JP3113232B2 - microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope for observing fluorescence generated from a sample.

[0002]

2. Description of the Related Art An epi-illumination type microscope has a structure in which an objective lens and an eyepiece are provided to enlarge a sample image, and an epi-illumination system is arranged between these lenses. The epi-illumination system irradiates the sample with light, and the sample is observed by its reflection image.

[0003]

However, the fluorescence obtained by irradiating the sample with the excitation light generally has a low light intensity level, and there is a problem that such weak light cannot be detected efficiently.

[0004] It is an object of the present invention to provide a microscope capable of efficiently detecting fluorescence generated from a sample.

[0005]

The microscope according to claim 1 is a microscope for observing fluorescence generated from a sample,
An epi-illumination optical system that irradiates the sample with light, an infinity correction objective lens that converts light transmitted through the sample into parallel light, and a corner cube reflector disposed at an exit pupil position of the infinity correction objective lens. And a fluorescence detection optical system having, for example, a filter, for detecting fluorescence generated from the sample among light emitted to the sample and fluorescence generated from the sample.

According to the microscope of the first aspect, the light transmitted through the sample passes through the infinity correcting objective lens and becomes parallel light. This collimated light is 3
The light is reflected twice, and again enters the infinity correction objective lens.
The light emitted from the infinity-correcting objective lens forms a spot at the same position as the position of the sample through which the light first transmitted. Therefore, since the irradiation light passes through the same position of the sample twice, the sample is excited twice by the irradiation light and the amount of fluorescence increases, so that weak fluorescence can be efficiently detected by the fluorescence detection optical system. .

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A confocal laser scanning microscope according to a first embodiment of the present invention will be described below with reference to FIG. In this confocal scanning microscope, a condenser lens 2, a pinhole plate 3 having a pinhole formed therein, and a half mirror 4 are arranged substantially in a line along the light irradiation direction of the laser light source 1. Therefore, the laser light emitted from the laser light source 1 is condensed by the condenser lens 2 and enters the pinhole of the pinhole plate 3. Since the mirror surface of the half mirror 4 is arranged at an angle of approximately 45 degrees with respect to the light irradiation direction of the laser light source 1, the light emitted from the pinhole plate 3 is substantially perpendicular to the incident optical path by the half mirror 4. Is reflected by

Along the traveling direction of the reflected light, a collimator lens 5 and an X-direction light deflector 6 are arranged substantially in a line. The light reflected by the half mirror 4 is collimated by the collimator lens 5 and enters the X-direction optical deflector 6. Since the X-direction optical deflector 6 has its rotation axis arranged in the Z-axis direction (direction orthogonal to the X-axis and the Y-axis), the irradiation light is swung in the X-direction with respect to the sample 12.

On the emission side of the X-direction light deflector 6, relay lenses 7, 8 and a Y-direction light deflector 9 are arranged in substantially one line. Therefore, the light emitted from the X-direction light deflector 6 enters the Y-direction light deflector 9 via the relay lenses 7 and 8. Since the rotation axis of the Y-direction light deflector 9 is arranged in the X-axis direction, the irradiation light is swung in the Y direction with respect to the sample 12. As a result, the light applied to the sample 12 is scanned at a high speed in the X direction and is scanned in the Y direction.

On the exit side of the Y-direction optical deflector 9, an imaging lens 10, an objective lens 11, and a sample 12 are arranged. The imaging lens 10 is arranged so as to form a spot on the front image plane of the objective lens 11, and light from a virtual light source, which is formed by this spot, is used as the objective lens 11.
Incident on. The light emitted from the objective lens 11 becomes a spot narrowed down to the diffraction limit, and
Scan two-dimensionally in the direction and the Y direction.

On the transmission side of the sample 12, an infinity correction objective lens 13, a telecentric imaging lens 14, and a plane mirror 15 are arranged. The infinity corrected objective lens 13 is
The sample 12 is located at the front focal point, and the front focal point of the telecentric imaging lens 14 is located at the rear focal point. Further, at the rear focal point of the telecentric imaging lens 14, a plane mirror 15 is arranged in a direction whose mirror surface is orthogonal to the optical axis. For this reason, the telecentric imaging lens 14 is
A light having a principal ray parallel to the optical axis is emitted toward the plane mirror 15, and the light emitted from the telecentric imaging lens 14 forms an actual image of the sample 12 on the plane mirror 15.

A dichroic mirror can be used as the half mirror 4, and a scanner (Galvano Metric Scanner) using a galvano mirror or a rotary polygon mirror can be used as the X-direction light deflector 6 and the Y-direction light deflector 9. A scanned scanner (Rotary Polygonal Scanner) can be used.

Next, the operation of the confocal laser scanning microscope according to the above embodiment will be described. The laser light emitted from the laser light source 1 is condensed by the condenser lens 2, converted into parallel light, and then oscillated in the X and Y directions by the X-direction light deflector 6 and the Y-direction light deflector 9. The light oscillated in the X and Y directions forms a spot on the front image plane of the objective lens 11 by the imaging lens 10. This can be called a virtual light source that is oscillated in the X and Y directions.
Incident on. The emitted light becomes a spot narrowed down to the diffraction limit and scans the sample 12 two-dimensionally.

The light transmitted through the sample 12 is converted into parallel light by the infinity correcting objective lens 13 and thereafter enters the telecentric imaging lens 14. The telecentric imaging lens 14 emits light toward the plane mirror 15 so that the principal ray of the light beam is parallel to the optical axis. The plane mirror 15 reflects the light emitted from the telecentric imaging lens 14 and makes the reflected light incident on the telecentric imaging lens 14 again. Light emitted from the telecentric imaging lens 14 enters the infinity correction objective lens 13,
The sample 12 is illuminated from the opposite direction by spot light formed at the same position on the sample 12 by the infinity correction objective lens 13. As described above, since the light passes through the sample twice, the contrast of the transmitted image becomes clearer, and the contrast of the image can be enhanced.

Further, the transmitted light that has passed through the sample 12 twice enters the objective lens 11, follows the path of the light through which the illumination light has passed in the opposite direction, and reaches the half mirror 4. The transmitted light passes through the half mirror 4 and is incident on a pinhole of a pinhole plate 16 placed at a position conjugate with the pinhole plate 3, and is thereafter detected by a photodetector 17.

According to the above-described embodiment, a two-dimensional confocal transmitted light image can be created based on the information scanned by the X-direction light deflector and the Y-direction light deflector and the output from the photodetector.

Further, by adding an infinity-correcting objective lens, a telecentric imaging lens and a plane mirror to a normal reflection-type confocal laser scanning microscope, it can be easily converted to a transmission-type confocal laser scanning microscope. Can be.

Next, a transmission microscope using an epi-illumination system according to a second embodiment of the present invention will be described with reference to FIG.

The configuration of the optical system for guiding illumination light to the sample 12 is different from that of the confocal laser scanning microscope according to the first embodiment.
3. Since the arrangement of the telecentric imaging lens 14 and the plane mirror 15 is the same, the description is omitted. According to this transmission microscope, an eyepiece 19 is provided between the sample 12 and the observer 18.
And the objective lens 20 are arranged, and the eyepiece 19
An epi-illumination system A is arranged between the lens and the objective lens 20. The epi-illumination system A includes, for example, a beam splitter 21, a lens 22, and an epi-illumination light source 23. Light from the epi-illumination light source 23 passes through the lens 22, and is directed to the objective lens 20 by the beam splitter 21. Reflected. In this case, a Koehler illumination system may be used as the epi-illumination system A in order to obtain uniform illumination.

Next, the operation of the transmission microscope according to the above embodiment will be described. Light from the epi-illumination system A is condensed by the objective lens 20 and is irradiated onto the sample 12. Also, the light transmitted through the sample 12 obtains light information on the sample 12,
The light enters the infinity correction objective lens 13 and is converted into parallel light by the infinity correction objective lens 13. Thereafter, the parallel light is incident on the telecentric imaging lens 14, and is incident on the plane mirror 15 so that the principal ray of the light beam is parallel to the optical axis of the telecentric imaging lens 14. After the incident light is reflected by the plane mirror 15, it is changed into substantially parallel light by the telecentric imaging lens 14. The parallel light is incident on the infinity correction objective lens 13, a spot is formed at the same position on the sample 12 by the infinity correction objective lens 13, and the same position where the illumination light first enters is irradiated from the opposite side.

The light transmitted through the sample 12 from the opposite side obtains optical information on the sample 12 again, and
Incident on. Since the light passes through the same position of the sample twice in this way, the contrast difference between the transmitted images is increased, and the contrast is enhanced. The light from the objective lens 20 passes through the beam splitter 21 and enters the eyepiece 19, and the observation light is observed by the observer 18. Although the eyepiece lens 19 is used in this embodiment, the eyepiece lens 1 is used.
A TV camera may be used instead of 9.

As described above, a normal epi-illumination type microscope is
By adding the infinity corrected objective lens 13, the telecentric imaging lens 14, and the plane mirror 15, it is possible to easily convert to a transmission microscope.

Next, a transmission microscope using an epi-illumination system according to a third embodiment of the present invention will be described with reference to FIG. The transmission microscope differs from the transmission microscope according to the second embodiment in the configuration of the optical system until the transmitted light of the sample 12 returns to the sample 12 again. However, the configurations of the epi-illumination system and the eyepiece are basically the same. Therefore, the description is omitted.

This transmission microscope is arranged so that the focal point of the infinity-correcting objective lens 24 coincides with the sample 12, and the exit pupil plane of the infinity-correcting objective lens 24 has a corner cube reflector (retro reflector) 25. Is characterized in that is arranged. Therefore, the light transmitted through the sample 12 is converted into parallel light by the infinity correction objective lens 24, reflected by the corner cube reflector 25, and irradiates the sample 12 from the back again.

The operation of the transmission microscope according to the above embodiment will be described below. Light from the epi-illumination system A is condensed by the objective lens 20 and is irradiated onto the sample 12. The light transmitted through the sample 12 obtains light information about the sample 12, enters the infinity correction objective lens 24, and is converted into parallel light by the infinity correction objective lens 24. After that, this parallel light is incident on the corner cube reflector 25,
The light is reflected twice and reenters the infinity correcting objective lens 24. The light emitted from the infinity correction objective lens 24 forms a spot at the same position on the sample 12, and irradiates the same position where the illumination light first enters from the opposite side. Light transmitted through the sample 12 from the opposite side obtains the same transmission information about the sample 12 again, and enters the objective lens 20.

As described above, the return light observed by the observer passes through the same position of the sample 12 twice, so that the difference in brightness of the transmitted image is increased and the contrast is enhanced. The light from the objective lens 20 passes through the beam splitter 21 and enters the eyepiece 19, and the observation light is
Observed by

According to the above-described embodiment, a transmitted image can be observed using a light source for reflected illumination. By adding an infinity correcting objective lens 24 and a corner cube reflector 25, ordinary incident light can be easily observed. The illumination microscope can be converted to a transmission microscope. Note that the present invention is not limited to the above embodiment. For example, the second
By using a laser as a light source of the transmission microscope according to the embodiment or the third embodiment and further adding a pinhole, the microscope can be converted to a confocal microscope.

The fluorescence generated when the irradiation light excites the sample is generated when the sample is excited twice by the transmission of the irradiation light twice, and both of them are captured by the objective lens. Therefore, the fluorescence whose light amount has been increased can be detected by a photodetector using a filter or the like. Furthermore, reflected light and scattered light generated by irradiating the sample with light are captured by the objective lens, and follow the path of the light that has passed the illumination light in the opposite direction to reach the half mirror 4, and are detected by the photodetector. can do.

[0029]

According to the present invention, the sample is excited twice by the irradiation light by transmitting the irradiation light through the same position of the sample twice, and the light such as the weak fluorescence can be detected efficiently.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a confocal laser scanning microscope according to a first embodiment of the present invention.

FIG. 2 is a schematic view showing an epi-illumination type microscope according to a second embodiment of the present invention.

FIG. 3 is a schematic diagram showing an epi-illumination type microscope according to a third embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser light source, 2 ... Condensing lens, 3 ... Pinhole, 4
... half mirror, 5 ... collimator lens, 6 ... X direction polarizer, 7, 8 ... relay lens, 9 ... Y direction polarizer, 10
... imaging lens, 11 ... objective lens, 12 ... sample, 13,
24: infinity corrected objective lens, 14: telecentric imaging lens, 15: plane mirror, 16: pinhole, 17 ...
Photodetector, 18: Observer, 19: Eyepiece, 2
0: Objective lens, 21: Beam splitter, 22: Lens, 23: Light source for epi-illumination, 25: Corner cube reflector (retro reflector)

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-58-145911 (JP, A) JP-A-6-18785 (JP, A) JP-A-3-188408 (JP, A) 131811 (JP, A) West German Patent Application Publication 2546079 (DE, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 21/00-21/01 G01N 21/62-21/74 G02B 21/00 JICST file (JOIS)

Claims (1)

(57) [Claims] 1. A microscope for observing fluorescence generated from a sample, an epi-illumination optical system for irradiating the sample with light, an infinity correction objective lens for converting light transmitted through the sample into parallel light, A corner cube reflector disposed at an exit pupil position of an infinity correction objective lens; and a fluorescence detection optical system that detects fluorescence generated from the sample among light emitted to the sample and fluorescence generated from the sample. A microscope, comprising: