JPH11223773A - Vertical fluorescent microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vertical fluorescent microscope which is rich in generality with plural independent optical paths in a vertical illuminating optical system in which the use of each vertical illuminating optical path is not limited. SOLUTION: The laser beam of a laser light source 3 for releasing the Caged reagent of a first vertical illuminating optical system optical path is introduced through a dichroic mirror DM1 of a cube CV1 to a sample 1 side, and the excitation light of a mercury lamp 9 for irradiating the excitation light of a third vertical illuminating optical system optical path is introduced through a dichroic mirror DM2 of a cube CV2 to the sample 1 side so that Caged reagent releasing test can be executed. On the other hand, the excitation light of a mercury lamp 6 for irradiating the excitation light of a second vertical illuminating optical system optical path is introduced through a dichroic mirror DM1' of a cube CV1' to the sample 1 side, and a laser beam from a laser light source 12 for the laser trap of a third vertical illuminating optical system optical path is introduced through a dichroic mirror DM2' of a cube CV2' to the sample 1 side so that laser trap test can be executed.

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 and, more particularly, to an epi-fluorescence microscope employing a laser trap method or a caged reagent release method used as a means for cell manipulation in the field of cell physiology.

[0002]

2. Description of the Related Art Epi-fluorescence observation using an epi-fluorescence microscope
In the field of biology, as a means for observing the morphology of a specific substance inside a cell, this method is widely and generally used at present.

In recent years, particularly in the field of cell physiology, laser trapping and caged methods have been used as methods for manipulating intracellular substances using the epi-illumination means of a microscope.
The reagent release method is widely used.

Here, the laser trap method (optical tweezer method) is a method of irradiating an arbitrary substance in a specimen with a laser beam and optically capturing the same, and measures the amount of force at the time of intracellular protein movement. As an irradiation means, an infrared laser is used. On the other hand, Cag
The ed reagent release method is a method in which a caged reagent is bound to a molecule having a physiological activity and administered to cells in a state where the activity is suppressed, and then the light is irradiated to release the bond and restore the activity of the molecule. In this case, ultraviolet light is used as the irradiation means.

As described above, the epi-fluorescence microscope employing the laser trapping method or the caged reagent releasing method,
In addition to ordinary epi-illumination illumination, various light sources for cell manipulation are required.

As shown in FIG. 12, an observation optical system optical path 103 that connects an objective lens 101 and an eyepiece 102 as shown in FIG.
An excitation light source 104 for fluorescence observation is arranged for a first dichroic mirror DM1 arranged together with a barrier filter BA therein, and a second dichroic mirror DM2 is arranged between the first dichroic mirror DM1 and the excitation light source 104. , A band-pass filter BP between the second dichroic mirror DM2 and the excitation light source 104.
And a sample operation light source 105 is disposed on an optical path orthogonal to the second dichroic mirror DM2.

Japanese Unexamined Patent Publication No. 8-234110 discloses a first dichroic mirror as an epi-illumination fluorescence microscope which introduces excitation light from an excitation light source 108 for fluorescence observation into an observation optical system optical path 107 as shown in FIG. There is disclosed one in which a DM1 and a second dichroic mirror DM2 for introducing a laser beam of a laser trap from a laser light source 109 are arranged in two stages.

[0008]

However, considering that the conventional light source shown in FIG. 12 performs fluorescence observation by switching the excitation light source 104 with the excitation wavelength, not only the bandpass filter BP but also the 1 dichroic mirror DM1
However, at this time, since the first dichroic mirror DM1 and the bandpass filter BP are separately arranged, they must be replaced separately, and the work for this is complicated. There is a problem that it becomes troublesome.

On the other hand, one disclosed in Japanese Patent Application Laid-Open No. 08-234110 discloses a first dichroic mirror DM.
When 1 is disposed on the specimen side with respect to the second dichroic mirror DM2, for example, when the laser wavelength range of the laser light source 109 is switched to the ultraviolet range and used for releasing the caged reagent, the laser reflected by the second dichroic mirror DM2 In order to reliably introduce light to the sample side, the first dichroic mirror DM1 and the barrier filter B
As A, it must have a property of transmitting a laser wavelength from the laser light source 109, and each time, it is necessary to perform a troublesome operation such as replacing the first dichroic mirror DM1 and the barrier filter BA with optimal ones. Feasibility is thin. Realistic laser light source 109
Cannot respond to the purpose of use other than the infrared region for the laser trap, and is poor in versatility. Further, since the second dichroic mirror DM2 is fixed in the observation optical system optical path 107, there is no problem in ordinary fluorescence observation,
In weak fluorescence observation such as photometry at the one-molecule level, light intensity is lost, which is not preferable.

The present invention has been made in view of the above circumstances, and provides a versatile epi-illumination fluorescence microscope having a plurality of independent optical paths of an epi-illumination optical system, and the use of each epi-illumination optical path is not limited. The purpose is to provide.

[0011]

According to one aspect of the present invention, an epi-illumination fluorescence microscope that enables simultaneous optical manipulation of a specimen and fluorescence observation by selectively irradiating a specimen with a plurality of epi-illumination lights. , A first epi-illumination optical system optical path disposed in a direction intersecting the observation optical system optical path with respect to the specimen via an intersection, and the same intersection as the first epi-illumination optical system optical path of the observation optical system optical path A second epi-illumination optical system optical path arranged in a direction intersecting with the observation optical system optical path, and intersects the observation optical system optical path via an intersection different from the first and second epi-illumination optical system optical paths. And an optical path of a third epi-illumination optical system arranged in the direction in which

Thus, for example, a laser light source for releasing a caged reagent is provided in the optical path of the first epi-illumination optical system, a mercury lamp for irradiating excitation light in the optical path of the second epi-illumination optical system, and a third epi-illumination optical system. A laser light source for a laser trap or a mercury lamp for irradiating excitation light is arranged on the optical path, and these first to third epi-illumination optical system optical paths are selected, and a laser light source for releasing a caged reagent and a laser light for excitation light irradiation Combination of a mercury lamp and a laser light source for a laser trap, it is possible to selectively perform a caged reagent release experiment by cage release and fluorescence observation, and a laser trap experiment such as fluorescence observation and capture of a small object by a laser trap and micro force measurement. be able to.

In the above-mentioned epi-illumination fluorescence microscope, it is preferable that a wavelength discriminating means is detachably disposed at an intersection of the observation optical system optical path and the third epi-illumination optical system optical path.

Thus, in the case where only normal fluorescence observation is performed without performing laser trapping, for example, a wavelength separation means such as a dichroic mirror is excluded from the observation optical path, thereby observing a light amount loss during weak fluorescence observation. An image can be obtained. Further, by inserting the wavelength separation means into the observation optical path, for example, a laser light source for a laser trap disposed in the optical path of the third epi-illumination optical system or a laser for a laser trap by a mercury lamp for irradiation of excitation light and a laser for fluorescence observation are provided. The excitation light can be selectively introduced into the optical path of the observation optical system. In this case, too, a laser trap experiment or a caged reagent release experiment can be selectively performed as necessary, thus constituting a versatile experimental system. be able to.

In the above-mentioned epi-illumination fluorescence microscope, the observation optical system optical path is further shifted in a direction intersecting with the first and second epi-illumination optical system optical paths and the third epi-illumination optical system optical path through a different intersection. May be arranged.

Thus, for example, a laser light source for irradiating excitation light in the first epi-illumination optical system optical path, a mercury lamp for irradiating excitation light in the second epi-illumination optical system optical path, and a third epi-illumination optical system A laser light source for a laser trap is arranged in the optical path, and Cag is provided in the optical path of the fourth epi-illumination optical system.
A laser light source for releasing the ed reagent is disposed, the first and second epi-illumination optical system optical paths are selected, and a mercury lamp and a laser light source as excitation light sources are selectively used. Irradiating an arbitrary protein in the cell with an ultraviolet laser to release the caged reagent, and irradiating the activated protein with an infrared laser to trap and perform micro measurement This makes it possible to construct a very effective experimental system in the field of cell physiology for analyzing the behavior of intracellular substances at the molecular level.

[0017]

Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 and FIG.
1 shows a schematic configuration of an inverted epi-illumination fluorescence microscope to which the present invention is applied. In the drawing, reference numeral 1 denotes a sample to be observed, an objective lens 2 is arranged below the sample 1, and a cube turret CT indicated by a broken line is provided on an optical axis 31 of an optical path of an observation optical system passing through the objective lens 2. .

The cube turret CT has an optical axis 31
Dichroic mirror DM with 45 ° inclination to
1 and 45 ° with respect to the optical axis 31
And a cube CV1 'having a dichroic mirror DM1', a barrier filter BA1 'and an excitation filter EX1'. ,
The cube CV1 or the cube CV1 'is selectively positioned at the point O1 where the optical axis 31 intersects.

The cube turret CT shown in FIG.
Although only two cubes are shown, at least four cubes having the same configuration and the same shape as the cubes CV1 and CV1 'can be inserted and removed, and the rotation of the turret and a positioning mechanism (not shown) allow any cube to be inserted. The reflection surface of the dichroic mirror can be adjusted to the point O1 where the optical axis 31 intersects. For example, FIG. 2 shows an example in which a dichroic mirror DM1 "and a cube CV1" having a barrier filter BA1 "are inserted into the CT inside the cube turret. When the wavelengths of a laser light source 3 and a mercury light source 6 described later are changed. Further, the cube CV1 'is a dichroic mirror DM1 such that the reflection surface of the dichroic mirror DM1' is oriented toward a mercury light source 6 described later when inserted into the optical axis 31.
1 'is held.

Then, the point O1 of the cube turret CT
The relay lens 5 and the collector lens 4 on the optical axis 32 of the optical path of the first epi-illumination optical system perpendicular to the optical axis 31
And the laser light source 3 are arranged on the same straight line, and a second epi-illumination optical system optical path perpendicular to the optical axis 31 from the point O1 and further perpendicular to the direction from the point O1 to the laser light source 3. The relay lens 8, the collector lens 7, and the mercury light source 6 are arranged on the same straight line on the optical axis 33.

Below the cube turret CT, a slider SV indicated by a broken line is provided so as to be movable along an optical axis 34 described later.

The slider SV includes a dichroic mirror DM2 having an inclination of 45 ° with respect to the optical axis 32, a cube CV2 having a barrier filter BA2 and an excitation filter EX2, and a dichroic mirror DM2 arranged in parallel with the dichroic mirror DM2. Are provided side by side in the direction in which the optical axis 32 is reflected by the dichroic mirror DM2 (the direction of the optical axis 34). These cubes CV2 and CV2 'can be inserted into and removed from the slider SV. An imaging lens 15 is disposed immediately below the cube CV2 of the slider SV, and an imaging lens 16 is disposed immediately below the cube CV2 '.

Then, by moving the slider SV along the direction of the optical axis 34, the reflecting surface of the dichroic mirror DM2 of the cube CV2 or the reflecting surface of the dichroic mirror DM2 'of the cube CV2' is selectively moved to the optical axis 31.
To the point O2 that intersects with

The switching stop position of the dichroic mirrors DM2 and DM2 'on the slider SV is set by a positioning mechanism (not shown).

The relay lens 11, the collector lens 10, and the mercury light source 9 are placed on the optical axis 34 of the optical path of the third epi-illumination optical system reflected from the point O2 of the slider SV.
Has been arranged. A point O intersecting with the optical axis 34 is provided on the optical axis 34 between the relay lens 11 and the collector lens 10.
3, a slider SV 'is arranged so as to be able to be inserted and removed, and the collector lens 13 and the laser light source 12 are arranged perpendicularly to the optical axis 34 via the slider SV'.

In this case, a total reflection mirror 14 is provided on the slider SV ', and the reflection surface of the total reflection mirror 14 is moved to a point O by moving the slider SV' in the direction of the arrow in the figure.
3 so that the light beam from the laser light source 12 can be guided in the direction of the point O2 intersecting with the optical axis 31.
The insertion / removal position of the slider SV 'is automatically set by a positioning mechanism (not shown).

On the other hand, on the optical axis 31 below the slider SV, a half prism 17 for dividing the optical path and a total reflection mirror 18 for guiding the optical axis 31 to the eyepiece are arranged.

The observation light transmitted through the imaging lens 15 or 16 is split into two by a beam splitter 17. The light reflected by the beam splitter 17 is guided to a photographing optical path, and forms an image on a light receiving surface Ph of a photographing device or a TV camera.

The light transmitted through the beam splitter 17 is reflected by a total reflection mirror 18 and guided to an observation optical path.
After forming an image once at the intermediate image forming point A on the observation optical path, the image is relayed by the relay lens R1 to form an image again at the point B, and this image is observed by the eyepiece Oc.

Next, the operation of the embodiment configured as described above will be described.

Now, an N ₂ laser with a wavelength of 337 nm for releasing the caged reagent is used as the laser light source 3, and a mercury light for extracting excitation light and a wavelength of 488 nm for fluorescence observation is used for the mercury light source 6. It is assumed that a mercury light source 9 is used for irradiating excitation light and a mercury light for extracting light having a wavelength of 532 nm for fluorescence observation, and a laser light source 12 is an IR laser having a wavelength of 1064 nm for a laser trap.

First, as shown in FIG. 1, the cube CV1 of the cube turret CT is positioned on the optical axis 31, the cube CV2 of the slider SV is inserted into the optical axis 31 of the optical path of the observation optical system, and the slider SV 'is moved. Optical axis 34
Remove from

In this state, the wavelength 337 of the laser light source 3
The N ₂ laser for releasing the caged reagent of nm is incident on the dichroic mirror DM1 of the cube CV1 via the collector lens 4 and the relay lens 5. Here, when a dichroic mirror DM1 that reflects light having a wavelength of 400 nm or less and transmits light exceeding 400 nm and has a spectral transmittance characteristic shown in FIG. 3 is used, the N ₂ laser of the laser light source 3 becomes a dichroic. Mirror DM
Then, the light is reflected by the objective lens 1 and is irradiated through the objective lens 2 to a predetermined position in the observation field of the specimen 1 to release the caged reagent.

On the other hand, in this state, the dichroic mirror DM2 of the cube CV2 of the slider SV moves the optical axis 31.
Is located at a point O2 where the optical axis 3
4, the mercury light having a wavelength of 532 nm from the mercury light source 9 is incident on the dichroic mirror DM2 'via the collector lens 10 and the relay lens 11. Here, the wavelength of the dichroic mirror DM2 'is 800n.
m and having a spectral transmittance characteristic shown in FIG. 4 that reflects light exceeding 800 nm.
The light from the mercury light source 9 is a dichroic mirror DM2 '.
, And is incident on the excitation filter EX2 of the cube CV2.
When a light having a spectral transmittance characteristic shown in FIG. 5 that transmits light of 10 nm or more and 550 nm or less is used, only the excitation light is transmitted and enters the dichroic mirror DM2. Further, a wavelength of 5 is added to the dichroic mirror DM2.
When a light having a spectral transmittance characteristic shown in FIG. 5 that reflects light of 70 nm or less and transmits light of more than 570 nm is used, the excitation light from the excitation filter EX2 is reflected upward by the dichroic mirror DM2 and becomes dichroic. The light passes through the mirror DM1 and is irradiated onto the observation field of the sample 1 via the objective lens 2.

When fluorescent light is emitted from the specimen 1 by the irradiation of the excitation light, the fluorescent light passes through the objective lens 2 and passes through the dichroic mirrors DM1 and DM2 to form the cube C.
The light enters the barrier filter BA2 of V2. Here, if a filter having the spectral transmittance characteristic shown in FIG. 5 that reflects light having a wavelength of 590 nm or less and transmits light having a wavelength exceeding 590 nm is used for the barrier filter BA2, the fluorescent light is transmitted and the half prism 17 is transmitted. Fluorescence observation is performed through the total reflection mirror 18.

Thus, the release of the caged reagent and the fluorescence observation can be performed simultaneously.

Here, a dichroic mirror D used for simultaneous release of caged reagent and fluorescence observation
The characteristics of M1 and DM2 are summarized as follows.

That is, as the dichroic mirror DM1, a laser beam (having a wavelength of 40
(Not more than 0 nm) while transmitting the excitation light from the mercury light source 9 and the light (wavelength exceeding 400 nm) emitted from the sample 1 (see FIG. 3). As the dichroic mirror DM2, one that reflects excitation light (wavelength of 570 nm or less) from the mercury light source 9 and transmits light (wavelength of more than 570 nm) emitted from the sample 1 is used (see FIG. 5). ).

Next, as shown in FIG. 2, the cube turret CT is rotated in the direction indicated by the arrow in the figure to obtain a cube CV1 '.
Is positioned on the optical axis 31, and by switching the slider SV, the cube CV 2 ′ is inserted into the optical axis 31, and further, the slider SV ′ is switched so as to switch the total reflection mirror 14.
Is inserted into the optical axis 34.

In this state, mercury light having a wavelength of 488 nm for fluorescence observation of the mercury light source 6 is passed through the collector lens 7 and the relay lens 8 to the excitation filter EX of the cube CV1 '.
1 '. Here, the light having a wavelength of not less than 470 nm and not more than 490 nm is transmitted through the excitation filter EX1 'in FIG.
Is used, only the excitation light enters the dichroic mirror DM1 ',
Further, the dichroic mirror DM1 'has a wavelength of 5
When a light having a spectral transmittance characteristic shown in FIG. 6 that reflects light of 00 nm or less and transmits light of more than 500 nm is used, the excitation light of the excitation filter EX1 ′ is reflected upward by the dichroic mirror DM1 ′, and The observation field of the specimen 1 is irradiated via the lens 2.

Then, when fluorescence is emitted from the specimen 1 by the irradiation of the excitation light, the fluorescence passes through the objective lens 2 and passes through the dichroic mirror DM1 ', and the cube CV1'
To the barrier filter BA1 '. Here, if a filter having a spectral transmittance characteristic shown in FIG. 6 that reflects light having a wavelength of 515 nm or less and transmits light having a wavelength of more than 515 nm is used for the barrier filter BA1 ', the fluorescent light is transmitted and the The light enters the dichroic mirror DM2 '. Since the wavelength of the fluorescent light at this time is 800 nm or less, the fluorescent light is transmitted through the dichroic mirror DM2 'and is observed through the half prism 17 and the total reflection mirror 18.

On the other hand, since the slider SV ′ is inserted on the optical axis 34, an IR laser having a wavelength of 1064 nm for laser trapping enters the laser beam source 12 through the collector lens 13 and the total reflection mirror 14. Here, the light is totally reflected and enters the dichroic mirror DM2 'of the cube CV2'. Then, the light is reflected upward by the spectral transmittance characteristic of the dichroic mirror DM2 'and is incident on the barrier filter BA1' of the cube CV1 '.
The light is transmitted by the spectral transmittance characteristic of the barrier filter BA1 ', further transmitted by the spectral transmittance characteristic of the dichroic mirror DM1', and is incident on the objective lens 2.

Then, the laser light incident on the objective lens 2 is condensed and radiated as a laser spot on a predetermined position on the sample 1, and a minute spot on the sample 1 is scattered near the irradiated laser converging point. Objects can be captured optically.

Thus, it is possible to simultaneously perform the observation of the minute object and the measurement of the minute force by the fluorescence observation and the laser trap.

Here, the characteristics of the dichroic mirrors DM1 'and DM2' used for simultaneously performing the observation of a minute object and the measurement of a minute force by the fluorescence observation and the laser trap are summarized as follows.

That is, the dichroic mirror DM1 '
As the excitation light from the mercury light source 6 (having a wavelength of 500 nm).
The laser beam transmitted from the laser light source 12 and the light emitted from the sample 1 (having a wavelength of 50
Those that transmit light (> 0 nm) are used (see FIG. 6).
Further, as the dichroic mirror DM2 ', a laser beam (having a wavelength of 800 nm or less) from the laser light source 12 is used.
While the light emitted from the sample 1 (having a wavelength of 80
Those that transmit light (> 0 nm) are used (see FIG. 4).

Therefore, with this configuration, the laser light from the laser light source 3 for releasing the caged reagent in the optical path of the first epi-illumination optical system is introduced into the sample 1 via the dichroic mirror DM1 of the cube CV1. ,at the same time,
The excitation light from the mercury lamp 9 for irradiating the excitation light in the optical path of the third epi-illumination optical system is introduced into the sample 1 through the dichroic mirror DM2 'of the cube CV2' and the dichroic mirror DM2 of the cube CV2.
An aged reagent release and a caged reagent release experiment by fluorescence observation can be performed. On the other hand, the excitation light from the mercury lamp 6 for irradiating the excitation light in the optical path of the second epi-illumination optical system is transmitted to the dichroic mirror DM1 ′ of the cube CV1 ′. At the same time, the laser light from the laser light source 12 for the laser trap on the optical path of the third epi-illumination optical system is applied to the dichroic mirror DM of the cube CV2 '.
By introducing the sample into the sample 1 via 2 ', it becomes possible to selectively perform laser trap experiments such as fluorescence observation, capture of a small object by a laser trap, and measurement of a small force. Two types of experiments can be selectively performed, and a versatile epi-fluorescence microscope can be provided.

Although the first embodiment has been described with reference to an inverted microscope, the present invention can be applied to a general upright microscope. Further, the type, wavelength, number, and arrangement of the light sources used this time need not always be the same.
Each illumination optical system optical path and observation optical system optical path (optical axis 31)
Does not need to be 90 ° and may be different from each other. Further, the characteristics of the dichroic mirror used this time are not necessarily the same, and a dichroic prism or other appropriate wavelength separating means may be applied as long as it is suitable for the desired light beam wavelength characteristics. The mechanism for inserting and removing the dichroic mirror from the optical axis includes a cube turret CT and a slider SV.
And may be any other insertion / removal mechanism. Then, the imaging lenses 15 and 16 are moved to the slider S
Instead of being attached to the V, one of them may be fixed at a predetermined position on the optical axis of the microscope main body.

(Second Embodiment) FIG. 7 shows a schematic configuration of a second embodiment of the present invention, and the same parts as those in FIG. 1 are denoted by the same reference numerals.

FIG. 7 shows a laser light source 12, a collector lens 13, a total reflection mirror 14,
The slider SV ', the dichroic mirror DM2', and the cube CV2 'are removed, and the rest is the same as FIG.

The second embodiment configured as described above has the following features.
In short, the laser light source 12 is removed from the configuration of FIG. 1 and the cube CV2 'is removed.

For this reason, the slider SV 'is switched to insert the imaging lens 16 into the optical axis 31, and the turret of the cube turret CT is rotated so that the portion without the cube is adjusted to the position of the optical axis 31. Or
By removing the cube CV1 and creating a state in which no extra optical element enters the parallel light beam path (between the objective lens 2 and the imaging lens 16) of the observation optical system, a normal inverted microscope can be obtained with only simple operations. Can be switched to

In this way, by removing the dichroic mirrors and barrier filters, which are not used in the ordinary fluorescence observation in which only a single excitation light is applied to the specimen, from the observation optical path, the unused cube enters the observation optical path. Therefore, it is possible to prevent an unexpected problem that fluorescence in a certain wavelength band is reflected from the observation optical path and is removed. In addition, by removing the extra optical element, it is possible to prevent the loss of light amount at the time of the weak fluorescence observation, and also to prevent the deterioration of the observation image obtained by combining the fluorescence observation and other microscopy.

When using a laser trap in the configuration shown in FIG. 7, the cube CV2 of the slider SV in FIG. 7 is changed to a cube CV2 'as shown in FIG. Instead, a laser light source 12 may be used.

With this configuration, the laser trap and the fluorescence observation can be performed simultaneously, and the same effects as described above can be expected. Further, in such an embodiment, the cube attached to the slider SV may be changed to another one according to the light source desired to be used.

In the second embodiment, an inverted microscope has been described, but the present invention can be applied to a general upright microscope. Further, the type, wavelength, number, and arrangement of the light sources used this time need not always be the same.
Each illumination optical system optical path and observation optical system optical path (optical axis 31)
Does not need to be 90 ° and may be different from each other. Further, the characteristics of the dichroic mirror used this time are not necessarily the same, and a dichroic prism or other appropriate wavelength separating means may be applied as long as it is suitable for the desired light beam wavelength characteristics. The mechanism for inserting and removing the dichroic mirror from the optical axis includes a cube turret CT and a slider SV.
And may be any other insertion / removal mechanism. Then, the imaging lenses 15 and 16 are moved to the slider S
Instead of being attached to the V, one of them may be fixed at a predetermined position on the optical axis of the microscope main body.

(Third Embodiment) FIG. 9 shows a schematic configuration of a third embodiment of the present invention, and the same parts as those in FIG. 1 are denoted by the same reference numerals.

In this case, FIG. 9 shows a case where the imaging lens 19 is extended by extending the slider SV instead of the slider SV shown in FIG.
Is provided, and the other components are the same as those in FIG.

The third embodiment configured as described above has the following features.
By switching the slider SV ", the cubes CV2 and CV2 'and the imaging lens 19 can be inserted into the parallel light beam path of the observation optical system by a positioning mechanism (not shown).

In this way, the effect described in the second embodiment can be further obtained without impairing the effect described in the first embodiment. That is, simply stated, it is possible to provide an inverted microscope that can selectively use various light beams only by simple switching and can be used as a normal inverted microscope.

In the third embodiment, the inverted microscope has been described. However, the present invention can be applied to a general upright microscope. Further, the type, wavelength, number, and arrangement of the light sources used this time need not always be the same.
Each illumination optical system optical path and observation optical system optical path (optical axis 31)
Does not need to be 90 ° and may be different from each other. Further, the characteristics of the dichroic mirror used this time are not necessarily the same, and a dichroic prism or other appropriate wavelength separating means may be applied as long as it is suitable for the desired light beam wavelength characteristics. The mechanism for inserting and removing the dichroic mirror from the optical axis includes a cube turret CT and a slider S.
V "may not be the same, and may be any other insertion / removal mechanism. The imaging lenses 15, 16, and 19 need not be attached to the slider SV". Any one of the lenses 15, 16, and 19 may be fixed on the optical axis of the microscope main body, and the other two may be excluded.

(Fourth Embodiment) FIG. 10 shows a schematic configuration of a fourth embodiment of the present invention, and the same parts as those in FIG. 2 are denoted by the same reference numerals.

In this case, FIG. 10 shows that a point O4 is provided on the optical axis 31 of the optical path of the observation optical system between the objective lens 2 having the structure shown in FIG. 2 and the point O1, and this point O4 corresponds to the reflection point of the optical axis. A cube CV1 having a dichroic mirror DM1 is arranged so as to form a relay lens 22, a collector lens 21, and a laser light source 20 on the optical axis 35 of the fourth epi-illumination optical system reflected by the dichroic mirror DM1. They are arranged on a line. Others are the same as FIG.

In the fourth embodiment configured as described above, when an N ₂ laser light source having a wavelength of 337 nm for releasing a caged reagent is used as the laser light source 20, the laser light oscillated from the laser light source 20 is collected by the collector lens. 21, the light is reflected upward by the above-described characteristics of the dichroic mirror DM1 through the relay lens 22, and irradiates a predetermined position in the observation field of the specimen 1.

That is, by doing so, it is possible to add the release of the caged reagent when using the fluorescence observation and the laser trap, and to make use of the wavelength characteristics of the respective optical elements described in the first embodiment to use the three light sources. Can be used simultaneously.

Thus, while irradiating the excitation light and observing the fluorescence inside the cell, N-protein can be added to any protein in the cell.
₂ Irradiate the laser to release the caged reagent, irradiate the activated protein with an IR laser to optically capture it, and perform micro force measurement.
It is very effective in the field of cell physiology where the behavior of intracellular substances is analyzed at the molecular level. In other words, in such a search for a specialized field, it is often desired to use various light sources at the same time, but by adopting a configuration as shown in FIG. 10, it is possible to provide a device having the above-mentioned great academic merits. Can be.

In the fourth embodiment, the dichroic mirror DM2 'for the laser trap is disposed at the lowest position among the dichroic mirrors on the other optical axes. Is also good. Further, a dichroic mirror on the optical axis may be added. Although the cube CV1 exists independently, a plurality of cubes may be inserted into and removed from the position of the cube CV1 by using some mechanism.

In the fourth embodiment, an inverted microscope has been described, but the present invention can be applied to a general upright microscope. Further, the type, wavelength, number, and arrangement of the light sources used this time need not always be the same.
Each illumination optical system optical path and observation optical system optical path (optical axis 31)
Does not need to be 90 ° and may be different from each other. Further, the characteristics of the dichroic mirror used this time are not necessarily the same, and a dichroic prism or other appropriate wavelength separating means may be applied as long as it is suitable for the desired light beam wavelength characteristics. The mechanism for inserting and removing the dichroic mirror from the optical axis includes a cube turret CT and a slider SV.
And may be any other insertion / removal mechanism. Then, the imaging lenses 15 and 16 are moved to the slider S
Instead of being attached to the V, one of them may be fixed at a predetermined position on the optical axis of the microscope main body.

(Fifth Embodiment) As described above, each embodiment described above is applicable to an erecting microscope. Therefore, an embodiment in which the present invention is configured by the upright microscope will be described with reference to FIG.

FIG. 11 shows a schematic configuration of an embodiment in which the present invention is configured by an upright microscope. In the figure, 1
Is a specimen to be observed, and an epi-illumination and an observation optical system including an objective lens are arranged above the specimen.

The symbols attached to the components of the epi-illumination optical system on the objective lens side of the imaging lens 15 shown here are as follows:
It shows that the components are the same corresponding to the symbols of the components shown in FIG. That is, the epi-illumination optical system in the inverted microscope of FIG. 1 shown in the first embodiment is configured to be approximately inverted.

An optical path splitting prism Pr is arranged above the image forming lens 15, and the observation light transmitted through the optical path splitting prism Pr forms an image on an imaging surface Ph of a photographic device, a TV camera, or the like. On the other hand, the observation light reflected by the optical path splitting prism Pr forms an image in front of the eyepiece Oc, and performs visual observation through the eyepiece Oc.

The operation of the embodiment configured as described above is as follows.
The operation is exactly the same as the operation described in the first embodiment, that is, the same effect can be obtained in an upright microscope.

The following is a summary of what has been described in each embodiment. The epi-illumination fluorescence microscope of the present invention selectively irradiates the specimen with a plurality of epi-illumination lights,
In an epi-illumination fluorescence microscope capable of simultaneously performing optical operation of a sample and fluorescence observation, a first epi-illumination optical system optical path arranged in a direction intersecting an observation optical system optical path with respect to the sample via an intersection, and the observation optical system A cube having a second epi-illumination optical system optical path and a first dichroic mirror disposed at a same intersection of the system optical path as the first epi-illumination optical system optical path and intersecting with the observation optical system optical path; 2
A cube having a dichroic mirror, and a first cube in which a desired cube is removably disposed at an intersection of the observation optical system optical path and the first and second epi-illumination optical system optical paths.
And a third epi-illumination optical system optical path arranged in a direction intersecting the observation optical system optical path via an intersection different from the first and second epi-illumination optical system optical paths; A cube having a dichroic mirror is provided, and a second movable body is provided so that the cube can be inserted and removed at an intersection of the observation optical system optical path and the third epi-illumination optical system optical path.

In the above-mentioned epi-illumination fluorescence microscope, the first dichroic mirror reflects light from the optical path of the first epi-illumination optical system toward the specimen, while transmitting fluorescence emitted from the specimen. Desirably. Further, it is preferable that the second dichroic mirror reflects the light from the optical path of the second epi-illumination optical system toward the sample, while transmitting the fluorescence emitted from the sample. Further, it is preferable that the third dichroic mirror reflects light from the optical path of the third epi-illumination optical system toward the sample, while transmitting fluorescence emitted from the sample.

In the epi-illumination fluorescence microscope, the first movable body is, for example, a cube turret. The second movable body is, for example, a slider.

The epi-illumination fluorescence microscope may further include a laser light source which forms the optical path of the first epi-illumination optical system. The epi-illumination fluorescence microscope may further include a mercury light source that forms the optical path of the second epi-illumination optical system. In addition, the epi-illumination fluorescence microscope is used for the third
And at least one of a laser light source and a mercury light source that form the optical path of the epi-illumination optical system.

The epi-illumination fluorescence microscope may further include a fourth epi-illumination optical system optical path arranged in a direction crossing the third epi-illumination optical system optical path via an intersection. In this case, the epi-illumination fluorescence microscope may further include a slider that disposes a total reflection mirror at an intersection of the third epi-illumination optical system optical path and the fourth epi-illumination optical system optical path. Good.

[0080]

As described in detail above, according to the present invention,
By having a plurality of independent optical paths of the epi-illumination optical system, selecting these epi-illumination optical system optical paths, combining a laser light source for releasing the caged reagent, a mercury lamp for irradiating the excitation light, and a laser light source for the laser trap, etc. It is possible to provide a versatile epi-illumination fluorescence microscope in which the use of each epi-illumination light path is not limited.

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of the first embodiment.

FIG. 3 is a diagram showing a spectral transmittance characteristic of a dichroic mirror DM1 used in the first embodiment.

FIG. 4 is a spectral transmittance characteristic diagram of a dichroic mirror DM2 ′ used in the first embodiment.

FIG. 5 shows a dichroic mirror DM2, an excitation filter EX2, and a barrier filter B used in the first embodiment.
FIG. 9 is a spectral transmittance characteristic diagram of A2.

FIG. 6 is a spectral transmittance characteristic diagram of a dichroic mirror DM1 ′, an excitation filter EX1 ′, and a barrier filter BA1 ′ used in the first embodiment.

FIG. 7 is a diagram showing a schematic configuration of a second embodiment of the present invention.

FIG. 8 is a diagram showing a schematic configuration in which the second embodiment is partially modified.

FIG. 9 is a diagram showing a schematic configuration of a third embodiment of the present invention.

FIG. 10 is a diagram showing a schematic configuration of a fourth embodiment of the present invention.

FIG. 11 is a diagram showing a schematic configuration of a fifth embodiment of the present invention.

FIG. 12 is a diagram showing a schematic configuration of an example of a conventional epi-fluorescence microscope.

FIG. 13 is a diagram showing a schematic configuration of another example of a conventional epi-illumination fluorescence microscope.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... sample, 2 ... objective lens, 3 ... laser light source, 4 ... collector lens, 5 ... relay lens, 6 ... mercury light source, 7 ... collector lens, 8 ... relay lens, 9 ... mercury light source, 10 ... collector lens, 11 ... Relay lens, 12 ... Laser light source, 13 ... Collector lens, 14 ... Total reflection mirror, 15, 16 ... Imaging lens, 17 ... Half prism, 18 ... Total reflection mirror, 19 ... Imaging lens, 20 ... Laser light source, Reference numeral 21: collector lens, 22: relay lens, 31, 32, 33, 34, 35: optical axis, BA1 ', BA1 ", BA2: barrier filter, CT: cube turret, CV1, CV1', CV1", CV2, CV2 ': Cube, DM1, DM1', DM2, DM2 ': Dichroic mirror, EX1', EX2: Excitation filter Oc ... eyepiece, Ph ... light-receiving surface, Pr ... optical path splitting prism, R1 ... relay lens, SV, SV ', SV "... slider.

Claims (3)

[Claims] 1. An epi-illumination fluorescence microscope in which a plurality of epi-illumination lights are selectively illuminated on a sample to enable optical operation of the sample and fluorescence observation at the same time. A first epi-illumination optical system optical path arranged in a direction intersecting with the first epi-illumination optical system optical path, and a first intersection epi-illumination optical system optical path intersecting with the observation optical system optical path at the same intersection. A third epi-illumination optical system arranged in a direction intersecting the second epi-illumination optical system optical path and the observation optical system optical path via an intersection different from the first and second epi-illumination optical system optical paths. An epi-illumination fluorescence microscope comprising an optical path. 2. An epi-illumination fluorescence microscope according to claim 1, wherein a wavelength discriminating means is detachably disposed at an intersection of said observation optical system optical path and said third epi-illumination optical system optical path. 3. The observation optical system according to claim 1, further comprising:
And the fourth epi-illumination optical system in a direction intersecting via a different intersection from the second epi-illumination optical system optical path and the third epi-illumination optical system optical path.
3. An epi-illumination fluorescence microscope according to claim 1, wherein the epi-illumination optical system optical path is arranged.