JP2008045982A - Drug discovery screening device - Google Patents

Abstract

Disclosed is a drug discovery screening apparatus that does not miss a sample that is a drug candidate.
An apparatus for performing drug discovery vinegar cleaning by irradiating a sample placed on a well plate with excitation light, performing image processing based on a fluorescence signal from the sample, and using a Nipow disk type confocal scanner. An optical image separation apparatus provided with return light separation means for taking out a focus image and separating return light from a sample is characterized in that an image coupling optical path based on light from a light source different from the excitation light is provided.
[Selection] Figure 1

Description

  The present invention relates to a drug discovery screening apparatus using a confocal image extraction port of a Niipou disc type confocal scanner in a confocal microscope observation system, and to a drug discovery screening apparatus capable of reliably acquiring a confocal image. It is.

  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 an optical image separation apparatus using a confocal scanner.

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

FIG. 2 is a configuration diagram showing a technique of a conventional optical image separation device described in Patent Document 1.
In FIG. 2, the confocal scanner 26 is connected to the microscope 5, and for example, the illumination parallel excitation light beam 1 (one-dot chain line) including the first fluorescence wavelength λ1 = 532 nm and the second wavelength λ2 = 647 nm is a microlens. The light is condensed into individual luminous fluxes by an 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 8 passes through individual pinholes of the Nipkow disk 4 and is placed on the well plate 7 by the objective lens 6 of the microscope. The sample is focused on each sample to excite the fluorescent reagent. The ML disk 2 and the nipou disk 4 rotate around the support shaft 25.

  In FIG. 2, reference numeral 20 denotes a spectroscopic optical unit connected to the confocal image extraction port 10 of the confocal scanner 26. The spectroscopic optical unit 20 includes a second DM 23 formed of a flat mirror as return light separating means, and bandpass filters 24a and 25b having spectral characteristics as stray light removing means.

  For example, fluorescent reagents CY3 and CY5 are added to each sample of the sample 8, and the fluorescent signal 12 emitted from each fluorescent reagent passes through the objective lens 5 again and is condensed on individual pinholes of the Nipkow disk 4. The fluorescence signals 12 that have passed through the individual pinholes are reflected by DM3, and confocal optical images of the selected wavelengths are connected to cameras 22a and 22b via relay lens 9, lens 21a, and bandpass filters 24a and 24b, respectively. Imaged.

  In the above-described configuration, the plane where the pinholes of the Niipou disc 4 are arranged, the plane to be observed on the sample 8, and the light receiving surfaces of the cameras 20a and 20b are arranged in an optically conjugate relationship with each other. An optical sectional image of the sample 8, that is, a confocal image is formed on 20a and 20b. Therefore, since the confocal image of the sample 8 can be simultaneously formed on the light receiving surfaces of the cameras 20a and 20b, the sample 8 in which a large number of specimens are arranged on the matrix is relative to the microscope and the confocal scanner. By moving to the position, it is possible to capture a confocal image obtained by performing arbitrary wavelength selective separation of all the samples at high speed.

  By the way, in the above-described conventional example, when acquiring an image, the well plate 7 is moved on an XY stage (not shown) so that the objective lens 6 is approximately at the center of each well in the well plate 7. The image of the sample 8 in the well is taken.

  However, in general, the sample (cell) does not necessarily collect in the center of the well. In particular, when the magnification of the objective lens 6 is increased, the field of view of the lens is narrowed, and cells at a location slightly away from the center of the well are not reflected on the cameras 20a and 20b. In such a case, there is a problem that an effective image used for image processing cannot be obtained and a sample that is a drug candidate is missed.

  The present invention has been made to solve the above-described problems of the prior art, and in a drug discovery screening apparatus, a camera is provided for taking a general-field distribution state of a sample (cell) in a well as a bright-field image. The purpose is to realize a drug discovery screening device that determines the range of samples (cells) from which a confocal image is taken from the distribution situation and does not miss a sample that is a drug candidate.

In the drug discovery screening device of the present invention, in claim 1,
A device that irradiates a sample placed on a well plate with excitation light and performs image processing based on a fluorescence signal from the sample to perform drug discovery vinegar cleaning, and extracts a confocal image from a Niipou disc confocal scanner An optical image separation apparatus including return light separating means for separating return light from the sample is provided with an image coupling optical path based on light from a light source different from the excitation light.

In claim 2, in the drug discovery screening device according to claim 1,
Light from a different light source incident on the image coupling optical path irradiates the sample from a direction opposite to the side irradiated with the excitation light.

In claim 3, in the drug discovery screening device according to claim 1 or 2,
The light from the different light sources incident on the image coupling optical path is transmitted through the sample, then branched by a dichroic mirror, and incident on the camera via a variable power lens.

In claim 4, in the drug discovery screening device according to claim 3,
The zoom lens magnification is a reduction system.

  According to the drug discovery screening apparatus of the present invention, a bright field optical system different from the confocal optical system is provided, and a place where a sample is appropriately present is determined based on a bright field image having a wider field of view than the confocal optical system. The location can be identified and imaged with a confocal optical system. As a result, a confocal image without failure can be obtained, the analysis accuracy of drug screening can be improved, and drug candidates can be reliably captured.

  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 bright field illumination is provided on the opposite side to which the excitation light beam is irradiated, and this bright field illumination observes the light transmitted through the sample to grasp the distribution state of the sample.

  As shown in FIG. 1, a sample 8 in one of the arrayed wells (holes) in the well plate 7 is irradiated with bright field illumination light 33 emitted from a halogen lamp (not shown). The irradiated sample 8 forms a bright field image by the objective lens 6 of the microscope 5. This image is formed again on the camera 22c by the variable power lens 34 disposed in the lens holder.

The magnification of the variable power lens 34 is a reduction system. Thus, the camera 22c becomes a bright field image obtained by imaging a wide range of the sample.
In order to separate it from the optical path of the confocal image described later, the optical path of the bright field image is reflected using the dichroic mirror 36 in the connecting member 37 disposed in the optical path. The wavelength characteristic of the dichroic mirror 36 is preferably a characteristic that reflects light having a long wavelength and transmits light having a short wavelength.

  As an example, if a dichroic mirror that reflects light having a wavelength of 680 nm or more is used, the fluorescence signal from the sample 8 is transmitted without passing through the dichroic mirror 36, and is configured by the confocal scanner 13 and the spectroscopic optical unit 20. Reach the focal optics.

  On the other hand, in the optical path for obtaining the confocal image, when excited with the excitation light beam 1 having a specific wavelength, the sample 8 emits a fluorescence signal 12 having a wavelength longer than that of the excitation light. The fluorescence signal is collected by the microscope 5 having the objective lens 6 and forms an image on the camera 22a and the camera 22b via the confocal scanner 26 and the spectroscopic optical unit 20. The spectroscopic optical unit 20 is an apparatus used to obtain a multi-wavelength image, and is unnecessary when the fluorescence signal has a single wavelength.

  In the present invention, the location of the well plate 1 is determined based on the bright field image so that a place where a sample (cell) is present in an appropriate quantity is identified and the sample can be imaged with a confocal optical system camera. Tweak the.

  As described above, according to the drug discovery screening apparatus of the present invention, a bright field optical system different from the confocal optical system is provided, and a sample is obtained based on a bright field image having a wider field of view than the confocal optical system. It is possible to identify the appropriate location. As a result, a confocal image without failure can be obtained, the analysis accuracy of drug screening can be improved, and drug candidates can be reliably captured.

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 3,23,36 Dichroic mirror 4 Pinhole array disk 5 Microscope 6 Objective lens 7 Well plate 8 Sample 9 Relay lens 10 Confocal image extraction port 12 Fluorescence signal 16 Lens actuator 20 Spectroscopic optical unit 21 Lens 22 Camera 23 Half mirror 24 Band pass filter 25 Support shaft 26 Confocal scanner 33 Bright field illumination 34 Variable lens 35 Lens holder 37 Connecting member

Claims (4)

  A device that irradiates a sample placed on a well plate with excitation light and performs image processing based on a fluorescence signal from the sample to perform drug discovery vinegar cleaning, and extracts a confocal image from a Niipou disc confocal scanner An optical image separation apparatus comprising return light separation means for separating return light from a sample, and further comprising an image coupling optical path based on light from a light source different from the excitation light.   The drug screening apparatus according to claim 1, wherein light from a different light source incident on the image coupling optical path irradiates the sample from a direction opposite to the side irradiated with the excitation light.   3. The structure according to claim 1, wherein light from different light sources incident on the image coupling optical path is transmitted through a sample, then branched by a dichroic mirror, and incident on a camera via a variable power lens. Drug discovery screening equipment.   The drug discovery screening apparatus according to claim 3, wherein the zoom lens magnification is a reduction system.

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