JPH0596816U - Confocal optical scanner - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] In a confocal fluorescence microscope, it is possible to search the position and focus of a sample without attaching or detaching a rotating pinhole substrate or an ND filter for adjustment, and a weak fluorescent sample. However, the confocal optical scanner that can obtain a semi-confocal image is provided. [Structure] Pinhole disk 112 having a large number of spirally arranged pinholes, condensing disk 112 having a lens array for focusing laser light on each pinhole, sample 3
The beam splitter 141 for the normal confocal optical scanner 10 including the filter 4, the beam splitter 12 that guides the reflected light to the eyepiece 5, and the drive motor 13.
The second branching optical system 14 including the mirror 142 and the mirror 143 is attached and detached so that a semi-confocal non-slice image can be obtained by attaching it and a confocal slice image can be obtained by detaching it.

Description

[Detailed description of the device]

[0001]

[Industrial applications]

 The present invention relates to a confocal optical scanner that scans a pinhole substrate used for a confocal fluorescence microscope, etc., and particularly makes it easy to find the position of a sample and to focus on it, and enables weak fluorescence measurement.

[0002]

[Prior Art]

FIG. 2 is a prior art example of a confocal optical scanner by the applicant of the present application, which is used for a confocal fluorescence microscope. In FIG. 2, reference numeral 1 denotes a confocal optical scanner. In this confocal optical scanner 1, for example, a plurality of Fresnel lenses formed on one surface of a glass substrate shown in FIG. A plurality of pinholes formed on the substrate shown in FIG. 3B and a collecting optical disk 111 formed by sequentially shifting the screen in the radial direction by Pr and P _{r in} the radial direction (Pθ in the circumferential direction). A pinhole disk 112, which is formed by sequentially shifting the pinhole disk, and a drum 113, which connects the condensing disk 111 and the pinhole disk 112 so that the pinholes are arranged at the focal positions of the Fresnel lens. Hole substrate 11, beam splitter 12 installed between condensing disk 111 and pinhole disk 112, and motor 13 for rotating pinhole board 11 at a constant speed. Composed of. Reference numeral 2 denotes an objective lens, which is installed at a position where the light emitted from the pinhole is applied to the sample 3 and the return light from the sample 3 is incident on the pinhole again. Reference numeral 4 is a filter, and 5 is an eyepiece.

In such a configuration, light emitted from a laser light source (not shown) is incident on the confocal optical scanner 1. The light incident on the confocal optical scanner 1 is condensed by a Fresnel lens formed on the condensing disk 111 of the pinhole substrate 11, passes through the beam splitter 12, and then on the pinhole disk 112. The light is focused on the pinhole formed on. The light passing through the pinhole is projected onto the sample 3 by the objective lens 2. The return light from the sample 3 passes through the objective lens 2 and the pinhole disk 112 again, and is reflected by the beam splitter 12 (bent in the direction orthogonal to the optical axis direction of the incident light), and the filter 4 and the eyepiece lens. The image of Sample 3 can be observed through 5. In this example, the pinhole substrate 11 is rotated at a constant speed by the motor 13, and the image of the pinhole scans the sample 3 by the rotation of the pinhole substrate 11. Further, the pinhole on the pinhole disk 112 and the light spot (image of the pinhole) on the sample 3 are in a confocal relationship, and the incident light from a laser light source (not shown) and the return from the sample 3 are returned. Since both the light pass through the pinhole, a high resolving power due to the confocal effect can be obtained, and since the return light from the sample 3 does not pass through the condensing disk 111, the confocal light obtained by the diameter of the pinhole is obtained. The resolution can be retained.

[0004]

[Problems to be solved by the device]

 In such a confocal fluorescence microscope using the confocal optical scanner shown in the above-mentioned prior art, when the position of the sample is searched or the focus is adjusted, the rotating pinhole substrate 11 is shifted to the right in the drawing to move the laser. The light was attenuated using an ND filter to prevent the fluorescence from fading, and was used as the optical system of a normal epifluorescence microscope. In that state, the pinhole substrate 11 was inserted by shifting it to the left in the figure, the ND filter was removed, the amount of light was increased, and a confocal fluorescence image was obtained. When a pinhole substrate is inserted, the light quantity decreases instead of obtaining a confocal slice image, so it is necessary to increase the light quantity.

Therefore, if the operability is poor and the amount of fluorescence is weak, it may not be possible to pass through the pinholes, making it impossible to observe.

The present invention has been made in view of the above-mentioned problems of the prior art, and it is possible to search the position of the sample and focus without attaching and removing the rotating pinhole substrate and ND filter. It is also an object of the present invention to provide a confocal optical scanner capable of obtaining a semi-confocal image even with a weak fluorescent sample.

[0007]

[Means for Solving the Problems]

 The configuration of the present invention for solving the above-mentioned problems is achieved by rotating a pinhole substrate and scanning a sample with irradiation light passing through the pinhole substrate. Means for collecting light, a pinhole disk for forming a plurality of pinholes, a light collecting disk and a pinhole disk, and the pinholes are respectively located at focal points of the plurality of light collecting means. A pinhole substrate composed of drums connected so as to be arranged, a rotating means for rotating the pinhole substrate at a constant speed, and a pinhole substrate installed between the condensing disc and the pinhole disc of the pinhole substrate. The emitted light from the first branch optical system, the pinhole substrate and the pinhole substrate is irradiated to the sample, and the return light from the sample is again emitted from the pinhole substrate. A second branching optical system that is installed between the objective lens and the objective lens that is installed at a position where the light enters the hole, and that reflects the return light from the sample to the side of the optical axis without passing through the pinhole substrate; Means for attaching and detaching the second branch optical system to and from the optical path, and a semi-confocal non-slice image in the state where the second branch optical system is attached, and the second branch optical system. It is characterized in that a confocal slice image can be obtained when the system is removed from the optical path.

[0008]

[Action]

 According to the present invention, by adding the second branching optical system, it is not necessary to attach and remove the pinhole substrate and the ND filter of the laser when the position of the sample is searched or the focus is adjusted. The image can be obtained also in.

[0009]

【Example】

 Hereinafter, the present invention will be described with reference to the drawings. FIG. 1 is a block diagram showing an embodiment in which the confocal optical scanner of the present invention is used in a confocal fluorescence microscope. In FIG. 1, the same elements as those in FIG. 2 are designated by the same reference numerals, and overlapping description will be omitted. 1 is different from FIG. 2 in that the confocal optical scanner 1a has an optical axis between the pinhole disk 111 and the objective lens 2 without returning the return light from the sample 3 to the optical axis. The second branching optical system 14 for reflecting the light to the side of is provided. In FIG. 1, the second branch optical system 14 is configured by using, for example, a beam splitter 141 and mirrors 142 and 143. The second branch optical system 14 is adjusted in advance, and can be removed from the optical path and attached to the optical path by a driving mechanism (not shown).

In such a configuration, the laser light is scanned by the pinhole substrate 11, passes through the beam splitter 12 and the beam splitter 141 of the second branch optical system 14, and is focused on the sample 3 by the objective lens 2. .. The return light (fluorescence) from the sample 3 is reflected by the beam splitter 141, the mirrors 142 and 143 of the second branch optical system 14 via the object lens 2, and the light amount does not decrease, and the eyepiece 5 The image of Sample 3 can be observed through. Therefore, in this state, it is possible to search the position of the sample and focus without attaching and removing the pinhole substrate 11 and the ND filter of the laser. In this state, a semi-confocal image is obtained, which is effective in the case of a weak fluorescent sample. On the other hand, by removing the second branch optical system 14 from the optical path by a drive mechanism (not shown), a confocal image similar to that of the conventional apparatus shown in FIG. 2 can be obtained.

As a result, by using the confocal optical scanner of the present invention, (1) a semi-confocal non-slice image: the amount of light is large and the entire sample can be seen, which is suitable for rough adjustment such as focusing. .. (2) Confocal slice image: Due to its high resolution, it is suitable for precise measurement, and fine adjustment such as focusing is easy. These two cases can be used by simply attaching and detaching the second branch optical system from the optical path.

In the above embodiment, a combination of a dichroic mirror, a half mirror, a polarization beam splitter and a quarter wave plate is used instead of the beam splitter 141 which constitutes the second branch optical system 14. The same effect can be obtained.

[0013]

[Effect of the device]

 As described above in detail with reference to the embodiments, according to the present invention, by simply attaching and detaching the second branch optical system to and from the optical path, it is possible to eliminate the decrease in the fluorescent light amount due to the pinhole, and to find the position of the sample. It is possible to realize a confocal optical scanner that is easy to focus and has the effect of obtaining a semi-confocal image even when it is difficult to observe a confocal image with a weakly fluorescent sample.

[Brief description of drawings]

FIG. 1 is a configuration diagram showing an embodiment in which a confocal optical scanner of the present invention is used in a confocal fluorescence microscope.

FIG. 2 is a configuration diagram showing an example of a confocal fluorescence microscope using a confocal optical scanner.

FIG. 3 is an example of a condensing disk and a pinhole disk used in a confocal optical scanner.

[Explanation of symbols]

 1a Confocal optical scanner 2 Objective lens 3 Sample 11 Pinhole substrate 12, 141 Beam splitter 13 Motor 14 Second branching optical system 111 Condensing disk 112 Pinhole disk 113 Drum 142, 143 Mirror

Claims (1)

[Scope of utility model registration request] 1. A confocal optical scanner that rotates a pinhole substrate and scans a sample with irradiation light that has passed through the pinhole substrate; From a pinhole disc having a plurality of pinholes formed thereon, and a drum connecting the condensing disc and the pinhole disc so that the pinholes are respectively arranged at the focal positions of the plurality of condensing means. A pinhole substrate, rotating means for rotating the pinhole substrate at a constant speed, and a first branching optical system installed between the condensing disc and the pinhole disc of the pinhole substrate, The sample is irradiated with light emitted from the pinhole substrate and the pinhole substrate, and the return light from the sample is installed at a position where it is incident on the pinhole of the pinhole substrate again. A second branching optical system installed between the objective lens and the objective lens to reflect the return light from the sample to the side of the optical axis without passing through the pinhole substrate, and an optical path of the second branching optical system. And a means for attaching and detaching from the optical path, and a semi-confocal non-slice image when the second branch optical system is attached, and a unit when the second branch optical system is removed from the optical path. A confocal optical scanner characterized in that a slice image of the focal point can be obtained.