WO2006037528A1

WO2006037528A1 - Fluorescence microscope comprising at least one absorption filter for preventing the reflection of undesired illumination light

Info

Publication number: WO2006037528A1
Application number: PCT/EP2005/010472
Authority: WO
Inventors: Reiner Mitzkus; Anke Vogelgsang
Original assignee: Carl Zeiss Jena Gmbh
Priority date: 2004-10-04
Filing date: 2005-09-28
Publication date: 2006-04-13
Also published as: DE102004048845A1

Abstract

The invention relates to the prevention of stray light in the detection beam path of fluorescence microscopes. To this end, an absorption filter (6) is applied to the reflector module or arranged in the vicinity thereof, said absorption filter effectively attenuating the excitation light (53) let through by the separating mirror (4).

Description

FLEURESCENCE MICROSCOPE WITH AT LEAST ONE ABSORPTION FILTER FOR REFLEX SUPPRESSION OF UNWANTED LIGHTING LIGHT

The invention relates to a microscope with at least one beam splitter. Such beam splitters are generally part of reflector modules for fluorescence microscopy. These reflector modules consist of two filters and the wavelength-selective splitter mirror / beam splitter.

The fluorescence of the specimen exciting light is provided by the excitation filter according to the selected application method narrowband and in the required wavelength available. The splitter mirror directs this light as efficiently as possible through the lens to the specimen. The light emitted by appropriate excitation of fluorescence in the specimen is shifted somewhat in the direction of its wavelength with respect to its wavelength (Stokes shift) and now passes through the splitter mirror. This passage through the splitter mirror must also be efficient in order to avoid losses. A blocking filter (emission filter) attached after the splitter mirror has the task of transmitting only the light emitted by the specimen, but blocking possible proportions of the excitation light as completely as possible.

In known reflector modules passing through the splitter mirror, non-reflected light at the opposite side of the excitation filter housing parts is at least partially reflected or scattered and thrown back into the beam path, in particular thrown with high efficiency in the direction of the emission filter, since the splitter mirror on its rear reflection layer reflects the excitation light well. A particular problem arises from the scattered light which strikes the emission filter at an angle other than 90 °, because the dichroic layers used for the filters can only realize their predicted filtering effect for vertical incidence of light; at oblique incidence, wavelengths other than those desired are also realized pass through.

Thus, in the detection beam path following the emission filter, which leads to observation by means of eyepieces or sensitive cameras, a background brightness directly caused by the excitation light is generated which reduces the contrast and in particular makes it impossible to study weak fluorescence. In DE 199 26 037 Al the Applicant has been proposed to solve this problem, to remove the back of the reflector module or make transparent and to direct the divider penetrating light on a reflective surface having at least partially conical and in this way from the beam path to remove. It But even with this solution can still happen that by reflection

Housing parts light is thrown back from the conical surface and thus reflected back into the detection beam path.

The invention has the object of known by the prior art

Suppression of unwanted excitation light in the detection beam path to further improve.

This object is achieved by a microscope with at least one beam splitter, wherein the

Reflex suppression unwanted illumination light is directed to this absorbent material.

It is particularly advantageous if light which still by the absorbent

Material passes on a light trap and is thus removed from the rays away.

A further advantage arises when the absorbent material is replaced by the

Excitation filter defined wavelength adapted and preferably mounted on the reflector module.

An alternative advantageous solution is to use the absorbent material on the

Mount microscope housing, wherein it is advantageously positioned obliquely to the incident light.

In order to avoid unwanted reflections on the absorbent material, it is advantageous to provide the splitter side of the absorbent material with a

To provide anti-reflex layer.

In addition, it is advantageous if, in addition, a scattered light sleeve is arranged in the detection beam path, which illuminates the light passing obliquely through the emission filter

Beam path away.

The invention will be explained in more detail below with reference to the drawings.

Show it:

Fig. 1 shows the known basic structure of a reflector module

Fig. 2, the emergence of false light in the emission beam path in a known

reflector module

Fig. 3 shows a first embodiment of the invention

Fig. 4 shows a second embodiment

Fig. 5 is a schematic representation of a microscope with a beam splitter

The reflector module 1 of Fig. 1 consists of an excitation filter 2, an emission filter 3 and a splitter mirror 4. The light of a light source (e.g.

Halogen lamp) meets the excitation filter 2, which only the desired part of Spectrum passes and is directed by the splitter mirror 4 in the direction of the object, not shown here (excitation beam Sl). After interaction of this light with the fluorescent parts of the object, the returning, somewhat shifted in its wavelength fluorescent light hits again on the splitter mirror 4, is transmitted by this and then strikes the Emissionsfϊlter 3, which has the task of transmitting only the desired fluorescent light and on to direct the detector, also not shown here (emission beam S2). The excitation light which has been reflected by the object must be blocked by the emission filter.

FIG. 2 shows how excitation light can enter the detection beam path S2 in the prior art. Since the splitter mirror 4 can not reflect the excitation light 100% in the direction of the object, a part of the excitation light reaches parts 5 of the microscope lying behind the table mirror (beam path S3) and is scattered or reflected by them in an undirected manner in general. A part of this scattered or reflected light can now fall obliquely on the emission filter 3 and is transmitted by this (beam path S4), since the emission filter unfolds its blocking effect for the excitation light only at normal incidence. This leads to a reduction in contrast in the detection beam path, which is undesirable.

In FIG. 3, according to the invention, an absorption filter 6 is mounted on the side of the reflector module 1 opposite the excitation filter 2, which absorbs the excitation light to a large extent. For this purpose it is favorable if the absorption filter is adapted to the excitation filter in order to absorb exactly the excitation wavelength. The absorption filter 6 extends over the entire side of the reflector module 1, so that also obliquely reflected light from the microscope parts 5 back to the absorption filter 6 again and is attenuated by this again (beam path S4). Instead of the microscope parts 5, a light trap may also be provided, as e.g. in DE 199 26 037 was proposed. To further improve the elimination of stray light in the detection beam path S2 can now be arranged a stray light sleeve, which preferably consists of a black anodized corrugated sleeve or a corrugated black plastic sleeve. By this, the incident light obliquely into the detection beam path is effectively eliminated.

In Fig. 4 is shown as an alternative that the absorption filter 6 can also be attached to the microscope part 5 or a possibly provided light trap according to DE 199 26 037. In any case, the absorption filter on its side facing the splitter mirror 4 on an anti-reflective coating on to effectively prevent the occurrence of reflections on its input side.

In Fig. 5, the total beam path is shown schematically in a microscope. From a light source 7, the light is directed onto the excitation filter 2 via a heat protection filter 8, the aperture diaphragm 9 and the field diaphragm 10. The splitter mirror 4 reflects the excitation light via the objective 11 onto the object 12. The fluorescence light generated by the excitation light in the object 12 again passes the objective 11 and is now transmitted by the splitter mirror 4 and through the emission filter onto the tube lens 13 and from there via a prism system the eyepieces 14 shown. Alternatively, the light can also be imaged by means of a camera attached to the phototube 15. Here, excitation filter 2, emission filter 3 and splitter mirror 4 are shown as separate components, but are usually combined to form reflector modules. The invention is not bound to the illustrative embodiments shown, expert developments do not lead to a departure from the scope defined by the claims. In particular, the invention can also be used in laser scanning microscopes in the suppression of stray light at the main or secondary color splitters.

Claims

claims

1. Microscope with at least one beam splitter, wherein for the reflection suppression unwanted illumination light is directed to a this absorbing material.

2. A microscope with at least one beam splitter according to claim 1, wherein light which passes through the absorbing material passes onto a light trap and is thus removed from the beam path.

3. A microscope with at least one beam splitter according to claim 1 or 2, wherein the absorbing material is adapted to the wavelength defined by the excitation filter.

4. A microscope with at least one beam splitter according to claim 1, 2 or 3, wherein the absorbing material is preferably mounted on the reflector module.

5. A microscope with at least one beam splitter according to claim 1, 2 or 3, wherein the absorbing material is attached to the microscope housing.

6. A microscope with at least one beam splitter according to one of claims 1 to 5, wherein the absorbing material is positioned obliquely to the incident light.

7. A microscope with at least one beam splitter according to one of claims 1 to 6, wherein the splitter side facing the absorbent material is provided with an anti-reflection layer.

8. microscope with at least one beam expensive according to one of claims 1 to 7, wherein in the beam path in addition a scattered light sleeve is arranged.