JP3339244B2 - Epi-fluorescence microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an epi-fluorescence microscope,
More particularly, the present invention relates to a microscope including an epi-illumination fluorescent illumination device and optical tweezers.

[0002]

2. Description of the Related Art As an observation method of a microscope, there is an epifluorescence observation method in which a short wavelength excitation light from a light source such as a mercury lamp is illuminated on an observation field of view on a sample, and long wavelength fluorescence emitted from the sample is observed. . Specifically, a dichroic mirror arranged in the parallel light beam path of the observation optical system from the objective lens to the enlarged image reflects excitation light of a short wavelength from a mercury lamp or the like, and the reflected light is sampled through the objective lens. To illuminate. Recently, an optical tweezer method using laser light has been known as a method for optically capturing a minute object such as a cell or a chromosome.

[0003] The principle of the optical tweezers method will be described below. In the optical tweezers method, an intense laser beam is focused on a sample via an objective lens having a large numerical aperture. At this time, laser light refraction occurs at the interface between the bead and water due to the difference in the refractive index between a minute object such as a bead in the specimen and a surrounding solution such as water. Utilizing the moment force acting on the beads by the refraction of the laser light, the beads are optically captured near the focal point of the laser light. The trappable object is not limited to beads, but can also be trapped by fine objects in biological specimens such as cells and chromosomes. In a microscope, various applications using the optical tweezers method can be considered.

In a conventional epi-fluorescence microscope incorporating optical tweezers, a laser beam is made incident on a sample using an optical path of an excitation light irradiation optical system for irradiating the sample with excitation light for epi-fluorescence observation. . That is, another second dichroic mirror is provided in the optical path between the light source for supplying the excitation light and the first dichroic mirror disposed in the optical path of the observation optical system, and the laser is transmitted through the second dichroic mirror. Light is taken into the optical path of the excitation light irradiation optical system.

[0005]

In the above-described conventional epi-illumination fluorescence microscope, the first dichroic mirror arranged in the optical path of the observation optical system is such that short-wavelength light such as excitation light and laser light are used. It is necessary to have a special property that both long-wavelength light is reflected and fluorescence from the specimen is transmitted. For example, when fluorescence observation is performed using the UV excitation method, light (excitation light) of 340 to 390 nm is used.
The first dichroic mirror must have a special property of reflecting light (laser light) of 800 nm or more and transmitting light (fluorescence) of 400 to 700 nm. This makes it difficult to manufacture the first dichroic mirror, and increases the manufacturing cost.

In a conventional epi-illumination fluorescence microscope in which optical tweezers are not incorporated, an excitation filter for selecting a wavelength of excitation light and a first dichroic mirror constitute a filter cassette which can be exchanged. And
When epifluorescence observation is performed by changing the excitation method, the first dichroic mirror and the excitation filter are integrally exchanged by exchanging an appropriate filter cassette.

However, in the above-described conventional epi-illumination fluorescence microscope, the excitation filter must be arranged closer to the excitation light source than the second dichroic mirror. Therefore, for example, in a case where epi-fluorescence observation is performed by changing from the UV excitation method to another excitation method such as the B excitation method, the first dichroic mirror and the excitation filter that are arranged apart from each other must be individually exchanged. No. As a result, not only is the configuration of the microscope complicated, but also the operability is poor.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and provides an epi-fluorescence microscope capable of performing epi-fluorescence observation and optical tweezers without using a dichroic mirror having special characteristics. The purpose is to:

[0009]

In order to solve the above-mentioned problems, in the present invention, an excitation light irradiation optical system for irradiating an excitation light for epi-illumination fluorescence observation to a sample, and an excitation light from the sample for the excitation light are provided. An observation optical system for forming an image of the fluorescence through an objective lens and observing an image of the specimen; and condensing a laser beam at a predetermined position of the specimen to optically move an object in the specimen at the predetermined position. In an epi-illumination fluorescence microscope provided with optical tweezers means for capturing, the excitation light irradiation optical system is disposed in a first light source for supplying the excitation light and an optical path of the observation optical system, and A first wavelength separation unit that guides excitation light from one light source to the sample and guides fluorescence from the sample to an optical path of the observation optical system, wherein the optical tweezers unit is configured to supply the laser light. The second light source and the front Disposed in the optical path of the observation optical system, a second that guides the laser light from the second light source to the specimen leads to fluorescence from the specimen to the optical path of the observation optical system
Provided is an epi-illumination fluorescence microscope characterized by having wavelength separation means.

According to a preferred aspect of the present invention, there is provided an optical path dividing means disposed in an optical path of the observation optical system for extracting a part of the fluorescent light from the sample, and the fluorescent light extracted through the optical path dividing means. A photographing optical system having photographing means for photographing the image of the specimen formed by the photographing apparatus.

[0011]

In the epi-fluorescence microscope of the present invention, the sample is irradiated with excitation light for epi-fluorescence observation through the first wavelength separation means such as a dichroic mirror disposed in the optical path of the observation optical system. The sample is irradiated with laser light for optical tweezers via a second wavelength separating means such as another dichroic mirror arranged in the optical path of the system. Specifically, for example, excitation light for fluorescence observation is totally reflected by the first dichroic mirror and guided into the optical path of the observation optical system, and laser light for optical tweezers is totally reflected by the second dichroic mirror for observation. Guide into the optical path of the optical system.

In this manner, the excitation light for fluorescence observation and the laser light for optical tweezers can be guided onto the sample via the objective lens with high irradiation efficiency without using a dichroic mirror having special characteristics. As a result, it is possible to minimize the output of laser light required for optically capturing a fine object in the sample. Alternatively, an optical path splitting unit such as a half prism may be arranged in the optical path of the observation optical system, and an image of a specimen formed by the fluorescent light taken out through the half prism may be taken by an imaging unit such as a video camera.

[0013]

Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 is a diagram schematically illustrating a configuration of an epi-illumination fluorescence microscope according to an embodiment of the present invention. The illustrated apparatus is an inverted microscope, and an objective lens 2 is arranged below the specimen 1 to be observed in the figure. Then, epi-illumination fluorescent illumination is performed from below the objective lens 2. The light beam from the epifluorescence light source 11 is condensed by the collector lens 12 to form an image once, and then enters the dichroic mirror DM1 via the relay lens 13 and the excitation filter EX1.

A field stop FS is arranged at an image forming position between the collector lens 12 and the relay lens 13. The dichroic mirror DM1 is arranged in the parallel light beam path of the relay lens system (3, 3 ') of the observation optical system of the microscope. The light beam for epi-illumination reflected upward by the dichroic mirror DM1 in the figure illuminates the observation field of the specimen 1 via the objective lens 2. The sample 1 and the field stop FS1 are at optically conjugate positions, and the observation field region is defined depending on the shape and size of the opening of the field stop FS.

In this embodiment, a UV excitation method is used, and the excitation filter EX1 has a characteristic of transmitting light having a wavelength of 340 to 390 nm, that is, excitation light. On the other hand, the dichroic mirror DM1 has a characteristic of reflecting light having a wavelength of 400 nm or less and transmitting light having a wavelength of 400 nm or more. As described above, the light source 11, the collector lens 12, the field stop FS, the relay lens 13, the excitation filter EX1, the dichroic mirror DM1, and the objective lens 2 constitute an excitation light irradiation optical system of the epi-fluorescence microscope.

As described above, the fluorescence having a wavelength exceeding 400 nm among the fluorescences generated in the sample irradiated with the excitation light from the light source 11 is condensed by the objective lens 2 and then transmitted through the relay lens system (3, 3 '). Dichroic mirror DM1
Through. Further, the light having a wavelength of 40 nm is transmitted through a barrier filter BA1 that transmits light having a wavelength of 420 nm or more.
Less than zero fluorescence is removed. Thus, after the light harmful to observation is removed, the fluorescence from the sample 1 enters the mirror 4 via the half prism 15. The light reflected obliquely to the upper left in the figure at the mirror 4 forms an enlarged image 5.

The light from the magnified image 5 is re-imaged via the relay lens system 6 and the prism 7, and an image 8 is formed.
The image 8 is observed by the observer's naked eye 10 via the eyepiece 9. As described above, the objective lens 2, the relay lens system (3, 3 '), the mirror 4, the relay lens system 6, the prism 7, and the eyepiece 9 constitute an observation optical system of the microscope. FIG. 2 shows the spectral transmittance characteristics of the dichroic mirror DM1, the excitation filter EX1, and the barrier filter BA1 for the UV excitation method in the present embodiment.

As shown by a broken line in FIG. 1, the dichroic mirror DM1, the excitation filter EX1, and the barrier filter BA1 form a filter cassette FC which is integrally insertable into and removable from the optical path of the observation optical system. are doing. The filter cassette FC is appropriately replaced with a filter cassette for another excitation method such as a B excitation method, a V excitation method, and a G excitation method.
Therefore, a desired excitation method can be selected with good operability only by exchanging the filter cassette that can be integrally inserted into and removed from the optical path of the observation optical system.

For example, when the B excitation method is used, a filter cassette F including a dichroic mirror DM1 ', an excitation filter EX1' and a barrier filter BA1 '.
Replace with C '. Here, the dichroic mirror DM
1 ′ reflects light having a wavelength of 510 nm or less, and 510n
It has the property of transmitting light exceeding m. In addition, the excitation filter EX1 ′ has a characteristic of transmitting excitation light having a wavelength of 410 to 460 nm. Further, the barrier filter BA
1 ′ has a property of transmitting light having a wavelength of 520 nm or more. The filter cassette F for the B excitation method
FIG. 3 shows the spectral transmittance characteristics of the dichroic mirror DM1 ', the excitation filter EX1', and the barrier filter BA1 'constituting C'.

The epi-fluorescence microscope of FIG. 1 further includes optical tweezers for optically capturing a fine object in the specimen 1. The optical tweezer means has, for example, a wavelength of 1.
It has a NdYAG laser light source 14 that emits 064 nm laser light. The laser light from the light source 14 is incident on a dichroic mirror DM2 arranged in the parallel light beam path of the relay lens system (3, 3 ') of the observation optical system. The dichroic mirror DM2 has a property of transmitting light having a wavelength of 800 nm or less and reflecting light having a wavelength of more than 800 nm so as to transmit visible light and reflect infrared light. FIG. 4 shows the spectral transmittance characteristics of such a dichroic mirror DM2.

The laser beam reflected upward in the figure by the dichroic mirror DM2 is applied to the barrier filters BA1 and BA1.
The light passes through the dichroic mirror DM1 and enters the objective lens 2. The laser light incident on the objective lens 2 is condensed and forms a laser spot at a predetermined position on the specimen 1. Thus, a minute object in the sample 1 can be optically captured near the laser focal point by the optical tweezers method. As described above, the laser light source 14, the dichroic mirror DM2, and the objective lens 2 constitute optical tweezers.

In this embodiment, an optical path dividing means such as a half prism 15 is provided in the optical path of the observation optical system. Therefore, a part of the fluorescence from the sample 1 is extracted from the observation optical system via the half prism 15, and the extracted fluorescence forms an enlarged image 5 ′ of the sample 1. In this manner, an enlarged image 5 'of the specimen 1 formed in the so-called side port can be photographed by photographing means such as a video camera (not shown).

In the above embodiment, the present invention is described by taking an inverted microscope as an example. However, the present invention can be applied to a general upright microscope. In the above embodiment, the dichroic mirror DM for fluorescence observation is used.
Although 1 is arranged on the specimen side with respect to the dichroic mirror DM2 for optical tweezers, the arrangement of these two dichroic mirrors may be reversed. Further, in the above-described embodiment, the configuration according to the UV excitation method is shown as the epi-fluorescence method, but another appropriate excitation method may be used.

Further, in the above-described embodiment, an example is shown in which a dichroic mirror that reflects excitation light and transmits fluorescence or a dichroic mirror that reflects laser light and transmits fluorescence is used. However, a dichroic mirror that transmits excitation light and reflects fluorescence or a dichroic mirror that transmits laser light and reflects fluorescence may be used. Instead of the dichroic mirror, a dichroic prism or other appropriate wavelength separating means can be used.

[0025]

As described above, according to the present invention, the excitation light for fluorescence observation and the laser light for optical tweezers are respectively placed in the optical path of the observation optical system via wavelength separation means such as individual dichroic mirrors. I'm taking it in. Therefore, the excitation light and the laser light can be guided onto the sample with high irradiation efficiency without using a dichroic mirror having special characteristics. As a result, the output of the laser beam can be minimized. Further, according to the present invention, a filter cassette in which an excitation filter for fluorescence observation and a dichroic mirror can be exchanged is constituted, and only by exchanging this filter cassette with a filter cassette for another excitation method, a desired excitation method can be achieved. Fluorescence observation with good operability.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a configuration of an epi-illumination fluorescence microscope according to an embodiment of the present invention.

FIG. 2 is a diagram illustrating spectral transmittance characteristics of a dichroic mirror DM1, an excitation filter EX1, and a barrier filter BA1 for a UV excitation method in the present embodiment.

FIG. 3 shows a dichroic mirror DM for the B excitation method
1 ′, excitation filter EX1 ′ and barrier filter BA
It is a figure which shows the spectral transmittance characteristic of 1 '.

FIG. 4 is a dichroic mirror DM2 for optical tweezers.
FIG. 3 is a diagram showing spectral transmittance characteristics of the present invention.

[Explanation of symbols]

 Reference Signs List 1 specimen 2 objective lens 3, 3 'relay lens system 4 mirror 5, 8 image plane 7 prism 9 eyepiece 10 naked eye 11 excitation light source 12 collector lens 14 laser light source 15 half prism EX1 excitation filter BA1 barrier filter DM1 dichroic mirror DM2 dichroic Mirror FC filter cassette FS Field stop

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-4-191720 (JP, A) JP-A-5-2137 (JP, A) JP-A-7-13083 (JP, A) JP-A-58-1983 25613 (JP, A) JP-A-5-296914 (JP, A) JP-A-2-91545 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G02B 12/00 G02B 12 / 16 G02B 12/18 G02B 12/32 G02B 12/36

Claims (4)

(57) [Claims] 1. An excitation light irradiating optical system for irradiating a sample with excitation light for epi-fluorescence observation, and forming an image of fluorescence from the sample with respect to the excitation light via an objective lens to observe an image of the sample. An epi-fluorescence microscope comprising: an observation optical system for; and optical tweezers for condensing a laser beam at a predetermined position of the sample and optically capturing an object in the sample at the predetermined position. An excitation light irradiating optical system is disposed in an optical path of the observation optical system, for supplying the excitation light, and guides the excitation light from the first light source to the sample, and emits fluorescence from the sample. And a first wavelength separation unit that guides the laser light to the optical path of the observation optical system, wherein the optical tweezers unit is disposed in an optical path of the observation optical system, and a second light source for supplying the laser light;
An epi-illumination fluorescence microscope comprising: a second wavelength separation unit that guides laser light from the second light source to the sample and guides fluorescence from the sample to an optical path of the observation optical system. 2. An optical path splitting means arranged in an optical path of the observation optical system for extracting a part of the fluorescence from the sample, and an image of the sample formed by the fluorescent light extracted via the optical path splitting means. 2. A photographing optical system further comprising photographing means for photographing a photograph.
Epi-fluorescence microscope according to 1. 3. The epi-fluorescence microscope according to claim 1, wherein the first wavelength discriminating means and the two wavelength discriminating means are arranged in a parallel light beam path of the observation optical system. 4. The first light source has an excitation filter for selectively transmitting the excitation light, and the first wavelength separation unit and the excitation filter are arranged with respect to an optical path of the observation optical system. The epi-illumination fluorescence microscope according to any one of claims 1 to 3, wherein the epi-fluorescence microscope is formed integrally and detachably.