JP2001228402A - Confocal optical scanner and confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a confocal optical scanner and confocal microscope capable of irradiating a sample with light uniform in light intensity without degrading its light quantity. SOLUTION: A light intensity uniformity lens 15 is arranged between a collimator lens 12 and a shielding plate 13. The incident light incident on the light intensity uniformity lens 15 has an incident light intensity distribution of a Gauss distribution in which the incident light intensity is strongest near the optical axis and is increasingly weaker the more distant from the optical axis. The light intensity uniformity lens 15 is a concave lens form to diffuse parallel light beams in the central part where the incident light is denser and a convex lens form to converge the parallel light beams in a peripheral part where the incident light is coarser. The light intensity uniformity lens 15 does not cut the light of a segment (the peripheral part of the lens) where the light intensity in the gauss distribution is low and is therefore capable of preventing the light quantity loss by maintaining the light quantity of the incident light at about 70 to 90%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of scanning a sample with light passing through a pinhole, passing fluorescence from a focus position of the sample through the pinhole, and excluding fluorescence from a position other than the focus position of the sample through the pinhole. The present invention relates to a confocal optical scanner and a confocal microscope capable of observing a three-dimensional image of a sample.

[0002]

2. Description of the Related Art A confocal optical scanner of this type has been well known. For example, Japanese Patent Application Laid-Open No. Hei 5-60980, entitled "Confocal Optical Scanner", filed by the present applicant, shows FIG. A confocal optical scanner having such a configuration is described.

[0003] The illustrated confocal optical scanner 1 scans a sample 8 with light passing through a pinhole 3a, passes fluorescence from the focal position of the sample 8 through the pinhole 3a, and scans the fluorescent light from a position other than the focal position of the sample 8. This is a device for eliminating the fluorescence from the pinhole 3a. The confocal optical scanner 1 is installed in a confocal microscope capable of observing a three-dimensional image of the sample 8. The confocal optical scanner 1 is formed such that a condensing disk 2 and a pinhole disk 3 are connected in parallel with a drum 4 interposed therebetween, and are rotated by a motor 5. Further, a beam splitter 6 is fixedly arranged between the condensing disk 2 and the pinhole disk 3.

A plurality of microlenses (for example, Fresnel lenses) 2a are formed on the light-collecting disk 2, and a plurality of pinholes 3a are spirally formed on the pinhole disk 3 in many rows. The condensing disc 2 and the pinhole disc 3 are connected so that each pinhole 3a is located at the focal position of each microlens 2a.

[0005] The laser beam incident on the focusing disk 2 is converged by the microlens 2a, passes through the beam splitter 6, and is focused on the pinhole 3a. The light passing through the pinhole is condensed by the objective lens 7 and is irradiated on the sample 8.
The return light from the sample 8 is reflected by the beam splitter 6 again through the objective lens 7 and the pinhole disk 3, and enters the camera 10 via the condenser lens 9. An image of the sample 8 is formed on an image receiving surface (not shown) of the camera 10.

In such a configuration, the condensing disk 2
When the sample surface 8a is optically scanned (raster-scanned) by the plurality of pinholes 3a, the surface image of the sample 8 can be observed by the camera 10. As described above, since the confocal microscope employs the point light source and the point light reception, it is possible to obtain a high-resolution three-dimensional image by excluding blur and scattered light other than the focal position.

FIG. 6 shows a specific example of a main part of a confocal optical scanner based on such a principle. The light from the point light source 11 emitted from the end of the optical fiber is collimated by the collimator lens 12 and is incident on the condenser disk 2. The point light source 11 is located at the front focal point (focal length f) of the collimator lens 12.

In this state, the divergence angle θ of the point light source 11
When the light inside is converted into a parallel light beam by the collimator lens 12, the light intensity distribution I on a plane perpendicular to the optical axis of the light passing through the collimator lens 12 becomes a Gaussian distribution as shown in FIG.

In order to cut out a uniform light beam near the optical axis (usually at least 10% in order to make distribution unevenness inconspicuous), a shielding plate 13 having an opening 14 is provided. (Hatched portion in FIG. 7B) is guided to the micro lens 2a. The hole in the opening 14 is usually circular.

Japanese Patent Application Laid-Open No. 11-95109 discloses a confocal optical scanner in which a light intensity distribution correction filter is arranged between a collimator lens 12 and a shielding plate 13 shown in FIG. This light intensity distribution correction filter is used to adjust the aperture 1 of the incident light whose light intensity distribution is a Gaussian distribution.
The intensity distribution of the light passing through 4 is flattened, and the other light is cut.

[0011]

As described above, in the conventional confocal optical scanner, the amount of light that can be used is small, and in order to illuminate the sample with sufficient intensity, it is necessary to use a light source with a correspondingly large output. there were. However, if a high-output light source is used, extra stray light increases, and there is a problem that it is not suitable for observation using weak light, particularly for fluorescence observation.

A fly-eye lens is generally known as a means for improving the non-uniformity and maintaining the light quantity. This fly-eye lens is used to apply a light beam parallel to the optical axis to a microlens array. It cannot be applied to a confocal optical scanner because it cannot be made incident.

An object of the present invention is to provide a confocal optical scanner and a confocal microscope capable of irradiating a sample with light having a uniform light intensity without reducing the light amount.

[0014]

The present invention solves the above-mentioned problems by the following means. Note that the description will be given with reference numerals corresponding to the embodiments of the present invention, but the present invention is not limited to this. According to the first aspect of the present invention, the sample (8) is scanned with light passing through the pinhole (3a), and fluorescence from a focal position of the sample passes through the pinhole;
A confocal optical scanner (1) for eliminating fluorescence from a position other than the focal position of the sample by the pinhole, while maintaining the amount of incident light and making the intensity of light passing through the pinhole uniform. A confocal optical scanner comprising a light intensity equalizing lens (15).

According to a second aspect of the present invention, in the confocal optical scanner according to the first aspect, the light intensity equalizing lens includes:
The confocal optical scanner is characterized in that a central part is a diverging lens part for diverging incident light, and a peripheral part is a converging lens part for converging incident light.

[0016] The invention of claim 3 is claim 1 or claim 2.
5. The confocal optical scanner according to claim 1, wherein a collimator lens (12) for converting light incident on the light intensity equalizing lens into parallel light, and an aperture (14) through which light transmitted through the light intensity equalizing lens passes. A light-collecting disk (2) having a plurality of microlenses (2a) through which light passing through the opening is transmitted, and a pinhole (3a) at a focal position of the microlenses. And a pinhole disk (3) for scanning the sample with light passing through the pinhole by rotating integrally with the light-collecting disk; and transmitting light traveling from the microlens toward the pinhole to the pinhole. And a beam splitter (6) for reflecting the fluorescent light passing through the light-receiving surface toward a light-receiving surface.

According to a fourth aspect of the present invention, there is provided the first or second aspect.
In the confocal optical scanner according to the, a collimator lens to collimate the light incident on the light intensity equalizing lens, a shielding plate having an opening through which the light transmitted through the light intensity equalizing lens, A pinhole disk that has a plurality of the pinholes through which the light passing through the opening passes and scans the sample by scanning the light passing through the pinhole by rotating; and from the opening toward the pinhole. A confocal optical scanner including a beam splitter that transmits light and reflects the fluorescence that has passed through the pinhole toward a light receiving surface.

The invention according to claim 5 is the invention according to claims 1 to 4.
The confocal optical scanner according to any one of the preceding claims, wherein a Kepler-type enlargement is provided between the light intensity equalizing lens and the shielding plate to increase the aperture of a light beam transmitted through the light intensity equalizing lens. A confocal optical scanner comprising an optical system (16) or a Galileo-type magnifying optical system (17).

The invention of claim 6 is the invention of claims 1 to 4
In the confocal optical scanner according to any one of the above, a Kepler-type reduction optical system that reduces the aperture of a light beam transmitted through the collimator lens between the collimator lens and the light intensity equalizing lens. 18) or a confocal optical scanner comprising a Galileo-type reduction optical system (19).

According to a seventh aspect of the present invention, there is provided a confocal microscope capable of observing a three-dimensional image of a sample, wherein the confocal optical scanner according to any one of the first to sixth aspects, A confocal microscope including an objective lens (7) that forms an image of light passing through a pinhole on the sample and a condensing lens (9) that forms an image of the fluorescence that has passed through the pinhole on an image plane. .

[0021]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (First Embodiment) Hereinafter, a first embodiment of the present invention will be described in more detail with reference to the drawings. FIG. 1 is a schematic configuration diagram of a confocal optical scanner according to a first embodiment of the present invention. FIG. 2 is a view for explaining a light intensity equalizing lens of the confocal optical scanner according to the first embodiment of the present invention. FIG. Shows the light intensity distribution,
FIG. 2B is a cross-sectional view illustrating a state in which the light intensity equalizing lens is cut along a plane including the optical axis, and FIG. 2C illustrates a light intensity distribution of light emitted from the light intensity equalizing lens. . The same members as those shown in FIGS. 5 and 6 are denoted by the same reference numerals, and detailed description thereof will be omitted.

The light intensity equalizing lens 15 is a lens that maintains the amount of incident light incident from the collimator lens 12 and equalizes the intensity of the incident light. The light intensity equalizing lens 15 is, for example, 15p-V-1 (15 Sep. 199).
8) An axisymmetric aspheric lens with properties known from the Japan Society of Applied Physics. As shown in FIG. 1, the light intensity equalizing lens 15 is disposed between the collimator lens 12 and the shielding plate 13. FIG.
As shown in (a), the incident light incident on the light intensity equalizing lens 15 has a Gaussian light intensity distribution, the intensity of the incident light is strongest near the optical axis, and the intensity of the incident light increases as the distance from the optical axis increases. Becomes weaker. As shown in FIG. 2B, the light intensity uniforming lens 15 is formed in a shape of a diffusion lens (concave lens) that diffuses parallel light at the center where the incident light is dense, and the incident light becomes sparse. The peripheral portion is formed in a converging lens (convex lens) shape for converging the parallel light. The light intensity equalizing lens 15 does not cut off light of a low light intensity portion (lens peripheral portion) in the Gaussian distribution.
The loss of light quantity can be prevented by maintaining the light quantity of the incident light at about 70 to 90%. As shown in FIG. 2C, the light emitted from the light intensity equalizing lens 15 is a parallel light having a substantially uniform light intensity distribution.

The confocal optical scanner according to the first embodiment of the present invention has the following effects. (1) In the first embodiment, the point light source 1 having a Gaussian distribution is provided because the light intensity equalizing lens 15 for maintaining the amount of incident light and for equalizing the intensity of the incident light is provided.
The light intensity distribution can be uniformly converted while maintaining almost all of the energy of No. 1 to guide the light to the microlens 2a.
As a result, the sample 8 can be uniformly illuminated by preventing a decrease in the light amount, as compared with, for example, a case where the vicinity of the center of the Gaussian distribution is widened and the peripheral portion is cut as in a conventional light intensity distribution correction filter. Can be.

(2) In the first embodiment, since the collimator lens 12 having a short focal length can be used, the distance between the point light source 11 and the collimator lens 12 is reduced. As a result, the size of the illumination optical system for irradiating the sample 8 with light is reduced, maintenance is facilitated, and the apparatus can be made compact and cost can be reduced.

(Second Embodiment) FIG. 3 shows a second embodiment of the present invention.
FIG. 3A is a diagram illustrating an arrangement of a magnifying optical system of a confocal optical scanner according to an embodiment. FIG. 3A is an example in which a Galileo-type magnifying optical system is arranged, and FIG. 3B is a Kepler-type magnifying optical system. Is an example in which.

The second embodiment of the present invention is an embodiment in which a Galileo type magnifying optical system 16 and a Kepler type magnifying optical system 17 are arranged between a light intensity equalizing lens 15 and a shielding plate 13. The Galileo type magnifying optical system 16 and the Kepler type magnifying optical system 17 shown in FIG. 3 are lens groups for expanding the aperture of the light beam transmitted through the light intensity equalizing lens 15. The Galilean magnifying optical system 16 shown in FIG. 3A includes a concave lens 16a on which parallel light from the light intensity equalizing lens 15 is incident, and a convex lens 16b on which light transmitted through the concave lens 16a is incident. . The Kepler-type magnifying optical system 17 shown in FIG.
5 is composed of a convex lens 17a on which the parallel light from 5 is incident and a convex lens 17b on which the light transmitted through the convex lens 17a is incident.

In the second embodiment, after the parallel light having a substantially uniform light intensity is formed by the small-intensity light intensity equalizing lens 15, the microlens 2 a is formed by the Galileo-type magnifying optical system 16 and the Kepler-type magnifying optical system 17. The diameter of the incident light beam can be increased. As a result, it is not necessary to use a large-diameter light intensity equalizing lens 15 which is difficult and expensive to manufacture, and the cost of the apparatus can be reduced.

(Third Embodiment) FIG. 4 shows a third embodiment of the present invention.
FIG. 4A is a diagram showing an arrangement of a reduction optical system of the confocal optical scanner according to the embodiment, FIG. 4A is an example in which a Galileo type reduction optical system is arranged, and FIG. 4B is a Kepler-type reduction optical system. Is an example in which.

The third embodiment of the present invention is an embodiment in which a Galileo type reduction optical system 18 and a Kepler type reduction optical system 19 are arranged between a collimator lens 12 and a light intensity equalizing lens 15. The Galileo type reduction optical system 18 and the Kepler type reduction optical system 19 shown in FIG. 4 are a lens group for reducing the aperture of the light beam transmitted through the collimator lens 12. Galileo type reduction optical system 1 shown in FIG.
Reference numeral 8 denotes a convex lens 18a on which parallel light from the collimator lens 12 is incident, and a concave lens 18b on which light transmitted through the convex lens 18a is incident. FIG.
The Kepler-type reduction optical system 19 shown in (b) includes a convex lens 19a on which parallel light from the collimator lens 12 is incident,
The convex lens 1 on which the light transmitted through the convex lens 19a is incident
9b.

In the third embodiment, the point light source 1 having a large divergence angle is provided by the large-diameter and short-focus collimator lens 12.
After converting the light from 1 into parallel light, the aperture of the light beam incident on the light intensity equalizing lens 15 is matched with the lens diameter of the light intensity equalizing lens 15 by the Galileo type reduction optical system 18 and the Kepler type reduction optical system 19. Can be done.

The present invention is not limited to the embodiment described above, but various modifications or changes can be made as described below, and these are also within the scope of the present invention. In this embodiment, the condensing disk 2 having the microlenses 2a and the pinhole disk 3 are integrally described, and the Nipkow plate type confocal microscope with the microlenses is rotated as an example. However, the condensing disk 2 is omitted. The present invention can be applied to a Nipkow plate confocal microscope.

In this embodiment, the case where the modularized confocal optical scanner 1 is installed in a confocal microscope has been described as an example. However, this confocal optical scanner 1 is unitized to form an optical microscope. It may be detachable.

In this embodiment, a laser beam is used as the point light source 11, but a tungsten lamp, a mercury lamp, a xenon arc lamp or the like may be used. Alternatively, the curvature of the light intensity equalizing lens 15 may be changed according to the wavelength of the laser light, and the sample 8 may be irradiated with a laser light suitable for the fluorescent dye.

[0034]

As described above, according to the present invention, since the light intensity equalizing lens for maintaining the light amount of the incident light and equalizing the intensity of the incident light is provided, the light intensity can be reduced without reducing the light amount. Can irradiate the sample with uniform light.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram of a confocal optical scanner according to a first embodiment of the present invention.

FIG. 2 is a view for explaining a light intensity equalizing lens of the confocal optical scanner according to the first embodiment of the present invention;
(A) shows the intensity distribution of the incident light incident on the light intensity equalizing lens, (b) is a cross-sectional view showing a state in which the light intensity equalizing lens is cut along a plane including the optical axis, and (c) is 5 shows an intensity distribution of light emitted from the light intensity equalizing lens.

FIGS. 3A and 3B are diagrams showing an arrangement of a magnifying optical system of a confocal optical scanner according to a second embodiment of the present invention, wherein FIG. 3A is an example in which a Galileo-type magnifying optical system is arranged, and FIG. This is an example in which a Kepler-type magnifying optical system is arranged.

4A and 4B are diagrams showing an arrangement of a reduction optical system of a confocal optical scanner according to a third embodiment of the present invention, wherein FIG. 4A is an example in which a Galileo-type reduction optical system is arranged, and FIG. This is an example in which a Kepler-type reduction optical system is arranged.

FIG. 5 is a diagram illustrating a configuration example of a conventional confocal optical scanner.

FIG. 6 is a configuration diagram showing a main part of a conventional confocal optical scanner.

FIG. 7 is a diagram showing a light intensity distribution of light passing through a collimator lens of a conventional confocal optical scanner.

[Explanation of symbols]

 Reference Signs List 1 confocal optical scanner 2 focusing disk 2a micro lens 3 pinhole disk 3a pinhole 11 point light source 12 collimator lens 13 shielding plate 14 aperture 15 light intensity equalizing lens 16 Galileo type magnifying optical system 17 Kepler type magnifying optical system 18 Galileo -Type reduction optical system 19 Kepler-type reduction optical system

Claims (7)

[Claims] 1. A method in which light passing through a pinhole is scanned on a sample, fluorescence from a focal position of the sample is passed through the pinhole, and fluorescence from a position other than the focal position of the sample is eliminated by the pinhole. An optical scanner for focusing, comprising: a light intensity equalizing lens that maintains the amount of incident light and equalizes the intensity of light passing through the pinhole. 2. The confocal optical scanner according to claim 1, wherein the light intensity equalizing lens is a diverging lens portion having a central portion that diverges incident light and a converging lens having a peripheral portion that converges incident light. A confocal optical scanner. 3. The confocal optical scanner according to claim 1, wherein the collimator lens converts light incident on the light intensity equalizing lens into parallel light, and transmits through the light intensity equalizing lens. A shielding plate having an opening through which light passes; a light-collecting disk having a plurality of microlenses through which light passing through the opening passes; and the light-collecting disk having the pinhole at a focal position of the microlens. A pinhole disk that scans the sample with light that has passed through the pinhole by rotating together, transmitting light traveling from the microlens to the pinhole, and transmitting the fluorescence that has passed through the pinhole to a light receiving surface; And a beam splitter for reflecting light toward the confocal optical scanner. 4. The confocal optical scanner according to claim 1, wherein the collimator lens converts light incident on the light intensity equalizing lens into parallel light, and transmits through the light intensity equalizing lens. A shielding plate having an opening through which light passes, and a plurality of pinholes through which light passing through the opening passes, and a pinhole for scanning the sample with light passing through the pinhole by rotating. A disc, transmitting light from the opening toward the pinhole,
A beam splitter that reflects the fluorescent light passing through the pinhole toward a light receiving surface. 5. The method according to claim 1, wherein:
In the confocal optical scanner according to the paragraph, between the light intensity equalizing lens and the shielding plate, a Kepler-type magnifying optical system or a Galileo type that enlarges the aperture of the light beam transmitted through the light intensity equalizing lens. An optical scanner for confocal, comprising a magnifying optical system. 6. One of claims 1 to 4
In the confocal optical scanner according to the paragraph, between the collimator lens and the light intensity equalizing lens, a Kepler-type reduction optical system or a Galileo-type reduction optical system that reduces the aperture of a light beam transmitted through the collimator lens. A confocal optical scanner, comprising: 7. A confocal microscope capable of observing a three-dimensional image of a sample, wherein the confocal optical scanner according to claim 1 has passed through the pinhole. A confocal microscope including: an objective lens that forms an image of light on the sample; and a condenser lens that forms an image of the fluorescence that has passed through the pinhole on an image forming surface.