JP2008064671A - Drug discovery screening system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug discovery screening system capable of acquiring confocal images with a high S/N ratio and improving precision in image processing. <P>SOLUTION: The system screens drug discovery by irradiating a sample mounted to a well plate with excited light and processing images on the basis of fluorescence signals from the sample. The system comprises both a fluorescence image acquisition means for acquiring fluorescence signals from the sample as fluorescence images and a confocal image acquisition means for acquiring fluorescence signals from the sample as confocal images and includes an optical path switching means for acquiring either the fluorescence images or the confocal images. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug discovery screening apparatus using a fluorescence microscope system, and relates to a drug discovery screening apparatus for acquiring both a confocal image and a fluorescence image.

  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 an optical image separation device using a confocal scanner as such a drug discovery screening device.

JP 2000-062480 A JP 2005-095012 A Japanese Patent Laying-Open No. 2005-098722

FIG. 2 is a block diagram showing the technology of the conventional optical image separation apparatus described in Patent Document 1, and shows an example of a Niipou disc type confocal scanner used with a microscope.
In FIG. 2, the confocal scanner 100 is connected to a microscope 200, and an excitation light beam 1 (dotted line) is condensed into individual light beams by a microlens array disk (hereinafter referred to as an ML disk) 2, and a dichroic mirror (hereinafter referred to as a dichroic mirror). , DM, and after passing through each pinhole of a pinhole array disk (hereinafter referred to as “Nipou disk”) 4, it is placed in the well hole of the well plate 60 by the objective lens 5 of the microscope 200. The sample 6 is condensed.

  Each sample 6 of the well plate 60 is added with a fluorescent reagent, and the fluorescent signal 7 (solid line) emitted from each fluorescent reagent passes through the objective lens 5 again and is condensed on each pinhole of the Niipou disc 4. .

  The fluorescence signal 7 that has passed through each pinhole is reflected by the DM 3, passes through the relay lens 9, and forms an image on the two-dimensional sensors 10 a and 10 b (camera) via the barrier filters 20 a and 20 b. The light is emitted from a confocal image extraction port (not shown). The DM 3 is designed to transmit the excitation light beam 1 and reflect the desired fluorescence signal 7.

  The ML disk 2 and the nipou disk 4 rotate around the rotation center axis 11 in a state where they are mechanically connected to each other by a connecting member 8. In this case, the individual microlenses and pinholes are arranged so that the individual pinholes formed on the Niipou disk 4 sweep the surface of the sample 6.

  Further, since the plane on which the pinholes of the Niipou disk 4 are arranged, the surface of the sample 6 and the light receiving surface of the two-dimensional sensor 10 are arranged in an optically conjugate relationship with each other, the two-dimensional sensor (camera) 10a. , 10b, an optical cross-sectional image of the sample 6, that is, a confocal image is formed.

  As described above, by rotating the ML disk 2 and the Niipou disk 4 at high speed, a confocal image of the sample 6 can be formed on the light receiving surfaces of the two-dimensional sensors (cameras) 10a and 10b in a short time. As shown in the figure, it is possible to capture a confocal image of all the samples at high speed while moving a sample 60 in which a large number of samples to be inspected are arranged in a matrix relative to the microscope and the confocal scanner.

  In the optical image separation device 300 connected to the confocal image extraction port, the fluorescent signal 7 reflected by the DM 3 described above reaches the second DM 30. Since the DM 30 has spectral characteristics, a signal having a predetermined wavelength passes through the DM 30, passes through the barrier filter 20a having the spectral characteristics, and reaches the two-dimensional sensor 10a. On the other hand, signals of wavelengths of other fluorescent components are reflected and pass through the barrier filter 20b having spectral characteristics and reach the two-dimensional sensor 10b. The confocal optical image formed on the Nipkow disk 4 is formed on the two-dimensional sensors 10a and 10b by the relay lens 9.

As described above, since the confocal image of the sample 60 can be simultaneously formed on the light receiving surfaces of the two-dimensional sensors 10a and 10b, the sample 6 in which a large number of specimens are arranged in a matrix is used as a microscope and a confocal scanner. By moving it relatively, it becomes possible to capture a confocal image obtained by performing arbitrary wavelength selective separation of all the samples at high speed.
Such confocal scanners are used in screening methods for new drug development.

By the way, in application for drug discovery screening, fluorescent images are better for translocation to analyze the transition from the cell membrane to the cytoplasm, and calcium signals, and confocal images when high image quality is required, such as neurite outgrowth measurement. Is suitable.
Therefore, in order to obtain the fluorescence image and the confocal image with the configuration of the prior art in FIG. 2, it is considered that the confocal scanner is shifted laterally and the fluorescence image is reflected by the dichroic mirror 3. It is difficult to move. There was a problem.

In addition, conventional confocal scanners use a pinhole-only tip plate. This nipow plate has low excitation light efficiency, and an image with a high S / N ratio cannot be obtained. There was a problem.
The present invention has been made to solve the above-described problems of the prior art,
A multi-color fluorescence image and a multi-color confocal image are configured to be obtained, and a confocal image with a high S / N ratio is obtained using a pinhole array disk (Nipou plate) with a microlens. An object is to provide an apparatus with improved processing accuracy.

In the drug discovery screening device of the present invention, in claim 1,
A fluorescence image in which a fluorescence signal from a sample is acquired as a fluorescence image in an apparatus for performing drug discovery screening by irradiating a sample placed on a well plate with excitation light and performing image processing based on the fluorescence signal from the sample. It comprises an acquisition means and a confocal image acquisition means for obtaining the fluorescence signal from the sample as a confocal image, and comprises an optical path switching means for obtaining either the fluorescence image or the confocal image. To do.

In claim 2, in the drug discovery screening device according to claim 1,
A pinhole array disk with a microlens is used as a Nipo disk constituting a confocal scanner for obtaining the confocal image.

In claim 3, in the drug discovery screening device according to claim 2,
It has a mirror moving means for switching the optical path.

In claim 4, in the drug discovery screening device according to claim 3,
A slider for fixing the mirror and a motor for moving the slider are provided.

  According to the drug discovery screening apparatus of the present invention, comprising a fluorescence image acquisition means for acquiring a fluorescence signal from a sample as a fluorescence image, and a confocal image acquisition means for acquiring a fluorescence signal from the sample as a confocal image, A pinhole array disk with a microlens was used as a NiPow disk comprising an optical path switching means for obtaining either a fluorescent image or a confocal image and constituting a confocal scanner for obtaining a confocal image.

  That is, the confocal optical system and the epifluorescence optical system are provided in the drug discovery device, and the two optical systems are selectively used depending on the drug discovery application, so that images suitable for various applications can be obtained. And since the pinhole array with a micro lens was utilized when obtaining a confocal image, a confocal image with a high S / N ratio can be obtained, and the accuracy of analysis can be improved.

  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 in the conventional example shown in FIG. The difference from the conventional example is that the optical path of the fluorescence signal condensed by the objective lens is switched so as to be incident on the optical image separation device without passing through the confocal scanner.

  Reference numeral 50 denotes an optical path switch formed in an L-shape, and one piece of the L-shaped portion is inserted between the microscope 200 and the confocal scanner 100, and this portion intercepts the optical path of the fluorescent signal 7 and reflects it at 90 degrees. A reflecting mirror 51a is disposed. The reflected light reflected by the reflecting mirror 51a is further reflected at a right angle by the dichroic mirror 3c disposed at the subsequent stage and enters the relay lens 9c.

On the other hand, the other piece of the L-shaped portion is inserted between the microscope 200 and the optical image separation device 300, blocks the output light from the confocal scanner 100, and converts the light collected by the relay lens 9c into the optical image separation device 300. To enter. Although not shown in the figure, the reflecting mirrors 51a and 51c are installed on, for example, an electric slider (not shown) and moved in the direction of the optical axis or perpendicular to the optical axis by moving the slider directly using a motor (not shown). Shall be allowed to.
Here, the conventional optical path is referred to as a first optical path A, and the optical path when reflected by the reflecting mirror 51a is referred to as a second optical path B.

  In the above-described configuration, when the fluorescent signal 7 from the sample 6 is acquired as a confocal image, the reflection mirror 51a is in a state (first optical path A) out of the optical path of the fluorescent signal, and the confocal scanner is similar to the conventional one. A confocal image is acquired via 100 and the optical image separation device 300.

  Next, when a fluorescent image is to be obtained, the reflecting mirror 51a is moved on the optical axis of the fluorescent signal using an electric slider or motor (not shown) and the reflecting mirror 51c is moved on the optical axes of the relay lenses 9a and 9b. Switch to the second optical path B. In this case, the sample is excited by the epifluorescence excitation light beam 1b (one-dot chain line). The dichroic mirror 3 c in the second optical path B has a characteristic of transmitting the excitation light beam and reflecting the fluorescence from the sample 6.

  As a result, the fluorescent signal is reflected by the reflecting mirror 51a and travels in the right angle direction, reflected by the dichroic mirror 3c and traveled in the right angle direction, collected by the relay lens 9c, and further reflected by the reflecting mirror 51c and reflected by the optical image. The light enters the separation device.

In the above-described configuration, in order to obtain a confocal image for the sample 6 in one of the arrayed wells (holes) in the well plate 60, the laser light source (not shown) in which a plurality of wavelengths are mixed is emitted. Irradiation using excitation light beam 1. Further, when it is desired to obtain a fluorescent image, for example, irradiation is performed using an excitation light beam 1b emitted from a mercury lamp (not shown).
As a result, fluorescent signals 7 of a plurality of bands are emitted from the irradiated sample, and are collected by the microscope 200 to obtain a fluorescent image.

The fluorescent image is divided into one of the following two optical paths depending on various applications of drug discovery.
The first optical path A is obtained after optically processing the fluorescent image into a confocal image, and the second optical path B is for capturing the fluorescent image as it is.

  In the case of the second optical path B, the fluorescent image is reflected by the reflection mirror 51a, passes through the dichroic mirror 3c and the relay lens 9c, is reflected again by the reflection mirror 51c, and enters the optical image separation device 300. The optical image separation device 300 divides the fluorescent image into a plurality of colors (the example is two colors in the figure), and is captured by the cameras 10a and 10b as fluorescent images for each color.

  In the case of the first optical path, the reflection mirrors 51a and 51c are retracted from the optical path. At this time, the fluorescent image goes straight without being reflected by the reflecting mirror 51a, reaches the pinhole disk array in the confocal scanner 100, and passes through the pinhole disk to become a confocal image. The confocal image is reflected by the dichroic mirror 11 and enters the spectroscopic optical unit 20. The confocal image is spectrally divided into a plurality of colors by the spectroscopic optical unit and picked up by the cameras 21a and 21b as confocal images for each color.

  As described above, according to the drug discovery screening apparatus of the present invention, the fluorescence image acquisition means for acquiring the fluorescence signal from the sample as a fluorescence image, and the confocal image for acquiring the fluorescence signal from the sample as a confocal image Comprising an acquisition means, comprising an optical path switching means for obtaining either the fluorescence image or the confocal image, and a pinhole array disk with a microlens is used as the Nipo disk constituting the confocal scanner, It is possible to realize a drug discovery screening apparatus that acquires a high confocal image and increases the accuracy of image processing.

The above description merely shows a specific preferred embodiment for the purpose of explanation and illustration of the present invention.
Therefore, the present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.

It is a block diagram which shows the structure of one Example of 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 3a-3c Dichroic mirror 4 Pinhole array disk (Nipou disk)
4a Pinhole array disk with microlens 5 Objective lens 6 Sample 7 Fluorescent signal 8 Connecting member 9a-9e Relay lens 10a, 10b Camera 11 Rotation center axis 20a, 20b Barrier filter 50 Optical path switcher 51a, 51c Reflective mirror 100 Confocal scanner 200 Microscope 300 Optical image separation device

Claims (4)

  A fluorescence image in which a fluorescence signal from a sample is acquired as a fluorescence image in an apparatus for performing drug discovery screening by irradiating a sample placed on a well plate with excitation light and performing image processing based on the fluorescence signal from the sample. It comprises an acquisition means and a confocal image acquisition means for acquiring a fluorescence signal from the sample as a confocal image, and comprises an optical path switching means for obtaining either the fluorescence image or the confocal image. Drug discovery screening device.   The drug discovery screening apparatus according to claim 1, wherein a pinhole array disk with a microlens is used as a Nipo disk constituting a confocal scanner for obtaining the confocal image.   The drug discovery screening apparatus according to claim 2, further comprising a mirror moving unit that switches the optical path.   The drug discovery screening apparatus according to claim 3, further comprising a slider for fixing the mirror and a motor for moving the slider.

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