JP2001305430A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize high-resolution microscopic technology in a conventional microscope, for example, in a confocal laser scanning type microscope especially even by additional equipment. SOLUTION: An objective lens 2 and a beam splitter/beam integrator 5 are formed in one module type assembly 8. The assembly 8 is provided with an interface 9 for connecting to an illumination/detection optical path 4 of the microscope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention comprises at least one light source, at least one detector, and two objective lenses, one of which is arranged on each side of the surface of the object to be inspected. The two objective lenses are oriented in opposite directions to each other and have a common focal point, and at least one beam splitter for distributing the illumination light to the objective lens in the illumination / detection optical path and coming from the objective lens The present invention relates to a microscope provided with a beam integrator for integrating detection light, particularly to a confocal laser scanning microscope.

[0002]

2. Description of the Related Art Microscopes of this kind, in particular those equipped with two objective lenses which are oriented in opposite directions and have a common focus in the image plane, have been known for some time. One example is EP 0 492 289. From this document, a double confocal scanning microscope having the configuration described in the above technical field is known. Specifically, a non-deflection type beam splitter device for dividing illumination light into coherent components is provided. The beam splitter arrangement is used to illuminate two objectives pointing in opposite directions and to integrate the mutually coherent light beams of these objectives. High resolution can be realized by the optical element realized here.

The microscope known from EP 0 491 289 is a so-called "high-end" microscope, in which the interferometer optical path is formed. Such microscopes are very complicated in construction and are therefore already more expensive in terms of basic construction than conventional microscopes. Also, this "high-end" microscope is special in construction, has considerable space on the optical bed, and is therefore very susceptible to external influences. In particular, the “high-end” microscope method set in this type of microscope requires a special microscope, and a microscope using the conventional microscope method cannot be used.

[0004]

The object of the present invention is to realize such a high-resolution microscope technology known from the prior art in a conventional microscope, for example, in a confocal laser scanning microscope, It is to be able to be realized by equipment.

[0005]

According to the present invention, in order to solve the above-mentioned problems, an objective lens and a beam splitter / beam combiner are combined into one modular assembly, and the assembly is connected to an illumination / detection optical path of a microscope. It has an interface for connection.

According to the present invention, first, a conventional microscope, for example, a confocal laser scanning microscope, can be additionally provided with a high-resolution microscope technique, and additionally equipped while maintaining the characteristics of the conventional microscope. It was confirmed that it was possible. In addition, the realization of high-resolution microscopy technology of conventional microscopes is possible after the fact that the key components required for high-resolution microscopy technology can be integrated into retrofitable assemblies. confirmed. Thus, according to the invention, the objective lens and the beam splitter / beam combiner are integrated into one modular assembly. This assembly has an interface for connecting to the illumination / detection optical path of the microscope. The modular assembly can be operated independently when viewed by itself, and its interface allows it to be connected to a microscope, with the
Connection to the detection optical path is absolutely necessary.

It is advantageous to insert the interface of the assembly into a microscope stand instead of a conventional objective lens or objective revolver, in which case it is connected to the microscope (to its illumination and detection beam path). The modification is thus simple, that is to say after removing the objective lens or the objective lens revolver, whereby the connection of the high-end assembly in question here is possible.

Referring to the specific configuration of the assembly including the optical components, the components of the assembly are mounted on a substrate. In order to avoid temperature-dependent changes in the optical path, it is particularly advantageous if the substrate is made of a material with a low coefficient of thermal expansion. Therefore, materials such as invar or soup line bar are problematic. Since this material hardly undergoes thermal expansion even when the generated temperature changes, a change in the optical path due to the temperature or a corresponding poor adjustment is almost prevented.

[0009] With regard to the positioning of the optical assembly in particular, it is advantageous if the optical elements of the assembly, which are advantageously arranged on a substrate, are arranged in one casing. The casing may be hermetically sealed to avoid external influences. It is advantageous for the casing to be at least sufficiently thermally insulated in order to effectively avoid external influences on the alignment.

[0010] In order to effectively avoid poor adjustment, the inside of the casing can be set to a predetermined temperature, and the optical path is adjusted in this temperature range. To maintain this temperature, the optical assembly includes at least a sufficiently constant predetermined heat-dissipating component. This heat dissipation component
If possible, the size must be selected in view of the thermal insulation to be achieved, so as to be suitable for keeping the assembly at a constant operating temperature. The heat-dissipating component is a laser light source, preferably a diode laser.

As a possibility to avoid misalignment due to thermal expansion, the optical elements of the assembly are constructed so that expansion due to temperature is offset so that the optical position of the assembly is not affected, and In some cases, it can be mounted and arranged by means of a special carrier, so that there is no effect on the optical adjustment of the assembly. This can be achieved, in particular, in that the individual components are designed to have exclusively linear expansion, ie to expand in one direction. Since a suitable holding portion is provided on the end side, and the expansions are in opposite directions, mutual cancellation due to temperature change is performed, and the optical path does not change. With this measure, the misalignment is sufficiently avoided.

The optical assembly may have other optical elements besides the objective lens and the beam splitter / beam combiner pointing in opposite directions. Another component, which is also preferably a built-in component of the assembly, is a sample table located between the objective lenses. When the assembly is connected to the microscope or the casing is connected, the sample table is oriented in the horizontal direction, so that the object to be inspected can be placed on the table as usual.

[0013] Advantageously, the sample table is accessible from outside the casing. Therefore, the sample table can be operated from the outside, and in particular, an object to be inspected can be set, and further, it is not necessary to open the casing. In the range of such an arrangement, the sample table and the object to be inspected thereon are protected by the casing.

The sample table can be set or displaced from the outside of the casing, as well as the object to be inspected can be set from outside the casing. For this purpose, the sample table can be motor-driven, in which case an operating mechanism, preferably a joystick or trackball, can be provided for operating the sample table.

Further, the assembly including the optical element may have an optical system for displacing the pupil of the objective used in the assembly. This allows adaptation to the light path extended by the assembly. An optical system for displacing the pupil is realized by a virtual image or an intermediate real image. Similarly, pupil displacement may be achieved by exchanging the barrel lens of the microscope. It is therefore important that the objective lens is further away from the microscope stand in the assembly, for example in the interferometer module, than in an ordinary working module with an objective revolver, After the assembly has been mounted on the microscope stand instead of the revolver, the pupil can be displaced for adaptation, i.e. the pupil position of the objective can be adapted. It is also basically possible to exchange a lens barrel arranged on a suitable lens wheel.

Further, an assembly is disposed within the illumination and detection optical path for diverting the illumination and detection optical path,
It may have at least one mirror. By using such a mirror to achieve turning of the optical path, a compact configuration of the assembly can be realized.

A shutter is arranged between the coupling position in the illumination and detection beam path and the beam splitter, preferably between the coupling position and the mirror, ie a shutter for fading out the light beam or at least one partial light beam. Have been.

A mirror for turning the illumination / detection light may be provided immediately in front of the two objective lenses. Both mirrors are also used to achieve a compact configuration of the entire assembly, which reduces the required casing size.

[0019] In response to specific application requirements, lighting
A shutter may be provided between the beam splitter and the mirror in the detection optical path. This shutter is also used to fade out or partially fade out the light beam.

Further, the assembly controls the interference phenomenon.
With an optical element for adjusting the phase, in particular
May be. That is, this is to realize the interferometer optical path
It is. For this purpose special interferometers may be incorporated,
For example, Sagnac interferometer, Michelson interferometer, Twy
Mann-Green interferometer or Mach-Zehnder interference
A meter may be incorporated. These interferometers are
Particularly suitable for forming. In addition, the assembly
Microscope technology 4Pi, SWFM, I²M, I³M, I ⁵Real M
It has an optical element for applying. This is a completely different
"High-end" microscopy technology using microscopy. This
For these microscopy methods, the objectives are oriented in opposite directions.
It is important that lenses are used.

[0021] The main components of the assembly can be positioned for alignment, that is to say by attaching actuators to the individual components. These actuators can be controlled by a control unit, in which case the control unit may be provided outside or inside (integrated with) the assembly. Since the actuator is advantageously controlled by a computer, the position can be adjusted using a suitable computer program. A suitable alignment guide for the user is also conceivable.

Further, a detector may be provided for detecting various characteristic quantities of the operating state of the assembly.
In this case, the detector is a built-in element of the assembly. The detectors are mechanical, electronic and / or optical sensors suitable for detecting the respective characteristic curves (detection of the operating state of the assembly). This allows for automatic positioning of the system or module.

As already mentioned, the assembly can have its own light source, which light source can at the same time be used for adjusting the temperature of the assembly. In addition, the light source can be used to create an auxiliary light path for aligning components. In this case, the auxiliary light path is generated as an interferometer light path and is a built-in element of the assembly. Also, the light source may be capable of being coupled to the assembly as an external light source, and any arbitrary light source is possible (without considering its size). Coupling by light guiding fibers is conceivable.

The light source can be implemented as a solid state laser, a diode laser or a gas laser. It should be noted that the assembly may have an interface to a fluorescent epi-illumination unit, a binocular tube and / or a confocal unit. Other configurations utilizing conventional microscope technology are also possible.

There are various possibilities to advantageously structure and improve the teachings of the present invention. This includes the dependent claims of claim 1, while the following description of embodiments of the invention with the aid of the drawings is pointed out. Universally advantageous configurations and variants of the invention will be described in connection with the description of advantageous embodiments of the invention with the aid of the drawings.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the drawings, the region relating to the conventional microscope 1 is only suggested. The microscope according to the present embodiment is a confocal laser scanning microscope. The confocal laser scanning microscope has a light source, not shown, a detector, also not shown, and at least two objective lenses 2. One objective lens 2 is arranged on each side of the sample surface 3. Both objective lenses 2 are directed in opposite directions and have a common focal point.

Further, as shown in the figure,
Provided in the detection optical path 4 are at least one beam splitter 5 for distributing the illumination light 6 to the objective lens 2 and a beam combiner 5 for integrating the detection light 7 coming from the objective lens 2. Note that the beam splitter and the beam combiner 5 may be a single optical element as shown in the figure.

According to the invention, the objective lens 2 and the beam splitter / beam combiner 5 are combined in one modular assembly 8. Assembly 8
Has an interface 9 for connecting to an illumination / detection optical path 10 of the microscope 1.

In the microscope 1 according to the invention, the relatively complex part of the interference microscope, ie the assembly containing the corresponding optical elements, is formed in a modular manner, so that the basically modular assembly 8 is usually Can be connected to a microscope. Thus, generally expensive laboratory microscopes can be used not only for normal modes of operation, but also for high resolution operation, i.e., with modifications using connectable modular assemblies. In this regard,
The normal assembly of certain microscopes can also be used for high resolution module operation. For example, illumination can be realized using an existing high-pressure vapor light source for a microscope. It is also conceivable that the specimen is inspected in a normal fluorescence epi-illumination optical path, and thereafter the parameters of the entire microscope system for data acquisition are set using a high-resolution module. Further, an important part of the sample to be inspected can be recorded or restricted. The modular assembly 8 can basically be used for all microscopy methods using two objective lenses pointing in opposite directions. Particularly important microscopy techniques are 4Pi, SWFM, I ²
M, I ³ M and I ⁵ M. Regarding these microscope techniques, for example, European Patent No. 0491289, US Pat. No. 4,621,911 and US Pat. No. 5,671,108.
No. 5 is pointed out. In particular, with this module, it is possible to record a sample in two or three dimensions by means of a sample raster or by a beam scanning method.

In the embodiment of the microscope according to the invention or of the modular assembly 8 important in the context of the invention, illustrated in the drawing, the individual optical elements are
1 is mounted on. This substrate 11 is made of a material having a small coefficient of thermal expansion. In the case of this embodiment,
The material of the substrate 11 is Suprainvar.

The modular assembly 8, or the substrate 11 with the individual optical elements, is arranged in a casing 12, which is thermally insulated.
By using the substrate 11 which does not expand much due to a temperature change, and by applying an appropriate treatment to the casing 12, if the temperature fluctuation around the microscope is limited, the modular assembly 8 or the interferometer realized therein can be used. It does not interfere with the adjusted light path.

To further reduce the effects of temperature fluctuations around the microscope, the individual optical elements are fixed or positioned by special holding parts, which are almost completely offset by the material expansion due to temperature fluctuations. And the substrate 11
It is advantageous to fix to In this way, even if the temperature changes, there is no effect on the optical positioning state of the assembly 8.

As already mentioned, the casing may be hermetically sealed so that the air flow around the microscope 1 does not adversely affect the optical path of the interferometer. To this extent, a member that heats the assembly 8 irregularly and strongly as a heat source should not be mounted inside the assembly 8 and operated. This is because the optical path of the interferometer is affected. However, it is advantageous to provide a diode laser which emits a predetermined, constant heat radiation as an integral part of the assembly 8. This is because the diode laser operates on the assembly 8 with a stable temperature. Therefore, the assembly 8 can be maintained at a preferable operating temperature including the test sample 13, for example, at 37 ° C. for a so-called in-vivo sample.

As can be seen from the figure, an improved sample table 14 is provided. The sample table 14
It extends horizontally between the two objective lenses 2. Therefore, a use condition that is easy for the operator to operate as in a normal microscope is realized. Since the sample table 14 is positioned relatively to the objective lens 2, the operator can manually or externally operate the sample table 14 with a motor. Operating elements not shown in the figures include joysticks or trackballs, which can operate and control the motor control.

As can be further seen, the assembly 8 or the optical member comprising (at least partially) the assembly 8 comprises an actuator 15 for controlling the interferometer. The actuator 15 is used for positioning the sample table 14, the mirror 16 provided thereon, and the beam splitter / beam combiner 5 in the illumination / detection optical path 4. The actuator for operating the sample table is not shown, and the optical axis 17 of the illumination / detection optical path 4 is shown for easy understanding of the drawing. Although not shown, the operation of the objective lens 2 may be performed using an actuator.

The control unit of the actuator 15 is arranged outside the assembly 8, and the control of the actuator 15 is performed by a computer. The illustrated shutter 18 is used to fade out the light beam 19 or at least one partial light beam 20. The shutter 18 may also be attached to the modular assembly 8.

The specific embodiment illustrated in the drawings is a high resolution 4Pi microscope module (modular assembly 8) and a conventional confocal laser scanning microscope (CLSM).
Is a combination of In this case, the modular assembly 8 is provided in the casing 12 instead of the objective lens revolver not shown in the drawing,
It is inserted into the microscope 1 using a compatible interface 9 or connected to a microscope stand.
Therefore, it is advantageous to use an eyepiece in a normal microscope module, especially for finding or locating the object to be inspected. Further, when the arrangement according to the present invention is applied, a two-dimensional or three-dimensional image can be obtained with a normal confocal laser scanning microscope.

As can be seen from the figure, an optical system 21 for displacing the pupil with respect to the objective lens 2 used in the assembly 8 is provided. Assembly 8
In order to attach to the stand of the microscope instead of attaching to the objective lens revolver of the microscope, it is necessary to adjust the position of the pupil of the objective lens 2. This is because the objective lens 2 in the interferometer module shown here (modular assembly 8) is farther from the microscope stand than in the normal operating mode using an objective lens revolver. Such a pupil displacement can be realized by an intermediate real image or an intermediate virtual image. Similarly, it is possible to exchange barrel lenses which can be placed on a suitable lens wheel (not shown) in the microscope stand. Therefore, the pupil can be virtually displaced by exchanging the barrel lens, and it is not necessary to take further measures.

The above embodiment is merely used for explaining the gist of the present invention, and the present invention is not limited to this arbitrarily selected embodiment.

[Brief description of the drawings]

FIG. 1 shows the basic configuration of an optical assembly that includes optical components and that can be additionally connected to a microscope (only suggested) because of its modular configuration.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Microscope 2 Objective lens 3 Surface of an object to be inspected 4 Illumination / detection optical path 5 Beam splitter / beam combiner 6 Illumination light 7 Detection light 8 Assembly 9 Interface 10 Microscope illumination / detection optical path 11 Substrate 12 Casing 13 Sample to be inspected 14 Sample table 15 Actuator 16 Mirror 17 Optical axis of illumination / detection optical path 18 Shutter 21 Optical system for displacing pupil

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Joachim Braddle DE 69198 Schliersheim Max Planck-Strasse 33 F-term (reference) 2H052 AA08 AB14 AB17 AB26 AC04 AC05 AC34 AD01 AD16 AD18 AD31 AF04

Claims (43)

[Claims] 1. An objective (2) comprising at least one light source, at least one detector and two objectives (2), one on each side of the surface (3) of the object to be inspected. Are arranged, the two objective lenses (2) are oriented in opposite directions to each other and have a common focal point, and the illumination light (6) is passed through the objective lens (2) in the illumination / detection optical path (4). A microscope provided with at least one beam splitter (5) for distributing the light to a beam and a beam integrator (5) for integrating the detection light (7) coming from the objective lens (2); And a beam splitter / beam combiner (5) are integrated into one modular assembly (8), which assembles an interface (9) for connection to the illumination and detection optical path (4) of the microscope. A microscope, comprising: 2. An interface (9) of an assembly (8) can be attached to a stand of a microscope instead of a conventional objective lens / object lens revolver, in which case it can be connected to the microscope (1). And
The microscope according to claim 1. 3. The optical element of the assembly (8) is mounted on one substrate (11).
The microscope according to claim 1. 4. The microscope according to claim 3, wherein the substrate is made of a material having a low coefficient of thermal expansion. 5. The substrate (11) is manufactured from Invar,
5. The microscope according to claim 4, wherein the microscope is advantageously manufactured from soup line bar. 6. The microscope according to claim 1, wherein the optical elements of the assembly are arranged in a housing. 7. The microscope according to claim 6, wherein the casing (12) is hermetically sealed. 8. The microscope according to claim 1, wherein the housing is at least sufficiently thermally insulated. 9. The microscope according to claim 1, wherein the optical assembly has a member having a predetermined heat radiation at least sufficiently constant. . 10. The microscope according to claim 9, wherein the size of the heat dissipating member is selected so as to be suitable for maintaining the assembly at a constant operating temperature. . 11. The heat dissipating member is a laser light source, preferably a diode laser.
Or the microscope according to 10. 12. The optical element of the assembly (8) is constructed such that expansion due to temperature is offset so that the optical position of the assembly (8) is not affected and the optical element may be placed on the substrate (11). The microscope according to claim 1, wherein the microscope is mounted and arranged using a special holder. 13. The method according to claim 1, wherein the optical assembly has a sample table arranged between the objective lenses. Microscope as described. 14. When the casing (12) is connected to the microscope (1), the sample table (14) is connected to the objective lens (2).
14. The microscope according to claim 13, characterized in that the microscope is directed horizontally between. 15. The microscope according to claim 13, wherein the sample table is accessible from outside the casing. 16. The sample table (14) can be adjusted in position from outside the casing (12).
A microscope according to any one of claims 13 to 15. 17. The microscope according to claim 13, wherein the sample table is driven by a motor. 18. To operate a sample table (14),
18. Microscope according to claim 17, characterized in that the operating mechanism is advantageously provided in the form of a joystick or a trackball. 19. An assembly (8) comprising an optical system (21) for displacing a pupil of an objective lens (2) used in said assembly (8). Item 19. The microscope according to any one of Items 1 to 18. 20. An optical system (2) for displacing the pupil.
20. The microscope according to claim 19, wherein 1) is realized by a virtual image. 21. An optical system (2) for displacing the pupil.
20. The microscope according to claim 19, wherein 1) is realized by an intermediate real image. 22. The microscope according to claim 19, wherein the displacement of the pupil is realized by exchanging the lens barrel of the microscope (1). 23. The pupil displacement is realized by an optical system (21) arranged in the illumination / detection optical path (4) so as to be located near the coupling position.
A microscope according to claim 19. 24. The assembly (8) has at least one mirror (16) arranged in the illumination and detection path (4) for deflecting the illumination and detection path (4). The microscope according to any one of claims 1 to 23, characterized in that: 25. A shutter (1) between the coupling position in the illumination and detection light path (4) and the beam splitter (5), preferably between the coupling position and the mirror (16).
The microscope according to any one of claims 1 to 24, wherein 8) is arranged. 26. The device according to claim 1, further comprising a mirror provided in front of the objective lens for turning the illumination / detection light. The microscope according to 1. 27. An assembly (8) comprising a beam splitter (5) and a mirror (16) in an illumination / detection optical path (4).
Microscope according to claim 26, characterized in that it has a shutter (18) between them. 28. The device according to claim 1, wherein the assembly has an optical element for controlling the interference phenomenon, in particular for adjusting the phase. Microscope as described. 29. An assembly (8) comprising a Sagnac interferometer, a Michelson interferometer, a Twyman-Green interferometer or a Mach-Zehnder interferometer to form an interfering light path. The microscope according to 28. 30. The assembly (8) is a microscope technology 4Pi,
^{SWFM, I 2 M, I 3} M, characterized in that it comprises an optical element for performing the ^{I 5} M, claims 1 2
9. The microscope according to any one of 9 to 9. 31. An actuator according to claim 1, wherein each of said individual elements is provided with an actuator (15) for positioning said individual elements or for adjusting the position of said assembly (8). A microscope according to any one of the preceding claims. 32. The actuator according to claim 31, wherein the actuator is controllable via a control unit.
The microscope according to 1. 33. Microscope according to claim 32, wherein the control unit is provided outside the assembly (8). 34. The actuator according to claim 31, wherein the actuator is controlled by a computer.
3. The microscope according to any one of 3 above. 35. Microscope according to claim 1, further comprising a detector for detecting various characteristic quantities of the operating state of the assembly (8). . 36. Microscope according to claim 35, wherein the detector is a built-in element of the assembly (8). 37. Microscope according to claim 35, wherein the detector is implemented as a mechanical sensor, an electronic sensor and / or an optical sensor. 38. At least one light source is provided for the assembly (8), which provides an auxiliary light path for adjusting the position of the component. The microscope according to one. 39. An auxiliary optical path is generated as an interferometer optical path,
39. The microscope according to claim 38, wherein the position adjustment is performed automatically. 40. Microscope according to claim 38, wherein the light source is a built-in element of the assembly (8). 41. Microscope according to claim 38, wherein the light source is coupleable to the assembly (8) as an external light source. 42. The microscope according to claim 38, wherein the light source is implemented as a solid-state laser, a diode laser or a gas laser. 43. A fluorescence epi-illumination unit, a binocular tube,
43. The microscope according to claim 1, wherein an interface (9) to a confocal unit is provided.