JP4110473B2 - Screening method and screening apparatus - Google Patents

Description

  The present invention relates to a screening method and apparatus used in drug discovery or drug discovery for developing new types of pharmaceuticals.

As this kind of screening apparatus, for example, an apparatus for screening cells treated with a fluorescent reagent or the like is well known (for example, Patent Document 1).
FIG. 3 is a configuration diagram of the screening apparatus described in Patent Document 1. This device is a Zeiss Axiovert inverted fluorescent that uses a standard objective lens with a magnification of 1-100 times for the camera and a light source with a power source 2 (eg, 100 W mercury-arc lamp or 75 W xenon lamp). An inverted fluorescent microscope 1 such as a microscope is used. There is an XY stage 3 for moving the plate 4 in the XY directions on the microscope objective lens.

Plate 4 is a standard 96 well plate. Cells are injected into each well and reagents are added. After the plate 4 is placed on the microscope assembly 1, the plate 4 is moved in the XY direction by the XY stage 3, and the fluorescence images emitted from the cells in each well are received by the camera 7. The output signal of the camera is input to the central processing unit 10, processed, and displayed on the display 12 in a display format corresponding to the fluorescent image. Further, a hard copy record can be printed on the printer 13.
In this way, the effect of the reagent on the cells can be detected.

  The Z-axis focus drive mechanism 5 is a Z-axis direction moving mechanism for adjusting the focus of the objective lens. The joystick 6 is for manually moving the stage in the XYZ directions. In addition, a power supply 8 for the camera and an automatic controller 9 are provided.

Special Table 2002-525603

However, such a screening apparatus has the following problems.
(1) With an optical fluorescence microscope, it is not possible to obtain an image in which all the portions from the top to the bottom of the cell are in focus at the same time, and only a blurred image with a defocused image can be obtained. Therefore, it is not possible to obtain a clean and accurate image with good signal-to-noise ratio that can grasp the morphology and activity (ADMETOX: absorption, distribution, metabolism, excretion and toxicity) throughout the cell. .
(2) The thermal energy of the light source is strong and adversely affects cells.
(3) Prior to observation, it is necessary to adjust the focal position of the objective lens.

The object of the present invention is to solve such problems, and by using a Nipo type confocal scanner and observing an image of the cell while changing the focal position of the objective lens in the direction of the optical axis, It is an object of the present invention to provide a screening method and apparatus capable of obtaining an image as a high-speed, beautiful and accurate (good SN ratio) image.
Another object of the present invention is to provide a screening apparatus in which the irradiation light quantity of excitation light is reduced so that the adverse effect of excitation light does not occur on cells.

In order to achieve such a subject, in the invention according to claim 1 of the present invention,
Preparing a plate in which a reagent and fluorescently labeled cells are injected into a plurality of wells, irradiating excitation light to the well to receive fluorescence generated from the cells, and obtaining a fluorescent image of the cells; An image processing unit that performs appropriate processing on the fluorescence image signal from the sensor unit, and a display unit that displays the output of the image processing unit, and a screening device that observes cell dynamics and the like,
The sensor unit includes a Niipou confocal scanner, an objective lens, a focal position variable means comprising a multi-head that has different focal positions and moves the focal position of the objective lens by switching, and an output of the confocal scanner A camera that captures an image, the focal position of the objective lens is moved in the optical axis direction by the focal position changing means, and at each focal position, the cell is irradiated with excitation light from the confocal scanner through the objective lens; A plurality of slice images differing in the optical axis direction are obtained.

As described above, the present invention has the following effects.
(1) A Nipo-type confocal scanner is used, and a sample image is observed by changing the focal position of the objective lens in the direction of the optical axis. Therefore, a clean and accurate (good SN ratio) image is obtained at high speed. be able to.
(2) The thermal energy of excitation light is weaker than in the past, and observation is possible without adversely affecting cells.
(3) There is no need to adjust the focal position of the objective lens prior to observation as in the prior art.

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing an embodiment of a screening apparatus according to the present invention. In FIG. 1, 100 is a sensor unit, 200 is a mechanism control unit, 300 is an image processing unit, 400 is a plate (corresponding to plate 4 in FIG. 3), 500 is a central processing unit, and 600 is a display unit.

The sensor unit 100 includes a laser light source 110, a Nipo type confocal scanner 120, an objective lens 130, a focal position varying unit 140, and a camera 150.
Laser light as excitation light generated from the laser light source 110 passes through the Nipo type confocal scanner 120 and is focused on the sample 401 of the plate 400 by the objective lens 130. Fluorescence from the sample excited and emitted by the laser light returns to the confocal scanner 120 via the objective lens 130 and is input to the camera 150. The camera 150 obtains a fluorescent image of the sample 401 on the XY plane. In this case, since a Nipo type confocal scanner is used, a high-speed, clean and accurate image (with a good SN) can be obtained.

The Niipou confocal scanner 120 is configured as shown in FIG. 2, for example. However, in FIG. 2, the relationship between the upside and the downside of FIG. 1 is shown, and the objective lens 130 and the light receiving element 151 of the camera 150 are also shown.
In FIG. 2, a laser 121 that is excitation light is condensed into individual light beams by each microlens 123 disposed on the microlens disk 122, passes through a dichroic mirror 124, and then enters a pinhole disk (also referred to as a “Nipou disk”) 125. The light passes through the individual pinholes 126 and is focused on the sample 401 by the objective lens 130.

The fluorescence generated from the sample 401 passes through the objective lens 130 again and is condensed on each pinhole of the pinhole disk 125. The fluorescence that has passed through each pinhole 126 is reflected by the dichroic mirror 124, and a fluorescent image is formed on the light receiving element 151 of the camera by the relay lens 129.
The dichroic mirror 124 used here is designed to transmit the excitation light 121 and reflect the fluorescence from the sample 401.

  The microlens disk 122 and the pinhole disk 125 are mechanically connected by a member 127 and rotate integrally around the rotation shaft 128. The individual microlenses 123 and the pinholes 126 are arranged so that the excitation light from the individual pinholes 126 formed on the pinhole disk 125 scans the observation plane of the sample 401. In addition, since the plane in which the pinholes 126 are arranged, the observation plane of the sample 401, and the light receiving element 151 of the camera are arranged in an optically conjugate relationship with each other, the optical of the sample 401 is placed on the light receiving element 151. A cross-sectional image, that is, a confocal image is formed.

  Returning to FIG. 1, the focal position changing unit 140 drives the objective lens 130 or the entire sensor unit 100 to move the focal position of the objective lens 130 continuously or discontinuously in the Z-axis direction (optical axis direction). (Hereinafter also referred to as scanning). For example, a piezo actuator (simply referred to as an actuator) is used as the focal position changing means. Hereinafter, an actuator will be described as an example.

  The mechanism control unit 200 places a substrate stage (not shown) on which the plate 400 is placed at a predetermined position with respect to the sensor unit 100 and appropriately moves the substrate stage in the XYZ directions in order to sequentially observe each well. XYZ direction driving and XY direction driving for adjusting the position of the sensor unit in the XY direction are performed.

The image processing unit 300 receives an image signal from the camera 150 and performs appropriate image processing and data processing for representing cell activity and the like. The processing includes, for example, processing for performing statistical analysis such as cell fluorescence intensity and kinetics, and histograms and correlation diagrams.

Unlike the conventional apparatus, this apparatus can obtain a high-speed, clean and accurate image with a good S / N ratio, so that the dynamics of each cell can be easily obtained accurately.
The processed image is displayed on the display unit 600 by the central processing unit 500.
The central processing unit 500 also appropriately controls the mechanism control unit 200, the actuator 140, and the image processing unit 300.

The operation in such a configuration will be described next. Each well of the plate 400 is pre-injected with a sample, ie, a live cell and a reagent that are fluorescently labeled. The plate 400 is driven by the mechanism control unit 200 and installed at a predetermined position on the sensor unit 100.
The sample is irradiated with laser light (excitation light) from the sensor unit 100, and fluorescence is generated from the cells. This fluorescence passes through the objective lens 130 and the actuator 140 and forms an image on the confocal scanner 120. The image (cell image) is read by the camera 150.

  At this time, the objective lens 130 is continuously or discontinuously scanned in the Z-axis direction by the actuator 140. According to the Nipo type confocal scanner, a slice image of a cell can be obtained at high speed, and the camera 150 can capture a slice image of a cell at each of different cross-sectional positions from the top to the bottom in the Z-axis direction.

In the image processing unit 300, predetermined image processing is performed based on a plurality of slice images obtained by the camera 150 to obtain an image such as activity over the entire cell.
In the image processing, for example, a plurality of slice images obtained by the camera 150 by three-dimensional data processing are used as one two-dimensional image (superimposed image), or the image is color-coded according to the difference in the amount of fluorescence generated. It also performs processing such as. The image processing can also include a Hough conversion process.

The image processed by the image processing unit 300 is stored in a storage unit (not shown) as necessary, and is displayed on the display unit 600 by the central processing unit 500.
In this way, it is possible to observe a clean and accurate image with a good S / N ratio that cannot be obtained by a conventional apparatus using a fluorescence microscope.

The present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.
For example, in the embodiment, the three-dimensional objective lens 130 is scanned in the Z-axis direction by the piezo actuator 140, but three-dimensional processing may be performed using a multihead such as far , middle, and near.
Alternatively, the objective lens 130 is not driven by a piezo actuator, but a voltage-controlled variable focus lens is inserted between the objective lens 130 and the confocal scanner 120, and the focal position of the objective lens on the sample is controlled by voltage control. You may make it move.

  In the embodiment, excitation is performed by laser light. However, two-photon absorption may be used, infrared light may be irradiated as excitation light, and two-photon absorption may be induced to generate fluorescence from the sample. . According to this, the cells need only be irradiated with infrared light with little light energy, and the influence of light on the cells can be almost eliminated.

Alternatively, the cells may be stained with quantum dots, excited with a short-wavelength laser beam, and the filter of the camera may have spectral characteristics having an identification function so that an image of the sample is captured by the camera. Thereby, sensitivity and identification ability can be improved easily.
Further, in a Nipo type confocal scanner, the output image may be detected using a line sensor. According to this, simultaneity and further high resolution can be achieved simultaneously. In this case, a multi-color line sensor may be used as the line sensor.

  The image used in the image processing unit 300 is obtained by exposing image information of a cross section of a sample (cell) obtained by scanning the objective lens by a necessary depth in the optical axis direction within the same frame period of the camera. Alternatively, an image (an image with a long focal depth corresponding to the scanned thickness, which is referred to as a long focal image) may be used.

It is a block diagram which shows one Example of the screening apparatus based on this invention. It is an example figure of a confocal scanner. It is a block diagram which shows an example of the conventional screening apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Sensor part 110 Laser light source 120 Confocal scanner 121 Laser 122 Micro lens disk 123 Micro lens 124 Dichroic mirror 125 Pin hole disk 126 Pin hole 127 Member 128 Rotating shaft 129 Relay lens 130 Objective lens 140 Actuator 150 Camera 151 Light receiving element 200 Mechanism control Section 300 Image processing section 400 Plate 401 Sample 500 Central processing unit 600 Display section

Claims (5)

Preparing a plate in which a reagent and fluorescently labeled cells are injected into a plurality of wells, irradiating excitation light to the well to receive fluorescence generated from the cells, and obtaining a fluorescent image of the cells; An image processing unit that performs appropriate processing on the fluorescence image signal from the sensor unit, and a display unit that displays the output of the image processing unit, and a screening device that observes cell dynamics and the like,
The sensor unit includes a Niipou confocal scanner, an objective lens, a focal position variable means comprising a multi-head that has different focal positions and moves the focal position of the objective lens by switching, and an output of the confocal scanner A camera that captures an image, the focal position of the objective lens is moved in the optical axis direction by the focal position changing means, and at each focal position, the cell is irradiated with excitation light from the confocal scanner through the objective lens; A screening apparatus configured to obtain a plurality of slice images different in the optical axis direction.   The screening apparatus according to claim 1, wherein the image processing unit has a function of obtaining a cell image based on the plurality of slice images or long-focus images.   The screening apparatus according to claim 1, wherein the sensor unit uses infrared light as excitation light and induces two-photon absorption to generate fluorescence from the cells. Cells injected into the well are pre-stained with quantum dots,
The sensor unit is configured to irradiate the cell with a laser beam having a short wavelength and capture an output image of the confocal scanner with a camera through a filter having a spectral characteristic having an identification function. The screening apparatus according to any one of claims 1 to 3.   The screening apparatus according to claim 1, wherein the sensor unit is configured to detect an output image of the confocal scanner using a line sensor.

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