DE4323129C2 - Inverted microscope - Google Patents

Description

The present invention relates to an inverted microscope the preamble of claim 1. Such an inverse Microscope is known from DE 26 40 974 A1. A Laser illumination is in this well-known inverted microscope not provided.

From DE 35 27 322 C2 a microscope is known in which Laser light in the incident light beam path for the conventional Lighting is coupled. Here, however, the laser light serves not to illuminate the object itself. With the help of Rather, an autofocus signal is generated by laser light. The Lasers are therefore very poorly designed, so that their Radiation does not damage the eyes.

From US 5 032 720 and WO 92/02839 A1 Laser scanning attachments known for conventional microscopes, at which a laser beam for object lighting at confocal microscopy is used. The coupling of the However, the laser beam comes from above into the photo output of the microscope. In addition to being very high and slightly unstable structure arises, have such additional systems the disadvantage that the photo output for conventional Applications is no longer available.

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 Incident light beam path is coupled into the microscope. to Avoiding eye damage is the key with Laser Scan Bettieb blocked visual observation beam path. By which The visual observation beam path is blocked, and whether or how a possible misuse such that the observation beam path is free despite laser scan operation given, is avoided, the essay cannot be deduced.  

EP 0 101 572 B1 describes a microscope with a laser Micromanipulator known. The manipulation beam is over the conventional reflected light beam path into the microscope coupled. Is between the laser and the reflected light reflector arranged a shutter, but its functionality is not is described in more detail. The reflected light reflector is a partial mirror educated. As a result, the or scattered on the object is always reflected laser light reflected in the eyepieces. at There is a focus on highly reflective object structures therefore a significant risk of eye damage in the Eyepieces of the observer looking in.

The present invention is intended to be an inverted microscope with a Laser lighting or another eye-damaging Create illuminating radiation in which eye damage to the Users through the laser radiation both when looking into that Eyepiece as well as when handling the object is prevented.

This goal is achieved through an inverted microscope with the features of claim 1 solved. Advantageous embodiments of the Invention result from the features of the dependent Expectations.

The microscope according to the invention has an inverse tripod an observation tube arranged on its front. There are several beam splitters or on a slide or turret Mirror for deflecting radiation that damages the eyes, for example laser radiation, towards a lens intended. The laser radiation is coupled in by the the front opposite the back in the for conventional reflected light illumination provided beam path.

The slide or revolver of the reflected light reflector contains in a switching position a full mirror. The full mirror steers then all the laser light to the lens, and that at the Sample reflected light back into the incident light beam path.  

This will prevent laser light from entering the observation tube avoided.

Furthermore, there is a sensor for detecting the switching position, in the full mirror is switched on in the beam path, intended. By coupling this sensor with a Shutter arranged between the laser and the full mirror then ensured that only then laser radiation into the Microscope is coupled when the full mirror in the Beam path is switched on. In a different switch position of the slider, in which a beam splitter for the visual Observation in the beam path is then the Laser lighting switched off. This ensures that the beam path to the eye is always blocked when the Laser beam is released.

There is also a above the object level Transmitted light illumination unit available. The housing wall this unit is a shield. For a good one Accessibility of the object space is the Transmitted light illumination device can be swiveled via an arm Tripod arranged. At the swivel joint of the arm are in turn Sensors are provided which are used to interrupt the shutter Laser beam are coupled. With the arm swung away from the object the laser is then also interrupted. The sensors on Swivel joint and on the reflected light slide are over one logical AND circuit coupled with the shutter control, see above that the laser beam is only released when the sensor of the reflector slide and the sensor of the swivel joint generate a signal at the same time.

The inverted microscope is preferably a laser scan microscope educated. Between the laser and the beam splitter or Mirror is a beam deflection unit that the laser beam in deflects two mutually perpendicular directions.  

So that no interventions on the microscope stand are necessary, the beam deflection unit is preferably in a separate, arranged behind the microscope stand arranged housing part.

Since the laser radiation also in the conventional Incident light beam is coupled in, exist none of the application possibilities of the microscope Limitations. The coupling of the laser lighting from the Back of the tripod also causes the Accessibility of the microscope stage in no way is restricted. The space to the side of the microscope stand stands fully for the positioning of micromanipulators etc. for Available. As for the reflected light path on the microscope stand Usually an interface for coupling the Incident lighting is provided, are not structural Tripod changes required.

For visual incident light exams should go beyond that between the beam splitter or mirror and the deflector a conventional lighting device, such as that Light from a halogen lamp in the incident light beam path can be coupled. This can be done using a Folding mirror done.

For microscopy with conventional lighting can instead of the full mirror one of the beam splitters in the Beam path can be switched. The object is then also through the eyepieces are observable.

In the following, details of the invention are based on the in the Illustrations illustrated in the drawings.

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 39 38 412 A1. 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 switch 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, essentially consists of a scanning unit, e.g. B. two galvanometer scanners, which deflects the incident laser beam 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 mirrored onto a detector below the drawing plane for scanning microscopy. 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 an unintentional view 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), for applications with several excitation wavelengths, in principle any number of additional external laser modules can be coupled to the detector module (C). 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. However, there are numerous variations are also possible. In particular can use other sensor types for generating the shutter signals can be used, for example microswitches or simple electrical contacts. In addition, in the detector module (C) multiple lasers or more than three parallel detection channels be provided.

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 (4)

1. Inverse microscope, with
a tripod ( 1 ),
one arranged at the front side (2) of the stand (1) ocular tube (3),
a nosepiece ( 4 ) with an attached lens ( 5 ),
an incident light reflector inserted into the beam path between the objective ( 5 ) and the eyepiece tube ( 3 ) for deflecting an illumination beam path ( 18 ) incident from the rear of the inverted microscope in the direction of the objective ( 5 ),
and with a transmitted light illumination device ( 24 , 28 ) which can be pivoted out of its operative position,
characterized by
that a laser ( 31 , 51 , 61 ) is provided for the illumination beam path,
that the incident light reflector has a full mirror ( 10 ) which can be introduced into the illumination beam path and which deflects the laser beam in the direction of the object,
that a sensor ( 20 , 21 ) registering the position of the full mirror ( 10 ) is provided, which controls a shutter ( 32 , 52 , 62 ) arranged at the laser output and only releases the laser beam when the full mirror ( 10 ) is in the beam path is introduced
and that the transmitted-light illumination device ( 24 , 28 ) is connected to a further sensor ( 26 , 27 ) which also controls the shutter ( 32 , 52 , 62 ) and only releases the laser beam by means of the shutter when the transmitted-light illumination device ( 24 , 28 ) is in the active position. 2. Inverse microscope according to claim 1, characterized in that a scanning device ( 13 , 14 ) is provided for scanning the object to be examined. 3. Inverse microscope according to claim 2, characterized in that the beam deflection unit ( 13 , 14 ) of the scanning device in a behind the microscope stand ( 1 ) arranged, separate housing part ( 12 ) is arranged. 4. Inverse microscope according to one of claims 1-3, characterized in that a conventional lighting device is provided and that the full mirror ( 10 ) is interchangeable with a partially transparent beam splitter.