JPH11149046A - Epi-illumination type fluorescent illumination device for microscope and filter unit thereof - Google Patents

Abstract

(57) [PROBLEMS] To provide an inexpensive epi-illumination type fluorescent lighting device that does not move an image due to switching of a filter unit and that does not require any trouble in manufacturing, and a filter unit thereof. A light source that emits illumination light, a collector lens that collects the illumination light, excitation filters 11a to 11c that transmit light of a desired wavelength in the illumination light, and light that has passed through the excitation filters 11a to 11c. A dichroic mirror 12 that reflects the light and transmits the fluorescence excited by the sample;
In an epi-illumination type fluorescent illumination device for a microscope comprising an absorption filter 13 that transmits only fluorescence, a dichroic mirror 12 is formed on one glass blank 20, and a plurality of thin films formed on the glass blank 20 and having mutually different wavelength selection characteristics. 20a to 20c, the absorption filter 13 is formed by adding one glass blank 30 and a plurality of thin films 30a to 30c formed on the glass blank 30 and having mutually different wavelength selection characteristics.
0c, 30a to 30c can be selectively inserted into the optical path of the microscope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an epi-illumination type fluorescent illumination device for a microscope and a filter unit therefor.

[0002]

2. Description of the Related Art An epi-illumination type fluorescent illuminating device for a microscope comprises a light source for emitting illumination light, a collector lens for condensing the illumination light,
An excitation filter that transmits light of a desired wavelength among the illumination light, a dichroic mirror that reflects light passing through the excitation filter, and transmits fluorescence excited by the sample, and an absorption filter that transmits only the fluorescence. Is provided.

In recent years, in observation of a fluorescence image with a fluorescence microscope, fluorescence observation using excitation light having a plurality of wavelengths has been frequently performed.

For example, an excitation filter, a dichroic mirror, and an absorption filter are used as filter units, a plurality of filter units are mounted on a slider or the like, and the sliders are moved to selectively place each filter unit on the optical path of the objective lens. I can do it.

FIG. 4 is a perspective view of a conventional filter unit.

[0006] The filter units 110A, 110B, 1
10C is linearly mounted on one slider (not shown).

[0007] Filter units 110A, 110B, 1
Illumination light emitted from the light source 101 can enter the 10C via the collector lens 102 and the heat ray absorption filter 103.

The filter unit 110A includes an excitation filter 111a, a dichroic mirror 112a, and an absorption filter 113a.

Similarly, the filter unit 110B includes an excitation filter 111b, a dichroic mirror 112b, and an absorption filter 113b.
10C includes an excitation filter 111c, a dichroic mirror 112c, and an absorption filter 113c.

The slider moves linearly in a plane perpendicular to the optical path L of the objective lens (not shown), and
10A, 110B and 110C can be moved linearly as shown by arrow a.

Therefore, the filter units 110A,
110B and 110C can be selectively arranged on the optical path L of the objective lens, and it becomes possible to observe a fluorescent image by a plurality of excitation lights.

Although not shown, a disk is provided in a plane perpendicular to the optical path of the objective lens, a plurality of filter units are arranged on the same circumference of the disk, and this disk is parallel to the optical path of the objective lens. The configuration may be such that the filter unit is switched by rotating the filter unit about an appropriate axis.

[0013]

Incidentally, the dichroic mirrors 11 each made of separate flat glass.
2a, 112b, 112c and the absorption filter 113a,
Since 113b and 113c have a manufacturing error, there is a problem that the parallelism of the front and back surfaces changes every time the filter units 110A, 110B and 110C are switched, and the image forming position in the eyepiece lens field of view changes.

FIG. 5 is a diagram showing a cross-sectional shape of the flat glass.

When a flat glass having a parallelism error between the front and back surfaces shown in FIG. 5 is inserted into the optical path, the mutual deflection angle γ between the light beam incident on the flat glass and the light beam emitted therefrom is γ = sin (n · sin α) −α (1) Here, α is the parallelism error between the front and back surfaces of the flat glass, and n is the refractive index of the flat glass.

Since the dichroic mirror and the absorption filter are arranged on the optical path between the objective lens and the eyepiece (see FIG. 1), the declination becomes 2.multidot..gamma.

Further, when the filter unit is switched to another filter unit (for example, when the filter unit is switched from the filter unit 110a to the filter unit 110b), the declination between the filter units is 4 · γ at the maximum.

In the equation (1), α = 10 ′, n = 1.
If 52, γ = 5 ′, so that the declination associated with the switching of the filter unit is 20 ′ at the maximum.

Normally, since the space between the objective lens and the lens barrel is afocal, the moving amount δ of the image in the field of the eyepiece due to the switching of the filter unit is δ = f · 4 · γ (2) Is required. Here, f is the focal length of the lens barrel (see FIG. 1).

In the equation (2), when f = 200 mm, δ is 0.9 mm, and it is difficult to accurately grasp the positions of the two fluorescences in the cell.

In order to reduce the amount of movement δ, it is necessary to reduce the parallelism error between the front and back surfaces of the flat glass constituting the dichroic mirror and the absorption filter, but it takes time and effort to manufacture and increases the manufacturing cost. There is a problem.

The present invention has been made in view of such circumstances, and an object thereof is to provide an inexpensive epi-illumination type fluorescent lighting device which does not require movement of an image due to switching of a filter unit and which does not require much time for manufacturing, and a filter unit therefor. It is to provide.

[0023]

In order to solve the above-mentioned problems, the invention according to claim 1 comprises a light source that emits illumination light, a collector lens that collects illumination light, and a desired wavelength of the illumination light. An incident-light fluorescent illumination device for a microscope, comprising: an excitation filter that transmits light; a dichroic mirror that reflects light passing through the excitation filter and transmits fluorescence excited by the sample; and an absorption filter that transmits only the fluorescence. Wherein the dichroic mirror is a single first mirror.
, And a plurality of thin films formed at different positions on the first light-transmitting substrate and having different wavelength selection characteristics, wherein the absorption filter is a single second light-transmitting substrate. And a plurality of thin films formed at different positions on the second light-transmitting substrate and having mutually different wavelength selection characteristics. The dichroic mirror and the absorption filter selectively apply the plurality of thin films, respectively. And is configured to be insertable into a microscope optical path.

The dichroic mirror is composed of one first light-transmitting substrate, and a plurality of thin films formed at different positions on the first light-transmitting substrate and having mutually different wavelength selection characteristics. Is composed of one second light-transmitting substrate and a plurality of thin films formed at different positions on the second light-transmitting substrate and having mutually different wavelength selection characteristics,
Since the dichroic mirror and the absorption filter are each configured so that a plurality of thin films can be selectively inserted into the optical path of the microscope, the optical axis due to the parallelism error between the front and back surfaces of the first light transmitting substrate and the second light transmitting substrate. Does not occur. Further, the dichroic mirror and the absorption filter can be easily manufactured.

According to a second aspect of the present invention, there is provided an excitation filter that transmits light of a desired wavelength out of illumination light, and a dichroic that reflects light passing through the excitation filter and transmits fluorescence excited by a sample. In a filter unit of an epi-illumination type fluorescent lighting device including a mirror and an absorption filter that transmits only the fluorescent light, the dichroic mirror includes one first light-transmitting substrate and different positions on the first light-transmitting substrate. And a plurality of thin films having wavelength selection characteristics different from each other, wherein the absorption filter is formed in one second light-transmitting substrate and at different positions on the second light-transmitting substrate. , And a plurality of thin films having mutually different wavelength selection characteristics.

The dichroic mirror is composed of one first light-transmitting substrate and a plurality of thin films formed at different positions on the first light-transmitting substrate and having mutually different wavelength selection characteristics. Is composed of one second light-transmitting substrate and a plurality of thin films formed at different positions on the second light-transmitting substrate and having mutually different wavelength selection characteristics. No declination of the optical axis occurs due to a parallelism error between the front and back surfaces of the substrate and the second translucent substrate. Further, the dichroic mirror and the absorption filter can be easily manufactured.

According to a third aspect of the present invention, in the epi-illumination type fluorescent illuminating apparatus for a microscope according to the first aspect, a housing for integrally fixing the dichroic mirror and the absorption filter is further provided. I do.

[0028] Since there is further provided a housing for integrally fixing the dichroic mirror and the absorption filter, the dichroic mirror and the absorption filter can be securely held.

The term "thin film" described in this specification includes a film formed by laminating a plurality of thin films having different characteristics in order to transmit (or reflect) light in a desired wavelength range.

[0030]

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

FIG. 1 is a configuration diagram showing an optical system of a fluorescence microscope using an epi-illumination type fluorescent lighting device according to a first embodiment of the present invention, and FIG. 2 is a perspective view of a filter unit of the epi-illumination type fluorescent lighting device.

The optical system of the fluorescence microscope includes a light source 1, a collector lens 2, a filter unit 10, and an objective lens 4.
, A lens barrel 5, a prism 6, and an eyepiece 7.

The filter unit 10 includes a heat ray absorbing filter 3, an excitation filter 11, and a dichroic mirror 12.
And an absorption filter 13 and are integrally fixed to the housing 15.

The excitation filter 11 includes three excitation filters 1
1a, 11b, and 11c.
Reference numerals a, 11b, and 11c are provided at predetermined intervals on the side surface of the housing 15 orthogonal to the optical path of the illumination optical system from the light source 1. Each of the excitation filters 11a, 11b, and 11c excites excitation light having a different wavelength.

The dichroic mirror 12 is provided at an angle of 45 ° with respect to the excitation filters 11a, 11b, 11c and the absorption filter 13, and is arranged so as to be insertable into and removable from the optical path of the objective lens 4. The dichroic mirror 12 reflects the excitation light and transmits the fluorescence.

The dichroic mirror 12 includes one glass blank (first light-transmitting substrate) 20 and three thin films 20a, 20b, and 20c formed on the glass blank 20 and having mutually different wavelength selection characteristics. You.

The three thin films 20a, 20b, 20c are provided on the surface of the glass blank 20 with the excitation filters 11a, 11b,
11c are formed at the same interval.

The thin films 20a, 20b, and 20c are formed by evaporating a metal compound such as magnesium fluoride or aluminum oxide, and have different wavelength selection characteristics depending on the type of metal to be evaporated.

The absorption filter 13 is provided on the upper surface of the housing 15 and is arranged so as to be insertable into and removable from the optical path of the objective lens 4 together with the dichroic mirror 12. This absorption filter 1
3 cuts the excitation light mixed with the fluorescence.

The absorption filter 13 is composed of one glass blank (second light transmitting substrate) 30 and three thin films 30 formed on the glass blank 30 and having different wavelength selection characteristics.
a, 30b, and 30c.

The three thin films 30a, 30b, 30c are provided on the surface of the glass blank 30 with the excitation filters 11a, 11b,
11c are formed at the same interval.

The thin films 30a, 30b, and 30c are formed by evaporating a metal compound, such as magnesium fluoride or aluminum oxide, and have different wavelength selection characteristics depending on the type of metal to be evaporated.

Next, the operation of the epi-illumination type fluorescent illumination device of the microscope will be described with reference to FIGS.

The illumination light emitted from the light source 1 is condensed by the collector lens 2, and the long-wavelength light serving as a heat source is cut off by the heat ray absorbing filter 3, and then enters the filter unit 10.

As for the illumination light, only the excitation light having a desired wavelength is transmitted by the excitation filter 11a and is incident on the thin film 20a of the dichroic mirror 12.

In the thin film 20 a, only the excitation light having a desired wavelength is further deflected by 90 ° and guided to the objective lens 4. The excitation light having passed through the objective lens 4 illuminates a sample 8 placed on the optical path of the objective lens 4.

The excitation light excites the fluorescent substance in the sample 8 and generates fluorescence having a longer wavelength than the excitation light.

The fluorescent light is guided again to the objective lens 4 through the reverse optical path together with the excitation light. The fluorescence that has entered the objective lens 4 enters the thin film 20a of the dichroic mirror 12, passes through the thin film 20a, and enters the absorption filter 30a.

In the absorption filter 30a, the excitation light mixed with the fluorescent light is cut off, and only the fluorescent light enters the eyepiece 7 through the lens barrel 5 and the prism 6, and is observed as a fluorescent image.

When illuminating the sample 8 with excitation light of another wavelength, the housing 15 is moved in a plane orthogonal to the optical path of the objective lens 4 to switch the filter unit 10.

For example, when using the excitation filter 11b, the filter unit 10 (casing 15) is moved in the direction shown by the arrow b so that the excitation filter 11b and the optical path of the illumination optical system are made to coincide.

With the switching of the filter unit 10, the thin film 20b of the dichroic mirror 12 and the thin film 30b of the absorption filter 13 corresponding to the excitation filter 11b
Is set on the optical path of the objective lens 4.

Further, when illuminating the sample 8 with excitation light of another wavelength, the filter unit 10 may be moved in the direction shown by the arrow c so that the excitation filter 11c and the optical path of the illumination optical system coincide.

According to this embodiment, the thin films 20a, 20a
Since b, 20c, 30a, 30b, and 30c are each constituted by one dichroic mirror 12 and glass blanks 20 and 30 of the absorption filter 13, high-precision processing for obtaining parallelism between the glass blanks 20 and 30 is performed. The filter unit 10 can be manufactured at a low cost without the necessity, and the declination γ becomes constant,
The movement of the image within the eyepiece field of view due to the switching of 0 is eliminated.

FIG. 3 is a diagram showing an optical system of a confocal microscope using an epi-illumination type fluorescent illumination device according to a second embodiment of the present invention. Is omitted.

The optical system of the confocal microscope includes a light source 5
1, a relay lens 52, and a dichroic mirror 12
XY scanner 53, objective lens 4, collimating lens 54, absorption filter 13, collector lens 5
5 and a fluorescence detector 56.

The light source 51 is a laser light source having a built-in excitation filter, and irradiates the sample 8 placed on the stage 60. As the light source 51, a multi-line type light source that can emit a plurality of laser beams is used.

The XY scanner 53 two-dimensionally scans the sample 8 with the excitation laser beam at a predetermined scanning speed.

The objective lens focuses the laser beam reflected by the XY scanner 53 on the sample.

The collector lens 55 is disposed between the absorption filter 13 and the fluorescence detector 56 and collects the fluorescence.

The fluorescence detector 56 is arranged at a position conjugate with the focal plane of the objective lens 4, detects the fluorescence passing through the collector lens 55, and converts it into an electric signal representing the light intensity.

The dichroic mirror 12 and the absorption filter 13 are placed on the optical path of the objective
And the two thin films 20a, 20b, 20
c, 30a, 30b, and 30c can be independently switched on the optical path of the objective lens 4.

The operation of the confocal microscope having the above configuration will be described with reference to FIG.

The excitation laser beam is transmitted through the relay lens 52 and is reflected by the thin film 20a of the dichroic mirror 12. This laser light is two-dimensionally scanned by the XY scanner 53 and guided to the objective lens 4. The laser light that has passed through the objective lens 4 is applied to a sample 8 placed on the optical path of the objective lens 4 and excites a fluorescent substance in the sample 8 to generate fluorescence.

The fluorescent light is emitted from the objective lens 4 together with the excitation laser light.
From the laser beam to the XY scanner 53, and is separated from the excitation laser beam by the thin film 20a of the dichroic mirror 12.

The fluorescent light transmitted through the thin film 20a passes through the collimator lens 54 and the absorption filter 13 and the collector lens 55
And is detected by the fluorescence detector 56.

When illuminating the sample 8 with excitation light of another wavelength, for example, the dichroic mirror 12 is
Is moved in a direction orthogonal to the optical path of the objective lens 4 and the thin film 20a located on the optical path of the objective lens 4 is switched to the thin film 20b.

According to the second embodiment, the dichroic mirror 12 and the absorption filter 13 can be independently switched on the optical path of the objective lens 4, so that three dichroic mirrors 12 and three absorption filters 13 are provided. Thin films 20a, 20b, 20c and thin film 30
When a, 30b, and 30c are formed, nine combinations can be performed, and many fluorescent observations can be performed with a small number of dichroic mirrors 12 and the like.

In the first embodiment, the excitation filter 11
Although a, 11b and 11c are separate bodies, they may be formed of three thin films on one glass blank like the dichroic mirror 12 and the absorption filter 13.

[0070]

As described above, according to the epi-illumination type illumination device for a microscope according to the first aspect of the present invention, the light due to the parallelism error between the front and back surfaces of the first light transmitting substrate and the second light transmitting substrate. When the dichroic mirror and the absorption filter are switched and inserted into the optical path of the microscope, no image shift occurs in the field of view of the eyepiece when there is no declination of the axis. Further, the production of the dichroic mirror and the absorption filter becomes easy, and the production cost is reduced.

According to the filter unit of the epi-illumination device according to the second aspect of the present invention, no declination of the optical axis occurs due to the parallelism error between the front and back surfaces of the first and second light transmitting substrates. When the dichroic mirror and the absorption filter are switched, the image does not move within the eyepiece field.
Further, the production of the dichroic mirror and the absorption filter becomes easy, and the production cost is reduced.

According to the epi-illumination device for a microscope according to the third aspect of the present invention, the dichroic mirror and the absorption filter can be securely held, the switching operation can be performed quickly, and the device is easy to use.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing an optical system of a fluorescence microscope using an epi-illumination type fluorescent lighting device according to a first embodiment of the present invention.

FIG. 2 is a perspective view of a filter unit of the epi-illumination type fluorescent lighting device.

FIG. 3 is a diagram showing an optical system of a confocal microscope using an epi-illumination type fluorescent lighting device according to a second embodiment of the present invention.

FIG. 4 is a perspective view of a conventional filter unit.

FIG. 5 is a cross-sectional view of the flat glass.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Light source 2 Collector lens 11, 11a-11c Excitation filter 12 Dichroic mirror 13 Absorption filter 15 Case 20 Glass blank (1st light transmission board) 20a-20c Thin film 30 Glass blank (2nd light transmission board) 30a-30c Thin film

Claims (3)

[Claims] A light source that emits illumination light, a collector lens that collects the illumination light, an excitation filter that transmits light of a desired wavelength of the illumination light, and a light that passes through the excitation filter is reflected. A dichroic mirror that transmits fluorescence excited by the sample and an epi-illumination type fluorescent illumination device of a microscope including an absorption filter that transmits only the fluorescence, wherein the dichroic mirror includes one first light-transmitting substrate,
A plurality of thin films formed at different positions on the first light-transmitting substrate and having mutually different wavelength selection characteristics, wherein the absorption filter comprises one second light-transmitting substrate;
And a plurality of thin films having different wavelength selection characteristics formed at different positions on the light-transmitting substrate. The dichroic mirror and the absorption filter each selectively insert the plurality of thin films into a microscope optical path. An epi-illumination fluorescent illumination device for a microscope, characterized in that it is configured to be capable of being used. 2. An excitation filter for transmitting light of a desired wavelength of illumination light, a dichroic mirror for reflecting light passing through the excitation filter and transmitting fluorescence excited by a sample, and In a filter unit of an epi-illumination type fluorescent lighting device comprising: an absorption filter that transmits light, the dichroic mirror includes one first light-transmitting substrate;
A plurality of thin films formed at different positions on the first light-transmitting substrate and having mutually different wavelength selection characteristics, wherein the absorption filter comprises one second light-transmitting substrate;
And a plurality of thin films having different wavelength selection characteristics formed at different positions on the light-transmitting substrate. 3. The epi-illumination type fluorescent illumination device for a microscope according to claim 1, further comprising a housing for integrally fixing said dichroic mirror and said absorption filter.