JP3093009B2 - 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 used for observing a state of a biological tissue or the like in the fields of medicine, biology, etc., and more particularly to the intensity of two or more types of fluorescent images emitted from a specimen. Field of the Invention The present invention relates to an epi-illumination fluorescence microscope having an improved technique for adjusting the fluorescence.

[0002]

2. Description of the Related Art In general, this type of fluorescence microscope is widely used in medicine and biology as well as in other fields for the purpose of detecting fluorescently labeled proteins and genes on biological tissue cells. I have. In particular, in recent years, even a substance that emits only weak fluorescence has been frequently used for examining the positional relationship with other substances by multiple staining with a plurality of fluorescent labels.

[0003] Conventionally, when observing a multi-stained fluorescent specimen using such an epi-fluorescent microscope, a fluorescent filter set (excitation filter, dichroic mirror, absorption filter) for observing a single fluorescent dye is switched. Therefore, one fluorescent dye must be observed one by one, and in order to examine the positional relationship between the fluorescent dyes, each fluorescent image must be multiplex-recorded and displayed in a photograph or video memory.

Therefore, in the above-described epi-illumination fluorescence microscope, not only is it time-consuming to switch the fluorescent filter set, but also, for example, it is necessary to detect the positional relationship between a fluorescent dye that is fast-fading or a fluorescent dye using time as a parameter. Is unsuitable. In addition, this observation method has a problem in that, when the fluorescent filter set is switched, misalignment of the observation optical system occurs due to the precision of components such as a dichroic mirror and an absorption filter, and an error occurs in a positional relationship detected from a fluorescent image. There is.

Therefore, recently, in order to improve the above disadvantageous problems, a fluorescent filter set having characteristics as shown in FIG. 7 has been used. That is, this fluorescent filter set includes an excitation filter 1 having two high transmittance regions λ _EA and λ _EB that efficiently excite two kinds of fluorescent dyes A and B, and these two regions λ _EA and λ _EB . By combining the dichroic mirror 2 and the absorption filter 3 having two high transmittance regions in the wavelength region longer than this region and the wavelength region λ _EB , two types of fluorescent dyes A and B are simultaneously observed. .

[0006]

Therefore, according to the above-mentioned fluorescent filter set, it is possible to eliminate the misalignment of the observation optical system and the error of the positional relationship, but the difference in the fluorescence intensity for each fluorescent dye and When there is a difference in the fading speed of the fluorescence, it is difficult to make these two types of fluorescence intensity the desired brightness. As a result, there is a problem that one of the fluorescent images recorded in observation or photography is too dark or too bright.

The present invention has been made in view of the above circumstances, and enables observation of a multi-stained fluorescent sample without switching a fluorescent filter set, and further, by appropriately changing the fluorescence intensity for each excitation by a simple operation. It is an object of the present invention to provide an epi-illumination fluorescence microscope that obtains a fluorescence image that can be easily observed.

[0008]

First, in order to solve the above-mentioned problems, the invention corresponding to claims 1 and 2 is for converting a light beam projected from an epi-illumination light source into a plurality of narrow-band excitation light wavelengths. An excitation filter, a dichroic mirror that reflects a plurality of narrow-band excitation light wavelengths converted by the excitation filter, irradiates the specimen with light, and transmits a plurality of types of fluorescence emitted from the specimen, and a dichroic mirror. An epi-fluorescence microscope having an absorption filter that absorbs an extra wavelength band from the transmitted light and transmits the wavelength of the fluorescence image, wherein the plurality of narrow-band excitation light wavelengths include any two of the narrow-band excitation light wavelengths. On the other hand, a state where the transmittance is partially low on the short wavelength side in the short wavelength side narrow band pumping light wavelength region and high in the long wavelength side narrow band pumping light wavelength region. From the long wavelength side narrow band pumping light wavelength region has a partially low transmittance on the long wavelength side of the long wavelength side, and so as to be in a state having a high transmittance in the short wavelength side narrow band pumping light wavelength region, the said. By holding it rotatably with the axis perpendicular to the optical axis in the epi-illumination light path before the dichroic mirror and tilting it by an appropriate angle with respect to the optical axis,
An interference filter that shifts a transmission wavelength band and varies a light amount ratio of the plurality of narrow band excitation lights is arranged.

Next, according to the first and third aspects of the present invention, an excitation filter for converting a light beam projected from an epi-illumination light source into a plurality of narrow-band excitation light wavelengths, and the excitation filter is used for the conversion. A dichroic mirror that reflects light of a plurality of narrow-band excitation light wavelengths and irradiates the sample with a plurality of types of fluorescent light emitted from the sample, and absorbs an extra wavelength range from light transmitted from the dichroic mirror. In an epi-fluorescence microscope having an absorption filter that transmits the wavelength of the fluorescence image, focusing on any two narrow-band excitation light wavelengths among the plurality of narrow-band excitation light wavelengths,
It has high transmittance in the short wavelength side narrow band excitation light wavelength region,
A first filter having a low transmittance in a long wavelength side narrow band excitation light wavelength region; and a first filter having a low transmittance in a short wavelength side narrow band excitation light wavelength region and having a long wavelength side narrow band excitation light wavelength. The transmission wavelength band is shifted by selectively inserting and removing the first and second filters in the incident illumination light path before the dichroic mirror in the region. Then, the light amount ratio of the plurality of narrow band excitation lights is varied.

[0010]

Therefore, the inventions corresponding to the first and second aspects of the present invention take the above-mentioned means, so that the interference filter can be rotatably held in the incident light path before the dichroic mirror with the axis perpendicular to the optical axis. By tilting the filter with respect to the optical axis, a part of the long wavelength side narrow band is gradually cut in the transmission wavelength band, and the filter is shifted to have a high transmittance in the low wavelength side band. The light intensity of multiple narrow-band excitation lights from the sample changes, and the ratio of the multiple fluorescence intensities emitted from the sample changes accordingly, so that the brightness of multiple types of observed fluorescence images can be easily adjusted and the efficiency is improved. Good excitation can be performed.

The invention according to claims 1 and 3 is characterized in that the epi-illumination light path before the dichroic mirror has a high transmittance in a short wavelength side narrow band excitation light wavelength region and a long wavelength side A first filter having a low transmittance in the narrow band excitation light wavelength region, a low transmittance in the short wavelength side narrow band excitation light wavelength region, and a high transmittance in the long wavelength side narrow band excitation light wavelength region; Since the transmission wavelength band is shifted by selectively inserting and removing the second filter having the above, the respective light amounts of the plurality of narrow band excitation lights from the excitation filter can be changed in the same manner as described above.

[0012]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a configuration diagram showing one embodiment of an optical system of an epi-illumination fluorescence microscope according to the present invention. In FIG.
Is a light source such as a mercury lamp, 12 is a collector lens for collecting the light projected from the light source 11, and 13 is a transmission wavelength shift rotatably held with an axis perpendicular to the output side optical axis of the collector lens 12. This is an interference filter as a filter. Further, on the output side of the interference filter 13, an aperture stop 14, a field stop 15, and FIG.
The excitation filter 16 having the transmittance shown in FIG. 7 and the dichroic mirror 17 having the transmittance shown in FIG.

Reference numeral 18 denotes an objective lens, and 19 denotes a vertically movable stage on which a sample 20 is placed. The reflected light from the dichroic mirror 17 irradiates the sample 20 via the objective lens 18, and this sample The two types of fluorescence emitted from 20 are guided again to the dichroic mirror 17 through the objective lens 18.

On the emission side of the dichroic mirror 17, an absorption filter 21 having a transmittance shown in FIG. 7 is disposed so as to be insertable into and removable from the optical path for switching the optical path to an observation system or a photographic system as required. A beam splitter 22 is disposed, and an eyepiece optical system 23 is disposed on the optical path side of the observation system of the beam splitter 22, while an eyepiece lens 24 for photographing is disposed on the optical path side of the photographing system.

FIG. 2 is a sectional view showing a rotation holding mechanism of the interference filter 13 applied to the epi-illumination fluorescence microscope according to the present invention, which is provided in a part of the epi-illumination light projection tube. That is, the rotation holding mechanism includes the cylindrical body 31 that forms a part of the light emitting tube main body, and the cylindrical body 3 that rotatably holds the interference filter 13 about an axis perpendicular to the optical axis.
1, a filter frame 32, a pin 33 operable to press the filter frame 32, a coil spring 34 for pressing the pin 33, the pin 33 and the coil spring 34 fixed to the cylindrical body 31. And a spring receiver 35 for supporting the spring.

Therefore, in the rotation holding mechanism, the interference filter 13 is rotated from the outside by electric or mechanical means, and receives the pressing force of the coil spring 34 so that the pin 3
Numeral 3 is for applying a pressing force to fix the filter frame 32, that is, the interference filter 13 at an arbitrary rotational position.

Next, the operation of the epi-illumination fluorescence microscope configured as described above will be described.

First, when the interference filter 13 is not present, the light projected from the light source 11
After being condensed by the light 2, the light is introduced into the excitation filter 16, where only light having a plurality of narrow-band excitation light wavelengths is transmitted as shown in FIG. 3 and further reflected by the dichroic mirror 17 to be sampled on the stage 19. Is irradiated. Since the sample 20 has been previously stained with two types of fluorescent dyes, the sample 20 emits two types of fluorescence in two excitation wavelength bands λ _EA and λ _EB as shown in FIG.

The fluorescent light emitted from the specimen 20 passes through the objective lens 18 and the dichroic mirror 17, and light in an unnecessary wavelength range is absorbed by the absorption filter 21, so that only the wavelength of the fluorescent image is observed by the eyepiece. Or you are led to a photography system.

Conventionally, it has been difficult to balance two types of fluorescent intensities to a desired brightness due to differences in the fluorescent intensities of the fluorescent dyes. When adjusting the wavelength, for example, the characteristics of the absorption filter 21 in the observation light path are changed or an auxiliary filter is inserted in the observation light path to partially cut off the fluorescence emitted from the specimen 20; A light intensity adjusting unit that adjusts the intensity of the illumination light by inserting a neutral density ND filter that does not exhibit selective absorption or adjusting the aperture stop 14 may be considered.

However, in the former fluorescence cutting means, the excitation light that excites the fluorescence in the cut portion is wasted, and the utilization efficiency of the fluorescence deteriorates, and the specimen 20 is also damaged extra. Absent. On the other hand, the latter light intensity adjusting means cannot independently adjust the intensity of the excitation light of the two wavelengths, and therefore cannot change the intensity ratio of the two types of fluorescence.

Therefore, in the epi-illumination fluorescence microscope according to the present invention, a transmission wavelength shift filter 13 having a rotation holding mechanism as shown in FIG. 2 is inserted into the epi-illumination light path, and a wavelength band as shown in FIG. Is obtained. In other words, the transmission wavelength shift filter 13 is an interference filter, and the interference filter 1 is shifted in a direction perpendicular to the output optical axis of the collector lens 12 (inclination 0 °).
4 can be obtained as shown in FIG. 4 (a). On the other hand, when inserted at an angle of 45 ° with respect to an axis perpendicular to the output optical axis of the collector lens 12,
The transmittance as shown in FIG. 4B can be obtained.

In general, the interference condition of the interference filter 13 is such that the maximum transmittance wavelength is λ and the optical thickness of the dielectric (the phase difference generated at the boundary of the dielectric is also included in the optical path length).
Is represented by t and the angle of refraction by the boundary is represented by Φ.

2t · cosΦ = mλ Here, when the order m is fixed and the interference condition is fixed, the wavelength λ is proportional to cosΦ. This Φ is a refraction angle, which can be considered to be equivalent because it is conjugate to the incident angle according to Snell's law. This means that as the incident angle increases, cosΦ decreases, and at the same time, the wavelength λ also decreases, so that the maximum transmittance portion gradually shifts to a lower wavelength side. For this reason, when gradually inclined from the axis in the direction perpendicular to the optical axis to 45 °, the light continuously transmits from the transmittance wavelength range as shown in FIG. 4A to the transmittance wavelength range as shown in FIG. 4B. The rate wavelength range can be shifted.

Furthermore, if the excitation filter 16 is used in combination with the interference filter 13 set in a direction perpendicular to the optical axis (inclination 0 °), the excitation light will be the hatched portion in FIG.
As is clear from this figure, a part of the excitation light obtained from the excitation filter 16 on the shorter wavelength side is cut off in the excitation wavelength band λ _EA on the shorter wavelength side, whereas, in the excitation wavelength band λ _EB on the longer wavelength side, it is cut off. Since it is not affected by the interference filter 13, the intensity ratio between the two excitation lights λ _EA and λ _EB changes. Therefore, the intensity ratio of the two types of fluorescence excited by the two excitation lights λ _EA and λ _EB can be changed.

Next, 45 degrees from the axis having a direction perpendicular to the optical axis.
If the interference filter 13 and the excitation filter 16 set at an angle are used together, the excitation light will be the shaded portion in FIG. 4B. In this case, of the excitation light obtained from the excitation filter 16, a part on the longer wavelength side is cut off in the excitation wavelength band λ _EB on the longer wavelength side, while the interference filter 13 is cut off in the excitation wavelength band λ _EA on the shorter wavelength side. , The intensity ratio between the two pump lights λ _EA and λ _EB can be changed in the same manner as described above. Therefore, two excitation lights λ _EA and λ
The intensity ratio of the two types of fluorescence excited by _EB can be changed.

Further, the inclination of the interference filter 13 is set to 0 °.
Between the two states of 45 ° and 45 °, ie the interference filter 13
4A gradually tilts in the direction of 45 ° from the state of 0 ° shown in FIG. 4A, the transmittance band of the interference filter 13 shifts to the shorter wavelength side according to the tilt angle.
At this time, in the excitation wavelength band λ _EA on the short wavelength side, the cut area gradually decreases, and the intensity of the excitation light increases. In addition, in the excitation wavelength band λ _EB on the long wavelength side, since the light is overlapped with the opaque area of the interference filter 13, the area that is gradually cut from the long wavelength side increases, and the intensity of the excitation light decreases accordingly. I will do it.

[0028] Therefore, As is clear from the above description, the excitation light lambda _EA, but the intensity ratio of lambda _EB can be continuously changed, the excitation light lambda _EA Accordingly, the 2 excited by lambda _EB The intensity ratio of the types of fluorescence can also be continuously changed. Therefore, if the inclination of the interference filter 13 is set to an arbitrary angle between the two states of 0 ° and 45 °, the ratio of the intensities of the two types of fluorescence excited by the excitation lights λ _EA and λ _{EB becomes} desired. It can be adjusted to the value you want.

Therefore, according to the configuration of the embodiment described above, when a difference occurs in the fluorescence intensity between the two types of fluorescent dyes, or when a difference occurs in the fading speed between the two types of fluorescent dyes,
Although the balance of the brightness of the two types of fluorescent images is different, in such a case, the inclination of the interference filter 13 is changed, that is, the ratio of the two types of fluorescent intensity can be easily adjusted by a simple operation. The problem that one fluorescent image is too bright or too dark can be solved. In addition, since the dimming is performed on the illumination side, the excitation efficiency is not deteriorated as described above, and there is no fear that the specimen 20 is damaged extra.

FIG. 5 is a view showing another embodiment of the present invention, in which a filter switching mechanism is used in place of the filter holding mechanism shown in FIG. In FIG.
Reference numeral 1 denotes a cylindrical body that constitutes a part of the light emitting tube main body, reference numeral 42 denotes a slider for inserting and removing a hole and a plurality of filters in and from an optical path, reference numeral 43a denotes a hole, and reference numerals 43b and 43c denote the present embodiment described later. The filter used, 44a-44c is
3a, a positioning click groove of the filters 43b, 43c, 45 is a pin operable to fit in the click grooves 44a to 44c to position and fix the slider 42, and 46 is a coil spring for pressing the pin 45. , 4
Reference numeral 7 denotes a spring receiver fixed to the cylinder 41 for supporting the pin 45 and the coil spring 46.

Accordingly, when the filters 43b and 43c having the above-described filter mechanism and the excitation filter 16 are combined, the obtained excitation light appears as shown by hatched portions in FIGS. 6 (a) and 6 (b).

In particular, in FIG. 6A, the filter 43b has a high transmittance on the short wavelength side and a low transmittance on the long wavelength side with respect to the two types of excitation wavelength bands λ _EA and λ _EB by the excitation filter 16. Therefore, the intensity of the two types of excitation wavelength bands λ _EA and λ _EB is lower in the long wavelength side excitation wavelength band λ _EB than in the short wavelength side excitation wavelength band λ _EA . As a result, the ratio of the two types of fluorescence intensities excited by the excitation lights λ _EA and λ _EB is constant compared to the intensity ratio when the filter 43b is not inserted into the optical path according to the change in the intensity ratio on the excitation light side. Varies by percentage.

Next, in FIG. 6B, the filter 43c has a low transmittance on the short wavelength side and a high transmittance on the long wavelength side with respect to two types of excitation wavelength bands λ _EA and λ _EB by the excitation filter 16. Therefore, the intensity of the two types of excitation wavelength bands λ _EA and λ _EB is higher in the longer wavelength side excitation wavelength band λ _EB than in the shorter wavelength side excitation wavelength band λ _EA . As a result, the ratio of the two types of fluorescence intensities excited by the excitation lights λ _EA and λ _EB is constant compared to the intensity ratio when the filter 43c is not inserted into the optical path according to the change in the intensity ratio on the excitation light side. Varies by percentage.

Therefore, the hole 43 is formed by the slider 42.
a, by appropriately switching the filters 43b and 43c, the intensity ratio of the excitation lights λ _EA and λ _EB is changed from the state of the excitation light by only the excitation filter 16 to the excitation light as shown by the hatched portion in FIG. Can be appropriately changed at a constant rate to the excitation light as indicated by the hatched portion, and the excitation light λ _EA , λ
Excitation light lambda _EA pursuant to the intensity ratio of the _EB, the intensity ratio of the two fluorescent by lambda _EB can be adjusted at an arbitrary constant rate.

Since the inclination of the transmittance of the filters 43b and 43c can be arbitrarily designed, the fluorescent dye and the sample 2 can be used.
According to the state of 0, the intensity ratio of the two types of fluorescence by the excitation lights λ _EA and λ _EB can be freely set by designing the filters 43b and 43c to be used.

Therefore, according to the configuration of this embodiment, when the difference in the fluorescence intensity between the two types of fluorescent dyes and the state of fading are known, only the switching of the slider 42 is performed.
It is possible to quickly change the intensity ratio of the types of fluorescent light, and to obtain a fluorescent image that is easy to observe by eliminating variations in the brightness of the fluorescent image for each fluorescent dye during observation and photographing. In addition, since the dimming is performed on the illumination side, there is no deterioration in excitation efficiency observed in the adjustment of the fluorescence intensity in the observation optical path, and no extra damage is given to the observation sample 20.

In the above-described embodiment, two types of filters 43b and 43c are used in the slider 42. However, for example, one or two or more types of filters and a slider corresponding thereto are used, so that an observer can be observed. It is possible to cope with a plurality of requests according to the preference and the state of the microscope.

In addition, the present invention can be variously modified and implemented without departing from the gist thereof.

[0039]

As described above, according to the present invention, in the multi-stained fluorescent specimen, the excitation light amount ratio of each fluorescent dye emitted from this specimen is gradually reduced by the interference filter from the direction perpendicular to the optical axis on the excitation light side. It can be adjusted by a simple operation such as tilting the filter in a predetermined direction, or switching the filter in and out with a slider, so that the difference in the fluorescence intensity and the fading speed of each fluorescent dye and the preference of the observer can be adjusted. To change the ratio of the brightness of the fluorescent image of multiple staining, and to correct the difference in the brightness of the fluorescent image due to the difference in fluorescent dye during observation or photographing, to obtain a fluorescent image that is easy to observe Can be.

Further, since the excitation light is adjusted on the irradiation light side,
The intensity of the fluorescence emitted from the sample and guided to the observation optical path is not weakened by the control filter.Therefore, in order to increase the fluorescence intensity, it is not necessary to irradiate the sample with more excitation light than usual, causing damage to the sample. I will not give.

Therefore, efficient excitation can be performed without impairing the fluorescence by light emission, and no extra damage is given to the specimen.

[Brief description of the drawings]

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

FIG. 2 is a cross-sectional view showing a rotation holding mechanism of an interference filter applied to the epi-fluorescence microscope according to the present invention.

FIG. 3 is a diagram showing a transmission wavelength range of an excitation filter without an interference filter.

FIG. 4 is a diagram showing the transmittance when the tilt angle of the interference filter is changed and the wavelength of the excitation light when the interference filter and the excitation filter are used together.

5A and 5B are diagrams showing a filter switching mechanism used in the epi-illumination fluorescence microscope according to the present invention, wherein FIG. 5A is a diagram for explaining the positioning of the filter, and FIG. 5B is a cross-sectional view of the filter switching mechanism.

FIG. 6 is a diagram illustrating transmittance when a filter is changed and wavelength of excitation light when a filter and an excitation filter are used in combination.

FIG. 7 is a diagram showing transmittance characteristics of a conventional fluorescent filter set.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 11 ... Light source, 12 ... Collector lens, 13 ... Transmission wavelength shift filter (interference filter), 16 ... Excitation filter, 17 ... Dichroic mirror, 18 ... Objective lens, 20 ... Sample, 21 ... Absorption filter, 22 ... Beam splitter Reference numeral 23, an eyepiece optical system, 24, an eyepiece for photographing, 31, 41, a cylindrical body, 32, a filter frame,
42: slider, 43b, 43c: filter.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Kazuo Kajiya 2-43-2 Hatagaya, Shibuya-ku, Tokyo Inside Olympus Optical Co., Ltd. (56) References JP-A-3-81648 (JP, A) JP JP-A-50-67689 (JP, A) JP-A-50-99779 (JP, A) JP-A-5-501458 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G02B 21 / 00 G02B 21/06-21/36 G01N 21/62-21/74

Claims (3)

(57) [Claims] An excitation filter for converting a light beam projected from an epi-illumination light source into a plurality of narrow-band excitation light wavelengths, and reflecting light of a plurality of narrow-band excitation light wavelengths converted by the excitation filter. A dichroic mirror that transmits a plurality of types of fluorescent light emitted from the sample, and an absorption filter that absorbs an extra wavelength region from light transmitted from the dichroic mirror and transmits the wavelength of the fluorescent image. In the epi-illumination fluorescence microscope, an epi-illumination fluorescence characterized in that a transmission wavelength shift filter that shifts a transmission wavelength band and changes a light amount ratio of the plurality of narrow-band excitation lights is arranged in an epi-illumination light path before the dichroic mirror. microscope. 2. The transmission wavelength shifting filter is arranged such that, for any two of the plurality of narrow-band excitation light wavelengths, a short-wavelength-side narrow-band excitation light wavelength region has a short wavelength side narrow-band excitation light wavelength region. From a state having a high transmittance in the long wavelength side narrow band pumping light wavelength region to a partially low transmittance in the long wavelength side narrow band pumping light wavelength region in the long wavelength side. Having, and in a short wavelength side narrow band excitation light wavelength region, so as to have a high transmittance, in the epi-illumination light path before the dichroic mirror, held rotatably with the axis in the direction perpendicular to the optical axis, 2. The epi-illumination fluorescence microscope according to claim 1, wherein the interference filter shifts a transmission wavelength band by inclining the optical axis by an appropriate angle. 3. The transmission wavelength shift filter focuses on any two narrow-band excitation light wavelengths among the plurality of narrow-band excitation light wavelengths, and provides a high transmittance in a short wavelength side narrow-band excitation light wavelength region. A first filter having a low transmittance in the long wavelength side narrow band excitation light wavelength region, and a low transmittance in the short wavelength side narrow band excitation light wavelength region, and a long wavelength side narrow band excitation light wavelength region. A second filter having a high transmittance in an excitation light wavelength region, and selectively inserting and removing these first and second filters in an epi-illumination light path before the dichroic mirror, thereby forming a transmission wavelength band. 2. The epi-illumination fluorescence microscope according to claim 1, wherein