DE4323129A1 - Microscope with laser illumination - has laser beam input via conventional light inlet and slider with mirror for deflecting light to objective - Google Patents

Abstract

The laser beam is input (18) from the rear (11) of the microscope stand (1). The direct light reflectors are accommodated in a slider (9) which can assume several switchable positions. In one position, the slider is equipped with a fully reflecting mirror (10), and the position of the mirror detected by sensors (20, 21). The sensors are coupled to a shutter which ensures that the laser beam is only allowed to propagate when the mirror is switched in. Above the object table (6) of the microscope, a screen (22) is provided which is non-transparent to eye-damaging radiation. The screen is connected via sensors (26, 27) to the beam interrupting shutter. USE/ADVANTAGE - For inverse-type laser scan microscope. High safety w.r.t. eyes of operator.

Description

The present invention relates to a microscope in which radiation that damages the eyes for object lighting, such as B. laser radiation is used.

From DE-PS 35 27 322 a microscope is known in which Laser light in the incident light beam path for the conventional lighting is coupled. Serves here however, the laser light is not used to illuminate the object per se. Rather, with the help of laser light Auto focus signal generated. The lasers are therefore very poorly designed so that their radiation does not is harmful to the eyes.

From US Pat. No. 5,032,720 and WO 92/02839 Known laser scanning attachments for conventional microscopes, where a laser beam for object lighting is used. The laser beam is coupled in but here from above into the photo output of the microscope. Aside from being a very tall and light unstable structure arises, have such additional systems the disadvantage that the photo output for conventional Applications is no longer available.

The latter disadvantages are in the laser scanning microscope Applicant, for example in prospectus No. 42-920-d is described with the printing note AW-H-VII / 88 Uoo, avoided. With this microscope, the laser is vertical arranged behind the actual microscope stand and is coupled into the microscope via a beam path, the above the conventional incident light illumination Beam path and runs parallel to this. The Coupling itself takes place via a in a slide  arranged full mirror. There is a slide position bare, so that in this switch position Observation light enters the photo output. Disadvantageous is here, however, that the entire laser scanning unit constructively on a special tripod, in particular with regard to the tripod height. An adaptation of the Laser lighting on different tripods is therefore only possible with a redesign.

From the essay "Laser Scan Microscope - Setup and Applications "in GIT Fachz. Lab. 28, (1984) is a laser Scanning microscope known in which the laser beam over the conventional reflected light beam path into the microscope is coupled. To avoid eye damage is at Laser scan operation of the visual observation beam path blocked. By what means the visual Observation beam path is blocked, and whether or how a possible incorrect operation such that the Observation beam path free despite laser scan operation is given, is avoided, the essay is not removable.

EP-B1-0 101 572 describes a microscope with a laser Micromanipulator known. The manipulation beam is over the conventional incident light beam path into the microscope coupled. Between the laser and the reflected light reflector a shutter is arranged, the way it works but is not described in detail. The reflected light reflector is designed as a partial mirror. This will always be the Scattered or reflected laser light into the object Mirrored eyepieces. When focusing on strong reflective object structures therefore exist considerable risk of eye damage to the eyepieces insightful observer.

The present invention is intended to be a microscope with a Laser lighting or another eye-damaging Create illuminating radiation in which none Limitations on conventional use occur. The laser lighting should be as simple as possible different microscope stands can be adapted. In addition, eye damage is said to be in the eyepieces insightful observer be excluded.

This is done through a microscope with the characteristics of Claim 1 reached.

The microscope according to the invention consists of a tripod an observation tube arranged on its front. There are one or more on a slide or revolver Beam splitter and a mirror to deflect one Incident light path towards a lens intended. The coupling of the laser radiation or one other eye-damaging radiation is emitted by the Front opposite back in the for conventional incident lighting provided Incident light beam path.

Since the laser radiation also in the conventional Incident light beam path is coupled regarding the possible uses of the microscope no restrictions. The coupling of the Laser lighting from the back of the tripod causes moreover, that the accessibility of the microscope stage is not restricted in any way. The room to the side of the Microscope stands stand for the positioning of Micromanipulators etc. available. As for the Incident light beam path on the microscope stand usually anyway an interface for coupling the Incident lighting is provided, are not structural Tripod changes required.  

Contains the slider or revolver of the reflected light reflector a full mirror in a switch position. The full mirror then redirects all of the laser light to the lens, and that light reflected from the sample into the incident light beam path back. This will prevent laser light from entering the Observation tube avoided. For microscopy with one conventional lighting, e.g. B. by means of a Halogen lamp, can be one of the full mirror instead Beam splitter can be switched into the beam path. The The object can then also be observed through the eyepieces.

Furthermore, a sensor for detecting the Switch position in which the full mirror in the beam path is switched on, be provided. Through a coupling this sensor with one between the laser and the Full mirror arranged shutter is then ensured that only then laser radiation is coupled into the microscope will when the full mirror in the beam path is switched on. In another switching position of the Slider in which a beam splitter for observation is switched on through the eyepieces in the beam path, the laser lighting is then switched off. This is ensures that the beam path to the eye always is interrupted when the laser beam is released.

In an advantageous embodiment, this is Microscope a laser scanning microscope. In this case between the laser and the beam splitter or mirror Beam deflection unit, which the laser beam in two to each other deflects vertical directions, provided. So that none Interventions on the microscope stand should be required the beam deflection unit in a separate, behind the Microscope stand arranged housing part can be arranged.  

For confocal microscopic fluorescence examinations should also focus on the focus level of the Objective confocal planes each arranged an aperture his. These multiple confocal planes are over color dividers with different spectral Transmission characteristics parallel to each other switched. As a result, fluorescence studies are included possible simultaneously according to different wavelengths. Each of the bezels should be independent of the others centerable and variable in their opening diameter his. As a result, the confocality of the measuring arrangement for each wavelength separately adjustable and to the respective Fluorescence intensity at the wavelength concerned customizable.

For visual incident light exams should go beyond that between the beam splitter or mirror and the Deflection device a conventional one Lighting device, for example the light of a Halogen lamp can be coupled into the incident light beam path. This can be done, for example, using a switching mirror, e.g. B. a folding mirror.

In a particularly advantageous embodiment the inverted microscope stand, i.e. with under the Object arranged lens. Because inverted microscopes very much are often used in the micro-biological field It is particularly important here that the room to the side of the Microscope stands for micromanipulators and Microinjection facilities are fully available. For Shielding by a transparent object passing through or with a removed object from the Laser radiation emerging should be above the lens Object table one impervious to laser light Shielding may be provided. This shield should moreover also via a sensor with a  Laser interrupting shutter are connected. This ensures that when the shield is removed likewise, no laser light falls into the surgeon's eye can.

Such shielding is also given by the fact that a transmitted light illumination unit above the object level is available. The housing wall of this unit provides then the shield at the same time. For a good one Accessibility of the object space is the Transmitted light illumination device can be swiveled via an arm arranged on the tripod. Are on the swivel joint of the arm again sensors are provided which are used with the shutter Interruption of the laser beam are coupled. At from The laser then becomes the object that is swung away interrupted. The sensors on the swivel joint and on Reflected light slides are connected via a logical AND Link coupled with the shutter control, so that the Laser beam is only released if the sensor of the Reflector slide and the sensor of the swivel joint at the same time a signal indicating the safety position produce.

In the following details of the invention are based on the in the embodiment shown in the drawings explained. In detail show:

Fig. 1 shows a laser scanning microscope of inverted design according to the invention in a section containing the optical axis of the lens and

Fig. 2 is a schematic diagram of the optical path in the microscope of FIG. 1.

The inverted microscope shown in FIG. 1 has a stand ( 1 ), on the front ( 2 ) of which a binocular tube ( 3 ) is arranged. A plurality of objectives ( 5 ) are accommodated on a nosepiece ( 4 ) under the object table ( 6 ). The observation beam path running along the optical axis ( 7 ) of the objective ( 5 ) is directed obliquely upwards to the eyepiece tube ( 3 ) via a mirror arranged below the objective. The reflected-light reflector slide ( 9 ) is arranged between the objective ( 5 ) and the mirror ( 8 ). The structure described so far corresponds to the inverse microscope known from DE-OS 39 38 412. With regard to the other components arranged in the observation beam path and the reflection into the phototube, reference is therefore expressly made to this published specification.

The reflected light slide valve ( 9 ) has at least three switch positions. One of these three switch positions is free and is used for visual transmitted light microscopy. In the second switch position, a 50% beam splitter is provided for visual reflected light microscopy and in the third switch position a full mirror ( 10 ) is provided for confocal microscopy. In another version for fluorescence applications, the reflector has four switching positions, one of which is equipped with the full mirror for confocal microscopy and the other two with fluorescence filter sets. The fourth position is empty for visual transmitted light microscopy or equipped with another set of fluorescent filters.

A scan module ( 12 ) is arranged on the back ( 11 ) of the microscope stand facing away from the eyepiece tube ( 3 ). The laser scanning module ( 12 ), which has its own housing, consists essentially of a scanning unit, e.g. B. two galvanometer scanners, which deflects the laser beam incident perpendicular to the plane of the drawing in two mutually perpendicular directions. A lens ( 15 ) arranged behind the scanning unit forms, together with the tube lens ( 17 ) arranged in the microscope stand, a relay lens system which images the two scanning mirrors ( 13 , 14 ) into the pupil of the objective ( 5 ) on the illumination side.

The laser beam is coupled into the stand ( 1 ) via a mirror ( 16 ) through the incident light beam path ( 18 ), which is already present in the microscope stand. Since such an incident light beam path ( 18 ) is generally present in all microscope stands, the scan module ( 12 ) can be easily adapted to a wide variety of microscope stands.

A folding mirror ( 19 ) is also provided between the deflecting mirror ( 16 ) and the coupling of the laser beam into the microscope stand ( 1 ), via which a conventional microscope lamp (not shown here) can be coupled into the incident light beam path instead of the laser beam for visual reflected light microscopy.

The position of the full mirror ( 10 ) is marked on the reflector slide ( 9 ) by a sensor. This sensor consists here specifically of two magnets ( 21 ), which are accommodated in two small bores in the reflector slide ( 9 ), and two probes opposite them, which are received on the guide of the reflector slide. If the magnets ( 21 ) and the probes ( 20 ) face each other, the resulting signal triggers a shutter (not shown here) in the beam path of the laser light and releases the beam path. Since only the switch position of the full mirror ( 10 ) is marked by magnets ( 21 ), the laser beam path is interrupted for any other switch position of the reflector slide ( 9 ) or in the absence of this slide. This prevents damage to the eyes when looking into the ocular tube ( 3 ). The design of the safety device with two magnets allows the failure of a sensor to be determined, so that the laser radiation is interrupted even if a sensor malfunctions.

Above the object table ( 6 ), a transmitted light condenser ( 24 ) is arranged on an arm ( 22 ) which can be pivoted about a horizontal axis ( 23 ) and a folding mirror ( 25 ) is arranged above it. Via the folding mirror ( 25 ), the light of a conventional transmitted light illumination which is not shown here and which is above the drawing plane can either be reflected or can be reflected for a scanning microscope on a detector below the drawing plane. The housing, in which the transmitted light condenser ( 24 ) and the folding mirror ( 25 ) as well as the detector and the transmitted light illumination device are arranged, serves at the same time to protect against unintentional insight into the laser light emerging from the objective ( 5 ). A sensor ( 26 , 27 ) is also provided so that such an unintentional view is avoided when the arm ( 22 ) is folded away. The signals of this sensor consisting of magnets ( 26 ) and probes ( 27 ) also control the shutter in the laser beam path. For this purpose, the signals from the sensors ( 20 , 21 ) and ( 26 , 27 ) are linked to one another in the sense of a logical AND circuit, so that the laser beam path is only released when both sensors indicate the safe state.

As can be seen from FIG. 2, the inverse laser scanning microscope has a modular structure. It consists of three standard blocks (A, B and C) and, in principle, any number of additional laser modules (D and E). Module (A) represents the microscope stand and module (B) the scanning module. The individual optical components in FIG. 2 are given the same reference numerals as in FIG. 1. All components are shown in FIG. 2 for simplification shown in one plane, although they are in different planes in the microscope actually realized.

Since the components of the modules (A and B) have already been discussed in connection with FIG. 1, these components will not be described again.

The detector module (C) is connected to the scanning module (B). This detector module contains a laser ( 31 ) with an upstream shutter ( 32 ). The shutter ( 32 ) is driven by an electromagnet. A color splitter ( 33 ) directs the laser beam emerging from the laser ( 31 ) onto a beam expansion ( 34 , 35 ). Another color splitter ( 36 ) directs the collimated laser beam emerging from the beam expansion ( 34 , 35 ) to the deflection unit ( 13 , 14 ). A neutral splitter or a polarization splitter can also be used here for incident light microscopy.

After passing through the scanning device ( 13 , 14 ), the collimated laser beam is deflected from the full mirror ( 10 ) to the objective ( 5 ) and from there focused on the specimen (not shown here).

The fluorescent light or the reflected light excited by the laser light in the preparation passes through the same beam path in the opposite direction between the objective and the divider ( 36 ). Since the fluorescent light differs in wavelength from the laser light, the fluorescent light transmits the color splitter ( 36 ). The fluorescent light is fed to three parallel confocal detection channels via two further color dividers ( 37 , 38 ) with different spectral transmission properties and a full mirror ( 39 ). Each of these confocal detection channels contains an objective ( 40 , 41 , 42 ), a confocal diaphragm ( 46 , 44 , 45 ) and a photodetector ( 47 , 48 , 49 ) for converting optical radiation into electrical signals. The confocal diaphragms ( 46 , 44 , 45 ) are each arranged in a plane conjugate to the focal plane of the objective ( 5 ). Each of these confocal diaphragms can be centered independently of the other by means of corresponding externally accessible adjusting screws (not shown) and can be varied in terms of their opening diameter. This enables the depth resolution of the microscopic image to be set separately for each fluorescence wavelength.

The divider ( 36 ), which separates the illuminating beam path from the measuring beam path, and the color dividers ( 37 , 38 ) for dividing the measuring light into the different detection channels are each accommodated in reflector slides, not shown here. Due to the different combinations of the reflector slides, the most varied wavelength combinations can therefore be set and registered simultaneously. The reflector slides in which the color dividers ( 37 , 38 ) are accommodated have an empty switch position. This makes it possible to switch a reflector slide to full passage when measuring very weak fluorescence, so that there is no additional weakening of the already weak fluorescent light.

As is further indicated by the modules (D and E), in principle, any number of additional external laser modules can be coupled to the detector module (C) for applications with several excitation wavelengths. Each of these additional laser modules (D and E) essentially consists of a laser ( 61 , 51 ), its own shutter ( 62 , 52 ), optionally filters for line selection (not shown here) for multiline lasers and adjustable coupling optics ( 63 , 53 ). Each of these adjustable coupling optics ( 63 , 53 ) consists of two adjustable mirrors, of which only one is shown here.

Based on the figures is a particularly advantageous one Embodiment of the invention described. There are however, numerous possible variations are also possible. In particular, can be used to generate the shutter signals other types of sensors are used, for example Microswitches or simple electrical contacts. Furthermore can also use several lasers or more in the detector module (C) be provided as three parallel detection channels.

It is essential for the invention that the full mirror ( 10 ) does not transmit any laser light. It is therefore not absolutely necessary for the full mirror ( 10 ) to completely reflect any wavelength. Rather, it is entirely sufficient if the full mirror ( 10 ) completely reflects light of the laser wavelength, or in the case of several lasers of different wavelengths, the light of all laser wavelengths.

Claims (10)

1. Microscope consisting of a tripod ( 1 ), an eyepiece tube ( 3 ) arranged on the front ( 2 ) of the tripod ( 1 ) and a slide ( 9 ) or revolver in which at least one beam splitter and a mirror ( 10 ) for deflecting one Incident light beam path ( 18 ) is recorded in the direction of the objective ( 5 ), a laser beam or another radiation damaging to the eyes being coupled from the rear side ( 11 ) opposite the front side ( 2 ) into the incident light beam path ( 18 ) provided for conventional incident light illumination. 2. Microscope according to claim 1, characterized in that a sensor ( 20 , 21 ) for detecting the switching position in which the mirror ( 10 ) is switched on in the beam path is provided, and that the sensor ( 20 , 21 ) with a between the laser ( 31 , 61 , 51 ) and the mirror ( 10 ) arranged shutter ( 32 , 62 , 52 ) is coupled. 3. Microscope according to one of claims 1 or 2, characterized in that between the laser ( 31 , 61 , 51 ) and the beam splitter or mirror ( 10 ) a beam deflection unit ( 13 , 14 ) in a separate, behind the microscope stand ( 1 ) arranged housing part ( 12 ) is arranged. 4. Microscope according to one of claims 1-3, characterized in that in several to the focal plane of the lens ( 5 ) confocal planes an aperture ( 44 , 45 , 46 ) is arranged. 5. Microscope according to claim 4, characterized in that the diaphragms ( 44 , 45 , 46 ) can each be centered separately and that the opening diameter can be varied independently of one another. 6. Microscope according to one of claims 3-5, characterized in that a conventional lighting device between the beam splitter or mirror ( 10 ) and the deflection device ( 13 , 14 ) in the incident light beam path ( 18 ) can be coupled. 7. Microscope according to any one of claims 1-6, characterized in that the stand ( 1 ) is an inverse type. 8. Microscope according to claim 7, characterized in that above the object table ( 6 ) an opaque to harmful radiation shield ( 22 ) is provided, and that the shield ( 22 ) via a sensor ( 26 , 27 ) with a shutter interrupting the laser beam ( 32 , 62 , 52 ) is connected. 9. Microscope according to claim 8, characterized in that above the object table ( 6 ) a transmitted light illumination unit ( 28 ) and a transmitted light detector ( 19 ) are provided, which are connected to the stand ( 1 ) via a pivotable arm ( 22 ), and that sensors ( 26 , 27 ) are provided on the swivel joint ( 29 ) of the arm and are coupled to a shutter ( 32 , 62 , 52 ) for interrupting the laser beam. 10. Microscope according to claim 9, characterized in that the laser beam is only released when the sensor ( 20 , 21 ) for detecting the switching position of the slide ( 9 ) or turret and the sensor ( 26 , 27 ) arranged on the swivel joint ( 23 ) ) generate a signal at the same time.