JP2002031762A - Lighting equipment for microscope - Google Patents

Abstract

(57) [PROBLEMS] To provide a microscope illuminating device capable of easily and accurately adjusting an incident angle of illumination light to a sample when illuminating the sample via an objective lens. SOLUTION: Emission means 11-1 emits condensed light along a direction O2 intersecting an optical axis O3 of an objective lens 21.
5, an optical member 1 that reflects condensed light in the direction of the objective lens 21 along the optical axis O3 and transmits observation light from the sample 31.
7 is provided. The emission units 11 to 15 and the optical member 16 are arranged such that the focal point of the collected light coincides with the rear focal plane of the objective lens 21. The emission units 11 to 15 include a laser light source 11, magnifying optical systems 12, 13, a reflecting optical system 14, and a condensing optical system 15. The optical axis of the condensing optical system 15 is parallel to the above-described direction O2. The reflection optical system 14
It is rotatable about an axis perpendicular to the optical axis of the condensing optical system 15 and the optical axes of the magnifying optical systems 12 and 13.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an illumination device for a microscope which is incorporated in a microscope for observing a specimen and illuminates the specimen by using total reflection of light.

[0002]

2. Description of the Related Art Conventionally, a microscope is used to illuminate a sample in a medium having a low refractive index by irradiating illumination light from a medium having a high refractive index and totally reflecting the light at a boundary surface with a medium having a low refractive index. An illumination device (a kind of dark field illumination device) is known. Since light leaks out of the interface into the medium having a low refractive index, the sample can be illuminated by the light that has leaked out.

As an example of such a microscope illuminating device, there is shown a configuration shown in FIG. 4, that is, a condenser lens 53 arranged on the opposite side of an objective lens 52 with a sample 51 interposed therebetween.
(For example, Kanji Yoka, Basics of Biological Microscopy, P.88-89, Baifukan (1973)). In this lighting device, the central part of the condenser lens 53 is shielded, and a slit 54 is provided in the peripheral part. The light L11 that has passed through the slit 54 is
(Light L12), and then tries to enter the water 57 via the oil 55 and the slide glass 56. Here, since the refractive index of the slide glass 56 is higher than that of the water 57, when the incident angle of the light L12 is equal to or larger than the critical angle, the slide glass 56 is totally reflected at the boundary surface between the slide glass 56 and the water 57 (light L13). ).

[0004] The specimen 51 is illuminated by light that has permeated into the water 57 from the boundary surface. Light and fluorescence irregularly reflected by the specimen 51 are generated when the objective lens 52 is an oil immersion objective lens.
The light enters the objective lens 52 via the water 57, the cover glass 58, and the oil 59. According to this lighting device, the light L13 totally reflected at the boundary surface between the slide glass 56 and the water 57
Passes through the condenser lens 53 again, and only the light generated from the sample 51 enters the objective lens 52.
Only the irregularly reflected light and fluorescence from No. 1 can be observed.

[0005]

However, in the above-described illumination device (FIG. 4), since the sample 51 is observed through the water 57, there is a problem that a clear image with large aberration cannot be obtained. Further, when light that permeates the water 57 from the boundary surface between the slide glass 56 and the water 57 passes through the sample 51, this transmitted light also enters the objective lens 52, so that an image with good contrast is obtained. I can't.

On the other hand, a microscope illuminating device for illuminating a specimen by guiding illumination light from the objective lens side through the objective lens has recently been proposed. For example, M. Tokunaga et a
l, Biochemical and Biophysical Research Communicat
In the lighting device shown in ions 235, p.49 (1997), the light emitted from the laser light source (YAG532) is N
D filter, lens (L1), λ / 4 plate, aperture
(A), the lens (L2), the mirror (M), the dichroic mirror (DM), and the objective lens (OL) are guided to the sample.

[0007] According to this illumination device, the sample can be observed without passing through water, and even if light that has permeated the water passes through the sample, it does not enter the objective lens (OL). However, since the angle of incidence of light guided to the sample side through the objective lens (OL) is adjusted by the parallel movement of the mirror (M), there is a problem that fine adjustment is difficult. SUMMARY OF THE INVENTION It is an object of the present invention to provide a microscope illuminating device that can easily and accurately adjust an incident angle of illumination light on a sample when illuminating the sample via an objective lens.

[0008]

According to the present invention, there is provided a microscope illuminating apparatus which emits condensed light along a predetermined direction intersecting an optical axis of an objective lens, and a condensed light emitted from the emitting means. An optical member that reflects light in the direction of the objective lens along the optical axis of the objective lens and transmits observation light generated from the sample and passing through the objective lens. further,
The emission means and the optical member are arranged such that the focal point of the condensed light coincides with the rear focal plane of the objective lens.

The emission means includes a laser light source, an enlargement optical system for expanding the diameter of light emitted from the laser light source, and a reflection optical system for reflecting the light whose diameter has been enlarged by the expansion optical system. And a condensing optical system for condensing the light reflected by the reflecting optical system. The optical axis of the converging optical system of the emitting means is parallel to the above-mentioned predetermined direction. The reflecting optical system is rotatable about an axis perpendicular to the optical axis of the condensing optical system and the optical axis of the magnifying optical system.

According to this illumination apparatus for a microscope, the condensing point of the condensed light is moved within the rear focal plane of the objective lens by the rotation of the reflection optical system, so that it is guided to the specimen side through the objective lens. The incident angle of the reflected light can be easily adjusted with high accuracy.

[0011]

Embodiments of the present invention will be described below in detail with reference to the drawings.

This embodiment corresponds to claims 1 to 4. As shown in FIG. 1, a microscope illumination device 10 of the present embodiment includes a laser light source 11, a fiber 12, a collimating lens 13, a mirror 14 (reflection optical system), and a condenser lens 15 (condenser optical system). ) And dichroic mirror 1
6 (optical member) and a band-pass filter 17. Fiber 12 and collimating lens 13
Corresponds to the “magnifying optical system” in the claims.

The laser light source 11 is an argon laser that emits parallel light having a wavelength of 488 nm. Fiber 12 is
This is a single mode fiber having a diameter of 50 μm. The collimating lens 13 is disposed at a point away from the end face 12a of the fiber 12 by a focal length d1 (100 mm) of the collimating lens 13.

The mirror 14 is a plane reflecting mirror. The reflection surface 14a of the mirror 14 includes an intersection C1 between the optical axis O1 of the collimator lens 13 and the optical axis O2 of the condenser lens 15. This mirror 14 is perpendicular to the optical axes O1 and O2,
1 is rotatable about an axis that includes one. The rotation angle of the mirror 14 can be adjusted with high precision by a screw mechanism (not shown). The condenser lens 15 is disposed at a point separated by a distance d2 from the reflection surface 14a (intersection C1) of the mirror 14. This distance d2 is equal to the focal length of the condenser lens 15 (160 m
m). The optical axis O2 of the condenser lens 15 is perpendicular to the optical axis O1 of the collimator lens 13. The optical axis O2 of the condenser lens 15 is also perpendicular to the optical axis O3 of the objective lens 21 of the microscope (not shown). Condensing lens 1
The distance from 5 to the optical axis O3 of the objective lens 21 is d3. This distance d3 is smaller than the focal length d2 of the condenser lens 15.

The dichroic mirror 16 reflects the light included in the wavelength region (around 488 nm) of the light emitted from the laser light source 11, and this wavelength region (around 488 nm).
It transmits light included in a longer wavelength region (515 nm to 550 nm). On the reflecting surface 16a of the dichroic mirror 16, the optical axis O2 of the condenser lens 15 and the objective lens 2
An intersection C2 with one optical axis O3 is included.

The dichroic mirror 16 has an optical axis O
2, It is arranged at an angle of 45 ° from O3. The distance from the reflection surface 16a (intersection C2) of the dichroic mirror 16 to the rear focal plane 21a of the objective lens 21 is d4. The sum (d3 + d4) of the distance d4 and the distance d3 is equal to the focal length (d2) of the condenser lens 15. The bandpass filter 17 blocks light included in the wavelength region (around 488 nm) and transmits light included in the wavelength region (515 nm to 550 nm). The bandpass filter 17 is disposed on the opposite side of the dichroic mirror 16 from the objective lens 21.

In the illumination device 10 for a microscope configured as described above, the parallel light emitted from the laser light source 11 is focused on one end face of the fiber 12 and
After propagating through No. 2, the light is emitted from the other end face 12a (diffused light L1). Then, the diffused light L1 is condensed by the collimating lens 13 and becomes parallel light L2. As described above, by passing through the fiber 12 and the collimating lens 13, the diameter of the parallel light emitted from the laser light source 11 is enlarged.

Fiber 12 and collimating lens 13
The parallel light L2 whose diameter has been enlarged by this is guided to the mirror 14 and reflected on the reflection surface 14a (parallel light L3). The traveling direction of the parallel light L3 reflected by the mirror 14 is determined by the rotation angle of the mirror 14. In the present embodiment, the parallel light L
The traveling direction of 3 is inclined from the optical axis O2 toward the optical axis O1.

The parallel light L3 is condensed by the condensing lens 15 and becomes condensed light L4. Here, the distance d from the reflection surface 14a (intersection C1) of the mirror 14 to the condenser lens 15
Since 2 is equal to the focal length of the condenser lens 15, the optical path of the condensed light L4 obtained through the condenser lens 15 is parallel to the optical axis O2 of the condenser lens 15. That is, the condensed light L
4 is emitted along a direction (predetermined direction) parallel to the optical axis O2 of the condenser lens 15.

Then, the condensed light L4 is guided by the dichroic mirror 16, and is reflected on the reflecting surface 16a toward the objective lens 21 (condensed light L5). Dichroic mirror 1
6 is reflected by the objective lens 21.
Is parallel to the optical axis O3. Here, the distance d3 from the condenser lens 15 to the intersection C2 and the distance from the intersection C2 to the objective lens 21
(D3 + d) with the distance d4 to the rear focal plane 21a
4) is equal to the focal length of the condenser lens 15 and the dichroic mirror 16 is arranged at an angle of 45 ° from the optical axes O2 and O3. It coincides with the side focal plane 21a.

Since the traveling direction of the parallel light L3 reflected by the mirror 14 is inclined from the optical axis O2 to the optical axis O1, the focal point P1 of the condensed light L5 is shifted to the rear focal plane 2 of the objective lens 21.
1a, it is located at a peripheral portion separated from the optical axis O3 by a distance d (see FIG. 2). Incidentally, the spot diameter of the condensed light L5 on the rear focal plane 21a is obtained by multiplying the diameter of the light beam emitted from the fiber 12 by the ratio of the focal length of the condenser lens 15 to the focal length of the collimator lens 13. The diameter of the fiber 12 is 50 μm, the focal length of the collimator lens 13 is 100 mm, and the focal length of the condenser lens 15 is 160 m
In the case of m, the spot diameter of the condensed light L5 is 80 μm.

As described above, the light (condensed light L5) condensed on the rear focal plane 21a of the objective lens 21 by the microscope illumination device 10 of the present embodiment is directed to the specimen 31 via the objective lens 21. It is guided and becomes parallel light that illuminates the sample 31. That is, the sample 31 is illuminated by the parallel light passing through the objective lens 21. Here, the specimen 31 is immersed in water 34 held between the cover glass 35 and the slide glass 33 as shown in FIG. In observing such a specimen 31, an oil 32 (refractive index 1.5 at d-line 587 nm) is applied to the objective lens 21.
The oil immersion objective according to 2) is used. Incidentally, the numerical aperture NA of the objective lens 21 is 1.4, and the magnification is 60.
Times or 100 times.

Therefore, the parallel light L 6 having passed through the objective lens 21 is transmitted through the oil 32 to the slide glass 33.
Attempts to enter the water 34 from. However, since the refractive index (1.52) of the slide glass 33 is higher than the refractive index (1.33) of the water 34, when the incident angle θ of the parallel light L6 is larger than the critical angle, the parallel light L6 slides. Glass 3
The light is totally reflected at the boundary surface between 3 and water 34.

In general, the parallel light L6 is emitted from the slide glass 33.
The condition of the incident angle θ at the time of total reflection at the boundary surface between the water and the water 34 is expressed by the following equation (1). Here, n1 is the refractive index of the slide glass 33 (1.52), and n2 is the refractive index of the water 34 (1.33). n1 · sin θ> n2 (1) In the present embodiment, the numerical aperture NA (= n1 · sinα) is 1.4.
Is used, the incident angle θ of the parallel light L6
Satisfies the following relational expression (2), the slide glass 33
Total reflection can occur at the interface between the water and the water 34.

1.33 <(n1 · sin θ) <1.4 (2) By the way, the angle of incidence θ of the parallel light L6 depends on the position of the converging point P1 on the rear focal plane 21a of the objective lens 21 (light Axis O3
Is uniquely determined according to the distance d) from the distance. The relationship between the incident angle θ and the distance d is expressed by the following equation (3). Here, n1 is the refractive index (1.52) of the slide glass 33, and f is the focal length of the objective lens 21.

N1 · sin θ = d / f (3) Therefore, the parallel light L 6 is transmitted to the slide glass 33 and the water 34.
In order to make total reflection at the boundary surface with the lens, the position of the focal point P1 is moved in the rear focal plane 21a of the objective lens 21, and the distance d from the optical axis O3 satisfies the following relational expression (4). It may be adjusted so that 1.33f <d <1.4f (4) Incidentally, the range satisfying the above relational expression (4) is the peripheral portion (the radial width is 0 in the rear focal plane 21a of the objective lens 21). .15mm). As described above, since the spot diameter of the condensed light L5 on the rear focal plane 21a of the objective lens 21 is about 80 μm, the condensing point P1 of the condensed light L5 is set to a range satisfying the above relational expression (4). High precision is required to adjust within.

In the microscope illumination device 10 (FIG. 1) of the present embodiment, the traveling direction of the parallel light L3 is changed according to the rotation angle of the mirror 14, and the condensed light L4 is changed according to the traveling direction of the parallel light L3. The point P2 where the light enters the dichroic mirror 16 can be moved. Further, when the point P2 of the condensed light L4 incident on the dichroic mirror 16 moves,
The optical path of the condensed light L5 reflected by the dichroic mirror 16 also moves.

Since the rotation axis of the mirror 14 is perpendicular to the optical axes O1 and O2, the point P2 on the dichroic mirror 16
Moves along the radial direction of the objective lens 21. For this reason, the optical path of the condensed light L5 reflected by the dichroic mirror 16 also moves in the radial direction.

Note that, even if the traveling direction of the parallel light L3 changes, the condensed light L4 is always emitted along the optical axis O2 of the condensing lens 15. For this reason, the condensed light L5 reflected by the dichroic mirror 16 always travels along the optical axis O3 of the objective lens 21. As described above, in the microscope illumination device 10 of the present embodiment, by adjusting the rotation angle of the mirror 14, the focal point P 1 of the focused light L 5 on the rear focal plane 21 a of the objective lens 21 is moved in the radial direction. Can be done.

Therefore, by adjusting the rotation angle of the mirror 14, the converging point P1 of the condensed light L5 is set within a range satisfying the above-mentioned relational expression (4), that is, within the rear focal plane 21a of the objective lens 21. (The radial width is about 0.15mm)
Can be positioned with high accuracy. At this time, the parallel light L6 guided to the specimen 31 side through the objective lens 21
(FIG. 2) is totally reflected at the boundary surface between the slide glass 33 and the water 34, passes through the objective lens 21 again (return light L
7), proceed to the dichroic mirror 16.

On the other hand, at a portion of the boundary between the slide glass 33 and the water 34 where the parallel light L6 is totally reflected, a part of the light leaks from the boundary to the water 34 side. The specimen 31 immersed in the water 34 is illuminated by the exuded light. By the way, the illumination range is that the magnification of the objective lens 21 is 6
In the case of 0 times, it is about 160 μm. The sample 31 illuminated using the totally reflected parallel light L6 generates fluorescence in all directions. The wavelength of the fluorescence from the specimen 31 is longer than the wavelength of the parallel light L6 (488 nm), and is 515 nm to 550 n.
m. The fluorescence from the specimen 31 is
And is condensed to become parallel observation light L8 (FIG. 1).

The observation light L8 is transmitted through the dichroic mirror 16 and then guided to the band pass filter 17, and is transmitted through the band pass filter 17 so as to pass through the microscope.
(Not shown). Therefore, image plane 3
An image (specimen image) is formed on 1A by the fluorescence (observation light L8) generated from the specimen 31.

The return light L7 that has passed through the objective lens 21 is reflected by the dichroic mirror 16. Further, even if there is a component of the return light L7 transmitted through the dichroic mirror 16, the component is blocked by the bandpass filter 17. As described above, in the microscope illumination device 10 of the present embodiment, the dichroic mirror 16 and the bandpass filter 17 are provided in the optical path of the fluorescence (observation light L8) generated from the sample 31, and the wavelength is selected. The return light L7 and the reflected light on the lens surface used in the above are not mixed with the weak observation light L8 to reach the imaging lens 23. Therefore, a sample image with a large S / N ratio and very good contrast is obtained on the image plane 31A.

Further, the illumination device 1 for a microscope according to the present embodiment.
At 0, the focus point P1 of the condensed light L5 is moved within the rear focal plane 21a of the objective lens 21 by rotation of the mirror 14 (that is, the incident angle θ of the parallel light L6 passing through the objective lens 21 is adjusted). Therefore, highly accurate positioning (adjustment) can be easily performed. Normally, since the objective lenses 21 having different magnifications have different focal lengths, when the objective lens 21 is replaced with one having a different magnification, the total reflection condition of the parallel light L6 (the above relational expression (4)) is used. The focus point P1 of the condensed light L5 (the incident angle θ of the parallel light L6) must be readjusted.

In the microscope illumination device 10 of the present embodiment,
Since high-precision adjustment can be easily performed by rotating the mirror 14, the magnification of the objective lens 21 can be easily changed, and illumination of the sample 31 using the parallel light L6 that is totally reflected can be easily realized. The replacement of the objective lens 21 is performed using the body surface 22 as a reference surface. Next, an inverted microscope 20 incorporating the above-described illumination device 10 for a microscope will be briefly described. As shown in FIG. 3, in the inverted microscope 20, a mirror 24 is arranged between the imaging lens 23 and the image plane 31A. The sample image formed on the image plane 31A via the mirror 24 is transferred to the image plane 31B via the relay optical system 25.
Alternatively, it is guided to the image plane 31C. The relay image formed on the image plane 31B is picked up by an image pickup tube (a high-sensitivity image pickup tube with an image intensifier) 26 or a TV camera. The relay image formed on the image plane 31C is the binocular unit 2
7 allows visual observation.

In the inverted microscope 20, another illuminating device 40 for transmitting and illuminating the sample 31 is arranged on the opposite side of the objective lens 21 with the sample 31 interposed therebetween. This lighting device 4
Reference numeral 0 denotes a lamp light source 41, a collector lens 42, a diffusion plate 43, an aperture 44, and a condenser lens 45. When the specimen 31 is transmitted and illuminated using the illuminating device 40, the microscope illumination device 10 of the present embodiment, which illuminates the sample 31 with incident light, is excluded from the optical path of the observation light L8.

In the above-described embodiment, the return light L7 is blocked by the dichroic mirror 16 and the bandpass filter 17, but a stopper may be provided on the optical path of the return light L7 to block the return light L7. In that case, a half mirror (semi-transparent mirror) can be used instead of the dichroic mirror 16. Further, the band pass filter 17 can be omitted.

Further, in the above embodiment, the return light L7 is blocked by the dichroic mirror 16 and the bandpass filter 17, but if a high-performance bandpass filter 17 can be prepared, a half mirror is used instead of the dichroic mirror 16. If a high-performance dichroic mirror 16 can be prepared, the bandpass filter 17 may be omitted.

In the above-described embodiment, the configuration (the dichroic mirror 1) for observing the fluorescence of the specimen 31 is used.
6, having a band pass filter 17).
When observing light diffusely reflected by the sample 31, a half mirror may be used instead of the dichroic mirror 16. Further, the band pass filter 17 can be omitted.

Further, in the above embodiment, the parallel light from the laser light source 11 is diffused by the fiber 12, but a diffuser may be used instead of the fiber 12. In this case, by disposing the diffusion plate on the end face 12a,
The collimated lens 13 can emit the parallel light L2 whose diameter is enlarged. Further, a beam expander may be used instead of the fiber 12 (diffusion plate) and the collimator lens 13 to increase the diameter of the parallel light from the laser light source 11. The larger the diameter of the parallel light L2, the wider the illumination range for the sample 31 can be.

In the above-described embodiment, the example in which the microscope illumination device 10 is incorporated in the microscope for observing the sample 31 in the state of being immersed in the water 34 has been described. Can also be applied to a microscope for observing in the air. In this case, the total reflection condition of the parallel light L6 (the above relational expression (4)) may be obtained by replacing 1.33 corresponding to the refractive index of water 34 with 1.0 corresponding to the refractive index of air. The present invention is applicable not only to the oil immersion objective lens but also to a water immersion objective lens (the oil immersion objective lens and the water immersion objective lens are collectively referred to as a liquid immersion objective lens).

Further, the illumination device 1 for a microscope according to the present embodiment
0 was incorporated into the inverted microscope 20 (FIG. 3).
However, it can also be incorporated into an upright microscope. In order to simplify the configuration, the collimator lens 13 and the condenser lens 1 are used.
5 is a parallel system, but may be a non-parallel system.

[0043]

As described above, according to the illumination device for a microscope according to any one of the first to fourth aspects, the position of the condensing point of the condensed light in the rear focal plane of the objective lens is determined by the reflection optics. Since the adjustment is made by the rotation of the system, the angle of incidence of light (illumination light to the sample) that has passed through the objective lens and guided to the sample can be easily adjusted with high accuracy.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a microscope illumination device 10 of the present embodiment.

FIG. 2 shows an objective lens 21 provided by a microscope illumination device 10.
The state where the light (L5) condensed on the rear focal plane 21a is guided to the sample 31 side through the objective lens 21 and the illumination of the sample 31 using the parallel light (L6) totally reflected will be described. FIG.

FIG. 3 is a diagram showing a configuration of the inverted microscope 20 and a state where the microscope illumination device 10 is incorporated in the inverted microscope 20.

FIG. 4 is a diagram illustrating an example of a conventional lighting device.

FIG. 5 is a diagram showing another example of a conventional lighting device.

[Explanation of symbols]

 Reference Signs List 10 Illumination device for microscope 11 Laser light source 12 Fiber 13 Collimating lens 14, 24 Mirror 15 Condensing lens 16 Dichroic mirror 17 Bandpass filter 20 Inverted microscope 21 Objective lens 22 Body-mounted surface 23 Imaging lens 31 Sample 32 Oil 33 Slide glass 34 Water 35 Cover glass

Claims (4)

[Claims] An emission unit that emits condensed light along a predetermined direction that intersects an optical axis of the objective lens; and a light source that emits the condensed light from the emission unit along an optical axis of the objective lens. An optical member that reflects in the direction of the objective lens and transmits observation light generated from a sample and passing through the objective lens; The emission unit is disposed so as to coincide with a rear focal plane of the objective lens. The emission unit includes a laser light source, an enlargement optical system that enlarges a diameter of light emitted from the laser light source, and a diameter that is increased by the enlargement optical system. A reflecting optical system that reflects the enlarged light, and a condensing optical system that condenses the light reflected by the reflecting optical system, wherein the condensing optical system has an optical axis parallel to the predetermined direction. The reflection optical system, the optical axis of the condensing optical system and the Microscope illuminating device, characterized in that is rotatable about an axis perpendicular to the optical axis of a large optical system. 2. The illumination device for a microscope according to claim 1, wherein the optical member reflects light in a wavelength region of the condensed light and transmits light in a wavelength region longer than the wavelength region. And a lighting device for a microscope. 3. The illumination device for a microscope according to claim 1, wherein the bandpass filter blocks light in a wavelength region of the condensed light in an optical path of the observation light transmitted through the optical member. An illumination device for a microscope, comprising: 4. The illumination device for a microscope according to claim 1, wherein a distance from the reflection optical system to the light collecting optical system is equal to a focal length of the light collecting optical system. An illumination device for a microscope, characterized by being equal.