JPH06337358A - Vertical fluorescence microscope - Google Patents

Abstract

PURPOSE:To obtain a vertical fluorescence microscope capable of suppressing a time for irradiating a sample with exciting light as little as possible, safely replacing the optical member for a vertical illumination optical system and performing the synchronization operation with the other external device. CONSTITUTION:The sample S is irradiated with a luminous flux emitted from a vertical lighting source 2 arranged on a part of the microscope body 1 through optical members 10-12 which are supported attachably/detachably to/from the body 1, and the microscope is provided with a shutter 16 arranged between the lighting source 2 and the members 10-12 and for interrupting and transmitting the luminous flux from the source 2, a solenoid 21 for driving the shutter 16 so as to be opened and closed, an attachment/detachment detecting means for emitting an attachment signal when the members 10-12 are attached with the body 1 and a 1st control means for giving an operation command to transmit the luminous flux to the solenoid 21 when the attachment signal is emitted from the means and for giving the command to interrupt the luminous flux to the solenoid 21 when the members 10-12 are detached from the body 1 and the detachment signal is emitted.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used in the fields of medicine, biology, and the like, and is used to irradiate a specimen with a light beam from an epi-illumination light source arranged in a part of a mirror body through an optical member. Regarding a fluorescence microscope.

[0002]

2. Description of the Related Art Conventionally, a fluorescent microscope for detecting a gene or the like is widely known as a sample of a protein labeled with fluorescence on living tissues or cells. In particular, in recent years, even for substances that emit only weak fluorescence, multiple-staining can be used to examine the positional relationship with other substances, and to determine in which part of the cell structure the fluorescently stained substance exists. I am trying to investigate in combination with the phase difference spectroscopic method and the differential interference spectroscopic method. Further, in the field of biology, research using a membrane potential sensitive dye whose fluorescence intensity changes according to the potential of the cell membrane has been actively conducted.

[0003]

When observing living tissues or cells using a fluorescence microscope, the damage caused by the excitation light to the sample becomes a problem, and the time during which the excitation light is irradiated to the sample is as short as possible. Better.

Further, in this type of fluorescence microscope, it is required that the optical members of the epi-illumination optical system be exchanged to be flexible enough to meet various applications.
For example, the aperture stop (AS) position is a position conjugate with the light source, and when the optical member of this part is exchanged, harmful ultraviolet rays or high temperature air may be irradiated if the illumination light flux is passed through. , Very dangerous.

Furthermore, this type of fluorescence microscope requires a system that operates in synchronization with other external devices.
The light-shielding operation of the above-mentioned epi-illumination optical system is the same. Also,
In the field of biology, it is required to be usable even when the microscope is not energized due to the necessity of noise removal.

However, in the prior art, as a means for blocking the excitation light, a light shielding plate is manually inserted in the epi-illumination optical system, or a mechanical shutter or an electromagnetic shutter is provided between the microscope frame and the lamp house. However, in any case, it has not yet met all the aforementioned requirements.

The present invention has been made based on the above-mentioned circumstances, and the time for which the excitation light is applied to the sample is minimized, and it is safe to replace the optical member of the epi-illumination optical system. Yes, it can be synchronized with other external devices,
It is another object of the present invention to provide an epi-illumination fluorescence microscope that enables epi-illumination fluorescence observation even if a drive means such as a solenoid is not supplied with an energization command.

[0008]

In order to achieve the above object, the invention corresponding to claim 1 is an epi-illumination fluorescence microscope for irradiating a specimen with a light beam from an epi-illumination light source arranged in a part of a mirror body. In the above, a light flux opening / closing means provided between the epi-illumination light source and the optical member, capable of blocking and passing a light flux from the epi-illumination light source, a drive means for opening / closing driving the light flux opening / closing means, and the optical member are provided. An attachment / detachment detecting means for detecting whether or not it is attached to the mirror body, and an operation command for giving the light flux to the driving means based on an arrival signal from the attachment / detachment detecting means, and the attachment / detachment detecting means. And a control means for giving a command to the driving means so as to cut off the light flux based on a de-signal from the epi-fluorescence microscope.

In order to achieve the above object, the invention according to claim 2 is the same as the invention according to claim 1, in which, in addition to a command from the first control means to the driving means, This is an epi-illumination fluorescence microscope with the addition of a second control means for controlling the opening / closing operation.

[0010]

According to the invention corresponding to claim 1, the time during which the sample is irradiated with the excitation light is minimized, and it is safe to replace the optical member of the epi-illumination optical system. It is possible to synchronize with and to observe epifluorescence. According to the invention corresponding to claim 2, in addition to the operation of claim 1, epifluorescence observation is possible even if the drive command is not given from the first control means to the drive means.

[0011]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a diagram showing a schematic configuration of one embodiment of the present invention, in which a lamp house 2 is attached to an upper rear surface side of a microscope main body 1 having a channel-shaped outer shape and a light passage formed inside. Inside the lamp house 2, an epi-illumination light source 3 made of, for example, a mercury lamp and a collector lens 4 are housed.

An observation cylindrical mirror 6 having an imaging lens 5 is attached to the upper surface of the front side of the microscope main body 1, and between the observation cylindrical mirror 6 and an arm portion 1a and a base portion 1b of the microscope main body 1 described below. In addition, a stage 7 on which the sample S is placed is provided so as to be vertically movable.

A revolver 8 is rotatably attached to the bottom surface of the arm portion 1a of the microscope body 1 at a position facing the stage 7, and the revolver 8 has a plurality of objective lenses 9.
Are supported on the sample S so as to be switchable.

Between the revolver 8 and the observation cylinder mirror 6,
An excitation filter 10, a dichroic mirror 11, and an absorption filter 12 that transmits fluorescence from the sample S are arranged inside the arm portion 1 a of the microscope body 1.

Between the collector lens 4 and the excitation filter 10 and inside the arm portion 1a, an aperture stop 13 and a field stop 14 are arranged on the optical path, and a field stop 14 is provided.
A collector lens 15 is arranged between the pump lens 10 and the excitation filter 10, and a light-flux opening / closing means such as a shutter 16 operated by a driving means such as a solenoid 21 described later is arranged between the aperture stop 13 and the collector lens 4. ing.

In such a structure, the light flux emitted from the epi-illumination light source 3 is condensed by the collector lens 4 and is incident on the excitation filter 10 via the aperture stop 13, the field stop 14 and the collector lens 15. To be done. The light flux that has passed through the excitation filter 10 becomes excitation light having a predetermined excitation wavelength, is incident on the dichroic mirror 11 that is arranged at an angle of about 45 degrees with respect to the observation optical axis, and is reflected to the sample S side. It

Since the distance between the objective lens 9 and the sample S is adjusted by vertically moving the stage 7 on which the sample S is placed, the objective lens 9 and the sample S can be focused.

FIG. 2 is a sectional view showing the structure of the shutter 16 shown in FIG. 1 and a driving means for driving the same, for example, a solenoid 21. In the shutter 16, the solenoid 21 is fixed to the inner bottom surface of the microscope body 1. The solenoid 21 is a rotary shaft 26 of the shutter 16 and an illumination optical axis 2 of the shutter 16.
It is provided to insert and remove in 2. By energizing the solenoid 21, the plunger 24 is pulled,
Along with this, the shutter 16 moves from the solid line position to the broken line position, so that the shutter 16 deviates from the illumination optical axis 22 and epi-illumination becomes possible. When the solenoid 21 is de-energized, the shutter 16 returns from the broken line position to the solid line position by the reaction force of the compression spring 23 wound around the outer circumference of the plunger 24, and is inserted into the illumination optical axis 22 to allow the epi-illumination. Is cut off.

A knob 25 is screwed onto the wall surface of the microscope body 1 so as to be coaxial with the plunger 24 of the solenoid 21, and the tip of the knob 25 is screwed into the plan The jar 24 acts against the reaction force of the compression spring 23, and the shutter 16 can be removed from the epi-illumination optical axis 22 regardless of the energization of the solenoid 21, and epi-illumination is possible. The knob 25 constitutes the second control means of the present invention.

FIG. 3 is a view showing a state in which the aperture diaphragm 13 or the field diaphragm 14 of FIG. 1 is attached to the microscope main body 1, and the aperture diaphragm unit 30, the knob 31, the fixing screw 32, and the magnet 33 are provided. , Hall element 34. In the aperture stop unit 30, the aperture stop 13 is opened and closed by operating the knob 31. The aperture stop unit 30 is guided from the side surface of the microscope body 1 and is fixed by fixing screws 32. In addition, the aperture stop unit 30
Has a magnet 33 at its tip, and the Hall element 34 of the microscope body 1 outputs a predetermined signal depending on whether the aperture stop unit 30 is attached or not.

FIG. 4 shows the aperture stop 13 shown in FIGS.
2 is a block diagram showing a control circuit of the present invention, which comprises a central processing unit (CPU) 40, a driver 41, an external controller 42, a communication interface 43, a memory 44, and a unit attachment / detachment detector 45 which constitute the first control means of the present invention. Has been done.

The unit attachment / detachment detector 45 is the magnet 3 shown in FIG.
3 and the Hall element 34, and when the aperture stop unit 30 is attached to the microscope main body 1, a predetermined arrival signal CP
It is configured to output to U40 and to stop energizing the solenoid 21 at all times when the aperture stop unit 30 is not attached to the microscope body 1.

The CPU 40 controls a system including a solenoid 21 for driving the shutter 16, and is connectable from an external controller 42 such as a PC via a communication interface 43.
The drive of No. 1 can be operated in synchronization with an external device. The memory 44 is a storage device of the CPU 40,
The sequence operation with the external controller 42, the operation condition of the solenoid 21, and the like are stored. The driver 41 is for driving the solenoid 21.

According to the embodiment described above, since the shutter 16 for cutting off the epi-illumination light beam is provided, the opening / closing operation of the shutter 16 is operated by, for example, a switch (not shown) or synchronized with an external device. By blocking the epi-illumination light flux from the epi-illumination light source 3 by the shutter 16 except when necessary, the time during which the sample S is irradiated with the excitation light can be minimized.

Further, even if the fixing screw 32 shown in FIG. 3 is loosened and the aperture stop unit 30 is removed from the microscope main body 1 while the epi-illumination light source 3 is turned on, the excitation light is blocked by the shutter 16. , Safe.

Further, the shutter 16 can be opened when necessary without energizing the solenoid 21. That is,
By screwing the knob 25 of FIG. 2 into the microscope body 1, the tip of the knob 25 abuts on the plunger 24 and is moved in the axial direction against the reaction force of the compression spring 23. , Returns from the solid line position to the broken line position and deviates from the illumination optical axis 22.

FIG. 5 is a cross-sectional view showing only the main part of the second embodiment of the present invention. The excitation filter 10, the dichroic mirror 11 and the absorption filter 12 shown in FIG. 1 are constructed as follows. is there. That is, the fluorescent filter cube 51, the rotary turret 52, the rotary shaft member 54, the magnet 55, the cover 56, the hall element 57, and the fixing screw 58 are arranged and fixed on the rotary turret 52 in the circumferential direction. It was done. The rotary turret 52 is rotatably supported by a rotary shaft member 54 fixed to a cover 56 by fixing screws 58. A dovetail 54a is formed at the lower end of the rotary shaft member 54, and the dovetail 54a is fitted in a dovetail groove 1a formed at the end of the inner bottom surface of the microscope body 1. Ant groove 1
A magnet 55 is embedded in a at a position where a hall element 57 is provided and a dovetail 54a is formed on the bottom surface of the a, the magnet 55 facing the hall element 57 at the time of fitting.

The rotary turret 52 is provided with a positioning mechanism (not shown) so that when the rotary turret 52 is rotated, the target fluorescent filter cube 51 is stopped at the observation optical axis 53. .
Further, in each fluorescent filter cube 51, the excitation filter 10, the dichroic mirror 11, the absorption filter 1 are provided.
One set of two optical elements is housed, and the characteristics of the optical elements are different for each fluorescent filter cube 51.

In this embodiment as well, as in the above-described embodiments, the solenoid control circuit is constructed in the same manner as in FIG. That is, the magnet 55 and the hall element 57 constitute the unit attachment / detachment detector 45 of FIG. 4, and the unit is provided only when the magnet 55 and the hall element 57 face each other, that is, when the rotary shaft member 54 is fitted to the microscope body 1. Detachment detector 45 to CPU 40
A signal is output to the solenoid 21, which energizes the solenoid 21 and opens the shutter 16. On the contrary, when the rotary shaft member 54 is removed from the microscope main body 1, since no signal is generated from the unit attachment / detachment detector 45, the solenoid 21 is deenergized and the shutter 16 is closed. Is safe from passing. further,
Since the rotary turret 52 is detachable from the microscope body 1, it is convenient. In order to remove the rotary turret 52 from the microscope body 1 in the state of FIG. 5, the fixing screw 58 is loosened, and then the rotation shaft member 54 and the microscope body 1 are fitted.

[0030]

As described above, according to the present invention, the time during which the excitation light is applied to the sample is minimized, and it is safe to replace the optical member of the epi-illumination optical system. It is possible to provide an epi-illumination fluorescence microscope that can be synchronized with an external device and can perform epi-illumination fluorescence observation even if a drive means such as a solenoid is not supplied with an energization command, for example.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of a first embodiment of an epi-fluorescence microscope according to the present invention.

FIG. 2 is a cross-sectional view showing the configuration of the shutter shown in FIG.

FIG. 3 is a cross-sectional view showing the configuration of the aperture stop shown in FIG.

FIG. 4 is a block diagram showing a schematic configuration of a control circuit for controlling opening / closing of the shutter of FIG.

FIG. 5 is a sectional view showing only a main part of a second embodiment of an epi-illumination fluorescence microscope according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Microscope main body, 2 ... Lamp house, 3 ... Epi-illumination light source, 4 ... Collector lens, 5 ... Imaging lens, 6 ... Observation cylinder mirror, 7 ... Stage, 8 ... Revolver, 9 ... Objective lens, 10 ... Excitation filter, 11 ... Dichroic mirror, 12 ... Collector lens, 13 ... Aperture stop, 14 ...
Field stop, 15 ... collector lens, 16 ... shutter,
S ... Specimen, 21 ... Solenoid, 22 ... Illumination optical axis, 23 ...
Compression spring, 24 ... Plunger, 25 ... Knob, 26 ...
Rotating shaft, 30 ... Aperture stop unit, 31 ... Knob, 32
... Fixed screws, 33 ... Magnet, 34 ... Hall element, 40 ... Central processing unit (CPU), 41 ... Driver, 42 ... External controller, 43 ... Communication interface, 44 ... Memory, 51 ... Fluorescent filter cube, 52 ... Rotating turret , 53 ... Observation optical axis, 54 ... Rotating shaft member, 55 ... Magnet, 56 ... Cover, 57 ... Hall element.

Claims (2)

[Claims] 1. An epi-illumination fluorescence microscope for irradiating a specimen with a light flux from an epi-illumination light source arranged in a part of a mirror body, the epi-illumination light source being provided between the epi-illumination light source and the optical member. Light flux opening / closing means for blocking and passing the light flux of, the driving means for opening / closing the light flux opening / closing means, the attachment / detachment detection means for detecting whether or not the optical member is attached to the mirror body, and the attachment / detachment detection An operation command is given to the drive means based on an arrival signal from the means so that the light flux passes, and the light flux is cut off to the drive means based on a de-signal from the attachment / detachment detection means. And a control means for giving a command as described above. 2. An epi-illumination fluorescence microscope which irradiates a specimen with a light flux from an epi-illumination light source disposed in a part of a mirror body, wherein the epi-illumination light source is provided between the epi-illumination light source and the optical member. Light flux opening / closing means for blocking and passing the light flux of, the driving means for opening / closing the light flux opening / closing means, the attachment / detachment detection means for detecting whether or not the optical member is attached to the mirror body, and the attachment / detachment detection Based on the arrival signal from the means, gives an operation command to the drive means so that the light flux passes,
First control means for giving a command to the drive means to cut off the light flux based on a de-signal from the attachment / detachment detection means; and a command from the first control means for the drive means. Separately, a second control means for controlling the opening / closing operation of the luminous flux opening / closing means, and the epi-illumination fluorescence microscope.