JP2008267842A - Biological observation container and biological microscope and biological observation apparatus using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a well plate, a biological microscope, and a biological observation apparatus capable of easily acquiring an image by facilitating an autofocus operation, and a sample placed inside a container via a bottom surface 62. The biological observation container 60 observed with a biological microscope is characterized in that an antireflection film 65 is formed on the surface 63 of the bottom surface 62.
[Selection] Figure 1

Description

  The present invention relates to a biological microscope and a biological observation apparatus that perform observation based on a biological observation container such as a well plate and a fluorescent signal from a sample in the biological observation container.

  3A and 3B show a conventional well plate used in a drug discovery device or the like, in which FIG. 3A is a plan view and FIG. The well plate 6 includes a plurality of wells 61 into which samples (observation targets) are injected and a bottom surface 62 made of optical glass.

In a high content screening (HCS) device for drug discovery, cells cultured in advance are dispensed together with a culture solution into a well 61 in a well plate 6 in an appropriate number, and different concentrations, amounts, or types of reagents for each well 61 are added. Drip to prepare a test sample. Next, these samples are excited by irradiation with light, and a fluorescent image emitted from the excited sample is captured by a camera via a microscope system. At that time, in order to acquire fluorescent images from all the wells 61, the well plate is moved on the XY stage. 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 objective lens of the microscope is designed to eliminate aberrations in consideration of the thickness of the sample cover glass 0.17 mm. Therefore, in order to obtain a high-quality image, t 0.17 mm is provided on the bottom surface of the well plate 6. The one using the glass is best.

  Generally, the bottom surface 62 of the well plate 6 is not a perfect plane but has a distortion. There is also variation in the thickness of the material (glass) of the bottom surface 62. Due to such non-uniform thickness of the bottom surface, the distance between the sample in each well 61 and the objective lens of the microscope is not the same, and some wells cannot acquire images. In order to acquire an image reliably, in the drug discovery device, the objective lens of the microscope is provided with an autofocus function, and the focal position of the objective lens is adjusted according to the distortion of the bottom surface of the well plate.

  FIG. 4 is a block diagram showing a conventional example of a biological microscope used in the drug discovery screening apparatus as described above. As shown in FIG. 4, the biological microscope of this embodiment includes an optical system 1 including an observation optical system 10 and a focus error detection optical system 20, and a control circuit 3 for controlling the position of the objective lens 11. And a focus adjustment unit 4 as a focus adjustment means for adjusting the focus position of the observation optical system.

  The optical system 1 has passed through an objective lens 11 disposed in the vicinity of the sample 5, a dichroic mirror 12 as a separating unit that separates light from the sample 5 side into observation light and focus error detection light, and the dichroic mirror 12. An observation unit 13 that receives observation light, a filter 14 for preventing incidence of focus error detection light on the observation unit 13, a laser diode 21 as a light source for focus error detection, and a focus error emitted from the laser diode 21 A lens group 22 that allows detection light to pass toward the sample 5, a half mirror 23 that bends part of the focus error detection light, and a mirror 24 that reflects the focus error detection light reflected by the sample 5 and bent by the half mirror 23. A quadrant photodiode 25 as a focus error signal generating means for receiving the focus error detection light, and a quadrant photodiode It comprises a collimator lens 26 and a cylindrical lens 27 to shape the beam shape of the focus error detection light incident on 25 into a predetermined shape, a filter 28 for cutting off the observation light bent by the dichroic mirror 12, a.

  As shown in FIG. 4, the objective lens 11 can be moved in the Z direction (optical axis direction) by an actuator 16. The actuator 16 is controlled by the control circuit 3.

  Next, the operation of the biological microscope of this embodiment will be described.

  Observation light from the sample 5 enters the observation unit 13 through the objective lens 11, the dichroic mirror 12, and the filter 14, and an observation image of the sample 5 is obtained in the observation unit 13. The objective lens 11, the dichroic mirror 12, the filter 14, and the observation unit 13 constitute an observation optical system 10.

  On the other hand, the focus error detection light emitted from the laser diode 21 is bent by the dichroic mirror 12 through the lens group 22, the half mirror 23, and the filter 28, and is irradiated onto the sample 5 through the objective lens 11. The focus error detection light reflected by the sample 5 returns to the dichroic mirror 12 through the objective lens 11, is bent here, and enters the filter 28. The focus error detection light that has passed through the filter 28 is folded back by the half mirror 23 and the mirror 24 and passes through the collimator lens 26 and the cylindrical lens 27. The focus error detection light that has passed through the collimator lens 26 and the cylindrical lens 27 is received by the quadrant photodiode 25.

  The lens group 22, the half mirror 23, the filter 28, the dichroic mirror 12, the objective lens 11, the mirror 24, the four-division photodiode 25, the collimator lens 26, and the cylindrical lens 27 constitute a focus error detection optical system 20.

  The filter 28 provided in the focus error detection optical system 20 blocks the observation light from the sample 5 side that could not be removed by the dichroic mirror 12. In the present embodiment, the observation light is blocked by the filter 28, and only the focus error detection light that has passed through the filter 28 enters the quadrant photodiode 25. For this reason, the quadrant photodiode 25 can generate an accurate focus error detection signal that is not affected by the observation light. Moreover, since the sensitivity of focus error detection can be improved by eliminating the influence of the observation light, the amount of focus error detection light irradiated on the sample 5 can be reduced, and the sample 5 is a living cell. In some cases, adverse effects on the sample 5 can be prevented.

  The collimator lens 26 and the cylindrical lens 27 have different focal lengths in two directions (x direction and y direction) orthogonal to the optical axis (z axis) and orthogonal to each other, and are based on the amount of light received by the quadrant photodiode 25. It is possible to detect a focus error using the astigmatism method. As will be described later, the focus error detection optical system 20, the control circuit 3, and the like function as focusing means.

  FIG. 5 shows the projected shape of the focus error detection light irradiated to the four-divided photodiode 25, FIG. 5 (A) is the shape when focused, FIG. 5 (B) is the shape when the focus is far, FIG. 5C shows the shapes when the focal points are close to each other. When the output level of the region 25a of the photodiode 25 is “A”, the output level of the region 25b is “B”, the output level of the region 25c is “C”, and the output level of the region 25d is “D”, “(A + C)” By calculating “− (B + D)”, a focus error detection signal can be obtained. In the so-called S-curve of the so-called focus error detection signal, a focused state is obtained at a point where the signal intensity is “0”. The focus error detection signal output from the four-divided photodiode 25 is given to the control circuit 3 via the focus adjustment unit 4. Since focus error detection by the astigmatism method is a well-known technique, detailed description thereof is omitted.

  Prior art documents related to microscope autofocus technology include the following.

JP-A-5-88072

  As shown in FIG. 6, in the above-described apparatus, utilizing the reflection of light due to the difference in refractive index at the boundary surface between the back surface 64 of the bottom glass 62 of the well 61 and the culture solution 51 (substantially water), The bottom of the well (back surface 64) is detected, and the focal position of the objective lens 11 is controlled to be constant with reference to this.

  However, in the conventional well plate 6, the focus error detection light 101 for autofocus is reflected from the surface 63 of the bottom glass 62 of the well 61 (surface reflected light 102), and the intensity thereof is higher than that of the reflected light 103 on the back surface. 10 times stronger. For this reason, as shown in the focus error detection signal chart of FIG. 7, the front surface reflection signal 201 deteriorates the SN ratio of the back surface reflection signal 202, which is a necessary signal, and as a result, the autofocus operation becomes difficult. is there.

This is because the degree of reflection of light differs depending on the difference in refractive index. About 4% of the incident light is reflected at the boundary between glass and air, and about 0.4% is reflected at the boundary between glass and water. This is because in the autofocus operation, the latter 0.4% reflected light is detected to detect the bottom of the well, that is, the back surface of the glass.

  An object of the present invention is to provide a well plate, a biological microscope, and a biological observation apparatus capable of easily obtaining an image by facilitating an autofocus operation using a microscope. To do.

In order to achieve such a problem, the invention according to claim 1 of the present invention is:
In the biological observation container where the sample in the container is observed through the bottom surface,
An antireflection film is formed on the bottom surface.

The biological microscope according to the invention of claim 2 is:
The focus position of the objective lens is adjusted to a focus position based on the back surface of the bottom surface of the biological observation container according to claim 1 based on the focus error signal.

The biological observation apparatus according to the invention described in claim 3 is:
The fluorescent image emitted from the sample in the biological observation container is subjected to image processing based on an image captured by a camera via the biological microscope according to claim 2.

The invention according to claim 4
The biological observation container according to claim 1,
The well plate is a biological observation container.

  As is apparent from the above description, according to the present invention, in the biological observation container in which the sample in the container is observed through the bottom surface, the anti-reflection film is formed on the surface of the bottom surface, so It is possible to provide a biological observation container, a biological microscope, and a biological observation apparatus that can easily operate and can reliably acquire an image.

Hereinafter, the present invention will be described in detail with reference to the drawings.
FIG. 1A is a plan view showing a first embodiment of a well plate according to the present invention, and FIG. 1B is a partially cutaway sectional side view. The same parts as those in FIG. The well plate 60 constitutes a biological observation container in which the sample in the container is observed through the bottom surface. The well plate 60 in FIG. 1 differs from that in FIG. 3 in that an antireflection film 65 is coated on the surface 63 of optical glass constituting the bottom surface 62 of the well plate 60.

The operation of the well plate of FIG. 1 is shown below. In FIG. 1, by coding an antireflection film 65 on the surface 63 of the bottom glass 62, the reflectance of the surface 63 can be suppressed to 1% or less as compared with the conventional 4%. As a result, as shown in the focus error detection signal chart of FIG. 2, the reflection of the front surface 63 (signal 203) can be prevented from affecting the reflection of the back surface 64 (signal 204).

According to the well plate having the above-described structure, the surface of the bottom glass of the well plate is coated with an antireflection film to suppress the surface reflection of the focus error detection light for autofocus. ) Can be detected with high accuracy, and an image of the sample can be obtained with certainty.

  The antireflection film can be formed by any method such as coating, vapor deposition, spraying, or the like.

Moreover, the object for forming the antireflection film is not limited to the well plate, and can be applied to all biological observation containers that are observed through the bottom surface by a biological microscope, such as a cover glass and a glass bottom dish.

  In addition, by using the well plate 60 of FIG. 1 instead of the well plate 6 of the biological microscope as illustrated in FIG. 4, an autofocus operation is easy, and a biological microscope that can reliably acquire an image of the sample is obtained. Can be provided.

  Furthermore, a fluorescent image obtained by such a biological microscope is enhanced by a confocal scanner into an image with a high S / N ratio, picked up with a camera, and then subjected to image processing with an image processing device, thereby performing an autofocus operation. Therefore, it is possible to provide a biological observation apparatus that can easily acquire an image. A drug screening apparatus having similar advantages can be provided by finding a sample that is a drug candidate based on the result of image processing (referred to as drug screening).

(A) which shows the 1st Example of the well plate which concerns on this invention is a top view, (B) is a partially notched cross-sectional side view. It is a chart which shows the focus error detection signal of the well plate of FIG. A conventional well plate is shown, (A) is a plan view and (B) is a partially cutaway sectional side view. It is a block diagram which shows the example of the conventional biological microscope. It is a figure which shows the projection shape of the focus error detection light in a conventional apparatus. It is explanatory drawing which shows the reflected light of the well plate bottom glass 62 in a conventional apparatus. It is a chart which shows the focus error detection signal in a conventional apparatus.

Explanation of symbols

60 Biological observation container 65 Antireflection film 62 Bottom surface 63 Front surface 64 Back surface

Claims (4)

In the biological observation container where the sample in the container is observed through the bottom surface,
A biological observation container, wherein an antireflection film is formed on the bottom surface. A biological microscope characterized in that the focal position of the objective lens is adjusted to an in-focus position with reference to the rear surface of the bottom surface of the biological observation container according to claim 3 based on a focus error signal. A biological observation apparatus that performs image processing on a fluorescent image emitted from the sample in the biological observation container based on an image captured by a camera via the biological microscope according to claim 2.   2. The biological observation container according to claim 1, wherein the well plate is a biological observation container.

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