DE4345538C2 - fluorescence microscope - Google Patents

Description

The present invention relates to a fluorescence microscope the preamble of claim 1.

Such a thing Fluorescence microscope is known from US 5,032,720. at this known fluorescence microscope Detection beam path divided by a color divider and that Wavelength-divided light subsequently detected. However, the wavelength-dependent division has an effect on weak fluorescence intensities disadvantageous because the weak fluorescence radiation through the color splitter and the Reflections arising in and out of the beam splitter is weakened further.

The present invention is intended to use a fluorescence microscope create a laser lighting that on the one hand Multi-channel detection of fluorescence radiation enables, in which but no additional for weak fluorescence intensities The fluorescence in the detection beam path is weakened.

This goal is achieved by using a fluorescence microscope Features of claim 1 solved. Advantageous configurations the invention result from the features of the dependent Expectations.

In the fluorescence microscope according to the invention, the color splitter is in a reflector slide added in a switch position is empty. This makes it with very faint fluorescence possible to switch the reflector slide to full passage, so that no additional weakening of the weak Fluorescent light takes place.

For confocal microscopic fluorescence examinations moreover in three confocal to the focal plane of the lens Levels can each be arranged an aperture. These three Confocal planes are over color dividers with different  spectral transmission characteristics parallel to each other connected. As a result, fluorescence studies are at three different wavelengths possible simultaneously. Any of the three Apertures should be centered and in independently of the others their opening diameter can be varied. This is the Confocality of the measuring arrangement for each wavelength separately adjustable and to the respective fluorescence intensity at the adjustable wavelength.

The fluorescence microscope preferably has a stand with a observation tube arranged on its front. On one slider or turret are multiple beam splitters or Mirror for deflecting the fluorescence stimulating Laser radiation, directed towards a lens. The The laser radiation can be launched from the front opposite back in for conventional Illuminated beam path provided.

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.

The slider or revolver of the reflected light reflector should be in a switching position contain a full mirror. The The full mirror then redirects all of the laser light to the lens, and the light reflected on the sample in the Reflected light path back. This will invade Avoid laser light in the observation tube.

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, consists essentially 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.

Claims (5)

1. Fluorescence microscope
with a laser arrangement ( 31 , 51 , 61 ) for exciting the fluorescence of a sample to be examined,
and a detector arrangement registering the fluorescent light with at least two detectors ( 47 , 48 , 49 ) in a detection beam path,
A color beam splitter (( 37 ) is provided, which splits the fluorescent light into the at least two detectors ( 47 , 48 , 49 ) depending on the wavelength,
and with a lens ( 5 ) for focusing the exciting laser radiation and for recording the fluorescent light,
characterized by
that the color beam splitter ( 37 ) is received on an reflector slide with an empty switching position,
the entire fluorescent light being applied to a detector ( 47 ) when an empty switching position is introduced into the detection beam path. 2. Fluorescence microscope according to claim 1, characterized characterized as a confocal fluorescence microscope is trained. 3. Fluorescence microscope according to claim 1 or 2, characterized in that each of the detectors ( 47 , 48 , 49 ) has an associated, separately centerable detector pinhole ( 44 , 45 , 46 ). 4. Fluorescence microscope according to claim 3, characterized in that the detector pin diaphragms ( 44 , 45 , 46 ) can be varied independently of one another in their opening diameters. 5. Fluorescence microscope according to one of claims 1 to 4, characterized in that there is a tripod ( 1 ), an eyepiece tube ( 3 ) arranged on the front ( 2 ) of the tripod, several beam splitters or mirrors accommodated in a slide or turret ( 4 ) for deflecting the laser beam provided by the laser arrangement in the direction of the objective and that the laser beam is coupled from the rear side opposite the front side ( 2 ) into the beam path ( 18 ) provided for conventional incident light illumination in the stand ( 1 ).