JP2002202459A - Dark visual field vertical illumination microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dark visual field vertical illumination microscope which quickly changes-over excitation wavelength and fluorescent wavelength, measures one tissue or one organelle by time-resolving, can be easily changed-over to a dark field and can obtain a clear image with low back ground. SOLUTION: The dark visual field vertical illumination microscope has a light source 1, a first filter 3 which selectively transmits irradiating light to a sample 20 and can be changed-over, an optical member for making the irradiating light to a ring-shaped luminous flux, a reflection mirror 5 for making the light transmitted through the member incident on a space between an outside lens barrel and an inside lens barrel of an objective lens 6, a second filter 8 which transmits only the light emitted from the sample and can be changed-over and a detecting means for detecting the light transmitted through the second filter. The objective lens has a mirror surface for converging the irradiating light to the sample at a top end part 17 of the outside lens barrel and is arranged so that the light from the sample passes in the inside lens barrel. Further a cylinder for preventing contamination of stray light is arranged between the objective lens and the second filter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dark-field epi-illumination microscope, and more particularly to a first filter disposed on an irradiation side and a second filter disposed on a measurement side, capable of obtaining a clear image with a low background. Can be easily switched, the wavelength of the irradiation light and the wavelength of the measurement light can be quickly switched, and the second filter can be switched and used as a normal dark-field microscope without substantially functioning the second filter. The present invention relates to a dark field epi-illumination microscope.

[0002]

2. Description of the Related Art In an epi-illumination type fluorescence microscope often used for measuring a biological sample, a dichroic mirror is generally used to efficiently guide excitation light and fluorescence to a sample and an observation optical system, respectively. An optical feature of the dichroic mirror is that it has a high reflectance for the wavelength of the excitation light and a high transmittance for the wavelength of the fluorescence. Therefore, the excitation light is almost reflected by the dichroic mirror, passes through the objective lens, and reaches the sample. on the other hand,
Fluorescence generated by the excitation light also reaches the dichroic mirror after being incident on the objective lens and almost passes therethrough. In addition, in the epi-illumination type fluorescence microscope, the excitation light is applied to the sample from the objective lens side, so that the fluorescence passing through the objective lens returns to the same optical path as the excitation light in reverse. However, in this case, 1) since the fluorescence emitted from the objective lens and its lens tube is mixed with the fluorescence emitted from the sample, noise is included in the measurement result and a clear image cannot be obtained. Or 2)
Since the switching of the dichroic mirror is complicated, the switching of the wavelengths of the excitation light and the fluorescence is complicated.

The problem described in 1) is particularly remarkable when using ultraviolet light as excitation light. Further, if the amount of the fluorescent dye is reduced in order to reduce the damage to the sample, the fluorescence from the objective lens and its lens barrel will interfere with the measurement even if excitation light other than ultraviolet light is used. The problem described in 2) becomes remarkable when it is desired to measure one sample while quickly switching the wavelength of the excitation light or the fluorescence light, for example, when measuring the lapse of time using a multi-stained sample. Further, even when a dichroic mirror for multiple staining can be used, it is inconvenient to prepare dichroic mirrors for the number of staining patterns.

[0004] As one of the methods for remedying such a drawback,
Objective lens having a double lens barrel used for dark-field illumination of a metal microscope (Japanese Patent Application Laid-Open No. 3-266809)
Can be diverted. However, since this microscope uses a dichroic mirror, there is a disadvantage that the above-mentioned problem 2) still remains.

As a microscope capable of suppressing the brightness of the background even when the sample is excited with ultraviolet light, there is a fluorescence microscope using an illumination method that does not allow excitation light to pass through an objective lens. this is,
For example, the illumination light (excitation light) from the light source is guided to the side of the objective lens by an optical fiber, and the sample is illuminated obliquely. However, this illumination method has a drawback that the operation of the sample, for example, the operation of microinjection or the like is difficult to perform because the component for introducing the excitation light (the end of the optical fiber) exists near the sample. Also,
Although two-photon excitation can be used instead of ultraviolet light excitation, the apparatus itself is very expensive, and since the laser is used as an excitation light source, the excitation wavelength cannot be quickly switched. There was a disadvantage that the wavelength was limited.

The inventor of the present invention has conducted intensive studies to solve the above-mentioned drawbacks. As a result, the wall surface of the lens barrel serving as the optical path of the excitation light is prevented so that the fluorescence from the lens barrel and the objective lens does not mix with the fluorescence emitted from the sample. By using an objective lens having a double lens barrel coated with a non-fluorescent paint and not using a dichroic mirror, not only a clear image with extremely low background can be obtained, but also the excitation light The present inventors have found that it is also possible to quickly switch between the wavelength and the wavelength of the fluorescence emitted from the sample, thereby simultaneously measuring a plurality of phenomena of one cell or organelle with time resolution.

Accordingly, a first object of the present invention is to provide a dark-field epi-microscope which can easily be switched to a dark-field microscope or from a dark-field microscope to an epi-fluorescence microscope. A second object of the present invention is to make it possible to time-resolve and measure one cell or organelle while quickly switching between an excitation wavelength and a fluorescence wavelength, and to obtain an epifluorescence that can obtain a clear image with a low background. It is to provide a microscope.

The above objects of the present invention are to provide a light source for irradiating a sample with light, a first filter switchably provided for selectively transmitting light from the light source to the sample, A slit for transmitting the irradiation light in a ring shape, a reflecting mirror for allowing the light transmitted through the slit to enter between the outer lens barrel and the inner lens barrel of the objective lens having the double lens barrel, and reflection and scattering from the sample. A dark-field epi-illumination microscope comprising: a switchable second filter that transmits only emitted light; and detection means for detecting light transmitted through the second filter. The objective lens having the lens barrel has a mirror surface at the tip of the outer lens barrel for condensing the irradiation light on the sample, and the light reflected, scattered or emitted from the sample passes through the inner lens barrel. Arranged A tube for preventing stray light from being mixed is disposed between the objective lens and the second filter so as to prevent mixing of light other than light reflected, scattered or emitted from the sample. Achieved by dark-field epi-microscopy.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The dark-field epi-illumination microscope of the present invention is mainly an epi-illumination fluorescence microscope, and therefore, a light source such as a mercury lamp used for a normal fluorescence microscope is used as a light source. The first filter is particularly important when used as an epi-illumination fluorescence microscope and selects the wavelength of the excitation light to be applied to the sample. Therefore, the first filter need not be used when simply used as a dark-field epi-illumination microscope. The first filter may be one that can be sequentially switched by a wheel, or may be a diffraction grating.

The optical member for converting the irradiation light into a ring-shaped light beam is for introducing the irradiation light only between two lens barrels constituting a double lens barrel to be described later. It has a ring shape. A slit is most commonly used as the optical member, but an axicon prism or a diffractive optical element can also be used. The optical member and the reflection mirror may be configured as an integrated box. In addition, it is preferable to provide a shutter between the optical member and the light source or between the first filter and the light source. At the center of the ring-shaped reflection mirror, a tube for preventing stray light from being mixed, which will be described later, is arranged. The reflection mirror and the cylinder may be integrated.

The objective lens according to the present invention includes a double lens barrel having a concentric cross section, a set of objective lenses incorporated in the inner lens barrel, and irradiation light (light) provided at the tip of the outer lens barrel. (Excitation light) on the sample. Preferably, the mirror surface is parabolic. Also, in order to ensure sufficient performance as an epi-fluorescence microscope, the outer wall of the inner lens barrel and the inner wall of the outer lens barrel, which form the optical path of the irradiation light, should be illuminated so that fluorescent light is not emitted from these locations. It is preferable to apply a fluorescent paint. It should be noted that the non-fluorescence here does not only mean that fluorescence is not substantially generated so as not to hinder the measurement, and that it does not completely generate fluorescence.

In the present invention, it is preferable that the non-fluorescent paint be applied not only to the wall surface of the lens barrel, but also to a portion facing the ring-shaped mirror (tip part 17 in FIG. 1). In this portion, since the excitation light is easily reflected toward the cylinder 7 (see FIG. 1), a paint for preventing reflection is applied. However, autofluorescence may be generated from the antireflection paint. Therefore, if a non-fluorescent paint is applied here, it is possible to suppress the generation of auto-fluorescence incident on the cylinder 7.

The less fluorescence emitted from the wall forming the optical path, the clearer the image obtained when used as an epifluorescence microscope with a low background. This makes it possible to clearly capture the fluorescence that was conventionally overlooked because it was buried in the brightness of the background, and the quality of the fluorescent image is greatly improved. Further, since the biological sample can be observed with a lower concentration of the fluorescent dye, there is a great advantage that the fluorescent measurement can be performed in a state close to the natural state with less influence of the fluorescent dye.

The second filter in the present invention is for using the microscope of the present invention as an epi-fluorescence microscope. This filter can be switched by a wheel like the first filter, or can be a diffraction grating. This filter is selected to determine the fluorescence wavelength to be measured. However, when the filter is simply used as a dark-field epi-illumination microscope, the second filter may not be provided, or the same filter as the first filter may be selected. It is sufficient that the second filter does not function substantially.

In the microscope of the present invention, the irradiation light (excitation light) does not pass through the inside of the inner lens barrel, and only the fluorescence to be measured or the reflected light or the scattered light passes through the inside of the inner lens barrel, and furthermore, the stray light is mixed. Cooling CCD camera and S
Since photometry or photographing is performed by a high-sensitivity image sensor such as an IT tube, a clear image with extremely low background can be obtained. In particular, by controlling the first and second filters and the imaging device and, if necessary, the shutter by computer, it is possible to perform measurement with a time resolution of less than a second for a plurality of wavelengths on a sample that has been multi-stained. In this case, the degree of freedom in selecting a dye is also increased.

For example, NADH (fluorescence wavelength: 460 nm)
Even when the fluorescence wavelength and the excitation wavelength of two substances overlap, such as flavin (excitation wavelength 460 nm), both can be measured with a time resolution of 100 milliseconds or less. That is, conventionally, the wavelengths of the excitation light and the fluorescence can be switched only within a range according to the characteristics of the dichroic mirror. However, in the present invention that does not use the dichroic mirror, there is no limitation by the dichroic mirror. As a result, the degree of freedom in selecting excitation light or fluorescence is increased, so that the present invention is particularly useful as a device for obtaining new knowledge on living bodies.

Next, the present invention will be further described with reference to examples. FIG. 1 is a principle diagram of the microscope of the present invention. In the figure, reference numeral 1 denotes a light source, 2 denotes a shutter, 3 denotes a first filter, 4 denotes a slit, 5 denotes a reflection mirror, 6 denotes an objective lens having a double lens barrel, 7 denotes a tube for preventing stray light from being mixed in, and 8 denotes a tube. A second filter, 9 is an imaging camera, 10 is a computer, 16 is a stage on which a cover glass is mounted, 17 is a tip of a lens barrel facing a reflection mirror, and 20 is a sample. FIG. 2 is a detailed view of the objective lens 6. In the figure, reference numeral 11 denotes an outer lens barrel,
12 is a parabolic mirror attached to the tip of the lens barrel, 13 is an inner lens barrel, and 14 is a (one set) objective lens for imaging. FIG. 3 is a plan view of the double-barrel objective lens.

The parallel excitation light from the light source 1 is passed through a ring-shaped slit 4 through a first filter 3, and the ring-shaped excitation light is passed through a ring-shaped reflection mirror 5 whose center is hollowed out in a circular shape. The light is reflected and introduced between the inner lens barrel 13 and the outer lens barrel 11 of the objective lens 6 having the double lens barrel.
The introduced excitation light is reflected by the parabolic mirror 12 attached to the tip of the outer lens barrel 11, and is focused on the sample 20. Since the excitation light is applied obliquely to the cover glass 15 so as not to cause total reflection, the cover glass 1
Excitation light reflected at 5 is prevented from entering the inner lens barrel.

Fluorescence emitted from the sample (reflected and scattered light from the sample when used as a dark field microscope)
Are converged by the objective lens 14 in the inner lens barrel 13 and become parallel light. The fluorescent light that has become parallel light passes through the cylinder 7 disposed at the center of the reflection mirror 5, and the second
The light passes through the filter 8 and enters the imaging camera 9. The first and second filters can be switched at a high speed of about 15 milliseconds by using a filter wheel. As a camera for fluorescence detection, a cooled CCD with 12-bit gradation and a maximum quantum efficiency of 50%
Preferably, a camera is used. By using such a camera, it is possible to detect a slight change in fluorescence intensity. An optical amplifying device can be provided before the fluorescence detection camera, if necessary.

By controlling the shutter 2, the first filter 3, the second filter 8, and the imaging camera 9 by computer, it becomes possible to perform fluorescence measurement at a plurality of wavelengths with a time resolution of 100 milliseconds or less. In addition, by emptying the second filter or making the same as the first filter, the microscope of the present invention is a dark-field epi-illumination microscope, for example,
The presence or absence of scratches on the metal surface can be checked.

[0021]

As described in detail above, the microscope of the present invention can do all the things that can be done with a conventional epi-illumination fluorescence microscope, and does not emit fluorescent light from the objective lens or the lens barrel, so that the background has a low background. A vivid image can be obtained, and an image conventionally buried in the background can also be obtained. Further, since it is easy to select the wavelengths of the excitation light and the fluorescence, for example, the production of NADH with one mitochondria, the reduction of flavin, the change in pH, the change in membrane potential, the ATP
The time change of a plurality of reactions such as the synthesis of Further, since the fluorescent filter can be substantially removed to be used as a dark-field epi-illumination microscope, the present invention is extremely significant.

[Brief description of the drawings]

FIG. 1 is a principle view of a dark-field epi-illumination microscope of the present invention.

FIG. 2 is a detailed principle diagram of an objective lens according to the present invention.

FIG. 3 is a plan view of a double-barrel objective lens of the present invention.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 light source 2 shutter 3 first filter 4 slit 5 reflecting mirror 6 objective lens 7 cylinder for preventing stray light from entering 8 second filter 9 imaging camera 10 computer 11 outer lens barrel 12 parabolic mirror 13 inner lens barrel 14 imaging (One set) objective lens for 15 cover glass 16 stage 17 lens barrel tip 20 sample

 ──────────────────────────────────────────────────続 き Continued on front page F term (reference) 2G043 AA03 BA16 CA03 EA01 FA02 GA04 GA07 GB01 GB07 GB17 HA01 HA02 HA09 KA02 KA05 LA03 MA01 2H052 AA09 AC04 AC06 AC08 AC17 AC27 AD29 AD34

Claims (9)

[Claims] 1. A light source for irradiating a sample with light, a first filter switchably provided to selectively transmit irradiation light to the sample from the light source, and a ring-shaped light beam for the irradiation light An optical member, a reflecting mirror for allowing light transmitted through the slit to enter between an outer lens barrel and an inner lens barrel of an objective lens having a double lens barrel, and light reflected, scattered, or emitted from the sample. A dark-field epi-illumination microscope comprising: a second filter switchably provided to transmit only light; and detection means for detecting light transmitted through the second filter. The lens has a mirror surface at the tip of the outer lens barrel for condensing the irradiation light onto the sample, and is arranged so that light reflected, scattered or emitted from the sample passes through the inner lens barrel, Reflection and scattering from the sample Alternatively, a dark-field epi-illumination microscope characterized in that a tube for preventing stray light from being mixed is disposed between the objective lens and the second filter so as to prevent mixing of light other than the emitted light. 2. The dark-field epi-illumination microscope according to claim 1, wherein a non-fluorescent paint is applied to a wall surface of the lens barrel forming an optical path of irradiation light of the objective lens having the double lens barrel. 3. The dark-field epi-illumination microscope according to claim 1, wherein the slit and the reflection mirror are formed as an integrated box. 4. The dark-field epi-illumination microscope according to claim 1, wherein switching between the first filter and the second filter is performed by a wheel. 5. The light detecting means is a high-sensitivity image sensor.
A dark-field epi-illumination microscope according to claim 1. 6. The dark-field epi-illumination microscope according to claim 1, wherein a shutter is arranged between the light source and the first filter. 7. The dark-field epi-illumination microscope according to claim 1, wherein the shutter, the first filter, the second filter, and the cooled CCD camera are all computer-controlled. 8. The dark-field epi-illumination microscope according to claim 7, wherein the focus control of the objective lens is performed by a computer. 9. The dark-field epi-illumination microscope according to claim 1, wherein a filter equivalent to the first filter can be selected as one of the options for switching the second filter.