JP5070995B2 - Confocal microscope - Google Patents

Description

  The present invention relates to a confocal microscope apparatus including an automatic focusing device that holds a focal plane of an objective lens constant in an optical axis direction.

  2. Description of the Related Art Conventionally, an autofocus device that performs automatic focusing control by driving an objective lens of a microscope with an actuator is known. In this apparatus, a deviation from the focal position of the objective lens of the microscope is detected, and the objective lens is moved and focused by an actuator such as a piezoelectric element in accordance with the detection signal of the deviation (see Patent Document 1). Such an autofocus device enables automatic focusing on the surface of an observation object when performing surface observation such as a metal microscope.

However, the biological microscope has the special feature of observing observation objects such as cells placed inside the cover glass, and automatic focusing on the observation objects such as cells placed inside the cover glass is necessary. It becomes. In particular, when an oil immersion lens is used as the objective lens, light reflection does not occur at the boundary between the cover glass and oil, and focusing on the surface of the cover glass becomes impossible, which is essential.

  In response to this requirement, as a first conventional technique, reflected light is generated on the front or back surface of a cover glass on which a sample such as a cell is placed by single beam focusing light, and the reflected light is reflected using a predetermined optical system. In some cases, astigmatism is generated from the image and the focal position is detected from the change in astigmatism.

  In addition, as a second conventional technique, a focused beam is applied to the sample, the reflected light on the surface of the sample is detected by a confocal method using a pinhole near the light receiving element, and the focal plane is detected from the intensity of the reflected light There is.

  As prior art of such a confocal confocal microscope apparatus, the following patent documents are known.

JP 2002-062480 A

  In the case of the first prior art, when a single beam of focusing light is focused on the front or back surface of the cover glass, that is, when the objective lens is focused on the front or back surface of the cover glass, astigmatism is substantially reduced. This value becomes 0, and this value is a reference for the position of the objective lens when the sample is observed.

  However, when the focusing light is focused on the front or back surface of the cover glass, the beam diameter of the focusing light is focused to a minimum and is easily affected by the state of the glass surface. For example, when dust, scratches or sample are present on the glass surface of the light condensing point, or when the surface is distorted or tilted, the astigmatism is not zero but becomes unstable and the observation reference position changes.

  In the case of the second prior art, since a single beam of focused light is collected and measured on the sample surface, the problem of “unstable” occurs in the same way, so the scanning range of the focused light is lined up. The method of spreading in a shape is used. However, since the scanning range is a one-dimensional line, this is not a fundamental solution, and there is a problem that the focusing speed is lowered due to this.

  The present invention has been made to solve the above-described problems of the prior art, and an object thereof is to provide a confocal microscope apparatus having a highly stable automatic focusing function.

In order to achieve such a problem, the invention according to claim 1 of the present invention is:
In a confocal microscope apparatus consisting of a Nipo Disc confocal scanner and a fluorescence microscope,
For a plurality of illumination beams passing through the pinhole of the Nipkow disk, a detection optical system for detecting light reflected from the rear surface or the surface of the cover glass for holding a sample after pinhole passage of the Nipkow disk,
Focusing means for adjusting the focal point of the objective lens of the fluorescence microscope to a focusing position based on the back surface or the surface of the cover glass based on the amount of the reflected light obtained by the detection optical system. Features.

The invention according to claim 2
The confocal microscope apparatus according to claim 1,
As a light source of the reflected light, a focusing light source that outputs laser light having a longer wavelength than excitation light and fluorescence generated from a sample excited by the excitation light; and
Mixing means for mixing the focusing light source with the excitation light and entering the confocal scanner;
It is provided with.

The invention described in claim 3
The confocal microscope apparatus according to claim 1 or 2,
The focusing means positions the focus of the objective lens at the focus position by deepening the focus of the objective lens by a predetermined amount from the front or back surface of the cover glass.

According to the drug discovery screening apparatus of the present invention, in the confocal microscope apparatus comprising the Niipou disc type confocal scanner and the fluorescence microscope, the reflection from the back surface or the surface of the cover glass holding the sample out of the excitation light that irradiates the sample. Based on the detection optical system for detecting light after passing through the pinhole of the Niipou disc, and the amount of the reflected light obtained by this detection optical system, the focal point of the objective lens of the fluorescence microscope is based on the back surface or the surface of the cover glass. Since the influence of the shape of the back surface or the front surface of the cover glass is averaged by a plurality of irradiation beams after passing through the pinhole, the automatic focusing with high stability is provided. A confocal microscope apparatus having a function can be provided.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a configuration block diagram showing an example of a confocal microscope apparatus according to an embodiment of the present invention. The confocal scanner 100 is connected to a microscope 200, and the parallel excitation light beam L1a emitted from the laser light source 20 is condensed into a plurality of individual light beams by a microlens array disk (referred to as ML disk) 3. After passing through a first dichroic mirror (DM) 7 made up of a flat mirror having spectral characteristics, each of a plurality of light beams is collected and passes through individual pinholes of the Niipou disk 4, and is received by the objective lens 8 of the microscope 200. The sample 2 is focused on one of the samples 2 placed on a sample (cell) holder (hereinafter referred to as a holder) 1 such as a dish or a well plate, and the fluorescent reagent of the sample 2 is excited, and the fluorescence signal from the sample 2 is excited. L2 (solid line in FIG. 1) occurs. Here, each of the light beams is condensed at one point at the focal position on the sample 2, but the observation surface made up of the entire light beam has a predetermined spread. The ML disk 3 and the nipou disk 4 rotate around the rotation center axis 6 while being mechanically connected by the connecting member 5. By this rotation, the light passing through the row of pinholes scans the sample surface.

  The fluorescent signal L2 emitted from the fluorescent reagent of the sample 2 passes through the objective lens 8 and is collected on each pinhole of the Niipou disc 4. The fluorescence signal L2 that has passed through each pinhole is reflected by the DM 7 and a confocal optical image is formed on the camera 300 via the relay lens 9 and the relay lens 12. The band-pass filter 11 between the relay lenses 9 and 12 has a spectral characteristic that transmits only the fluorescence L2 from the sample 2 and blocks light of other wavelengths. The dichroic mirror 10 has a spectral characteristic that reflects only the wavelength of the excitation light L1a and transmits light of other wavelengths.

  In the above-described configuration, the observation plane on the sample 2 and the light receiving surface of the camera 300 are arranged in an optically conjugate relationship with respect to the plane on which the pinholes of the Niipou disk 4 are arranged. An optical cross-sectional image of the sample 2, that is, a confocal image is formed on. Accordingly, since a confocal image of the sample 2 can be formed on the light receiving surface of the camera 300, the holder 1 in which a large number of samples to be inspected are arranged on a matrix is relative to the microscope 200 and the confocal scanner 100. The confocal images of all the samples can be captured at a high speed.

  Hereinafter, the automatic focusing mechanism will be described. When the excitation light beam L1a (the chain line in FIG. 1) is incident on the cover glass on the bottom surface of the holder 1, it is reflected by about 4% of the incident light quantity due to the difference in refractive index between air and glass. When the reflected light L1b of the excitation light reaches the dichroic mirror 7, most of the light is transmitted but partially reflected. An example of the spectral characteristics of the dichroic mirror 7 is shown in the chart of FIG. In this example, a laser having a wavelength of 488 nm is used as excitation light, and the dichroic mirror 7 has a characteristic of transmitting 95% of the excitation light and reflecting 5%.

  The excitation light L 1 b reflected by the dichroic mirror 7 passes through the relay lens 9, is reflected by the dichroic mirror 10, passes through the band pass filter 13 and the relay lens 14, and enters the light receiver 15. The bandpass filter 13 has a characteristic of transmitting only the wavelength of the excitation light L1b and blocking other wavelengths.

  The focus adjustment unit 16 differentiates the detection signal output from the light receiver 15 and compares the differential value with a predetermined target value. The control circuit 17 outputs a control signal corresponding to the deviation signal output from the focus adjustment unit 16 (by PID calculation or the like). The lens actuator 18 is controlled by a signal output from the control circuit 17 and moves the objective lens 8 in the Z direction (optical axis direction).

In the above, the dichroic mirror 10, the bandpass filter 13, the relay lens 14, and the light receiver 15 are configured to transmit the reflected light L1b from the back surface or the front surface of the cover glass holding the sample 2 out of the excitation light L1a that irradiates the sample 2. The detection optical system 110 that detects after passing through the 4 pinholes is configured.

Further, the focus adjustment unit 16, the control circuit 17, and the lens actuator 18 focus the objective lens 8 of the fluorescence microscope 200 on the back surface or the surface of the cover glass based on the amount of the reflected light L1b obtained by the detection optical system 110. A focusing means 400 is configured to match the reference focusing position.

  FIG. 3 is an explanatory diagram showing the relationship between the focal position of the objective lens 8 and the amount of light received by the light receiver 15. When the objective lens 8 is focused on the cover glass surface 1b, the reflected light L1b is the strongest. If the focus shifts, the reflected light L1b cannot pass through the pinhole of the Niipou disc 4 due to the confocal effect, and the reflected light L1b rapidly decreases. When the focus is on the back surface 1c of the cover glass, the reflected light becomes stronger again.

  In this way, by measuring the amount of reflected light, it can be detected that the focus of the objective lens 8 is aligned with the front and back surfaces 1b or 1c of the cover glass. Therefore, the amount of reflected light is measured to detect the front surface 1b or back surface 1c of the cover glass, and the objective lens 8 is moved by a certain amount d1 or d2 from the front surface 1b or back surface 1c as shown in FIG. It can be an observation surface to be observed. By setting the target value of the adjusting unit 16 to 0, the objective lens 8 can be controlled to the control reference surface (the front glass surface 1b or the back surface 1c), so that the movement of a certain amount d1 or d2 from the reference surface is a focal point. It can be set according to a target value given by the adjustment unit 16.

  According to the confocal microscope apparatus as described above, when the in-focus reference plane is determined from the light receiver 15, a value obtained by averaging information on many positions on the cover glass front surface (back surface) can be obtained. In other words, since the reflected light from the entire area of the observation surface is used instead of a single beam, the influence of the shape of the surface (back surface) of the cover glass is averaged, so the presence of dust, scratches and samples on the front and back surfaces This makes it less susceptible to surface distortion and tilt. Therefore, the focal position of the objective lens can be detected with high stability.

  When the objective lens 8 is a dry system, the cover glass surface 1b is used, and in the case of liquid immersion (in order to increase the resolution, the space between the objective lens 8 and the cover glass surface 1b is filled with liquid). Is used as the focusing reference plane.

In the above embodiment, the differential value of the received light amount is controlled to the target value. However, the present invention is not limited to this, and the reference surface (back surface or front surface of the cover glass) is detected by detecting the maximum value of the received light amount by the focus adjustment unit 16. And the deviation from the reference plane may be set according to the target value.
The detection optical system is not limited to that shown in FIG. 1, and any means capable of separating the reflected light of the excitation light from the fluorescent light can be used.

  FIG. 4 is a structural block diagram showing a modification of the confocal microscope apparatus of FIG. 1, which uses focusing light having a wavelength different from that of excitation light as focal plane detection light. The same parts as those in FIG. 1 are denoted by the same symbols, and redundant description is omitted.

The focusing light beam L3a is generated by using an infrared laser light source 120 that is on the longer wavelength side (for example, wavelength 780 nm) than excitation light and fluorescence that are generally used. The dichroic mirror 19 reflects the excitation light beam L1a and transmits the focusing light beam L3a, thereby mixing the focusing light beam L3a with the excitation light beam L1a and entering the confocal scanner 100. The dichroic mirror 70 is, for example, a type having the spectral characteristics shown in FIG. Transmission and reflection are 50:50 with respect to the focusing light of 780 nm. Therefore, a part of the focusing light beam L3a (chain line in FIG. 4) is reflected by the cover glass on the bottom surface of the cage 1, and when the reflected light L3b of the focusing light reaches the dichroic mirror 70, 50% is reflected. Is done. Thereafter, the light enters the light receiver 15 through the same path as the reflected light L1b of the excitation light in the case of FIG.

Here, the dichroic mirror 19 constitutes a mixing unit that mixes the focusing light source with excitation light and enters the confocal scanner.

According to the focus microscope apparatus having the configuration as shown in FIG. 4, in the configuration of FIG. 1, the excitation light itself is used, so that only a few percent of the excitation light is reflected by the dichroic mirror 7, and the light quantity at the light receiver 15 is small. Although it is small, the amount of focused light received by the light receiver 15 can be increased about five times. Therefore, the focusing operation is further stabilized. The other points are the same as in FIG.

  The mixing means is not limited to the dichroic mirror, and any means that can mix and synthesize two light beams can be used.

  The present invention is not limited to the above-described embodiments and modifications, and includes many changes and modifications without departing from the essence thereof.

It is a block diagram showing a configuration of an example of a confocal microscope apparatus according to an embodiment of the present invention. 3 is a chart showing an example of spectral characteristics of a dichroic mirror 7. It is explanatory drawing which shows the relationship between the focus position of the objective lens 8, and the light-receiving light quantity in the light receiver. It is a block diagram showing a modification of the confocal microscope apparatus of FIG. 3 is a chart showing an example of spectral characteristics of a dichroic mirror 70.

Explanation of symbols

2 Sample 4 Nipo disk 8 Objective lens 19 Mixing means 200 Fluorescence microscope 100 Nipo disk type confocal scanner 110 Detection optical system 120 Focusing light source 400 Focusing means L1a Excitation light 1c Back surface 1b Surface

Claims (3)

In a confocal microscope apparatus consisting of a Nipo Disc confocal scanner and a fluorescence microscope,
For a plurality of illumination beams passing through the pinhole of the Nipkow disk, a detection optical system for detecting light reflected from the rear surface or the surface of the cover glass for holding a sample after pinhole passage of the Nipkow disk,
Focusing means for adjusting the focal point of the objective lens of the fluorescence microscope to a focusing position based on the back surface or the surface of the cover glass based on the amount of the reflected light obtained by the detection optical system. A confocal microscope device. As a light source of the reflected light, a focusing light source that outputs laser light having a longer wavelength than excitation light and fluorescence generated from a sample excited by the excitation light; and
Mixing means for mixing the focusing light source with the excitation light and entering the confocal scanner;
The confocal microscope apparatus according to claim 1, further comprising: The focusing means positions the focal point of the objective lens at the in-focus position by deepening the focal point of the objective lens by a predetermined amount from the front surface or the back surface of the cover glass. The confocal microscope apparatus described in 1.

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