JP2008101968A - Screening apparatus for drug development, and development method for drug development - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening apparatus for drug development which eliminates the irregularities caused by the difference between operators, when the number of measuring targets is counted from the image of an acquired sample and has high reproducibility. <P>SOLUTION: An image processor 11 is the screening apparatus for drug development constituted so that the samples 6 placed on a well plate 60 are irradiated with exciting light 1 to perform image processing, on the basis of the fluorescence signals 7 from the samples 6 to screen a drug development. The image processor 11 for counting the number of measuring targets present in an image is equipped with a memory 13 for preliminarily storing the upper and lower limit thresholds of the sizes of the measuring targets is provided at each sample 6, to automatically count the number of the measuring targets on the basis of the upper and lower limit thresholds. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an image processing apparatus that counts the number of measurement objects from an acquired sample image in a drug discovery screening apparatus using a fluorescence microscope system.

  The drug discovery screening device excites a sample arranged in an array of wells (holes) on a well plate by irradiating it with light of a specific wavelength, and magnifies and expands the fluorescence image emitted from the excited sample with a microscope system. The image is captured with a camera. In that case, the well plate is moved on the XY stage in order to acquire fluorescent images from all wells.

  Next, image processing is performed on the image acquired by the camera, and a sample that is a drug candidate is found based on the result. In order to improve the image quality, a confocal scanner is installed between the microscope and the camera.

  The following patent documents are known as prior art of such a drug discovery screening apparatus using a confocal scanner.

JP 2002-062480 A JP 2005-095012 A Japanese Patent Laying-Open No. 2005-098722

  FIG. 5 is a block diagram showing the technology of the conventional drug discovery screening device described in Patent Document 1. The confocal scanner 100 is connected to a microscope 200, and the illumination parallel excitation light beam 1 (one-dot chain line) is condensed into individual light beams by a microlens array disk (referred to as ML disk) 2.

  After passing through a first dichroic mirror (DM) 3 composed of a flat mirror having spectral characteristics, the sample passes through individual pinholes of the Niipou disc 4 and is placed on the well plate 60 by the objective lens 5 of the microscope 200. 6 is condensed on each sample to excite the fluorescent reagent. The ML disk 2 and the nipou disk 4 rotate around the rotation center axis 12 while being mechanically connected by the connecting member 8.

  The fluorescent signal 7 emitted from the fluorescent reagent of each sample of the sample 6 passes through the objective lens 5 again and is collected on each pinhole of the Niipou disc 4. The fluorescence signals 7 that have passed through the individual pinholes are reflected by the DM 3, and a confocal optical image is formed on the camera 10 via the relay lens 9.

  In the above-described configuration, the plane on which the pinholes of the Niipou disc 4 are arranged, the plane to be observed on the sample 6, and the light receiving surface of the camera 10 are arranged in an optically conjugate relationship with each other. An optical cross-sectional image of the sample 6, that is, a confocal image is formed. Therefore, since a confocal image of the sample 6 can be formed on the light receiving surface of the camera 10, the sample 6 in which a large number of specimens are arranged on a matrix is relative to the microscope 200 and the confocal scanner 100. By moving, the confocal images of all the samples can be captured at high speed.

  By the way, in the above-described conventional example, the fluorescent image or the confocal image is selected by inserting and removing the Nipo disk 4. In drug discovery applications, fluorescent images are suitable for translocation to analyze the transition from the nucleus to the cytoplasm and calcium signals, and confocal images are suitable when high image quality is required, such as neurite outgrowth measurement. In these applications, various reactions are digitized by image processing, and the degree of reaction is normalized by the number of cells.

  In the conventional example, when counting the number of cells, an independent region in the acquired image is labeled and the area is calculated. For the area, the operator (or user) sets the upper and lower thresholds, and those below the lower threshold are not counted as noise, and those between the upper and lower thresholds are regarded as having one cell. When the number is equal to or greater than the upper threshold value, the number of cells is regarded as being in contact with each other.

  However, if the number of cells is calculated as described above, the area threshold value is set by the operator during image processing, so that variation occurs depending on the operator, affecting the reproducibility of the measurement results, and the measurement accuracy decreases. There was a problem of doing.

  The present invention has been made in order to solve the above-described problems of the prior art. When counting the number of objects to be measured from the acquired sample images, the present invention eliminates variations due to operator differences and has high reproducibility for drug discovery screening. It aims to realize.

In order to achieve such a problem, the invention according to claim 1 of the present invention is:
In the drug discovery screening apparatus that irradiates the sample placed on the well plate with excitation light and counts the number of measurement objects present in the image based on the fluorescence signal from the sample,
A memory in which upper and lower thresholds of the size of the measurement target are stored in advance is provided.

The invention according to claim 2
The drug discovery screening device according to claim 1,
The number of measurement objects is counted by adding the number of independent regions within the upper and lower threshold values and twice the number of independent regions larger than the upper threshold value.

The invention described in claim 3
The drug discovery screening device according to claim 1,
The memory is prestored with a composite upper limit threshold of the size of the plurality of measurement objects that are in contact with or overlapping, and the number of the measurement objects is counted by combining the upper and lower limit thresholds and the composite upper limit threshold. To do.

The invention according to claim 4
The drug discovery screening device according to claim 3,
The composite upper limit threshold is a plurality of times the upper limit threshold.

The invention according to claim 5
The drug discovery screening device according to any one of claims 1 to 4,
Using a Nipo type confocal scanner, the focal position is changed in the optical axis direction, and the confocal image is extracted by irradiating the sample with the excitation light at each focal position.

The invention described in claim 6
The drug discovery screening device according to any one of claims 1 to 5,
The measurement object is a cell or a cell nucleus.

The invention described in claim 7
In the drug discovery screening method of irradiating the sample placed on the well plate with excitation light and counting the number of measurement objects present in the image based on the fluorescence signal from the sample,
The upper and lower thresholds of the size of the measurement object are stored in advance in a memory.

The invention described in claim 8
The drug discovery screening method according to claim 7,
The number of measurement objects is counted by adding the number of independent regions within the upper and lower threshold values and twice the number of independent regions larger than the upper threshold value.

The invention according to claim 9
The drug discovery screening method according to claim 7,
The memory is prestored with a composite upper limit threshold of the size of a plurality of the measurement objects that are in contact with or overlapping, and the number of the measurement objects is counted by combining the upper and lower limit thresholds and the composite upper limit threshold. .

The invention according to claim 10 is:
The drug discovery screening method according to claim 9, wherein
The composite upper limit threshold is a plurality of times the upper limit threshold.

The invention according to claim 11
The drug discovery screening method according to any one of claims 7 to 10,
Using a Nipo type confocal scanner, the focal position is changed in the optical axis direction, and the confocal image is extracted by irradiating the sample with the excitation light at each focal position.

The invention according to claim 12
The drug discovery screening method according to any one of claims 7 to 11,
The measurement object is a cell or a cell nucleus.

The invention according to claim 13
In the drug discovery screening device or drug discovery screening method according to any one of claims 1 to 12,
The size is an area.

  According to the drug discovery screening apparatus of the present invention, information relating to the size of a sample to be used in advance is stored in a memory, an upper and lower limit threshold is set in advance for the size of the sample, and the value is obtained in an actually acquired image. By applying, it is possible to realize a drug discovery screening device that can eliminate variations due to operator differences and count the number of measurement objects with good reproducibility.

  Hereinafter, an embodiment of a drug discovery screening apparatus according to the present invention will be described with reference to FIG.

  In FIG. 1, the same elements as those of the conventional example shown in FIG. The difference from the conventional example is that information on the size of the sample to be used is stored in a database and stored in the memory, and upper and lower thresholds are set in advance for the sample size and automatically applied to the actually acquired image. It is in the point which was made to do.

  As shown in FIG. 1, the sample 6 in one of the arrayed wells (holes) in the well plate 60 is irradiated with an excitation light beam 1 emitted from a laser light source (not shown). A fluorescent signal 7 is emitted from the irradiated sample 6 and collected by the microscope 200 to obtain a fluorescent image. The confocal scanner 100 is used to obtain an image with a high S / N ratio. A single-color or multi-color fluorescent image that has passed through the confocal scanner 100 is captured by the camera 10, and image processing is performed by the image processing device 11. In the image processing apparatus 11, information regarding the size of the sample is stored in the memory 13 in advance as a database. By reading from the memory 13, the upper and lower thresholds of the sample size are set in the image processing apparatus 11 in advance.

The operation in the image processing apparatus 11 will be described below.
FIG. 2 shows a conceptual diagram of an image acquired by the camera 10. For example, when counting the number of cell nuclei (hereinafter referred to as nuclei) based on such an image 20, first, an independent region in the image, that is, a cell 21, a nucleus 22, noise 23, an overlapping cell nucleus 24, etc. Detect and label. Among the labeled independent regions, the number of independent regions is counted for each size of the measurement target (nucleus) to obtain a frequency distribution of the sizes. An example of the frequency distribution diagram of the size is shown in FIG. For this purpose, the lower limit threshold and the upper limit threshold are set in advance in the image processing apparatus 11 according to the size of the measurement target (core) based on the database in the memory 13. As an example, in a human-derived He-La cell widely used as a sample, the size of the nucleus is about 5 to 10 μm.

The number of nuclei is obtained as follows using upper and lower thresholds.
(1) The number of nuclei is 0 in the range below the lower threshold in FIG. (Example: Noise 23 in FIG. 2)
(2) In the range from the lower threshold value to the upper threshold value in FIG. 3, the number of nuclei is the sum of the frequencies according to size. (Example: Core 22 in FIG. 2)
(3) In the range above the upper limit in FIG. 3, the number of nuclei is set to be twice the total number of frequencies according to size. (Example: overlapping cell nucleus 24 in FIG. 2)
As a result, the number of nuclei is the sum of the above (2) and (3).

FIG. 4 shows a flowchart in which the image processing apparatus 11 automatically counts the number of nuclei.
Hereinafter, the detailed operation of the image processing apparatus 11 will be described with reference to FIG. When the sample is set, the number m of cell nuclei (hereinafter referred to as a count value) is set to an initial value 0 (step 301). Next, data corresponding to the sample is taken out from the database of the memory 13, and upper and lower thresholds Smax and Smin for the nucleus are set (step 302). An image is acquired with a camera (step 303), and independent regions 1 to N are labeled (step 304). Next, the areas Sn (n = 1 to N) are calculated for the independent regions 1 to N (step 305). Area 1 is selected by setting n = 1 (step 306), n is compared with the number N of independent areas (step 307), and if n is larger than the number N of independent areas, the process ends (step 313). If n is not larger than the number N of independent regions, the area S1 of the independent region 1 is compared with the lower threshold Smin (step 308), and if the area S1 of the independent region 1 is smaller than the lower threshold Smin, it is regarded as noise. Instead of adding to the numerical value m, n = n + 1 = 2 is set (step 312), and the next region 2 is counted. When the area S1 of the independent region 1 is not smaller than the lower limit threshold value Smin, the area S1 of the independent region 1 is compared with the upper limit threshold value Smax (step 309). When the area S1 of the independent region 1 is larger than the upper limit threshold Smax, m = m + 2 = 0 + 2 = 2 is set (step 310), and when the area S1 of the independent region is not larger than the upper limit threshold Smax (step 309), m = m + 1 = 0 + 1 = 1 (step 311). Thereafter, n = n + 1 = 2 is set (step 312), and the process shifts to the counting of the independent region 2, and the steps of 307 and below are repeated for the regions 2 to N to obtain the final nucleus count value m.

  According to the drug discovery screening device as described above, information on the area of the sample to be used in advance is stored in a memory and stored in a memory, and an upper and lower limit threshold is set in advance for the area of the sample, and an actually acquired image is obtained. By applying the value, it is possible to realize a drug discovery screening apparatus that can eliminate variations due to differences in operators and count the number of measurement objects with high reproducibility.

  In addition, since the upper and lower thresholds corresponding to the sample can be automatically set, the number of measurement objects can be counted efficiently.

  In the above embodiment, the area is used as the size of the sample.

  Further, the image to be counted is not limited to the confocal image, and the fluorescence image when the Nipo disk 4 is omitted may be counted.

In addition, in the case where a plurality of nuclei are in contact or overlap with each other in a nucleus region larger than the upper limit threshold in FIG. 3, a composite upper limit threshold that is a multiple of the upper limit threshold is provided, and the nuclei are counted more finely as follows. May be.
In the region from the upper limit threshold to twice the upper limit threshold, the number of nuclei is set to be twice the actual number (frequency).
In the region from 2 to 3 times the upper threshold, the number of nuclei is 3 times the actual number (frequency).
・ ・ ・ ・ ・ ・
In the region from n times to n + 1 times the upper threshold, the number of nuclei is set to n + 1 times the actually measured value (frequency).
Moreover, although the case where the number of nuclei was counted was shown in said Example, you may count a cell and others.

  The present invention is not limited to the above-described embodiments and modifications, and includes many changes and modifications without departing from the essence thereof.

It is a block diagram showing the configuration of an embodiment of the drug discovery screening apparatus of the present invention. It is a conceptual diagram of the image acquired with the camera 10 of the drug discovery screening apparatus of this invention. It is a frequency distribution diagram of the size of the measurement object It is a flowchart which counts the number of nuclei with the drug discovery screening apparatus of this invention. It is a block diagram which shows the structure of the conventional drug discovery screening apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Excitation light beam 2 Micro lens array disk 3 Dichroic mirror 4 Pinhole array disk 5 Objective lens 6 Sample 7 Fluorescence signal 8 Connecting member 9 Relay lens 10 Camera 12 Center of rotation axis 13 Memory 60 Well plate 100 Confocal scanner 200 Microscope

Claims (13)

In the drug discovery screening apparatus that irradiates the sample placed on the well plate with excitation light and counts the number of measurement objects present in the image based on the fluorescence signal from the sample,
A drug discovery screening apparatus comprising a memory in which upper and lower thresholds of the size of the measurement target are stored in advance. The number of the measurement objects is counted by adding the number of independent regions within the upper and lower thresholds and twice the number of independent regions larger than the upper threshold. Drug screening device. The memory is prestored with a composite upper limit threshold of a plurality of the measurement objects that are in contact with or overlapping, and the number of the measurement objects is counted by combining the upper and lower limit thresholds and the composite upper limit threshold. The drug discovery screening device according to claim 1.   4. The drug discovery screening device according to claim 3, wherein the combined upper limit threshold is set to the multiple of the upper limit threshold. The confocal image is extracted by irradiating the sample with the excitation light at each focal position by changing a focal position in an optical axis direction using a Nipo type confocal scanner. Item 4. A drug discovery screening device according to Item 4.   The drug discovery screening apparatus according to claim 1, wherein the measurement target is a cell or a cell nucleus. In the drug discovery screening method of irradiating the sample placed on the well plate with excitation light and counting the number of measurement objects present in the image based on the fluorescence signal from the sample,
A drug discovery screening method, wherein upper and lower thresholds of the size of the measurement target are stored in a memory in advance. The number of the measurement objects is counted by adding the number of independent areas within the upper and lower thresholds and twice the number of independent areas larger than the upper threshold. Drug screening method. The memory is prestored with a composite upper limit threshold value of a plurality of the measurement objects that are in contact with or overlapping, and the number of the measurement objects is counted by combining the upper and lower limit threshold values and the composite upper limit threshold value. The drug discovery screening method according to claim 7.   The drug discovery screening method according to claim 9, wherein the combined upper limit threshold is set to the multiple of the upper limit threshold. 8. A confocal image is extracted by irradiating the sample with the excitation light at each focal position by changing a focal position in an optical axis direction using a Nipo type confocal scanner. Item 10. A drug discovery screening method according to Item 10.   The drug discovery screening method according to claim 7, wherein the measurement object is a cell or a cell nucleus.   The drug discovery screening apparatus or drug discovery screening method according to claim 1, wherein the size is an area.

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