5a and a light receiving element 5b, and the well 8a can be imaged at a desired imaging magnification (observation magnification) by changing the zoom magnification of the zoom optical system 5a. it can. At the time of imaging, the light axis 5c is aligned with the well 8a to be observed, and the light transmitted through the well 8a from below in the state where the illumination device 19 disposed below the mounting portion 7 is turned on. Receives light.

  Next, the configuration of the control system will be described with reference to FIG. In FIG. 4, a rectangular frame 13 indicates a control treatment function of the control unit 13, and the control unit 13 includes a processing / calculation unit 20, an observation stage control unit 21, an imaging control unit 22, an image storage unit 23, and well information. A storage unit 24 and a plate information storage unit 34 are provided. The processing / arithmetic unit 20 indicates various processing functions realized by the CPU of the control unit 13 executing a processing program stored in a program storage device attached to the control unit 13. When executing these processes, the processing / arithmetic unit 20 captures input signals from the keyboard 10, the mouse 11, and the identification code reading unit 12 connected to the control unit 13, and sends a screen display signal to the display unit 9. Output.

  The function of each part of the control unit 13 will be described. The observation stage control unit 21 controls the operation of the observation stage 6 in accordance with a control command from the processing / calculation unit 20. Thereby, out of the plurality of wells 8 a of the plate 8, any well 8 a to be observed can be aligned with the imaging unit 5. The imaging control unit 22 controls the illuminating device 19 and the imaging unit 5 in accordance with a control command from the processing / calculating unit 20, and causes the light receiving element 5b to image the well 8a. The image acquired by the imaging unit 5 is stored in the image storage unit 23.

  Here, the types of images acquired and stored for the purpose of observation for protein crystal detection will be described. When imaging by the imaging unit 5, the zoom optical system 5a is set to a low magnification to obtain a first image (hereinafter, abbreviated as “well image”) in which the entire well 8a as a target is within the imaging field of view. Is done. This imaging process is performed by the well image imaging processing unit 25 of the processing / calculating unit 20 controlling the observation stage control unit 21 and the imaging control unit 22 based on the well arrangement information. The acquired well image is stored in the well image storage unit 23a of the image storage unit 23 in units of wells according to the address instructed from the well image imaging processing unit 25. The well image storage unit 23a is a first image storage area for storing a well image that is a first image.

  Therefore, the well image imaging processing unit 25 sets the imaging magnification of the imaging unit 5 to a low magnification, and controls the observation stage 6 that is a positioning mechanism based on the arrangement information of the wells 8a on the plate 8, thereby the imaging unit 5 Is aligned with the plate 8, the well 8 a is imaged by the imaging unit 5, a low-magnification image for each well 8 a is acquired as a well image, and the well image is stored in the first image storage area of the image storage unit 23. This is a first imaging processing unit that executes a process (first imaging process).

Then, the drop recognition processing unit 27 recognizes the well image stored in the well image storage unit 23a to detect the position of the drop 15 in the well image, and the detection result is the drop position storage unit of the well information storage unit 24. 24a is stored. The drop recognition processing unit 27 detects the position of the drop 15 from the well image, and stores the information as droplet position information for each drop position storage unit 24a which is a droplet position storage unit (droplet detection process). ).

  That is, in the present embodiment, the control unit 13 is connected to an input device for an operator to operate the droplet observation apparatus 1 and a display unit 9 for displaying predetermined display contents, and is acquired by the imaging unit 5. The image storage unit 23 stores an image and the drop position storage unit 24a that stores the position of the drop 15 for each well 8a.

  Further, by setting the zoom optical system 5a to a high magnification, a second image (hereinafter, abbreviated as “drop image”) in which the drop 15 of the imaging target portion in the well 8a is enlarged within the imaging visual field is acquired. . In this imaging process, the drop image imaging processing unit 26 of the processing / calculating unit 20 controls the observation stage control unit 21 and the imaging control unit 22 based on the droplet position information stored in the drop position storage unit 24a. Done. The acquired drop image is stored in the drop image storage unit 23b of the image storage unit 23 in units of wells according to an address instructed from the drop image imaging processing unit 25. The drop image storage unit 23b is a second image storage area for storing a drop image that is a second image.

  Therefore, the drop image imaging processing unit 26 sets the imaging magnification of the imaging unit 5 to a high magnification and controls the observation stage 6 based on the droplet position information, thereby aligning the imaging unit 5 and the plate 8. The imaging unit 5 captures the drop 15, acquires an enlarged image of the drop 15 as a drop image, and executes a process (second imaging process) for storing the drop image in the drop image storage unit 23 b of the control unit 13. The second imaging processing unit.

  The reduced image creation processing unit 28 has a function of reducing an image by digital image processing, whereby a reduced image obtained by reducing the drop image is created (see the unit cell 42 shown in FIG. 7) and further created. The reduced image is stored in the reduced image storage unit 23 c of the image storage unit 23. In other words, the reduced image creation processing unit 28 creates a reduced image obtained by reducing the drop image and stores it in the reduced image storage area of the image storage unit 23 (reduced image creation processing).

  The crystal determination processing unit 29 determines the state of protein crystallization in the well 8a, that is, whether or not a protein crystal has been generated in the drop 15 by recognizing the drop image. This determination may be performed by a known protein crystal detection method (for example, a method for determining the presence or absence of a protein crystal from a crystal feature image obtained by performing differentiation or binarization on a drop image (Japanese Patent Laid-Open No. 2004-345867). No.))). The determination result is stored as crystallization information in the well in the crystallization information storage unit 24b of the well information storage unit 24 in units of wells. That is, the control unit 13 is configured to include a crystal determination unit that determines whether a protein crystal is generated in the drop 15 by using the drop image that is the second image.

  The drop position correction processing unit 30 responds to the operation of instructing the correct position of the drop 15 in the first image displayed on the display unit 9 performed by the input device, and the droplet corresponding to the first image. Processing for correcting the position information is performed. This processing is executed based on an instruction input operation on the screen displayed on the display unit 9 by the display processing unit 31 described below.

Based on the image data stored in the image storage unit 23 and the well information stored in the well information storage unit 24, the display processing unit 31 performs a process for causing the display unit 9 to display an image described below. The list display unit 31a reads a plurality of reduced images from the reduced image storage unit 23c, and arranges the plurality of reduced images in an arrangement corresponding to the lattice arrangement of the wells 8a on the plate 8 in a predetermined display format. A list display process (reduced image list display process) is performed (see the unit cells 42 displayed in a grid array in the list display layout 41 in FIG. 7).

  The first image selection display unit 31b responds to the operation of selecting one of the plurality of reduced images displayed on the display unit 9 with the mouse 11 of the input device, and the selected reduced image. The well image corresponding to the well 8a (see the well image 43 shown in FIG. 8) is read from the first image storage unit 23a and displayed on the display unit 9 (first image selection display process). That is, in response to the operation of designating one of the unit cells 42 with the cursor 11a in FIG. 7, the well image 43 shown in FIG.

  At this time, the first image selection display unit 31b displays a graphic indicating the position based on the droplet position information stored in the drop position storage unit 24a on the displayed well image. That is, as shown in FIG. 8, in the well image 43, the cross cursor 44 is displayed based on the stored droplet position information. At this time, if there is a recognition error of the drop 15 by the drop recognition processing unit 27, the cross cursor 44 does not necessarily match the drop 15 as shown in FIG. A cross cursor 44 may be displayed on the screen.

  The command generation unit 32 generates a command for causing each processing function unit described above to execute a processing operation. There are two types of these commands: commands generated according to a processing program executed by the processing / arithmetic unit 20, and commands generated by manual input operations via the keyboard 10 and mouse 11. That is, an imaging process and a recognition process for acquiring an image by the imaging unit 5, and a crystal determination performed based on the recognition result are executed by a command automatically generated according to the processing program, and a display position indicating the position of the drop 15 The drop position correcting process for correcting the image on the screen is executed by a command generated when the operator inputs the image of the well 8a displayed on the display unit 9 while visually observing the image.

  The plate information registration unit 33 performs data processing for aggregating information stored in units of wells in the image storage unit 23 and the well information storage unit 24 into one plate unit and registering the information in the plate information storage unit 34. As a result, the crystallization plate information in which the well images, the drop images, the drop positions, and the crystallization information of all the wells 8a belonging to the plate 8 are compiled for one plate 8 is edited.

  This droplet observation apparatus 1 is configured as described above, and the protein crystal detection process performed by the droplet observation apparatus 1 will be described with reference to FIGS. It explains along. In FIG. 5, first, an observation magnification for imaging by the imaging unit 5 is set (ST1). Here, the zoom optical system 5a of the image pickup unit 5 is set to a low magnification, that is, a magnification at which an image clearly showing the entire well 8a occupies most of the imaging visual field. Next, the plate 8 is positioned with respect to the imaging unit 5, the leading well 8a is positioned at an observation position by the imaging unit 5 (ST2), an image of the well 8a is captured by the imaging unit 5, and the well image storage unit 23a. (ST3). Then, the drop recognition processing unit 27 recognizes the well image to perform drop recognition for detecting the position of the drop 15 (ST4), and stores the detected drop position in the drop position storage unit 24a.

Thereafter, it is determined whether or not there is a well that has not been subjected to drop recognition. If there is an unexecuted well, the observation stage 6 is moved and the plate 8 is positioned with respect to the imaging unit 5, whereby the next well 8 a is Positioning to the observation position (ST7), returning to (ST3), the subsequent processing is repeatedly executed. When it is determined in (ST6) that there is no unexecuted well, the process proceeds to the step shown in FIG.

  Here, first, the observation magnification for imaging by the imaging unit 5 is set to a high magnification, that is, a magnification such that an image clearly showing the entire drop 15 occupies most of the imaging visual field (ST8). Next, the plate 8 is positioned based on the drop position of the leading well 8a read from the drop position storage unit 24a (ST9), and an image of the drop 15 in the well 8a is picked up by the image pickup unit 5 to store the drop image. Store in the unit 23b (ST10). Next, a reduction image of the drop image is created by executing a process of reducing the stored image by the reduced image creation processing unit 28 (ST11). The created reduced image is stored in the reduced image storage unit 23c.

  Then, the stored reduced image is read out by the list display unit 31a and displayed on the well list display layout (ST12). Accordingly, as shown in FIG. 7A, in each unit cell 42 of the well list display layout 41 set on the display screen 40, the reduced image of the drop 15 is a position corresponding to the well 8a where the drop exists. Is displayed. Next, a crystal detection process is executed by the crystal determination processing unit 29 for the drop image acquired in (ST10) (ST13). Thereby, the presence or absence of protein crystals in the drop 15 in the target well 8a is determined, and the determination result is stored in the crystallization information storage unit 24b as crystallization information for each well 8a.

  Thereafter, it is determined whether or not there is an untreated well 8a. If there is an untreated well, the plate 8 is positioned based on the drop position of the next well 8a to be observed (ST15) and (ST10). Returning to, the subsequent processing is repeated in the same manner. When it is determined that there is no untreated well in (ST14), the protein crystal detection process for the plate 8 is completed.

  In the above-described processing, in the drop recognition executed in (ST4), a situation occurs in which the position of the drop 15 is not correctly detected due to various factors. For example, when the image of the drop 15 is thinly obtained, or when there is an image element in the well 8a that is confused with the outline of the drop 15, the position deviated from the center position of the true drop 15 is the drop recognition processing unit. 27 is erroneously detected as a drop position.

  Then, when imaging of (ST10) is performed based on such an incorrect drop position, it becomes an observation object like the drop position defective cell 42 * appearing at the position of (E6) in FIG. 7A. An abnormal drop image in which most of the power drop 15 is out of the imaging field of view is acquired. As a result, protein crystallization information for the well 8a corresponding to the drop position defective cell 42 * cannot be obtained correctly. In the present invention, as will be described below, a plurality of reduced images are displayed in a list on the display unit 9, so that the operator can know in a short time whether or not there is a false detection of the drop position. Can do.

  Then, in order to eliminate the problem due to the acquisition of the above-mentioned abnormal drop image, in the crystallization observation method shown in the present embodiment, the droplet position information correction process using a manual operation as described below. Therefore, the correct drop image is obtained. That is, as shown in FIG. 7A, when a drop position defective cell 42 * in which the drop 15 is not correctly imaged is recognized by visual observation, the cursor 11a displayed on the display screen 40 is moved to the mouse. 11 is executed to execute the following processing.

First, as shown in FIG. 7B, it is manually determined that the unit cell 42 located at (E6) is the drop position defective cell 42 * by moving to the position of the drop position defective cell 42 * and clicking. Enter according to instructions. Corresponding to the operation input for selecting the unit cell 42 with the mouse 11, as shown in FIG. 8A, the well image 43 corresponding to the drop position defective cell 42 * is displayed on the display screen 40. The well list display layout 41 is displayed in an overlapping manner. Here, in the well image 43, a cross cursor 44 indicating the drop position detected by the drop recognition processing unit 27 appears at a position shifted from the position of the drop 15 existing in the well 8a.

  When the operator visually confirms the deviation between the drop 15 and the cross cursor 44, the operator performs a click operation for teaching the drop position with the mouse 11 while the cursor 11a is moved to the center of the drop 15. As a result, the cross cursor 44 moves onto the drop 15 as shown in FIG. In this state, by moving the cursor 11a to the update button 43a and performing a click operation, the erroneous drop position of the well 8a stored in the drop position storage unit 24a is corrected to the correct position.

  Next, the processes from (ST9) to (ST12) are executed for this well 8a by manual operation or automatically. As a result, the drop position defective cell 42 * shown in FIG. 9A is in a state where the image of the drop 15 is correctly displayed in the unit cell 42 of (E6) as shown in FIG. 9B. Then, a normal crystal detection process is executed in (ST13) for the normal drop image acquired in this way.

  That is, in the above-described droplet observation method, the first imaging processing (ST1, ST2, ST3) by the well image imaging processing unit 25, the droplet detection processing (ST4, ST5) by the drop recognition processing unit 27, and the drop image Second imaging processing (ST8, ST9, ST10) by the imaging processing unit 26, reduced image creation processing (ST11) by the reduced image creation processing unit 28, reduced image list display processing (ST12) by the list display unit 31a, The first image selection display process by the first image selection display unit 31b and the droplet position information correction process by the drop position correction processing unit 30 are included.

  In the reduced image list display process, a plurality of reduced images are displayed on the display unit 8 in an arrangement corresponding to the lattice arrangement of the wells 8a in the plate 8, and are displayed in the first image selection display process. In this case, a figure (here, a cross cursor 44) indicating a position based on the droplet position information stored in the drop position storage unit 24a is displayed on the well image. In addition, for a protein solution, a crystal determination process for determining whether a protein crystal is generated in the drop 15 using the drop image 15 is included.

  By adopting the above-described method for observing a droplet for detecting a protein crystal, when the drop 15 existing in a random state in the well 8a is imaged at a high imaging magnification, the drop 15 is out of the imaging field. Even if the image is taken in an abnormal state and a correct image cannot be obtained, an abnormal image can be obtained by performing a predetermined manual operation using the function of the droplet observation device described above. You can easily re-acquire the correct image. Therefore, it is possible to efficiently and efficiently perform image re-acquisition work to replace abnormal images that are inevitably generated in protein crystal observation work for a large number of samples with good correct images. It has become.

(Embodiment 2)
FIG. 10 is a block diagram showing the configuration of the control system of the droplet observation apparatus according to the second embodiment of the present invention, and FIGS. 11 and 12 are flowcharts of protein crystal detection processing in the droplet observation method according to the second embodiment of the present invention. FIG.

  In the second embodiment, in the configuration of the control system shown in FIG. 4 of the first embodiment, a method for creating an image to be displayed as a list by the list display unit 31a is used to create a reduced image by reducing a drop image. Instead of the method, a substantially similar image is obtained by cutting out the range corresponding to the drop from the well image.

  In FIG. 10, the drop image cut processing unit 28A reads the well image stored in the well image storage unit 23a, and based on the drop position stored in the drop position storage unit 24a, the range corresponding to the drop 15 in the well image. Perform cutting process. The cut image that has been cut is stored in the drop cut image storage unit 23d of the image storage unit 23. That is, the drop image cut processing unit 28A is a cut image creation processing unit that creates a cut image obtained by cutting a predetermined range based on the droplet position information of the well image from the well image, and the drop cut image storage unit 23d. Is a cut image storage unit for storing a plurality of cut images,

  Then, the list display unit 31a performs a process of reading a plurality of cut images from the drop cut image storage unit 23d and displaying them on the display unit 9. Further, the first image selection display unit 31b responds to the operation of selecting one of the plurality of cut images displayed on the display unit 9 performed by the input device, and corresponds to the selected cut image. The well image 8a of the well 8a is read from the well image storage unit 23a and displayed on the display unit 9. The configuration other than the above is the same as the configuration shown in FIG.

  Next, a droplet observation method will be described with reference to FIGS. FIG. 11 shows a drop position detection process in protein crystallization detection. (ST21) to (ST25) shown in FIG. 11 are the same as (ST1) to (ST5) shown in FIG. In the second embodiment, from the well image stored in the well image storage unit 23a, a drop cut image in which a portion corresponding to the position of the drop 15 is cut out by a drop image cut processing unit 28A is created, The image is stored in the drop cut image storage unit 23d (ST26) (image cut processing). Next, the drop cut image is displayed on the well list display layout as in the first embodiment (ST27) (cut image list display process).

  Thereafter, it is determined whether or not there is a well that has not been subjected to drop recognition. If there is an unexecuted well, the next well 8a is positioned at the observation position (ST29), and the process returns to (ST23) to repeat the subsequent processing. Execute. When it is determined in (ST28) that there is no unexecuted well, the drop position detection process is terminated. In this drop position detection process, if the well list display layout is displayed on the display unit 9 in (ST27), whether or not the drop position is correctly detected by visual observation by the operator as in the first embodiment. If a drop position detection failure is recognized, a drop position correction process is executed manually.

  Thereafter, the process proceeds to the protein detection process shown in FIG. 12, and the processing steps (ST30) to (ST35) are executed. (ST30), (ST31), (ST32), (ST33), (ST34), and (ST35) shown here are (ST8), (ST9), (ST10), (ST13), (ST13) shown in FIG. The content is the same as ST14) and (ST15).

That is, in the above-described droplet observation method, the first imaging process (ST21, ST22, ST23) by the well image imaging processor 25, the droplet detection process (ST24, ST25) by the drop recognition processor 27, and the drop image Image cut processing (ST26) by the cut processing unit 28A, cut image list display processing (ST27) by the list display unit 31a, first image selection display processing by the first image selection display unit 31b, and drop position correction processing unit 30 The droplet position information correction process by the second image pickup process by the drop image pickup processing unit 26 (ST28, ST29, S)
T30).

  In the second embodiment, the same effect as in the first embodiment is obtained. Further, in the second embodiment, the well list display can be performed only by acquiring the low magnification image of each well 8a. Therefore, before acquiring the high magnification drop image, the drop position correction process is performed. There is an advantage that can be done.

  The droplet observation apparatus and the droplet observation method of the present invention have an effect that a good image can be obtained with excellent operability, and are useful in detecting protein crystals generated in a protein solution.

The perspective view of the droplet observation apparatus of Embodiment 1 of this invention Sectional drawing of the plate used as the object of the droplet observation apparatus of one embodiment of this invention Partial sectional view of the droplet observation apparatus according to Embodiment 1 of the present invention. 1 is a block diagram showing the configuration of a control system of a droplet observation apparatus according to Embodiment 1 of the present invention. Flow chart of protein crystal detection processing in the droplet observation method of Embodiment 1 of the present invention Flow chart of protein crystal detection processing in the droplet observation method of Embodiment 1 of the present invention The figure which shows the display screen in the droplet observation apparatus of Embodiment 1 of this invention. The figure which shows the display screen in the droplet observation apparatus of Embodiment 1 of this invention. The figure which shows the display screen in the droplet observation apparatus of Embodiment 1 of this invention. FIG. 3 is a block diagram showing a configuration of a control system of a droplet observation apparatus according to a second embodiment of the present invention. Flow chart of protein crystal detection processing in the droplet observation method of Embodiment 2 of the present invention Flow chart of protein crystal detection processing in the droplet observation method of Embodiment 2 of the present invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Droplet observation apparatus 3 Observation part 4 Operation part 5 Imaging part 6 Observation stage (positioning mechanism)
7 Placement part 8 Plate 8a Well 9 Display part 13 Control unit 15 Drop (droplet)
23a Well image storage unit (first image storage area)
23b Drop image storage unit (second image storage area)
24a Drop position storage unit (droplet position storage unit)
25 well image imaging processing unit (first imaging processing unit)
26 Drop image capturing processor (second imaging processor)

Claims (16)

A droplet observation device for observing a droplet containing a protein solution existing in the crystal generation space on a plate having a plurality of crystal generation spaces in a lattice arrangement for generating protein crystals,
A mounting unit for mounting the plate, an imaging unit for imaging the crystal generation space at a desired imaging magnification, and a positioning mechanism for relatively positioning the plate on the mounting unit with respect to the imaging unit; An input device for an operator to operate the droplet observation device and a display unit for displaying predetermined display contents are connected, and an image storage unit for storing an image acquired by the imaging unit and a position of the droplet A control unit having a droplet position storage unit for storing each of the crystal spaces,
The control unit is
By setting the imaging magnification of the imaging unit to a low magnification and controlling the positioning mechanism based on the arrangement information of the crystal generation space in the plate, the imaging unit and the plate are aligned. A first imaging processing unit that images the crystal generation space, acquires a low-magnification image for each crystal generation space as a first image, and stores the first image in a first image storage area of the image storage unit;
A droplet detection processing unit that detects a position of the droplet from the first image and stores the information as droplet position information in the droplet position storage unit for each crystal generation space;
The imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information to align the imaging unit with the plate, and the imaging unit images the droplet. A second imaging processing unit that acquires an enlarged image of the lever droplet as a second image, and stores the second image in a second image storage area of the image storage unit;
A reduced image creation processing unit that creates a reduced image obtained by reducing the second image and stores the reduced image in a reduced image storage area of the image storage unit, and reads a plurality of reduced images from the reduced image storage unit and displays a list on the display unit A list display section to be
In response to an operation of selecting one of the plurality of reduced images displayed on the display unit by the input device, the first image of the crystal generation space corresponding to the selected reduced image is displayed as the first image. A first image selection display unit that reads from the first image storage area and displays on the display unit;
A droplet that corrects the droplet position information corresponding to the first image in response to the operation of instructing the correct position of the droplet in the first image displayed on the display unit being performed by the input device. A position information correction unit;
A droplet observation apparatus comprising:   2. The droplet observation apparatus according to claim 1, wherein the list display unit displays a plurality of reduced images in a list on the display unit in an arrangement corresponding to a lattice arrangement of a crystal generation space on the plate.   The first image selection display unit displays a figure indicating a position based on the droplet position information stored in the droplet position storage unit on the displayed first image. Or the droplet observation apparatus of 2.   The said control unit is provided with the crystal | crystallization determination part which determines whether the protein crystal | crystallization was produced | generated in the said droplet using the said 2nd image. Droplet observation device. A mounting unit for mounting a plate having a plurality of crystal generation spaces for generating protein crystals in a lattice arrangement, an imaging unit for imaging the crystal generation space at a desired imaging magnification, and a front of the imaging unit. A positioning mechanism that relatively positions the plate on the placement unit, an input device for an operator to operate the droplet observation device, and a display unit that displays predetermined display contents are connected, and the imaging unit A liquid comprising: an image storage unit that stores the acquired image; and a control unit that includes a droplet position storage unit that stores, for each crystal space, the position of a droplet containing the protein solution existing in the crystal generation space. A droplet observation method for observing the droplet on the plate by a droplet observation device,
By setting the imaging magnification of the imaging unit to a low magnification and controlling the positioning mechanism based on the arrangement information of the crystal generation space in the plate, the imaging unit and the plate are aligned. Imaging a crystal generation space, obtaining a low-magnification image for each crystal space as a first image, and storing the first image in a first image storage area of the image storage unit;
A droplet detection process for detecting the position of the droplet from the first image and storing the information as droplet position information in the droplet position storage unit;
The imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information to align the imaging unit with the plate, and the imaging unit images the droplet. A second imaging process of acquiring an enlarged image of the lever droplet as a second image and storing the second image in a second image storage area of the image storage unit;
Reduced image creation processing for creating a reduced image obtained by reducing the second image and storing the reduced image in a reduced image storage area of the image storage unit;
A reduced image list display process of reading a plurality of reduced images from the reduced image storage area and displaying a list on the display unit;
In response to the operation of selecting one of the plurality of reduced images displayed on the display unit being performed by the input device, the first image of the crystal generation space corresponding to the selected reduced image is selected from the first image. A first image selection display process for reading from the first image storage area and displaying on the display unit;
A liquid that corrects the droplet position information corresponding to the first image in response to the operation of instructing the correct position of the droplet in the first image displayed on the display unit being performed by the input device. Drop position information correction processing,
A droplet observation method comprising:   6. The droplet observation method according to claim 5, wherein, in the reduced image list display processing, a plurality of reduced images are displayed on the display unit in an arrangement corresponding to a lattice arrangement of a crystal generation space on the plate.   6. The graphic indicating the position based on the droplet position information stored in the droplet position storage unit is displayed on the displayed first image in the first image selection display process. Or the droplet observation method of 6.   The droplet observation according to claim 5, further comprising a crystal determination process for determining whether a protein crystal is generated in the droplet using the second image. Method. A droplet observation device for observing a droplet containing a protein solution existing in the crystal generation space on a plate having a plurality of crystal generation spaces in a lattice arrangement for generating protein crystals,
A mounting unit for mounting the plate, an imaging unit for imaging the crystal generation space at a desired imaging magnification, and a positioning mechanism for relatively positioning the plate on the mounting unit with respect to the imaging unit; An input device for an operator to operate the droplet observation device and a display unit for displaying predetermined display contents are connected, and an image storage unit for storing an image acquired by the imaging unit and a position of the droplet A control unit having a droplet position storage unit for storing each of the crystal spaces,
The control unit is
By setting the imaging magnification of the imaging unit to a low magnification and controlling the positioning mechanism based on the arrangement information of the crystal generation space in the plate, the imaging unit and the plate are aligned. A first imaging processing unit that images the crystal generation space, acquires a low-magnification image for each crystal generation space as a first image, and stores the first image in a first image storage area of the image storage unit;
A droplet detection processing unit that detects the position of the droplet from the first image and stores the information as droplet position information in the droplet position storage unit for each crystal generation space;
An image cutout processing unit that creates a cutout image obtained by cutting out a predetermined range based on the droplet position information of the first image from the first image, and stores the cutout image in a cutout image storage area of the image storage unit; ,
A list display unit that reads a plurality of cut images from the cut image storage area and displays a list on the display unit;
In response to an operation of selecting one of the plurality of cut images displayed on the display unit being performed by the input device, the first image of the crystal generation space corresponding to the selected cut image is displayed as the first image. A first image selection display unit that reads from the first image storage unit and displays on the display unit;
A liquid that corrects the droplet position information corresponding to the first image in response to the operation of instructing the correct position of the droplet in the first image displayed on the display unit being performed by the input device. A droplet position information correction unit;
The imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information to align the imaging unit with the plate, and the imaging unit images the droplet. A second imaging processing unit that acquires an enlarged image of the lever droplet as a second image, and stores the second image in a second image storage area of the image storage unit;
Droplet observation storage characterized by comprising.   10. The droplet observation apparatus according to claim 9, wherein the list display unit displays a list of the plurality of cut images on the display unit in an arrangement corresponding to a lattice arrangement of a crystal generation space on the plate.   The said 1st image selection display part displays the figure which shows the position based on the droplet position information memorize | stored in the said droplet position memory | storage part on the displayed 1st image. Alternatively, the droplet observation apparatus according to 11.   The said control unit is provided with the crystal | crystallization determination part which determines whether the protein crystal | crystallization was produced | generated in the said droplet using the said 2nd image. Droplet observation device. A mounting unit for mounting a plate having a plurality of crystal generation spaces for generating protein crystals in a lattice arrangement, an imaging unit for imaging the crystal generation space at a desired imaging magnification, and a front of the imaging unit. A positioning mechanism that relatively positions the plate on the placement unit, an input device for an operator to operate the droplet observation device, and a display unit that displays predetermined display contents are connected, and the imaging unit A liquid comprising: an image storage unit that stores the acquired image; and a control unit that includes a droplet position storage unit that stores, for each crystal space, the position of a droplet containing the protein solution existing in the crystal generation space. A droplet observation method for observing the droplet on the plate by a droplet observation device,
By setting the imaging magnification of the imaging unit to a low magnification and controlling the positioning mechanism based on the arrangement information of the crystal generation space in the plate, the imaging unit and the plate are aligned. Imaging a crystal generation space, obtaining a low-magnification image for each crystal space as a first image, and storing the first image in a first image storage area of the image storage unit;
A droplet detection process for detecting the position of the droplet from the first image and storing the information as droplet position information in a droplet position storage unit;
An image cutting process for creating a cut image obtained by cutting a predetermined range based on the droplet position information of the first image from the first image, and storing the cut image in a cut image storage area of the image storage unit;
A cut image list display process for reading a plurality of cut images from the cut image storage area and displaying a list on the display unit,
In response to an operation of selecting one of the plurality of cut images displayed on the display unit being performed by the input device, the first image of the crystal generation space corresponding to the selected cut image is displayed as the first image. A first image selection display process for reading from the first image storage unit and displaying on the display unit;
A liquid that corrects the droplet position information corresponding to the first image in response to the operation of instructing the correct position of the droplet in the first image displayed on the display unit being performed by the input device. Drop position information correction processing,
The imaging magnification of the imaging unit is set to a high magnification, and the positioning mechanism is controlled based on the droplet position information to align the imaging unit with the plate, and the imaging unit images the droplet. A second imaging process of acquiring an enlarged image of the lever droplet as a second image and storing the second image in a second image storage area of the image storage unit;
A droplet observation method comprising:   14. The droplet observation method according to claim 13, wherein, in the cut image list display processing, a plurality of cut images are displayed in a list on the display unit in an arrangement corresponding to a lattice arrangement of a crystal generation space on the plate.   14. In the first image selection display process, a graphic indicating a position based on droplet position information stored in the droplet position storage unit is displayed on the displayed first image. Or the droplet observation method of 14. The droplet observation according to claim 13, further comprising a crystal determination process for determining whether or not a protein crystal is generated in the droplet using the second image. Method.