WO2007136075A1

WO2007136075A1 - Fluorescent microscope

Info

Publication number: WO2007136075A1
Application number: PCT/JP2007/060466
Authority: WO
Inventors: Masayuki Syono; Michihiro Nakamura
Original assignee: The University Of Tokushima
Priority date: 2006-05-23
Filing date: 2007-05-22
Publication date: 2007-11-29
Also published as: JPWO2007136075A1; JP4998804B2

Abstract

Provided are a fluorescent microscope having an excellent operability, and an easily portable fluorescent microscope capable of observing an observation target such as a virus on site. The fluorescent microscope comprises a light source for generating an excited light, an illuminating optical system for irradiating a sample placed at an observation position, with the excited light from the light source, an observation system for acquiring the fluorescent light emitted from the sample by the irradiation of the excited light, thereby to form a sample image, and a shielding member disposed to cover an optical path between the objective lens and the sample. The shielding member is a cylindrical member, which is made of an optically transparent synthetic resin for absorbing the light of a predetermined wavelength and which is disposed to cover the generally cylindrical objective lens unit having the objective lens mounted therein.

Description

Specification

Fluorescence microscope

Technical field

The present invention relates to a fluorescence microscope that improves operability and an easily portable fluorescence microscope that can observe observation objects such as viruses on site.

Background art

[0002] Rapid detection of pathogenic viruses is extremely important in the treatment and prevention of infectious diseases. However, virus culture takes a long time and is optically invisible, making rapid detection difficult. As a result, even if a virus infection was suspected, the diagnosis could not be performed on the spot and appropriate processing could not be performed promptly.

[0003] For rapid measurement of virus, a method is known in which an antibody corresponding to the virus to be measured is fluorescently labeled and the virus aggregation is observed in a solution (see Patent Document 1). This method has the feature that an optically invisible virus can be aggregated in a few microliters in a few seconds with a force of several seconds, thereby allowing optical observation.

[0004] In addition, in order to observe a virus by the method described in Patent Document 1, a fluorescence microscope for observing the fluorescence emitted from the observation sample is required, but generally, the fluorescence emitted from the sample is extremely weak. The fluorescent microscope is desired to be used in a dark room where external light is blocked to some extent. For this reason, Patent Document 2 discloses a technique related to a light-shielding member so that stray light around a sample can be reliably blocked, and an observer who looks at an eyepiece can clearly observe fluorescence. Patent Document 1: International Publication No. 2003Z060519

Patent Document 2: JP 2005-345718

Disclosure of the invention

Problems to be solved by the invention

[0005] When there is a suspicion of virus infection, it is effective to perform virus detection on site.

For this purpose, it is preferable to use a virus measuring method described in Patent Document 1 and to provide a fluorescent microscope that is small and light and easy to carry to the site. Also the microscope It is preferable that the fluorescence microscope be capable of performing fluorescence observation even in a dark room and having good operability. In addition, it is desirable that the microscope has a means for transmitting the data of observation images at the site to experts.

[0006] According to the light-shielding member of the fluorescence microscope described in Patent Document 2, the fluorescence observation can be performed well even in a dark room. However, since the inside of the objective lens and the observation sample is completely covered by the light shielding member whose inside cannot be visualized, it is difficult to confirm the distance between the objective lens and the sample. In addition, it was necessary to extend and contract the light shielding member when exchanging samples.

[0007] In addition, in order to realize a fluorescent microscope that is small and light and easy to carry to the site, it is necessary to carefully select and limit functions and components. For example, in a fluorescent microscope using a mercury lamp as a light source, the parts constituting the lens and the optical filter become large. Even eyepieces take up space.

Therefore, the object of the present invention is a fluorescent microscope that is easy to carry with a small size and light weight that solves the above-mentioned problems, and has good operability, and is preferably used in combination with the virus measurement method described in Patent Document 1. The object is to provide an example fluorescence microscope.

Means for solving the problem

[0008] In a first aspect, the present invention provides a light source that generates excitation light, an illumination optical system that irradiates a sample placed at an observation position with excitation light from the light source via an objective lens, and the excitation In the fluorescence microscope, the shielding member includes an observation system that acquires the fluorescence generated by the sample by light irradiation and obtains a sample image, and a shielding member provided so as to cover an optical path between the objective lens and the sample. A fluorescent member characterized in that it is made of a light-transmitting synthetic resin that absorbs light of a specific wavelength, and is a cylindrical member provided so as to cover a substantially cylindrical objective lens unit in which the objective lens is provided. Provide a microscope.

In the fluorescence microscope according to the first aspect described above, the portion covered by the shielding member is a substantially cylindrical objective lens unit in which the objective lens is provided and the optical path between the objective lens and the sample, and completely covers the microscope stage. Therefore, the visibility of the microscope stage and the operability of the microscope can be improved.

The shielding member absorbs light of a specific wavelength but transmits light of other wavelengths, The transparency is such that the objective lens unit and the sample can be visually recognized through the shielding member. That is, since the position of the tip of the objective lens unit and the position of the sample can be confirmed through the shielding member, the operability of the microscope can be improved.

In addition, since the shielding member absorbs light of a specific wavelength, for example, by setting the reflected light of the excitation light irradiated on the sample and the wavelength of the fluorescence emitted from the sample to this specific wavelength, The reflected light and fluorescence of the excitation light can be blocked inside and outside the shielding member.

[0009] In a fourth aspect, the present invention provides the fluorescence microscope according to the first aspect, wherein the shielding member is movable up and down along the surface of the objective lens unit and is at an arbitrary position. And a fluorescence microscope characterized in that a fixing screw is provided for fixing to the objective lens unit.

In the fluorescence microscope according to the fourth aspect, the position of the shielding member relative to the objective lens unit can be arbitrarily adjusted. For this reason, even if the distance between the objective lens and the sample, that is, the focal length is changed, light of a specific wavelength can be appropriately blocked. In addition, when the shielding member is fixed to the objective lens unit at an appropriate position, it can function as a stopper when the objective lens unit is brought close to the microscope stage, and can be used as a reference for the focal length.

The fluorescence microscope according to the fifth aspect includes a microscope stage on which a sample is placed, and a sample mark for displaying the sample position is provided on the microscope stage. The fluorescence microscope according to the sixth aspect includes a slide glass on which a sample is placed, and a sample mark for displaying the sample position is provided on the slide glass. Further, in the seventh fluorescent microscope, the sample mark is a fluorescent mark, and in the fluorescent microscope according to the eighth aspect, the sample mark is a visible mark. This fluorescent microscope has a fluorescent mark and a visible mark, and the sample can be easily placed at an accurate position.

[0010] In a ninth aspect, the present invention provides the fluorescence microscope according to the first aspect, wherein the light source is a semiconductor laser.

In the fluorescence microscope according to the ninth aspect, the straightness of the excitation light can be improved by using a semiconductor laser as the light source. In addition, if the output intensity of the laser beam is about the same as that of a mercury lamp, the semiconductor laser is as small as or smaller than that of a mercury lamp. Can be typed. In addition, semiconductor lasers have a longer life and better energy efficiency in light emission than mercury lamps. In addition, mercury lamps are vulnerable to vibration and unsuitable for carrying, and there is a concern that high-pressure mercury vapor will cause debris to scatter when the mercury lamp is damaged. Is suitable.

[0011] In the fluorescence microscope according to the ninth aspect of the present invention, the semiconductor laser can include an irradiation intensity adjusting unit that can arbitrarily adjust the irradiation intensity of the laser. In this fluorescence microscope, since the irradiation intensity of the semiconductor laser can be adjusted by the irradiation intensity adjusting means, the intensity of the excitation light irradiated to the sample, that is, the intensity of the fluorescence emitted from the sample can be adjusted to an intensity suitable for observation. Further, when a mercury lamp is used as the light source, the illuminance adjustment of the light source is usually performed by replacing the ND filter (dark filter), so that the illuminance adjustment becomes stepwise. However, since the irradiation intensity of the excitation light can be adjusted steplessly in this viewpoint, it is possible to select an illuminance more suitable for observation.

[0012] In the fluorescence microscope according to the ninth aspect of the present invention, the semiconductor laser can be powered by a portable small battery.

In this fluorescent microscope, the portable microscope can be easily moved and carried by using a portable small battery as a power source for the semiconductor laser.

[0013] In the fluorescence microscope according to the first aspect of the present invention, the observation system can capture the sample image with a digital camera.

In this fluorescence microscope, a digital camera capable of converting a photographed image of a sample image into digital data, that is, electronic data, is used as an observation system. Digital cameras include digital still cameras that record still images or digital video cameras that record moving images. In addition, the digital camera means in a broad sense one that includes storage means for temporarily or permanently storing electronic data of a photographed image, and one that includes a sensor portion called an image sensor.

[0014] In the fluorescence microscope according to the first aspect of the present invention, the observation system can include transmission means for transmitting electronic data of a sample image photographed by the digital camera by radio waves. In this fluorescent microscope, the transmission means inputs the electronic data of the sample image taken by the digital camera into an apparatus that can restore the electronic data such as a display. Transmit by radio wave so that you can This transmission means is suitable when, for example, a device capable of recovering electronic data is located away from the fluorescence microscope. In addition, this transmission means is suitable, for example, when an expert who can determine an observation image is in a place away from the fluorescent microscope and transmits electronic data to the expert by radio waves.

[0015] In a tenth aspect, the present invention provides the fluorescence microscope according to the first aspect, wherein the observation system is configured to fluoresce a sample image photographing unit in which the digital camera and a display that projects the sample image are integrated. Provided is a fluorescent microscope characterized in that it can be removed from the microscope.

In the fluorescence microscope according to the tenth aspect, the digital camera and the display for projecting the sample image are integrated to form a sample image photographing unit, and the sample image photographing unit can be attached to and detached from the fluorescence microscope. Since this sample image capturing unit is considered to be a relatively expensive part, it is effective for the management, maintenance, and compatibility of the sample image capturing unit to enable removal of the sample image capturing unit with the help of a fluorescence microscope. It becomes.

According to the present invention, in the fluorescence microscope according to the first aspect, the number of the objective lens units can be one.

In a fluorescence microscope based on this viewpoint, by carefully selecting the objective lens to be installed at a single magnification, for example, the size of the microscope can be reduced compared to the case where an objective lens unit with multiple magnifications can be switched by a revolver. can do.

[0017] In the fluorescence microscope according to the first aspect of the present invention, the objective lens unit can be fixed to a lens barrel portion of the fluorescence microscope by screwing.

In the fluorescence microscope according to this viewpoint, the objective lens unit is fixed to the lens barrel portion of the microscope with a screw, and can be replaced with an objective lens unit of another magnification by loosening the screw. For this reason, even if it is a single fluorescence microscope and only one objective lens unit can be attached at a time, it is possible to observe various magnifications by replacing the objective lens unit.

[0018] In the fluorescence microscope according to any one of the first aspect of the present invention, the illumination optical system and the optical filter of Z or the observation system can be replaced with optical filters having different light transmission characteristics.

In this fluorescence microscope, the wavelength of the excitation light that is the optical filter of the illumination optical system is specified. Change the wavelength of the excitation light to be used and the fluorescent substance to be labeled on the sample by making it possible to replace the optical filter for specifying the wavelength of the fluorescence that is the optical filter for the observation system and the optical filter for the observation system It can respond to changes.

[0019] In the fluorescence microscope according to the first aspect of the present invention, the microscope stage on which the sample is placed can be a microscope stage that can adjust only the position in the two horizontal axes.

In this fluorescence microscope, the observation position of the sample can be finely adjusted by the microscope stage having the function of adjusting the position in the two horizontal axes. Also, in reducing the size of the microscope, a structure in which the objective lens unit is moved up and down is more suitable than a structure in which the focal length for sample observation is adjusted by moving the microscope stage up and down.

[0020] In the fluorescence microscope according to the first aspect of the present invention, the microscope stage on which the sample is placed can be provided with a groove on which the slide glass is placed.

In this fluorescent microscope, the position of the slide glass can be made almost constant by providing a groove for positioning the slide glass on the microscope stage.

[0021] In an eleventh aspect, the present invention provides the fluorescence microscope according to the first aspect, wherein the microscope stage on which the sample is placed is provided with means for detecting whether or not the sample is placed. Provide a microscope.

In the fluorescence microscope according to the eleventh aspect, by providing means for detecting the presence or absence of the sample on the microscope stage, for example, by linking with the switch of the light source, the excitation light is irradiated when the sample is not on the microscope stage. It is possible to stop.

[0022] In the fluorescence microscope according to the first aspect of the present invention, the sample may be a virus or an antibody that is labeled with a fluorescent dye and aggregates by an aggregation reaction. In this fluorescence microscope, the sample to be observed is labeled with a fluorescent dye and is a virus or an antibody that aggregates by an agglutination reaction. The present fluorescence microscope can be suitably used for observing viruses or antibodies that are labeled with a fluorescent dye and aggregate by an agglutination reaction. The invention's effect

[0023] According to the present invention, the shielding member has a cylindrical shape and covers the substantially cylindrical objective lens unit, and the fluorescent microscope can be miniaturized. The shielding member absorbs light of a specific wavelength, but is a light transmissive grease that can visually recognize the position of the objective lens unit and the sample. Therefore, it is easy to grasp the positional relationship between the objective lens unit and the sample, and the operability of the fluorescence microscope can be improved. Further, since it is a compact, lightweight, easy to carry, and good operability fluorescence microscope, it can be suitably combined with the virus measuring method described in Patent Document 1.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described. However, this does not limit the present invention.

[First Embodiment]

In the fluorescence microscope of the present invention, the shielding member can be, for example, an orange light-transmitting synthetic resin that absorbs light having a wavelength of 580 nm or less. For example, in the case of blue light having a wavelength of excitation light having a wavelength of S405 nm, the orange light shielding member can prevent excitation light diffusely reflected from the sample from entering the eyes of the observer. In addition, for the fluorescence emitted from the sample, for example, if a fluorescent label emitting green fluorescence of 530 nm is applied, the orange shielding member allows light having the same wavelength as the fluorescence of 530 nm to be shielded by the orange shielding member. Can be prevented from entering from outside. Thereby, the fluorescence emitted from the sample can be observed efficiently. The shielding member absorbs light with a wavelength of 580 nm or less but transmits light with a wavelength of 580 nm or more. Therefore, the objective lens unit and the sample to be measured are visually observed even though the shielding member is installed. This makes it possible to check the position of the microscope, improving the operability of the microscope.

In addition, if the shielding member is made of a synthetic resin such as acrylic, it can be lightweight and has a hardness that can withstand use, and can also be damaged by contact with the objective lens unit, slide glass, microscope stage, etc. No moderate elasticity can be obtained.

The shielding member has a cylindrical shape, but may have a cylindrical shape such as a perfect circle or an ellipse, or may have a cylindrical shape such as a polygonal cross section as long as it can cover the objective lens unit. Further, since the shielding member does not completely cover the microscope stage, the visibility of the microscope stage and the operability of the microscope can be improved.

Further, the shielding member is a cylindrical member mainly covering the objective lens unit, and does not enclose so much that light of external force does not enter the sample to be measured at all. Temporarily, light from the outside In order to realize a means for enclosing the microscope stage, the sample, and the objective lens unit as a whole, a large shielding means such as a box-shaped openable cover that covers them together is required. In addition, the opening / closing operation of the shielding means is required. For this reason, it can be said that the shielding member of the present invention contributes to miniaturization of the microscope and improvement of operability.

(Second embodiment)

In the fluorescent microscope of the present invention, a fixing screw having a screw hole and a knob combined therewith can be installed on the side surface of the cylindrical shielding member of the cylindrical shielding member. Since the shielding member can move up and down along the surface of the objective lens unit, by tightening the fixing screw, the tip of the fixing screw comes into contact with the side surface of the objective lens unit, and the shielding member can be moved to any desired position. It can be fixed to the objective lens unit at the position. This fixed position is an arbitrary force. When the objective lens unit is brought close to the microscope stage, the distance between the shielding member and the microscope stage is a guideline for the focal point in sample observation, that is, the objective lens unit and the sample. It can be used as a guide for the distance.

Further, the cylindrical shielding member can be produced with a length slightly shorter than the length of the objective lens unit. This light-shielding member is installed so as to cover the objective lens unit, and is slid and fixed to a position away from the sample-side tip of the objective lens unit. That is, this state is a state where the sample-side tip of the objective lens unit is not hidden by the shielding member when viewed from the side. Next, visually observe the objective lens unit as close as possible to the sample together with the shielding member. At this time, care should be taken so that the cover glass placed on the upper surface of the sample does not come into contact with the objective lens unit. Next, with the objective lens unit kept close to the sample, the fixing screw of the shielding member is loosened, and the shielding member is lowered until it comes into contact with the upper surface of the slide glass. In this lowered state, tighten the fixing screw again to re-fix the shielding member to the objective lens tube. Then, the objective lens unit is focused by separating the sample force with the shielding member.

Next, when the measurement of the sample is completed, the objective lens unit is raised to replace the sample. At this time, the shielding member remains fixed to the objective lens unit, and the fixing position remains unchanged. Then, after raising the objective lens unit together with the shielding member, the sample is exchanged, and the shielding member is lowered again to approach the sample. However, the sample should be set on the same standard slide glass before and after replacement. The objective lens unit is lowered until it comes into contact with the upper surface of the shielding member cast slide glass. Then, by gradually raising the objective lens, it is focused and observed. In other words, once the shielding member is positioned, attention when the objective lens unit is brought close to the sample can be reduced, and the measurement can be performed efficiently. (Third embodiment)

In the fluorescence microscope of the present invention, a semiconductor laser that emits ultraviolet laser light having a wavelength of 360 nm can be used as a light source that emits excitation light.

By using a semiconductor laser as the light source, the straightness of the irradiated light can be improved compared to the case where a mercury lamp is used as the light source. Therefore, an observation image with good contrast can be obtained. Also, the lifetime of the light source is about 200 hours with a mercury lamp, and there is a risk that it will break when the lifetime is reached, and fragments may scatter. In addition to its long life, there is no need to worry about parts being separated when it breaks!

In addition, if the output of the semiconductor laser used as the light source of the fluorescence microscope is about 40 to 80 mA, a semiconductor laser of that scale can reduce the power consumption more than the case of the mercury lamp, and it is related to the light source. The size of the portion can also be reduced by not requiring a cooling fan. For example, the size of the semiconductor laser can be set below the microscope stage or on the back of the microscope. As a result, the light source can be integrated with the fluorescent microscope, and the fluorescent microscope can be easily carried. At this time, if the fluorescent microscope is powered by a small portable battery such as a 9V battery, the fluorescent microscope can be easily carried.

Further, if the semiconductor laser is provided with irradiation intensity adjusting means such as a knob for arbitrarily adjusting the irradiation intensity of the laser, the intensity of the excitation light, that is, the intensity of the fluorescence emitted from the sample can be adjusted. As a result, the intensity of the fluorescence emitted from the sample can be adjusted to an intensity that allows easy observation. In addition, if the intensity of the excitation light emitted from the fluorescent label is too strong, the emitted fluorescence will be attenuated immediately, so the intensity of the excitation light needs to be adjusted appropriately. In addition, the use of ultraviolet laser light having a wavelength of 360 nm as excitation light can effectively excite blue fluorescence having a wavelength of around 470 nm from the sample when the fluorescent dye labeled on the sample is DAPI.

[0028] (Fourth embodiment)

In the fluorescence microscope of the present invention, a semiconductor laser that emits laser light having a wavelength of 490 nm can be used as a light source that emits excitation light. Using laser light with a wavelength of 490 nm as excitation light can effectively excite green fluorescence with a wavelength near 530 nm from the sample when the fluorescent dye labeled on the sample is FITC.

[0029] (Fifth embodiment)

In the fluorescence microscope of the present invention, a digital camera can be used for an observation system for observing a sample image. As a digital camera to be used, a digital still camera that records a still image of a sample image or a digital video camera that records a moving image of a sample image can be used. Specifically, for example, a CCD camera or a CMOS camera (CMOS image sensor) can be used. The use of a digital camera in the observation system means that the observed sample image can be recorded and transmitted as electronic data, which improves convenience for the observer. For example, data can be temporarily stored in a flash memory or the like, and compressed data of a captured image can be transmitted to other devices such as an image processing device via a LAN or radio wave. Means for transmitting electronic data by radio waves are useful, for example, for quickly transmitting observation data to an expert in an outdoor microscope observation.

When a semiconductor laser is used as the light source, using a digital camera for taking a sample image leads to avoiding the danger of directly viewing the laser beam during observation. Moreover, due to recent advances in electronic technology, it is easy to make the size of a digital camera smaller than the eyepiece part of a conventional fluorescent microscope. For this reason, it is possible to reduce the size of the microscope. In addition, the electronic data of the observation sample from the digital camera can be adjusted for image quality such as contrast, brightness, and gain when returning to the display. For this reason, when the observed sample image is unclear, it can be adjusted to an easy-to-confirm image.

[0030] (Sixth embodiment)

When adopting a digital camera in the observation system in the fluorescence microscope of the present invention, The camera and the display that projects the sample image can be integrated into the sample image capturing unit, and the microscope body force can be removed in place of the V-shaped eyepiece. For example, if this sample image photographing unit is standardized so that it can be used for other fluorescent microscopes, it is not necessary to provide an expensive digital color and display for each microscope. As a result, when the sample image capturing unit is out of order, the range of compatibility with the sample image capturing unit of other microscopes is widened, and it is convenient in terms of management and maintenance of the sample shadow unit.

In addition, the display of the sample image photographing unit may be a liquid crystal display for a small and light weight display. In the future, it is also possible to adopt displays such as organic EL. If the sample image capturing unit has the function of transmitting data by radio waves together with the camera part and the liquid crystal display part as if it were a camera-equipped mobile phone, sample images can be captured because there are more electronic circuits and more expensive. This will be convenient for storage, management and compatibility. Figure 7 shows a schematic diagram of the document image capture unit. In FIG. 7, 2 is a microscope stage, 3a is a slide glass on which a sample is placed, 7 is a lens barrel, 8 is an objective lens unit attached to the lower part of the lens barrel, and 6a is a focus by moving the objective lens unit 8 up and down. This is a focusing dial for adjusting the In the sample image photographing unit, the display 14 and the digital power camera 13 are integrated, and may be opened and closed in a so-called shell shape. FIG. 7 (a) shows a state in which the sample image photographing unit 17 is removed from the microscope body, and the lens part or sensor part of the digital camera 13 functions as an eyepiece lens of the microscope body. Connect as shown in (b). Further, the transmission antenna 18 may transmit the observation image electronic data. In FIG. 7, the illustration of the shielding member provided so as to cover the optical path between the objective lens and the sample is omitted.

(Seventh embodiment)

In the fluorescence microscope of the present invention, the objective lens unit can be one without any force. By using a single objective lens unit, the microscope can be reduced in size compared to the case where a plurality of objective lens units are attached using a so-called revolver. In addition, the magnification of the objective lens is preferably such that the state of the virus can be observed, the field of view is not too small, and the position adjustment of the observation site is about 20 times. In addition, the objective lens unit should be detachably attached to the microscope with screws. Then, it can replace | exchange for the objective lens unit of another magnification. At this time, it is preferable to replace the shielding member provided so as to cover the objective lens unit as appropriate in accordance with the size of the objective lens unit.

[Eighth Embodiment]

In the fluorescence microscope of the present invention, the illumination optical system and the observation system can be replaced with optical filters having different light transmission characteristics. For example, a fluorescent filter block that is a unit in which an excitation filter that is an illumination optical system filter and an absorption filter that is an observation system filter and a Dyke mouth is integrated is used, and this fluorescent filter block is optically transmitted. By changing to a fluorescence filter block with different characteristics, it is possible to cope with changes in the wavelength of the excitation light used and changes in the fluorescent substance labeled on the sample.

[0033] (Ninth embodiment)

In the fluorescence microscope of the present invention, the microscope stage is a so-called XY stage that can adjust the position only in two horizontal axes. At this time, the vertical adjustment for focusing the sample observation should be performed with a microscope having a structure in which the objective lens cue is moved up and down instead of moving the microscope stage up and down. If the so-called XYZ stage, in which the microscope stage moves up and down, leaves a lot of space under the microscope stage, creating a space unnecessary for miniaturization of the microscope.

In addition, if a groove for positioning the slide glass is provided on the microscope stage, the position of the slide gas can be made almost constant at every measurement. This can reduce the trouble of adjusting the horizontal orthogonal biaxial position of the slide glass according to the position of the objective lens. In addition, for example, if the slide glass is a special one in which the sample placement position is indicated by a circle, the sample position relative to the observation field of the objective lens can be made almost constant, and the microscope stage can be made fine. The amount of adjustment can be reduced. FIG. 6 shows a schematic diagram of a configuration example of the microscope stage 2 provided with the groove 2a for positioning the slide glass 3a. In Fig. 6, in order to adjust the position in the XY direction, a dial-type micrometer 2b is installed along two horizontal axes on each of two sides in the horizontal orthogonal biaxial direction on a rectangular parallelepiped microscope stage. For this reason, the vertical height of the microscope stage can be minimized, and the entire microscope can be The height of the body can be suppressed.

In addition, an optical sensor or a contact switch that detects the presence or absence of the slide glass can be installed on the part of the microscope stage where the slide glass is placed, and linked to the laser light emission of the semiconductor laser that is the light source. . Thus, for example, if the operation is performed so that the emission of the laser beam is stopped when the sample is not on the microscope stage, a safety device can be provided to prevent the laser operator from being mistakenly irradiated with the laser beam.

[0034] (Tenth embodiment)

The fluorescence microscope of the present invention is transported to a poultry farm where a virus is suspected to have occurred or to a marine area where a large number of fish have been killed, and the virus is observed in combination with the virus measuring method described in Patent Document 1. In the virus measurement method of Patent Document 1, the virus aggregates in about 1 to 60 seconds and becomes observable when irradiated with excitation light, so the result is immediately known. For this reason, the present invention, in combination with a fluorescence microscope that does not require much operation time due to good operability, enables rapid virus measurement.

In addition, the fluorescence microscope of the present invention is provided with a shielding member that absorbs light of a specific wavelength around the objective lens unit, so that it is not necessary to limit the observation place to a dark room, and a virus is generated. It can be suitably used on site.

In addition, when there is no expert who can judge viruses in the field where the fluorescence microscope of the present invention is being used to observe viruses, the observation data from the microscope is transmitted to the radio waves to the expert. If you attach a device that can be used for transmission, you can request instructions from experts more quickly. This can be useful for preventing the spread of virus infection.

Hereinafter, examples of the present invention will be described.

Example 1

FIG. 1, FIG. 2 and FIG. 3 show schematic views of the fluorescence microscope according to the first embodiment. Fig. 1 is a schematic diagram viewed from the left side of the fluorescence microscope, Fig. 2 is a schematic diagram when viewed from the front, and Fig. 3 is a diagram showing the optical path in the schematic diagram viewed from the front.

In FIG. 1, reference numeral 1 denotes a base, and a microscope stage 2 which is an XY stage is provided on the base 1. The microscope stage 2 is provided with a dial for moving the stage in two horizontal axes. The description is omitted in any of FIGS. microscope The sample 3 is placed on the stage 2, and the sample 3 is placed on the slide glass 3a and covered with the cover glass 3b. A groove 2a for placing the slide glass 3a is provided on the microscope stage 2, and the depth of the groove 2a is the same as that when the slide glass 3a is placed on the microscope stage 2 and viewed from the side. The upper surface of the glass 3a has a depth at which the upper surface force of the microscope stage 2 protrudes.

Further, if the sample 3 is placed at the center of the slide glass 3a, it can be positioned almost at the center on the microscope stage 2. Then, by rotating the dial of the microscope stage (not shown), the placement position of the sample 3 can be finely adjusted in two horizontal axes, the so-called XY direction.

[0038] Further, the base 1 is provided with upright columns 4 upright. A support arm 5 is provided on the support column 4 so as to be parallel to the base 1 through the focusing unit 6. The focusing unit 6 is provided on the support column 4 via an elevating mechanism including a pion and a rack (not shown), and the support arm 5 can be moved up and down along the support column 4 by operating the focusing dial 6 a. . In FIGS. 2 and 3, the focusing unit 6 and the support arm 5 are hidden by the lens barrel 7 and cannot be confirmed.

The lens barrel 7 is fixed to the tip of the support arm 5. A screw-in fixed objective lens unit 8 is attached to the lower end of the lens barrel 7 on the microscope stage 2 side. This object lens unit 8 moves in the direction of the observation optical axis a by the vertical movement of the support arm 5 by the operation of the focusing unit 6 and changes the relative distance from the sample 3 so that the sample 3 can be focused. It has become.

A side holder of the lens barrel 7 is provided with a fine holder 9 for fixing the optical fiber.

The fiber holder 9 is disposed along a direction parallel to the support arm 5. An optical fiber 9 a connected to the light source 10 is connected to one end of the fiber holder 9. The light source 10 emits excitation light in fluorescence observation and has a semiconductor laser power. For example, the semiconductor laser can emit excitation light having a wavelength of 405 nm. As the semiconductor laser, a 405 solid laser manufactured by Nichia Corporation equipped with means for adjusting the irradiation intensity of the laser was used.

[0041] In the hollow portion of the lens barrel 7, along the light source optical axis 10a of the light source 10, in FIG. 1 and FIG. In this case, a fluorescent filter block 11 (not shown) is arranged. FIG. 3 shows a fluorescent filter block 11 and shows that the fluorescent filter block 11 is composed of an excitation filter 11a, an absorption filter 1 lb, and a dichroic mirror 1 lc. . The excitation filter 11a selects light of a specific wavelength from the light source 10, and the dichroic mirror 11c reflects the light selected by the excitation filter 1 la as excitation light along the observation optical axis a. Irradiate Sample 3. Further, the dichroic mirror 11c transmits the fluorescence emitted from the sample 3 and the stray light of the excitation light reflected from the sample 3 through the objective lens unit 8 along the observation optical axis a. The absorption filter l ib selects and transmits only light having a specific wavelength in order to remove light having a wavelength unnecessary for observation from the light transmitted by the dichroic mirror 11c. The fluorescent filter block 11 is exchanged by opening and closing the fluorescent filter block exchange port 16 shown on the lens barrel 7 in FIGS. The fluorescent filter block used is a Nikon fluorescent filter block.

An imaging lens 12 (not shown in FIGS. 1 and 2) is disposed on the observation optical axis a in the hollow portion of the lens barrel 7 and above the fluorescent filter block 11. In FIG. 3, the imaging lens 12 and the state of the focal point are indicated by dotted lines. On the upper part of the lens barrel 7, a CCD camera 13a for taking an image that has passed through the imaging lens 12 is arranged on the observation optical axis a. As the CCD camera 13a, for example, a CCD camera 13a provided with means for outputting a photographed image as electronic data can be used. The captured image detected by the CCD camera 13a is displayed on the display 14 connected to the CCD camera 13a. The CCD camera 13a used is a DE GITAL SIGHT DS—: L1, which is a set with the display 14.

The objective lens unit 8 is provided with a cylindrical shielding member 15 so as to cover the substantially cylindrical objective lens unit 8. The shielding member 15 is shown in FIG. 1 and FIG. 2, and V is omitted in FIG.

A female screw is provided on the side surface of the shielding member 15, and a fixing screw 15a is assembled to the female screw. The shielding member 15 can be fixed on the objective lens unit 8 by tightening the fixing screw 15a. Here, since the shielding member 15 can be moved up and down at any position on the objective lens unit 8, it can be fixed at any position. The length of the shielding member 15 is preferably about the same as the length of the objective lens unit 8 to about half. Further, the shielding member 15 is made of an orange transparent acrylic resin, and the state of the objective lens unit 8 and the microscope stage 2 can be confirmed through the shielding member 15. Since the shielding member 15 is orange, it can absorb light having a wavelength of about 580 nm or less. That is, light having a wavelength of 580 nm or less can be blocked out of the light outside the cylinder inside the cylindrical shielding member 15, and the inside of the cylinder can be blocked outside the cylindrical shielding member 15. Can block light with a wavelength of 580 nm or less. FIG. 4 shows a schematic diagram of the shielding member 15 and the fixing screw 15a.

[0044] Next, a usage example of a fluorescence microscope will be described. Sample 3 that was fluorescently labeled using the method for measuring agglutination described in Patent Document 1 was placed on a slide glass 3a and covered with a cover glass 3b. Next, after raising the objective lens unit 8 away from the microscope stage 2, in a side view, the tip of the objective lens unit 8 on the sample 3 side is not covered by the shielding member 15. Then, the position of the shielding member 15 was raised above the objective lens unit 8 and fixed by tightening the fixing screw 15a. In this state, a slide glass 3a on which the sample 3 was placed was placed on the groove 2a on the microscope stage 2. FIG. 5 (a) shows a schematic view of the shielding member 15 pulled up.

[0045] Next, with the shielding member 15 fixed to the objective lens 8, the objective lens unit 8 is visually contacted with the cover glass 3b by rotating the focusing dial 6a. It went down to the last minute. A schematic diagram of the objective lens unit 8 brought close to the sample 3 is shown in FIG. 5 (b).

Next, the fixing screw 15a of the shielding member 15 is loosened, the fixation of the shielding member 15 and the objective lens unit 8 is released, and the lower end of the shielding member 15 is moved along the surface of the objective lens unit 8. The glass was lowered until it contacted the slide glass 3a. At this time, it is important that only the shielding member 15 is lowered and brought into contact with the slide glass 3a without moving the vertical position of the objective lens unit 8. At this position, the fixing screw 15a of the shielding member 15 is tightened again, and the objective lens unit 8 and the shielding member 15 are fixed. FIG. 5 (c) shows a schematic diagram in which the shielding member 15 is fixed again.

Next, the light source 10 was turned on and laser light was emitted. The laser beam is emitted from a blue laser with a wavelength of 405 nm and passes through the optical fiber 9a and the fiber holder 9 as excitation light. Is incident on the fluorescent filter block 11. The fluorescent filter block 11 reflected the excitation light and entered the sample 3, and the sample 3 emitted green fluorescence. At this time, the excitation light incident on the sample 3 is irregularly reflected in accordance with the uneven surface of the sample 3 which has a good linearity because it is a laser light source. If the shielding member 15 is not provided, the irregularly reflected excitation light may be directly incident on the observer's eyes, which is dangerous.

Next, the objective lens unit 8 and the lens barrel 7 are raised together with the shielding member 15 by the rotation of the focusing dial 6 a, and focused by moving away from the sample 3. Note that the magnification of the objective lens unit 8 used is 20 times, and the amount by which the objective lens unit 8 is raised for focusing is the interval generated between the cover glass 3b and the tip of the objective lens unit 8 is 0.5. The amount of increase is about lmm. For this reason, the gap generated between the slide glass 3a and the shielding member 15 is small, and the probability that the excitation light enters the human eye is extremely low.

[0049] Sample 3 emits fluorescence upon receiving excitation light. If sample 3 is labeled with FITC, fluorescence with a wavelength of about 530 nm is emitted by excitation light with a wavelength of 405 nm. However, when FITC is a label, light with a wavelength of 488 nm is the ideal excitation light. If sample 3 is labeled with DA PI (Hoechst 33258), fluorescence with a wavelength of about 470 nm is emitted. However, when DAPI is a label, the ideal excitation light is 365 nm light.

The fluorescence emitted from the sample 3 passes through the objective lens unit 8 along the observation optical axis a, passes through the fluorescence filter block 11, is imaged by the imaging lens 12, and is detected by the CCD camera 13a. It is. The image detected by the CCD camera 13a is displayed by the display 14 connected to the CCD camera 13a. In this embodiment, a semiconductor laser is used as the light source 10, and if an image formed by the imaging lens 12 is observed with an eyepiece, it is dangerous because high-intensity light is directly viewed. . For this reason, it is suitable not to look directly at the observation image using a CCD camera or the like.

In addition, observation through the CCD camera 13a and the display 14 makes it easier to check the image compared with direct observation using an eyepiece because the contrast can be adjusted if the color is shaded! Has the advantage of being adjustable.

[0050] Further, when observing the fluorescence emitted from the sample 3, light having a wavelength that is not absorbed by the light shielding member 15 enters from the outside of the shielding member 15 into the inside of the shielding member 15, that is, the observation region. Also, The sample 3 emits even stray light that is reflected light of the excitation light. However, since the light observed by the CCD camera 13a is light having a wavelength that passes through the fluorescent filter block 11, even light that has been absorbed by the shielding member 15 or stray light caused by excitation light can be used. If light of a wavelength that does not pass through 1 lb of the absorption filter of the fluorescent filter block 11 is cut, it is cut during observation. Therefore, a clear image can be obtained by the CCD camera 13a. In addition, when focusing, there is a slight gap between the shielding member 15 and the slide glass 3a as a distance of about 0.5 to Lmm is generated between the cover glass 3b and the tip of the objective lens unit 8. However, an interval occurs. Therefore, light having a wavelength that passes through the absorption filter ib of the fluorescent filter block 11 can enter the observation region from the gap between the shielding member 15 and the slide glass 3a. However, since the gap is small, the amount of intrusion light is small and does not interfere with observation unless the intensity of the intrusion light is extremely strong. FIG. 8 shows a schematic cross-sectional view of the tip of the objective lens unit 8. The objective lens unit 8a protrudes from the sample-side tip 8c of the substantially cylindrical objective lens barrel 8b at the tip of the objective lens unit 8. It is in a state of being hidden a little inside. This is also considered to be useful for blocking intrusion light having a gap between the shielding member 15 and the slide glass 3a.

Next, when exchanging the sample 3 placed on the microscope stage 2 with another sample, the objective lens unit 8 is attached to the focusing dial 6a without changing the fixed relationship between the objective lens unit 8 and the shielding member 15. Raise by rotation. At this time, the output of the excitation light from the light source 10 is stopped. A schematic diagram at this time is shown in Fig. 5 (d).

Next, the sample 3 is exchanged with another sample in a state where a sufficient gap is formed between the objective lens unit 8 and the microscope stage 2. However, the samples before and after replacement are both samples placed on the same standard slide glass, and the overall height and shape are almost the same.

Next, the objective lens unit 8 is lowered by the rotation of the focusing dial 6a until the lower end force S of the shielding member 15 fixed to the objective lens unit 8 comes into contact with the slide glass 3a. At this time, the shielding member 15 plays a role of a stopper by contacting the slide glass 3a. For this reason, in the operation of lowering the objective lens unit 8, the degree of careful operation can be reduced. The focal point is not much different from the position measured with the previous sample, so it can be easily adjusted by slightly raising the objective lens unit 8. Therefore, operation time I think that it can be realized significantly shortened. In the method for measuring the agglutination reaction described in Patent Document 1, since 1 to 60 seconds after stirring is the observable agglomeration reaction completion time, observation is performed while the observation sample is replaced and focused. I think it will be possible. Therefore, we think that quick measurement is possible. FIG. 9 shows the observed fluorescent aggregation image of batteriophage M13K07.

FIGS. 10 and 11 show microscope stages 102 and 112 provided with sample marks 104 and 114 and slide glasses 123a and 133a. The microscope stage shown in these figures is provided with a sample mark inside the positioning groove of the microscope stage. The sample mark consists of a visible mark that can be seen directly by the eye and a fluorescent mark that emits fluorescence when excited by the light source. In the sample mark 104 of FIG. 10, the visible mark 104a and the fluorescent mark 104b are displayed as concentric circles, and the visible mark 104a is displayed as a larger circle than the fluorescent mark 104b. Furthermore, the sample mark 114 in FIG. 11 is provided with a lattice-like fluorescent mark 114b inside a circular visible mark 114a.

In the microscope stage shown in these drawings, a sample mark is provided in the positioning groove, that is, in the center of the sample stage on which the slide glass is placed. The sample mark is provided with both a visible mark and a fluorescent mark. In this structure, the visual mark is first used to roughly align the sample visually, then the fluorescent microscope barrel is lowered to focus on the position of the fluorescent mark and the excitation laser beam is applied. For example, a 20x objective lens has a working distance of a few millimeters, which is sufficiently larger than the thickness of the slide glass (about 1.2 mm), so the sample can be aligned using this method. Sample marks displaying both fluorescent and sample marks can be used more conveniently. This is because the focus alignment can be confirmed by the visible mark even when the sample does not necessarily emit fluorescence, and the fluorescence is not visible. However, the sample mark can be either a visible mark or a fluorescent mark.

[0054] In addition, a fluorescence microscope in which a sample mark is provided on a microscope stage, which is a sample stage, is located at a position sufficiently away from the focus when the microscope is observed (when the sample is focused). Do not disturb the observation! ,.

In FIG. 12 and FIG. 13, sample marks 124 and 134 are provided on the slide glasses 123a and 133a. Trial The material marks are provided with both visible marks 124a and 134a and fluorescent marks 124b and 134b. The sample marks 124 and 134 provided on the slide glass can be used not only for sample positioning but also for focus positioning. The sample marks 124 and 134 on the slide glass are placed outside the field of view of the sample so as not to disturb the observation of the sample. For example, the sample mark can be placed outside the sample and placed outside the field of view of the fluorescence microscope. This slide glass is provided with a sample mark so that the sample can be easily placed at an accurate position, and there is no harmful effect of observation by the sample mark. The above glass slide is provided with a sample mark consisting of both a visible mark and a fluorescent mark. However, the sample mark can be either a visible mark or a fluorescent mark. The sample mark can be provided by printing.

Brief Description of Drawings

FIG. 1 is a schematic view of a fluorescence microscope according to Example 1 of the present invention.

FIG. 2 is a schematic view of a fluorescence microscope according to Example 1 of the present invention.

FIG. 3 is a schematic view of a fluorescence microscope according to Example 1 of the present invention.

FIG. 4 is a schematic view of a shielding member according to Embodiment 1 of the present invention.

FIG. 5 is a schematic operation diagram of the fluorescence microscope according to Example 1 of the present invention.

FIG. 6 is a schematic view of a microscope stage according to a ninth embodiment of the present invention.

FIG. 7 is a schematic view of a fluorescence microscope according to a sixth embodiment of the present invention.

FIG. 8 is a schematic cross-sectional view of the distal end of the objective lens unit according to Example 1 of the present invention.

FIG. 9 is a photographed image of fluorescent aggregated images of Pacteriophage M13K07 with a fluorescence microscope according to Example 1 of the present invention.

FIG. 10 is a schematic view of a microscope stage of a fluorescence microscope that works on an example of the present invention.

FIG. 11 is a schematic view of a microscope stage of a fluorescence microscope that works on an example of the present invention.

FIG. 12 is a schematic view of a slide glass used in an example of the present invention.

FIG. 13 is a schematic view of a slide glass used in an example of the present invention.

Explanation of symbols

[0057] 1 ... Base

2. Microscope stage, 2a ... groove, 2b ... micrometer

3 ··· Sample, 3a… Slide glass, 3b… Cover glass 4 ... post

5 ... Support arm

6 ··· Focusing part, 6a… Focus dial

7 -mm

8 ... Objective lens unit, 8a ... Objective lens group,

8b ... Objective lens barrel, 8c ... Sample side tip

9 ... Fiber holder, 9a ... Optical fiber

10 ... light source, 10a ... light source optical axis

11… Fluorescence filter block, 11a… Excitation filter, lib… Absorption filter lie- 'Dichroic mirror

12 ... imaging lens

13 ... Digital camera, 13a-"CCD camera

14… Display

15… Shielding member, 15a… Fixing screw

16 ··· Fluorescent filter block replacement port

17 ... Sample image photographing unit

18 ... Transmitting antenna

a '"¾l observing optical axis

102 ... Microscope stage, 102a ... groove

104 ... Sample mark

104a… Visible mark

104b ... Fluorescent mark

112 ... Microscope stage, 112a ... Groove

114 ... Sample mark

114a… Visible mark

114b ... Fluorescent mark

123a ... slide glass

124 ··· Sample mark 6

124a… Visible mark

124b ... Fluorescent mark

133a ... slide glass

134 ... Sample mark

134a… Visible mark

134b ... Fluorescent mark

Claims

The scope of the claims

[1] A light source for generating excitation light, an illumination optical system for irradiating the excitation light from the light source to a sample placed at an observation position via an objective lens, and a fluorescence emitted from the sample by the irradiation of the excitation light A fluorescence microscope including an observation system that obtains a sample image by obtaining a sample image and a shielding member provided so as to cover an optical path between the objective lens and the sample,

The shielding member is made of a light-transmitting synthetic resin that absorbs light of a specific wavelength, and is a cylindrical member provided so as to cover an objective lens unit in which the objective lens is installed. microscope.

[2] The fluorescence microscope according to claim 1, wherein the shielding member absorbs the reflected light of the excitation light irradiated onto the sample as light of a specific wavelength.

[3] The fluorescence microscope according to claim 1, wherein the shielding member absorbs a wavelength of fluorescence emitted from the sample as a specific wavelength.

[4] In the fluorescence microscope according to claim 1, the shielding member can be moved up and down along the surface of the objective lens unit and fixed to the objective lens unit at an arbitrary position. A fluorescent microscope characterized in that a fixing screw is provided.

[5] The fluorescence microscope according to claim 1! A fluorescence microscope comprising a microscope stage on which a sample is placed, and a sample mark for displaying the sample position is provided on the microscope stage.

[6] The fluorescence microscope according to claim 1, further comprising a slide glass on which the sample is placed, and a sample mark for displaying the sample position is provided on the slide glass. .

7. The fluorescent microscope according to claim 5 or 6, wherein the sample mark is a fluorescent mark.

[8] The fluorescence microscope according to claim 5 or 6, wherein the sample mark is a visible mark.

[9] The fluorescent microscope according to claim 1, wherein the light source is a semiconductor laser.

[10] The fluorescence microscope according to claim 1, wherein the observation system is connected to the digital camera. A fluorescent microscope characterized in that a sample image photographing unit integrated with a display for projecting a sample image is detachable from a fluorescent microscope.

The fluorescence microscope according to claim 1! A fluorescence microscope comprising a microscope stage on which a sample is placed, the microscope stage being provided with means for detecting whether or not the sample is placed.