JP2008185432A - Drug discovery screening device - Google Patents

Abstract

A drug discovery screening apparatus capable of selectively obtaining both a fluorescent image and a confocal image is realized with a simple configuration.
In a drug discovery screening apparatus for performing drug discovery screening based on fluorescence information generated from a sample by irradiating the sample placed on the well plate with excitation light, excitation for generating fluorescence in the sample An excitation light source that outputs light, a confocal scanner that converts the excitation light into an excitation light beam composed of a plurality of beam spots for scanning the sample, and the sample is irradiated with the excitation light beam supplied from the confocal scanner A microscope for observing the fluorescence, an optical image detection means for reading the confocal scanner or the confocal image or fluorescence image obtained through the microscope, and the fluorescence returned from the microscope in front of the confocal scanner. And an optical path switching unit that selectively bypasses and enters the optical image detection unit.
[Selection] Figure 1

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 the fluorescence image generated from the excited sample with a microscope system. The camera is taking a magnified image. Further, in order to acquire fluorescent images from all wells, the well plate or the objective lens is moved on the XY stage.

  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 (fluorescence information). In order to improve the image quality, a confocal scanner (Nipou disk) is installed between the microscope and the camera.

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

Japanese Patent Laying-Open No. 2005-098722 JP 2005-095012 A JP 2005-102629 A

  FIG. 3 is a block diagram showing an example of a conventional drug discovery screening apparatus described in Patent Document 1, and shows an example of a Niipou disc type confocal scanner used with a microscope.

  In FIG. 3, the confocal scanner 100 is connected to a microscope 200, and excitation light output from an excitation light source (not shown) is converted into a plurality of beam spots by a microlens array disk (hereinafter referred to as ML disk) 2. After passing through the dichroic mirror 3, it passes through individual pinholes of a pinhole array disk (hereinafter referred to as “Nipou disk”) 4, and the wells 60 of the well plate 60 as excitation light beams 1 (broken lines) by the objective lens 5 of the microscope 200 The sample 6 placed on is irradiated.

  Each sample 6 of the well plate 60 has a fluorescent reagent added thereto, and the fluorescence 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 fluorescent light 7 that has passed through each pinhole is reflected by the dichroic mirror 3, and enters the two-dimensional sensors (cameras) 10a and 10b via the relay lens 9, the dichroic mirror 30, and the bandpass filters 20a and 20b. Here, the dichroic mirror 3 is designed to transmit the excitation light beam 1 and reflect the fluorescence 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. For this reason, a plurality of beam spots (excitation light beams) formed by the ML disk 2 and the Niipou disk 4 scan the surface of the sample 6 according to the rotation thereof.

  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, the 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 by rotating the ML disk 2 and the Niipou disk 4 at high speed. As shown in the drawing, it is possible to capture a confocal image of all the samples at high speed while moving the well plate 60 in which a large number of samples to be inspected are arranged in a matrix relative to the microscope 200 and the confocal scanner 100. It becomes.

  Furthermore, by displacing the objective lens 5 in the optical axis direction, the observation position of the confocal image (the depth of focus with respect to the sample 6) can be changed, and a plurality of slice images can be obtained for one sample 6. .

  In addition, in the optical image detection unit 300 connected to the confocal image extraction port of the confocal scanner 100, the fluorescence 7 reflected by the dichroic mirror 3 reaches the second dichroic mirror 30. Since the dichroic mirror 30 can have arbitrary spectral characteristics, the fluorescence of a predetermined wavelength component passes through the dichroic mirror 30 and reaches the two-dimensional sensor 10a via the bandpass filter 20a. On the other hand, the fluorescence of other wavelength components is reflected and reaches the two-dimensional sensor 10b via the bandpass filter 20b.

As described above, according to the optical image detection means 300, the confocal image can be split into a plurality of colors, and the confocal image arbitrarily wavelength-separated can be captured at high speed with respect to the total number of samples 6. It becomes.
Such confocal scanners are used in screening methods for new drug development.

  By the way, in applications in drug discovery screening, fluorescent images are suitable for translocation to analyze the transition from the cell membrane to the cytoplasm and calcium signal, and when high image quality such as neurite outgrowth measurement is required, Confocal images are suitable.

  Therefore, in order to obtain a fluorescence image and a confocal image in the configuration of the prior art of FIG. 3, the confocal scanner 100 (ML disk 2 and Niipou disk 4) is shifted laterally and the fluorescence is reflected by the dichroic mirror 3. However, these optical components are precisely positioned, and it is not easy to move the confocal scanner 100.

  The object of the present invention is to realize a drug discovery screening apparatus capable of selectively obtaining both a fluorescent image and a confocal image with a simple configuration, eliminating the drawbacks of the conventional apparatus as described above. .

  The drug discovery screening device of the present invention is the drug discovery screening device according to claim 1, wherein the sample placed on the well plate is irradiated with excitation light and drug discovery screening is performed based on fluorescence information generated from the sample. An excitation light source that outputs excitation light for generating fluorescence in the sample, a confocal scanner that converts the excitation light into an excitation light beam that includes a plurality of beam spots for scanning the sample, and the confocal scanner A microscope for irradiating the sample with an excitation light beam supplied from the microscope and observing the fluorescence thereof, an optical image detecting means for reading a confocal image or a fluorescent image obtained via the confocal scanner or the microscope, and returning from the microscope Optical path switching means for selectively bypassing the incident fluorescence in front of the confocal scanner and entering the optical image detection means. Characterized in that it Bei.

  According to a second aspect of the present invention, in the drug discovery screening apparatus according to the first aspect, the optical path switching unit selectively selects a dichroic mirror that transmits only the wavelength component of the excitation light between the confocal scanner and the microscope. It is characterized by inserting.

  According to a third aspect of the present invention, in the drug discovery screening apparatus according to the second aspect, the optical path switching unit includes a moving unit that moves the dichroic mirror on the optical axis of the fluorescence.

  According to a fourth aspect of the present invention, in the drug discovery screening apparatus according to the third aspect, the moving means includes a slider that holds the dichroic mirror and a motor that moves the slider.

  The drug discovery screening device according to any one of claims 2 to 4, wherein the dichroic mirror has a spectral characteristic equal to that of a dichroic mirror used in the confocal scanner. .

  According to the drug discovery screening apparatus of the present invention, the optical path switching means is provided between the microscope and the confocal scanner, and the fluorescence returned from the microscope is selectively bypassed in front of the confocal scanner, to the optical image observation means. Since the light is incident, the fluorescence image and the confocal image can be selectively obtained by a simple switching operation, and images suitable for various drug discovery applications can be easily obtained.

  Also, since the spectral characteristics of the dichroic mirror used for the optical path switching means are aligned with the spectral characteristics of the dichroic mirror used in the confocal scanner, it is common for both fluorescence image observation and confocal image observation. Excitation light sources can be used.

  Furthermore, since a pinhole array with a microlens is used for the nipou disk constituting the confocal scanner, 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 the drawings.

  FIG. 1 is a block diagram showing an embodiment of the drug discovery screening apparatus of the present invention. In the figure, the same components as those shown in FIG. The apparatus shown in FIG. 1 differs from the conventional example in that, when observing a fluorescent image, the optical path of the fluorescent light collected by the objective lens 5 is switched so as to enter the optical image detection means 300 without passing through the confocal scanner 100. This is the point.

  Reference numeral 50 denotes an optical path switching means 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 selectively blocks the optical path of the fluorescence 7 by 90. A dichroic mirror 51a is arranged to reflect at regular intervals. The fluorescence reflected by the dichroic mirror 51a is further reflected at a right angle by the reflecting mirror 51b 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 confocal scanner 100 and the optical image detecting means 300, and the output light from the confocal scanner 100 is selectively blocked in this portion and collected by the relay lens 9c. A reflection mirror 51c for allowing the emitted fluorescence to enter the optical image detection means 300 is disposed. Although not shown in the figure, the optical path switching means 50 has a moving means composed of, for example, an electric slider and a motor. The dichroic mirror 51a and the reflecting mirror 51c are held on the electric slider, and the optical path is driven by the motor. The insertion position inside is controlled.

  Here, the position indicated by the solid line in the figure is the position when the fluorescent image is observed. A position indicated by a broken line is a position at the time of confocal image observation.

  In the above configuration, at the time of confocal image observation, the dichroic mirror 51a and the reflection mirror 51c of the optical path switching unit 50 are moved to the positions indicated by broken lines in the drawing.

  That is, the fluorescence emitted from the sample 6 is incident on the confocal scanner 100 without being reflected by the dichroic mirror 51a, reflected by the dichroic mirror 3 in the confocal scanner 100, and then blocked by the reflecting mirror 51c. Without incident on the optical image detection means 300.

  Therefore, the optical system at this time is the same as that of the apparatus shown in FIG. 3 described above, and the optical image detection means 300 can obtain a confocal image of the sample 6 that is appropriately dispersed into a plurality of colors.

  Next, at the time of fluorescent image observation, the dichroic mirror 51a and the reflection mirror 51c of the optical path switching means 50 are moved to the positions indicated by solid lines in the drawing by moving means (not shown) such as an electric slider or motor.

  Here, the dichroic mirror 51a has the same spectral characteristics as the dichroic mirror 3 in the confocal scanner 100, and FIG. 2 shows an example of the spectral characteristics.

  Assuming that the excitation light beam 1a has three wavelengths of 405 nm, 488 nm, and 635 nm, the spectral characteristics are selected so that the dichroic mirrors 3 and 51a also transmit these wavelength components.

  Accordingly, the excitation light output from the excitation light source is supplied to the microscope 200 as the excitation light beam 1a via the confocal scanner 100 (ML disk 2, dichroic mirror 3, and nipou disk 4) and the dichroic mirror 51a. The sample 6 is irradiated.

  At this time, the confocal scanner 100 (ML disk 2 and Nipou disk 4) operates (rotates) in the same manner as when confocal images are observed, and the excitation light beam 1a is irradiated onto the sample 6 as a plurality of beam spots. Is scanned and excited.

  As described above, in a state where the dichroic mirror 51a is inserted between the confocal scanner 100 and the microscope 200, the fluorescence emitted from the sample 6 is reflected by the dichroic mirror 51a, and the optical image is transmitted through the reflection mirrors 51b and 51c. Incident on detection means 300.

  For this reason, the optical image detection means 300 can obtain a fluorescent image without using the confocal scanner 100. In the optical image detection means 300, as in the case of confocal image observation, the fluorescent image is appropriately divided into a plurality of colors and photographed as color-specific fluorescent images by the cameras 10a and 10b.

  Note that when observing a fluorescent image, a plurality of images (slice images) are not observed with respect to one sample 6, and therefore the position of the objective lens 5 in the optical axis direction is fixed at an arbitrary position. . For example, when the focus is manually adjusted at the start of fluorescence image observation, the focus state at that time is maintained thereafter.

  As described above, according to the drug discovery screening apparatus of the present invention, the optical path switching means is provided between the microscope and the confocal scanner, and the fluorescence returned from the microscope is selectively bypassed in front of the confocal scanner. Since it is made incident on the optical image observation means, a fluorescence image and a confocal image can be selectively obtained by a simple switching operation, and images suitable for various drug discovery applications can be easily obtained. Can do.

  Also, since the spectral characteristics of the dichroic mirror used for the optical path switching means are aligned with the spectral characteristics of the dichroic mirror used in the confocal scanner, it is common for both fluorescence image observation and confocal image observation. Excitation light sources can be used.

  In the above description, the optical image detection unit that divides the image into a plurality of colors and captures the image as a color-specific image is exemplified as the fluorescence image and confocal image observation unit. It is not limited to.

  Further, the configuration of the confocal scanner is not limited to the illustrated one.

It is a block diagram which shows one Example of the drug discovery screening apparatus of this invention. It is a characteristic view which shows an example of the spectral characteristic in the dichroic mirrors 3 and 51a. It is a block diagram which shows an example of the conventional drug discovery screening apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 1a Excitation light beam 2 Micro lens array disk 3, 30 Dichroic mirror 4 Pinhole array disk 5 Objective lens 6 Sample 7 Fluorescence 8 Connecting member 9a-9e Relay lens 10a, 10b Camera 11 Rotation center axis 20a, 20b Band pass filter 50 Optical path switching means 51a Dichroic mirror 51b, 51c Reflection mirror 60 Well plate 100 Confocal scanner 200 Microscope 300 Optical image detection means

Claims (5)

  In a drug discovery screening device that irradiates a sample placed on a well plate with excitation light and performs drug discovery screening based on fluorescence information generated from the sample, the excitation light for generating fluorescence in the sample is output. An excitation light source, a confocal scanner that converts the excitation light into an excitation light beam composed of a plurality of beam spots for scanning the sample, and the sample is irradiated with the excitation light beam supplied from the confocal scanner to emit fluorescence. A microscope for observation, an optical image detection means for reading a confocal image or a fluorescence image obtained through the confocal scanner or the microscope, and a fluorescence selectively returned from the microscope in front of the confocal scanner And an optical path switching means for entering the optical image detection means.   The drug discovery screening apparatus according to claim 1, wherein the optical path switching unit selectively inserts a dichroic mirror that transmits only a wavelength component of excitation light between the confocal scanner and the microscope.   The drug discovery screening apparatus according to claim 2, wherein the optical path switching means includes a moving means for moving the dichroic mirror on the optical axis of the fluorescence.   The drug discovery screening apparatus according to claim 3, wherein the moving means includes a slider that holds the dichroic mirror and a motor that moves the slider.   The drug discovery screening apparatus according to any one of claims 2 to 4, wherein the dichroic mirror has a spectral characteristic equal to that of a dichroic mirror used in the confocal scanner.

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