KR101909938B1 - Auto focusing timelapse camera apparatus for cells in well of multi-well plate and, auto focusing methods thereof - Google Patents

Abstract

In the present invention, the wells of a multi-well plate are photographed at predetermined time intervals, and live cells in culture can be clearly photographed with a focused image, and the camera is photographed while moving in the horizontal direction in place of the multi- It is possible to eliminate or minimize the movement and entanglement of the object.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an autofocus time-lapse imaging apparatus and method for cells accommodated in a well of a multi-well plate,

The present invention relates to an apparatus for photographing cells contained in a well of a multi-well plate, and more particularly, to a device capable of capturing living cells in a well in a focused image with a focus image at a predetermined time.

In addition, the present invention also relates to a method of automatically acquiring a focus image when photographing using such a photographing apparatus.

Generally, when a cell is cultured in real time using a multi-well plate, it is necessary to photograph each well at predetermined time intervals to confirm the state of the cell.

However, when the focus is taken on the basis of the well and the image is photographed after moving to another well, the well is often out of focus. This is because the cells in the well are floating in the culture medium and therefore move continuously.

Therefore, it is necessary to newly adjust the focus for each well to obtain a sharp focus image. However, it is almost impossible for a researcher to focus manually on each well, as some cells take several days to incubate. Therefore, there is a need for an apparatus and method that can automatically focus on the wells of a multi-well plate.

On the other hand, in the conventional photographing apparatus, the multi-well plate is movable along the x, y axis (i.e., the horizontal direction) and the camera is fixed in position. Therefore, in order to capture the next well after photographing a specific well, the multi-well plate moves along the x and y axes so that the well to be photographed corresponds to the camera lens.

However, since the cells of each well are floated in the culture medium or reagent, the cells move and become entangled with each other as the multi-well plate moves along the x, y axis (i.e., the horizontal direction) There is a problem.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide an apparatus and a method for capturing living cells floating in a culture solution of each well with a focused image every predetermined time .

It is still another object of the present invention to provide an apparatus and a method capable of preventing cell movement and entanglement due to horizontal movement of a multi-well plate.

In order to solve the above problem, a photographing method according to the present invention includes the steps of: (a) moving one of the multi-well plate 1 and the camera in the horizontal direction so that the well to be photographed corresponds to the camera lens 20 ; (b) acquiring a plurality of images by photographing the wells at a predetermined height while vertically moving any one of the multi-well plate (1) and the camera after the step (a); (c) obtaining a pixel value (e.g., color intensity, 1 to 255) of each pixel in the photographed image; (d) arranging the pixel values obtained in the step (c) in rows and columns; (e) generating a group of two neighboring pixels in each row, subtracting the pixel value of the group to calculate a subtraction value, or grouping three or more neighboring pixels into one group, Subtracting the pixel value and calculating a subtraction value; (f) classifying the subtraction value into an edge when the subtraction value is equal to or greater than a predetermined reference value, and obtaining an edge number for each image; And (g) selecting an image having the largest number of edges from the plurality of images as a focus image.

Preferably, after step (g), (h) moving either the camera or the multi-well plate 1 in the vertical direction to the height of the focus image; And (i) moving any one of the camera and the multi-well plate 1 so that the well to be photographed next corresponds to the camera lens 20. The steps (h) and (h) of the step (i) may be performed first or the step (i) may be performed first.

In the above photographing method, it is preferable that the camera moves in the horizontal direction and the multi-well plate 1 moves in the vertical direction.

A photographing apparatus according to the present invention includes a frame (30) on which an illumination lamp (25) and a camera are installed; A lens 20 of the camera provided on the upper surface of the frame 30; An illumination lamp (25) provided on the opposite side of the camera lens (20) with the multi-well plate (1) as the center; A horizontal moving unit 40 installed below the frame 30 for moving the frame 30 horizontally; A vertical moving unit 60 installed in the lateral direction of the frame 30 for vertically moving the multi-well plate 1; A data storage unit for storing the photographed image; And a monitor for externally displaying the photographed image.

The vertically moving unit 60 includes a tray 68 provided on the vertical plate 63. The tray 68 includes a rectangular bracket 68a including a central through portion 68c; A stepped step 68b formed on an inner surface of the bracket 68a; A groove 69 formed in the upper portion of the step tang 68b in the inner surface of the bracket 68a; A spring 69a provided inside the groove 69 and a bearing ball 69b provided at the tip of the spring 69a.

The multi-well plate 1 is installed on the step jaw 68b so that the bearing ball 69b pushes the multi-well plate 1 toward the opposite step jaw 68b by the elastic force of the spring 69a, - The well plate (1) can be fixed.

On the upper surface of the frame 30, a through hole 34 is formed in a portion corresponding to the multi-well plate 1. [ It is preferable that the camera lens 20 is installed in the through hole 34 and the camera body 22 is installed on one side of the upper surface of the frame 30. [

The present invention has the following effects.

First, living cells floating in the culture medium in the wells can be photographed with focused images every predetermined time.

Second, by moving the camera in the horizontal direction and positioning the camera and the well to correspond to each other, the flow of the culture fluid and the cells contained in the well can be eliminated or minimized, thereby effectively obtaining the focused image.

1 is a perspective view showing a photographing apparatus according to a preferred embodiment of the present invention.
2 is a perspective view showing a state in which a cover of the photographing apparatus is partially removed.
3 is a configuration diagram showing each configuration of a photographing apparatus and its connection state.
4 is a front view showing a schematic configuration of a horizontal moving unit provided in the photographing apparatus;
5 is a plan view showing a schematic configuration of a vertical movement unit provided in the photographing apparatus;
6A and 6B are flowcharts showing a photographing method according to the present invention.
7 is a photograph showing a photographed image using a photographing apparatus.
8 is a photograph exemplarily showing pixels constituting the image of FIG. 7. FIG.
Figure 9 is a matrix of pixel values of the pixel of Figure 8;
10 is a diagram showing calculation of subtraction values in the matrix of FIG. 9;
11 is a photograph showing a focused image.
Figure 12 is a photograph showing an unfocused image.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined. Therefore, the embodiments described in the present specification and the configurations shown in the drawings are merely examples of the present invention and are not intended to represent all of the technical ideas of the present invention, so that various equivalents And variations are possible.

FIG. 1 is a perspective view showing a photographing apparatus according to a preferred embodiment of the present invention, FIG. 2 is a perspective view showing a state in which a cover of the photographing apparatus is partially removed, FIG. FIG.

Referring to the drawings, the photographing apparatus 100 includes a frame 30, a camera for photographing a well of the multi-well plate 1, a horizontal movement unit 40 for moving the camera in the horizontal direction, A control unit for controlling the movement of the horizontal movement unit 40 and the vertical movement unit 60 and for controlling the shooting and illumination of the camera, a control unit for controlling the movement of each pixel A central processing unit (including a CPU and an arithmetic unit) for calculating pixel values of each pixel and using the pixel values to calculate an edge, a data storage unit for storing photographed images, pixel values and edges, And a monitor for externally displaying the photographed image.

The frame 30 is installed on the horizontal moving unit 40 and can be moved in the horizontal direction (i.e., the x and y axis directions) by the horizontal moving unit 40. [ In the frame 40, a camera and an illumination lamp 25 are installed. Therefore, when the frame 30 is moved in the horizontal direction by the horizontal moving unit 40, the camera and the illumination lamp 25 are also moved in the horizontal direction accordingly.

Preferably, a through hole 34 is formed in the center of the frame 30, a camera lens 20 is provided in the through hole 34, and the camera body 22 is installed on one side of the upper surface of the frame. This arrangement can reduce the size of the frame 30, rather than installing the entire camera inside the frame 30.

The illumination lamp 25 is installed on the support base 32 and is installed on the opposite side of the camera lens 20 with the multi-well plate 1 as the center. The operation of the illumination lamp 25 is controlled by the illumination control unit.

The horizontal movement unit 40 includes a first horizontal movement unit 41 and a second horizontal movement unit 46 as shown in Fig.

When the ball screw 42a is rotated by the rotation of the servo motor 42, the first horizontal moving unit 41 horizontally moves the upper flat plate 42b in the x direction. At this time, since the legs 43b provided below the flat plate 42b are slid along the rails 43a provided on the floor, the movement is guided.

When the ball screw 47a is rotated by the rotation of the servo motor 47, the second horizontal moving unit 46 horizontally moves the upper flat plate 47b in the y direction (direction perpendicular to the x direction) . At this time, since the leg 48b provided under the flat plate 47b is slid along the rail 48a provided on the upper surface of the flat plate 42b, the movement is guided.

The vertically moving unit 60 moves the multi-well plate 1 up and down in the vertical direction (i.e., the z-axis direction). 5, when the servo motor 61 is rotated to rotate the ball screw 62, the vertical moving unit 60 vertically moves the front vertical plate 63 thereof. At this time, the legs 66 provided on the back surface of the vertical plate 63 slide along the rails 65 to guide the movement. The rails 65 are installed on both sides of the ball screw 62 in the vertical direction on the front surface of the flat plate 64.

A tray 68 is coupled to the vertical plate 63 and a multi-well plate 1 is installed in the tray 68. [ Therefore, the multi-well plate 1 can be moved up and down in the vertical direction (i.e., the z-axis direction) by the vertically moving unit 60. [

The tray 68 has a rectangular bracket 68a including a central penetrating portion 68c, a stepped step 68b formed inside the bracket 68a, a stepped step 68b on the inner surface of the bracket 68a, A groove 69 formed in the upper part of the groove 69 and a spring 69a provided in the groove 69 and a bearing ball 69b provided at the tip of the spring 69a.

Grooves 69 are formed on at least two sides of four sides of the tray 68. Then, the spring 69a provides an elastic force to the bearing ball 69b. Accordingly, when the multi-well plate 1 is installed on the tray 68, the multi-well plate 1 can be fixed to the opposite stepped portion 68b by the elastic force of the spring 69a.

The control unit includes a vertical movement unit control unit, a horizontal movement unit control unit, a camera control unit, and a lighting control unit.

The vertical movement unit control unit controls the movement of the vertical movement unit 60 according to the data previously input by the user, and the horizontal movement unit control unit controls the movement of the horizontal movement unit 40 according to the data previously inputted by the user. The camera control unit controls the operation of the camera in conjunction with the operation of the vertical and horizontal movement units 40 and 60, and the illumination control unit controls the operation of the illumination lamp 25. [

The user uses the data input unit to calculate the number of wells to be photographed (the position of the well), the photographing cycle, the vertical photographing interval, the illumination brightness, the reference value of edge selection, And the like (the edge and the subtraction value are described below).

Data such as the photographed images, pixel values, edge values, and edge counts are stored in a data storage unit, and the entire captured image or a selected image (e.g., a focus image) .

The central processing unit (including the CPU and the arithmetic unit) performs the determination and data processing of S41 to S49 (FIG. 6B) on the photographed image. The central processing unit (CPU) will be described further below.

As described above, in the present invention, since the camera is moved in the horizontal direction (x, y direction) and the multi-well plate 1 moves only in the vertical direction (z direction), the culture medium or reagent in the wells does not flow, do. Accordingly, in the present invention, the cells in the well are immobilized, movement is minimized, and cell entanglement is minimized or minimized.

On the other hand, the cells in the well are floating in the culture medium, but the conventional method can not be used to measure the exact distance to the cells in the culture medium. For example, an ultrasonic sensor can not measure an accurate distance to a cell because an ultrasonic wave is reflected on the surface of a culture liquid, and a laser sensor or a light sensor can not be used because it may affect a cell. In consideration of such specificity, in the present invention, a plurality of images are obtained for one well cell by photographing at a predetermined height while moving the multi-well plate 1 in the vertical direction, and automatically selecting a focus image among the plurality of images do.

Then, in order to select a focused image, a pixel intensity of each pixel is obtained in each image, the pixel values are arranged in a matrix, the subtraction values are calculated by subtracting the two neighboring pixel values from each other, When the value is greater than or equal to a predetermined reference value, the image is classified into an edge, the total number of edges is obtained from each image, and an image having the largest number of edges is selected as a focus image for the corresponding well.

This process will be described with reference to Figs. 6A to 6B.

First, the frame 30 is horizontally moved by the horizontal movement unit 40 so that the camera lens 20 corresponds to the well to be photographed (S10). Subsequently, the multi-well plate 1 is moved in the downward direction (-z axis direction) by -50 steps (S20), and then the multi-well plate 1 is moved upward by +10 steps, That is, moving the multi-well plate 1 upward by 10 steps after taking a picture is repeated 10 times (one step is 0.5 탆) (S30).

On the other hand, the photographing interval and the number of photographing times are not fixed, but can be increased or decreased to suit the characteristics of the cells and the culture purpose.

When the photographing is completed, the central processing unit (CPU) outputs the photographed image as shown in FIG. 7 (S41), measures the number of pixels of the rows and columns in the output image (S42) For example, color intensity (1 to 255) is measured (S43).

Subsequently, the pixel values of all the pixels are arrayed two-dimensionally as rows and columns as shown in Figs. 8 to 9 (S44), and the edges are sequentially analyzed from the first row to the last row (S45).

That is, two adjacent pixels in each row are grouped and the subtraction value is calculated by subtracting the pixel value of the group. If the subtraction value becomes equal to or greater than a predetermined reference value, for example, if the absolute value of the subtraction value becomes 100 or more, the edge is classified and the total number of edges is counted in the image (S46). FIG. 10 shows these subtraction values, and the case of the reference value (absolute value 100) or more is shown in yellow.

Meanwhile, the group may be composed of three or more neighboring pixels. In this case, the subtraction value is obtained by subtracting the minimum value from the maximum value of the pixels belonging to the group. The reference value may be set differently depending on the photographing purpose, the culture purpose, and the like.

Then, it is determined whether or not the next image exists (S47). If there is a next image, S41 to S46 are repeated. If there is no next image, the number of edges of each image is compared (S48) (S49). FIG. 11 shows the selected focus image through this process, and FIG. 12 shows the non-focused image.

When the focus image selection is completed, the multi-well plate 1 is vertically moved to the height at which the focus image is captured (S50). The step S50 is based on the fact that the focus height of the next well is likely to be the same as or similar to the focus height of the previously captured well, and it is possible to shorten the photographing time.

Next, it is determined whether there is a well to be photographed (S60). If there is a next well, the process moves to S20 to repeat imaging and analysis and focus image selection.

The above-described photographing steps (S10 to S60) are repeated at pre-input time intervals.

Although the method of acquiring the focused image based on the horizontal movement of the camera and the vertical movement of the multi-well plate 1 has been described above, the method of acquiring the focused image may be performed using an existing imaging apparatus. That is, the photographing apparatus in which the multi-well plate 1 is moved horizontally and the camera is vertically moved is used, or the camera is fixed and the multi-well plate 1 is moved horizontally and vertically, The focus image can be obtained using the focus image acquisition method of the present invention. In this case, the horizontal movement of the multi-well plate 1 has the disadvantage that cells in the culture fluid or reagent may move or tangle, but still have the advantage of automatically capturing sharp focus images. On the other hand, a photographing apparatus in which the multi-well plate 1 moves horizontally and the camera moves vertically, and a photographing apparatus in which the camera is fixed and the multi-well plate 1 moves horizontally and vertically are conventional ones, The description of the structure of the conventional photographing apparatus will be omitted.

1: multi-well plate 11: cover
20: camera lens 22: camera body
25: illumination lamp 30: frame
32: support 34: through-hole
40: Horizontal moving unit 60: Vertical moving unit
68: Tray 100: photographing device

Claims (7)

(a) moving one of the multi-well plate (1) and the camera horizontally so that the well to be photographed corresponds to the camera lens (20);
(b) acquiring a plurality of images by photographing the wells at a predetermined height while vertically moving any one of the multi-well plate (1) and the camera after the step (a);
(c) obtaining a pixel value of each pixel in the photographed image;
(d) arranging the pixel values obtained in the step (c) in rows and columns;
(e) generating a group of two neighboring pixels in each row, subtracting the pixel value of the group to calculate a subtraction value, or grouping three or more neighboring pixels into one group, Subtracting the pixel value and calculating a subtraction value;
(f) classifying the subtraction value into an edge when the subtraction value is equal to or greater than a predetermined reference value, and obtaining an edge number for each image; And
(g) selecting an image having the largest number of edges from among the plurality of images as a focus image. The method according to claim 1,
After the step (g)
(h) moving one of the camera and the multi-well plate (1) in the vertical direction to the height of the focus image; And
(i) moving either the camera or the multi-well plate (1) so that the well to be photographed next corresponds to the camera lens (20)
The autofocus time-lapse imaging method for cells housed in a well of a multi-well plate, wherein step (h) and step (h) of step (i) . 3. The method according to claim 1 or 2,
Characterized in that the camera moves in the horizontal direction and the multi-well plate (1) moves in the vertical direction. A frame 30 on which the illumination lamp 25 and the camera are installed;
A lens 20 of the camera provided on the upper surface of the frame 30;
An illumination lamp (25) provided on the opposite side of the camera lens (20) with the multi-well plate (1) as the center;
A horizontal moving unit 40 installed below the frame 30 for moving the frame 30 horizontally;
A vertical moving unit 60 installed in the lateral direction of the frame 30 for vertically moving the multi-well plate 1;
A data storage unit for storing the photographed image;
A monitor for displaying a photographed image on the outside;
A control unit for controlling the movement of the horizontal movement unit 40 and the vertical movement unit 60 and for controlling the photographing of the camera; And
And a central processing unit which obtains pixel values of each pixel in the photographed image, converts the pixel values of each pixel into a matrix, and calculates an edge using the pixel value,
After the camera moves horizontally by the horizontally moving unit 40 so as to correspond to the predetermined well, the camera moves vertically by the vertically moving unit 60 to move the cells in the well A plurality of images are acquired at each height,
The central processing unit calculates a subtraction value by creating two groups of neighboring pixels in each row of the matrix and subtracting the pixel value of the group or by grouping three or more neighboring pixels among the neighboring pixels, And selects the image having the largest number of edges from the plurality of images as the focus image. The image processing apparatus according to claim 1, Of the well of the multi-well plate. 5. The method of claim 4,
The vertical movement unit 60 includes a tray 68 provided on the vertical plate 63,
The tray 68,
A rectangular bracket 68a including a central penetrating portion 68c;
A stepped step 68b formed on an inner surface of the bracket 68a;
A groove 69 formed in the upper portion of the step tang 68b in the inner surface of the bracket 68a; And
A spring 69a provided in the groove 69 and a bearing ball 69b provided at the tip of the spring 69a,
The multi-well plate 1 is installed on the step jaw 68b so that the bearing ball 69b pushes the multi-well plate 1 toward the opposite step jaw 68b by the elastic force of the spring 69a, Characterized in that the well plate (1) is fixed. 6. The method of claim 5,
A through hole 34 is formed in the upper surface of the frame 30 at a portion corresponding to the multi-well plate 1. The camera lens 20 is installed in the through hole 34, Is provided on one side of the upper surface of the multi-well plate (30). delete

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