JP2000089124A - Compound microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound microscope capable of simultaneously observing the same part of the same sample by different methods. SOLUTION: In order to observe a transmitted image by a lower observation system, an exciting filter 33 is removed. And a mirror for transmitting only fluorescence and reflecting light whose wavelength is other than that of the fluorescence is used as a dichroic mirror 34. In a lower optical system, a dichroic mirror 14 for reflecting only excitating light for exciting the observation sample 10 and emitting the fluorescence and transmitting the light whose wavelength is other than that of the fluorescence is used, and a fluorescent filter 10 is removed. By having such a constitution, white light emitted from an illumination light source 31 is transmitted through the observation sample 10, then, the light reaches an eyepiece 22 or an image pickup device 25. Besides, light emitted from an illumination light source 11 is excited by the exiting filter 13, then, the observation sample 10 is illuminated by the exited light, then, the excited fluorescence reaches an eyepiece 42 or an image pickup device 45.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope, and more particularly, to a compound microscope capable of simultaneously observing a sample from above and observing a sample from below.

[0002]

2. Description of the Related Art FIG. 4 shows the configuration of an upright microscope generally used in the prior art. The one shown in FIG. 4 is a relatively high-end machine, which has two illumination systems and one imaging system in addition to one binocular observation system. The lower illumination system is used for observing a transmission image or a fluorescence image, and the upper (epi-illumination) illumination system is used for observing a reflection image or a fluorescence image. When the upper illumination system is used, the objective lens 35 also functions as an illumination lens. It is rare that the upper illumination system and the lower illumination system are used at the same time.

When observing a transmission image, an excitation filter 13, a dichroic mirror 34, a fluorescence filter 36
Remove. Then, the illumination light source 11 is turned on, and the light is condensed by the condenser lens 12, the reflection mirror 26,
The sample is guided to the observation sample 10 through 7 and the observation sample is illuminated from the back side. The transmitted light passes through the objective lens 35, its path is selected by the switchable mirror 38, and reaches the eyepiece 42 (43) via the reflection mirror 39 or reaches the imaging device 45 via the projection lens 44. Binocular optical system 28 if necessary
Are provided so that the image of the observation sample 10 can be observed with both eyes.

When observing a fluorescent image using the lower illumination system, the excitation filter 13 and the dichroic mirror 3 are used.
4. Attach the fluorescent filter 36 and turn on the illumination light source 11. After the light from the illumination light source 11 passes through the condenser lens 12, the light is converted into excitation light in a wavelength band in which the excitation filter 13 excites fluorescence, and this excitation light is transmitted through the reflection mirror 26 and the illumination lens 27 to the observation sample 10. Is irradiated. Fluorescence emitted from the observation sample 10 when excited by the excitation light;
The excitation light transmitted through the observation sample 10 passes through the objective lens 35 and reaches the dichroic mirror 34.

The dichroic mirror 34 is selected to transmit light on the long wavelength side to which the wavelength band of fluorescence belongs and reflect light on the short wavelength side to which the wavelength band of excitation light belongs. Therefore, of the light that has passed through the objective lens 35, the excitation light is reflected in a direction substantially different from the traveling direction by 90 degrees, and only the fluorescence and a very small amount of the excitation light pass through the dichroic mirror 34 and reach the fluorescent filter 36. The very small amount of excitation light that has passed through the dichroic mirror 34 is almost completely removed by the fluorescent filter 36, and only the fluorescence is selected in its path by the switchable mirror 38, and reaches the eyepiece 42 (43) via the reflection mirror 39. Alternatively, the light reaches the image pickup device 45 via the projection lens 44. Thereby, the fluorescence image of the observation sample 10 excited by the excitation light by the lower illumination system can be observed.

When observing a reflected image using the upper illumination system, the excitation filter 33 and the fluorescence filter 36 are removed, and the dichroic mirror 34 is changed to a beam splitter (half mirror). When the illumination light source 31 is turned on, the light from the illumination light source 31 is reflected by a beam splitter (half mirror) 34 via a condenser lens 32 and irradiates the observation sample 10 through an objective lens 35.
The reflected light from the observation sample 10 passes through the objective lens 35, passes through the beam splitter (half mirror) 34, reaches the eyepiece 42 (43) via the reflection mirror 39, or reaches the image pickup device 45 via the projection lens 44. Reach Thereby, the reflection image of the observation sample 10 can be observed.

When observing a fluorescent image using the upper illumination system, an excitation filter 33 and a fluorescent filter 36 are attached, and as a dichroic mirror 34, light on the long wavelength side to which the fluorescence wavelength band belongs is transmitted and excitation light is emitted. The one that reflects light on the short wavelength side to which the above wavelength band belongs is selected. Then, when the illumination light source 31 is turned on, the light from the illumination light source 31 reaches the excitation filter 33 via the condenser lens 32, and is converted into excitation light in a wavelength band for exciting fluorescence, and the excitation light is converted to a dichroic mirror 34. Reflected by the objective lens 35
The observation sample 10 is radiated through the.

[0008] The fluorescence emitted from the observation sample 10 when excited by the excitation light and the excitation light reflected by the observation sample 10 pass through the objective lens 35 and enter the dichroic mirror 3.
Reaches four. Of the light that has passed through the objective lens 35, the excitation light is reflected in a direction substantially different from the traveling direction by 90 degrees,
Only fluorescence and very little excitation light are dichroic mirror 3
4 and reaches the fluorescent filter 36. The very small amount of excitation light that has passed through the dichroic mirror 34 is almost completely removed by the fluorescent filter 36, and only the fluorescence is selected in its path by the switchable mirror 38, and reaches the eyepiece 42 (43) via the reflection mirror 39. Or the projection lens 44
And reaches the image pickup device 45 via. Thereby, the fluorescence image of the observation sample 10 excited by the excitation light by the lower illumination system can be observed.

A transmission image and a fluorescence image of the observation sample 10 can be observed simultaneously. In this case, the lower illumination system is arranged in the same manner as when observing a transmitted image. And
Put the excitation filter 33 in the upper illumination system,
Out of the light from the wavelength band that excites the fluorescence. Then, instead of the dichroic mirror 34, a beam splitter (half mirror) is used, or a 45-degree incident filter that reflects only light in the wavelength band of the excitation light and transmits light in other wavelength bands is used. As the fluorescent filter 36, a filter that passes light in a wavelength band other than the wavelength band of the excitation light is selected.

The light from the lower illumination system transmitted through the observation sample 10 is transmitted through the objective lens 35, the beam splitter (half mirror) or the filter 34, and
6 also passes through the eyepiece 42 via the reflection mirror 39.
(43) or the imaging device 4 via the projection lens 44
Reaches 5. Light from the illumination light source 31 is converted into excitation light by an excitation filter 33, reflected by a beam splitter (half mirror) or a filter 34, and irradiates the observation sample 10 via an objective lens 35. The fluorescence emitted from the observation sample 10 passes through the objective lens 35, passes through a beam splitter (half mirror) or a filter 34, passes through a fluorescence filter 36, and passes through a reflection mirror 39.
To the eyepiece 42 (43) via the projection lens 44 or to the imaging device 45 via the projection lens 44. In this way,
A fluorescence image and a transmission image can be observed simultaneously.

FIG. 5 shows a configuration diagram of an inverted microscope generally used conventionally. In this inverted microscope, the upper illumination system basically includes an illumination light source 31, a condenser lens 32, an excitation filter 33, a reflection mirror 46, and an illumination lens 47, and the lower optical system basically includes the illumination light source 11 , Condenser lens 12, excitation filter 13, dichroic mirror 14, fluorescent filter 16, reflection mirror 1
7, switchable mirror 18, reflection mirrors 19 and 20, binocular optical system 21, eyepiece 22 (23), projection lens 2
4. Consists of an imager 25. In order to reduce the height of the optical system provided below the observation sample 10, these optical systems are turned sideways by changing the optical axis by 90 degrees by the reflecting mirror 17, and the subsequent optical systems are provided in the horizontal direction. Point,
Except that an extra reflecting mirror 20 for inverting the image upside down is provided, it is basically the same as the upright microscope, and its use is basically the same as that of the upright microscope. Omitted. Such an inverted microscope is mainly used in the fields of medicine and biology for the purpose of observing a sample in a petri dish with an objective lens having a small working distance.

The arrangement of the optical elements of the upright microscope and the inverted microscope shown in FIGS. 4 and 5 includes a phase plate, a Nomarski prism,
By appropriately inserting a special optical element such as a polarizing plate, a phase difference image, a differential interference image, a polarization image, and the like can be observed.

[0013]

Conventionally, an upright microscope and an inverted microscope have been properly used according to practical purposes, and each of them has contributed in various fields. There is no difference in the various imaging principles used simply by the difference between observing the sample from above and observing from below.

There is a limitation in performing simultaneous observation based on a plurality of imaging principles with a conventional upright microscope or inverted microscope. For example, as described above, by using illumination for observing a transmission image and illumination for observing a fluorescence image at the same time, it is possible to observe the fluorescence image and the transmission image in a superimposed manner.
However, this reduces the contrast of the fluorescent image, so this method cannot be used when the fluorescence from the sample is weak. In such a case, the transmission image and the fluorescence image of the same sample must be observed at different times by changing the observation method.

However, there is a problem that it is difficult to accurately judge the correspondence between the position where the fluorescence is generated and the transmitted image due to the position change due to the movement of the sample.
In particular, when the sample is moving, it has been impossible to accurately determine the correspondence between the position where fluorescence is generated and the transmitted image.

The present invention has been made in view of such circumstances, and it is an object of the present invention to provide a compound microscope capable of simultaneously observing the same portion of the same sample by different methods.

[0017]

The above object is achieved by a compound microscope in which an upright microscope and an inverted microscope are combined with a common optical axis.

By combining an upright microscope and an inverted microscope with a common optical axis, not only the illumination optical system can be simplified, but also the same At the same time, the fluorescence image and the transmission image, and the phase difference image and the transmission image can be observed simultaneously. Further, by changing the combination of the optical systems, the differential interference image and the transmission image, and the polarization image and the transmission image can be simultaneously observed. In addition, depending on the combination of optical elements that can be adopted by those skilled in the art based on ordinary knowledge, various types of imaging elements based on these and other imaging principles may be used.
The two images can be combined and observed simultaneously.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a configuration diagram of an optical system according to the first embodiment of the present invention. In FIG.
10 is an observation sample, 11 and 31 are illumination light sources, 12 and 32 are condenser lenses, 13 and 33 are excitation filters, and 14 and 34.
Is a dichroic mirror, 15 and 35 are objective lenses, 1
6, 36 are fluorescent filters, 17 is a reflection mirror, 18,
38 is a switchable mirror, 19 and 39 are reflection mirrors, 20,
40 is a reflection mirror, 21 and 41 are binocular optical systems (image left-right reversal), 22, 23, 42 and 43 are eyepieces, 24 and 4
4 is a projection lens, and 25 and 45 are imaging devices. The configuration of this optical system is typical, and a filter may be removed or a dichroic mirror may be mounted depending on the purpose of use.
Use it by replacing it with a positive incidence filter or beam splitter (half mirror).

This configuration is basically a combination of the upright microscope shown in FIG. 4 and the inverted microscope shown in FIG. 5 with their optical axes aligned, and is required for the upright microscope. The lower illumination system and the upper illumination system required for the inverted microscope can be replaced with the upper and lower illumination systems in the compound microscope, and are unnecessary, and the configuration can be simplified.

In the present embodiment, the fluorescence image and the transmission image of the sample can be observed simultaneously and independently by combining filters. For example, to observe a fluorescence image with an upper observation system centered on the eyepiece lens 42 (43) and the imager 45 and a transmission image with a lower observation system centered on the eyepiece lens 22 (23) and the imager 25, First, from the request for observing the transmitted image with the lower observation system, illumination for the transmitted image is performed with the upper illumination system including the illumination light source 31 and the condenser lens 32. On this occasion,
The excitation filter 33 is removed to use white light.
Then, as the dichroic mirror 34, a 45-degree incident fluorescence transmission filter that transmits only fluorescence and reflects light of another wavelength is used (strictly, such a 45-degree incidence fluorescence transmission filter has band-pass characteristics). Is not a dichroic mirror, but in this specification,
For convenience, this is also called a dichroic mirror).

Then, the light from the illumination light source 31 is reflected by the dichroic mirror 34 and reaches the objective lens 35. The objective lens 35 functions as an illumination lens, and the observation sample 10 is illuminated. In the lower optical system, a 45-degree incident excitation light reflection filter that reflects only excitation light that excites the observation sample 10 and emits fluorescence and transmits light of other wavelengths is used as the dichroic mirror 14. 16 has been removed. The light transmitted through the observation sample 10 and passed through the objective lens 15 passes through the dichroic mirror 14 and is reflected by the reflection mirror 17, and then the transmitted light is observed by the switchable mirror 18 through the eyepiece 22 (23). , The observation by the image pickup device 25, or the observation by both of them with appropriate distribution.

When observing with the eyepiece 22 (23),
The top, bottom, left, and right of the image coincide with the top, bottom, left, and right of the sample 10. This is because the upper and lower parts of the sample 10 are turned upside down when the sample 10 is viewed directly from the lower part, and are turned forward by the objective lens, turned by the reflecting mirror 17, turned by the switching mirror 18, and turned on the reflecting mirror 1.
At 9, the light is inverted, and at the reflection mirror 20, the light is inverted. Regarding the left and right, when the sample 10 is viewed directly from below, the left and right are not inverted, but are inverted by the objective lens, and mirrors 17, 18, 19, 2
Even if the light is reflected at 0, there is no change in the left and right directions, and the light is guided to the binocular optical system 21 whose overhead view is shown in FIG.

In FIG. 2, reference numeral 51 denotes a half mirror;
56 is a reflection mirror. In the drawings including FIG. 2, the same reference numerals are given to the same components as those shown in the preceding drawings in the section of the embodiment of the invention, and the description thereof will be omitted. In FIG. 2, regarding the right eye system,
It is rotated forward by the half mirror 51 and inverted by the reflection mirror 52,
The light is rotated forward by the reflection mirror 53. The left-eye system is rotated forward by the reflection mirror 54, inverted by the reflection mirror 55, and rotated forward by the reflection mirror 56. In the binocular optical system 21, no upside down occurs. Further, the eyepieces 22 and 23 do not act in the vertical and horizontal directions.

When the image is observed by the image pickup device 25, the upper, lower, left, and right sides of the image can be matched with the upper, lower, left, and right sides of the sample 10. Because
Invert up, down, left, and right with the objective lens and project
4 to make a normal rotation. Since it is normal for the image pickup surface of the image pickup device to form an image by inverting up, down, left, and right, in this case, the image pickup device 25
Can be mounted upside down.

Next, observation of a fluorescent image by an upper observation system centered on the eyepiece 42 (43) and the image pickup device 45 using a lower illumination system including the illumination light source 11 and the condenser lens 12 will be described. . White light from the illumination light source 11 reaches the excitation filter 13 via the condenser lens 12. The excitation filter 13 is a filter that passes only light in a wavelength band necessary for exciting fluorescence. The excitation light that has passed through the excitation filter is reflected upward by the dichroic mirror 14. The objective lens 15 functions as an illumination lens, and the observation sample 10 is illuminated. Fluorescence from the observation sample 10 passes through the objective lens 35, passes through the dichroic mirror 34, and further passes through the fluorescence filter 36. Sample 10
The excitation light that has passed through the objective lens 35 passes through the objective lens 35, most of which is reflected by the dichroic mirror 34, and a small amount of excitation light that has passed therethrough is completely removed by the fluorescent filter 36. Whether the fluorescence is observed with the eyepiece 42 (43) or the imager 45 with the switchable mirror 38,
Or decide whether to distribute them appropriately and observe with both.

When observing with the eyepiece 42 (43),
The top, bottom, left, and right of the image coincide with the top, bottom, left, and right of the sample 10. This is because the upper and lower parts are inverted by the objective lens 35, rotated forward by the switchable mirror 38, inverted by the reflecting mirror 39, and rotated forward by the reflecting mirror 40. The left and right are inverted by the objective lens, and there is no change in the left and right even if they are reflected by the mirrors 38, 39 and 40, and the binocular optical system 4 shown in an overhead view in FIG.
It is led to 1. Then, in the binocular optical system 41, the left and right are rotated forward as described above, and the eyepieces 42 (43)
It is led to.

As described above, in the embodiment shown in FIG. 1, by appropriately selecting the optical element,
In the upper observation system, the fluorescence image of the observation sample can be observed without reversing the top, bottom, left and right, and in the lower observation system, the transmission image of the observation sample can be observed without reversing the top, bottom, left, and right. Can be performed simultaneously without moving the sample. Strictly simultaneous observation is not possible when humans observe through eyepieces,
It is difficult to exactly grasp the correspondence between the transmission image and the fluorescence image. However, the upper and lower images are simultaneously captured by the imaging devices 25 and 45 and simultaneously observed by a monitor, and the correspondence between the transmission image and the fluorescence image is accurately grasped by superimposing the images of the imaging devices 25 and 45 by image processing. can do. In any case, unlike the conventional case, even if the amount of fluorescence is weak, the observation of the transmission image and the fluorescence image are performed independently, so that the fluorescence image and the fluorescence image are not reduced without lowering the contrast of the fluorescence. Transmission images can be observed simultaneously.

In this embodiment, since the upper optical system and the lower optical system are substantially the same, a transmission image is obtained by the lower illumination system and the upper observation system, and a transmission image is obtained by the upper illumination system and the lower observation system. It is also possible to observe a fluorescent image.

FIG. 3 shows a configuration diagram of an optical system according to the second embodiment of the present invention. In FIG. 3, 57 is a ring slit, and 58 is a phase plate (1/4 wavelength plate). In this embodiment, by appropriately selecting each optical element, a fluorescent image can be observed by the lower illumination system and the upper observation system, and a phase difference image can be observed by the upper illumination system and the lower observation system.

In this case, a band-pass filter that passes monochromatic light having a wavelength different from that of the excitation light is used as the filter 33. Illumination light from the light source 31 passes through the condenser lens 32 to be converted into parallel rays, is then converted to monochromatic light by the bandpass filter 33, and passes through a ring slit 57 for phase difference observation.
As the dichroic mirror 34, a 45-degree incident fluorescence transmission filter that passes only the fluorescence from the observation sample 10 and reflects light of another wavelength is used.

Then, the illumination light is reflected downward by the dichroic mirror 34, the objective lens 35 works as an illumination lens, and the sample 10 is illuminated. Of the light that has passed through the sample 10 and passed through the objective lens 15, the light that has not been diffracted by the observation sample 10 undergoes phase modulation by passing through the phase plate 58. Light diffracted by the observation sample 10 passes without passing through the phase plate 58. As the dichroic mirror 14, a 45-degree incident excitation reflection filter having a characteristic of reflecting only excitation light for exciting fluorescence in the observation sample 10 and transmitting light of another wavelength is used. Then, the light diffracted or not received by the sample 10 passes through the dichroic mirror 14 and is reflected by the reflection mirror 17. Then, the transmitted light is observed by the switchable mirror 18 by the eyepiece 22 (23). It is determined whether the image is to be observed by the image pickup device 25 or to be appropriately distributed and observed by both.

When observing with the eyepiece 22 (23),
When the image is observed by the image pickup device 25, the upper, lower, left, and right of the image coincide with the upper, lower, left, and right of the sample 10, as in the first embodiment. Thus, the phase contrast image of the observation sample 10 can be observed using the upper illumination system and the lower observation system. Since the principle of this phase contrast microscope is well known,
The description is omitted.

The lower illumination system has the same configuration as that of the first embodiment except that a phase plate 58 is provided.
Therefore, the white light from the illumination light source 11 is used as the excitation light and illuminates the observation sample 10, but at this time, the operation of the phase plate 58 can be ignored. The fluorescence emitted from the observation sample 10 is observed in the upper observation system. Regarding the observation of this fluorescence, the configuration of the upper observation system is the same as that of the first embodiment, and thus the fluorescence of the observation sample 10 You can observe the image.

In the above embodiment, the simultaneous observation of the transmission image and the fluorescence image and the simultaneous observation of the fluorescence image and the phase contrast image have been described. Simultaneous observation of images or of these and other images is possible. Combinations of optical systems for achieving these are easily possible by those skilled in the art.

[0036]

As described above, according to the present invention, not only the illumination optical system can be simplified, but also two images based on various imaging principles can be simultaneously observed without reducing the contrast. be able to.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an optical system according to a first embodiment of the present invention. (Side view)

FIG. 2 is a diagram showing a configuration of an image left-right reversing binocular branch optical system. (Overview)

FIG. 3 is a configuration diagram of an optical system according to a second example of the embodiment of the present invention. (Side view)

FIG. 4 is a configuration diagram of a configuration diagram of an erect microscope generally used conventionally. (Side view)

FIG. 5 is a configuration diagram of a configuration diagram of an inverted microscope generally used conventionally. (Side view)

[Description of sign]

10: observation sample, 11: illumination light source, 12: condenser lens,
13 ... excitation filter, 14 ... dichroic mirror,
15 Objective lens, 16 Fluorescent filter, 17 Reflective mirror, 18 Switchable mirror, 19, 20 Reflective mirror, 21 Binocular optical system (image left / right reversal), 22, 23 Eyepiece lens, 24 Projection lens , 25 image pickup device, 26 reflection mirror, 27 illumination lens, 31 illumination light source, 32
Condenser lens, 33 ... Excitation filter, 34 ... Dichroic mirror, 35 ... Objective lens, 36 ... Fluorescent filter, 38 ... Switchable mirror, 19 ... Reflection mirror, 39 ... Reflection mirror, 40 ... Reflection mirror, 41 ... Binocular optical system (Image left / right reversal), 42: eyepiece, 43: eyepiece, 44
... Projection lens, 45 ... Imaging device, 46 ... Reflection mirror, 47
... Illumination lens, 51 ... Half mirror, 52-56 ... Reflection mirror, 57 ... Ring slit, 58 ... Phase plate

Claims (1)

[Claims] 1. A compound microscope wherein an upright microscope and an inverted microscope are combined with a common optical axis.