EP3455663A1

EP3455663A1 - Optical scanning microscope and examination method

Info

Publication number: EP3455663A1
Application number: EP17723981.1A
Authority: EP
Inventors: Christian Schumann; Albrecht Weiss; Tobias Bauer; Cornell Peter Gonschior
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2016-05-13
Filing date: 2017-05-12
Publication date: 2019-03-20
Also published as: US11630292B2; US20200319445A1; JP2019517027A; JP6945557B2; DE102016108987A1; WO2017194742A1; DE202016008489U1

Abstract

The invention relates an optical scanning microscope (1, 2, 3), which comprises an illumination system (20) having a light source portion (100-103) emanating from a light source (100), a first polarization-dependent beam splitter (105a) and a second polarization-dependent beam splitter (105b) and a first optical channel (21) and a second optical channel (22) between the first beam splitter (105a) and the second beam splitter (105b), wherein the light source portion (100-103) is configured to emit a first illumination light beam (203) with light with a first main polarization direction and a second main polarization direction, the first beam splitter (105a) is configured to at least predominantly guide the light with the first main polarization direction into the first channel (21) and the light with the second main polarization direction into the second channel (22), the second beam splitter (105b) is configured form a second illumination light beam (24) from the light with the first main polarization direction from the first channel (21) and from light with the second main polarization direction from the second channel (22), and the channels (21, 22) are configured to emit the light with the first main polarization direction from the first channel (21) and the light with the second main polarization direction from the second channel (22) with different convergent angles. A corresponding method is likewise subject matter of the invention.

Description

Optical scanning microscope and examination method

The invention relates to a scanning optical microscope and a corresponding

Investigation method according to the preambles of the independent claims.

PRIOR ART In modern functional biology, the investigation of dynamic processes in biological systems is frequently of interest. For these is a spatial and temporal Interaktionsbzw. Manipulation of microscopic samples of central importance. For this purpose, methods such as fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), uncaging and photoactivation are known. In such methods, the sample to be examined is typically scanned for manipulation by means of a focused laser beam, using a scanning device (used in an orthoscopic beam path).

At the same time, it is desirable to detect the course of the experiment in a wide-field microscope with an optical section. One possibility is, for example, TIRF (Total Internal Reflection Fluorescence Microscopy) microscopy, in which a

Laser beam is focused in the entrance pupil of the lens to obtain a flat illumination of the object field. This illumination occurs at an adjustable angle determined by the position of the laser beam in the entrance pupil. When illuminated at an angle that does not propagate through the interface between the specimen toughened glass and an aqueous sample, total reflection and a thin illumination of the interface by evanescent waves are obtained. For the exact setting of the

Lighting angle is a position control system of the laser beam in the entrance pupil needed. For this purpose, a (used in a conoscopic beam path) grid device can be used.

For further details, reference is made to relevant literature, for example with respect to the TIRF microscopy on D. Axelrod, Traffic 2, 764-774 (2001) and with respect to Raster-based Manipulation of Microscopic Samples Using Scanning Systems of Confocal Microscopes on EAJ Reits, Nat. Cell Biol. 3, E145-E147 (2001). to

Positioning of the laser beam in the entrance pupil is for example on the

DE 10 2006 033 306 A1.

For switching between orthoscopic and conoscopic beam path can be, for example, according to US 7 187 494 B2 a Bertrand lens arrangement in the

Beam path of an orthoscopic grid system drive to a conoscopic

Realize grid system. However, this has the disadvantage that the mechanical

Movement of mirrors, prisms and lens systems consuming and also incompatible with the required in the task switching times below the relevant biological time scales, e.g. less than 10 ms.

In US7573635B2 the switching is described by means of the grid system itself. However, the method described requires a complex mirror system because the raster mirror unit is used in the conoscopic beam path with multiple reflections, which makes a corresponding system complicated and expensive to handle.

A combination system for orthoscopic and conical illumination beam paths is known from EP 1 752 809 B2, which however has the disadvantage that only a part of the objective pupil is always accessible for a conoscopic illumination.

From DE 10 2013 222 562 A1, a lighting device is known, by means of which an orthoscopic and a conoscopic beam path can be provided. Here, a ring mirror is used. A centrally through the ring mirror, i. the non-mirrored region or a corresponding recess, passing illumination light beam is used to generate the orthoscopic beam path. A peripheral to the ring mirror, i. whose mirrored area, striking illumination light beam is used to generate the conoscopic beam path. Thus, only one part of the beam path cross-section can be used to generate the orthoscopic beam path or the conoscopic beam path.

Object of the present invention is, among other things, the control of a laser beam in the pupil and the scanning of a

Laser beam in the object field of a microscope by one and the same raster unit to accomplish. Furthermore, the switching between the two uses of Scanning unit quickly go so that at typical image acquisition rates of about 100 Hz in microscopic live cell experiments no interruptions.

Disclosure of the invention

According to the invention, a scanning optical microscope and a method for

Examining a sample using such a scanning microscope with the features of the independent claims proposed. Advantageous embodiments are the subject of the dependent claims and the following description.

The present invention is based on the fundamental idea, in one

Scanning microscope at any time both an orthoscopic and a conoscopic beam path available and to realize the switching between these two beam paths by means of polarizing beam splits. The combination of both beam paths is accomplished by polarization optical means.

Against this background, the present invention proposes a scanning optical microscope which comprises an illumination system with a light source emanating from a light source

Light source section, a first and a second polarization-dependent beam splitter and a first and a second optical channel between the first and the second beam splitter comprises. In the light source portion of the optical scanning microscope, a raster unit of known type is integrated, as it is basically known and will be explained below. Here is a "polarization-dependent beam splitter" (English: Polarizing Beam Splitter, PBS, commonly referred to in certain designs as "pole cube"), this is understood as an optical element, the light of different

Distract polarization directions differently. For example, a polarizing beam splitter can pass light of a first polarization direction without being deflected, while deflecting light of a second, different polarization direction in an angle defined by the design and the optical materials. With regard to expert knowledge of polarizing beam splitters and the underlying physical principles, for simplicity, reference is made to relevant specialist literature, eg Bennett, JM: Polarizers, Chapter 3 in: Bass, ME et al. (Ed.): Handbook of Optics. Fundamentals, Techniques & Design, Volume 2, New York: McGraw-Hill, 2nd edition, 1995, referenced. In the proposed scanning optical microscope, the light source section is configured to radiate a first illumination light beam with light of first and second main polarization directions. Under "light of a first Hauptpolarisationsrichtung" or "light of a second

Hauptpolarisationsrichtung "is understood in the context of the present invention, light comprising predominantly or exclusively light waves, which are present in a first polarization direction or a second polarization direction or their

Polarization directions are each in a narrow angular range of, for example, ± 10 ° ± 5 ° or ± 1 °. Due to an incomplete polarization, smaller fractions may also be present in one or more other polarization directions. The formulation according to which corresponding light comprises "mainly or exclusively" light waves which are present in the first polarization direction or the second polarization direction, indicates, for example, that less than 25%, 10%, 5% or 1% in a different

Polarization direction present. The first and second polarization directions are aligned orthogonal to each other. Here, the orthogonal orientation includes both circularly polarized light and linearly polarized light, which in two

Polarization directions is present The light of the first and the second Hauptpolarisationsrichtung can thereby according to the present invention at the same time, i. at one time in one and the same

Illumination beam, be provided. If this is the case, that includes

Illumination light beam corresponding illumination light of a polarization state, which corresponds to a linear combination of orthogonal Hauptpolarisationsrichtungen. Of the

However, illumination beam can also successively with the first

Hauptpolarisationsrichtung and then emitted with the second Hauptpolarisationsrichtung, as also explained below. In the latter case, the illumination light beam comprises light with predominantly or exclusively the first in a first period of time

Hauptpolarisationsrichtung and in a second period of light with predominantly or exclusively the second Hauptpolarisationsrichtung. However, the light does not necessarily have to be completely polarized here; For suppressing residual light of a different polarization direction, for example, an arrangement with optical shutters explained below can be used. The basic inventive idea, namely the orthoscopic and the

concomitant conoscopic beam path is achieved by the use of different optical channels between the first and the second beam splitter implemented. For this purpose, it is provided that the first beam splitter in the scanning microscope according to the invention is adapted to lead the light of the first illumination light beam with the first Hauptpolarisationsrichtung at least predominantly in the first channel and the light of the first illumination light beam with the second Hauptpolarisationsrichtung at least predominantly in the second channel. At the first beam splitter, therefore, a different "treatment" of the light with the first or the second takes place

Main polarization direction.

An illumination light beam that extends between the light source section and the first beam splitter in a common beam path section is therefore converted into the first or the second channel depending on polarization. In this way, the light of the first channel can be influenced differently from the light of the second channel, for example already alone by different optical lengths of the two channels and / or by different optical elements in the two channels.

The inventively guided in the two channels light with the first and the second Hauptpolarisationsrichtung is then back into a common

Beam path section performed. For this purpose, according to the invention, the second beam splitter is set up to form a second illumination light beam from light having the first main polarization direction from the first channel and from light having the second main polarization direction from the second channel.

In this case, for example, by means of the light source section in a first period of time the illumination light beam is predominantly or exclusively with light of the first

Main polarization emitted, this light enters predominantly or exclusively in the first channel and from this in the second beam splitter. The second beam splitter then forms the second illumination light beam predominantly or exclusively from the light from the first channel. The second illumination light beam comprises in this way predominantly or exclusively the light with the first main polarization direction. In a second period in which by means of the light source section of the

Illuminating light beam predominantly or exclusively with light of the second

Hauptpolarisationsrichtung is radiated, this light enters via the first beam splitter predominantly or exclusively in the second channel and from this in the second beam splitter. The second beam splitter forms the second in this way

Illuminating light beam mainly or exclusively from the light of the second

Hauptpolarisationsrichtung from the second channel. If, however, the illumination light beam is emitted by the light source section in such a way that it simultaneously comprises light of the first and the second main polarization direction, the light of the first main polarization direction passes predominantly or exclusively into the first and the light with the second via the first beam splitter

Hauptpolarisationsrichtung predominantly or exclusively in the second channel via. In order to prevent both the light of the first main polarization direction and the light of the second main polarization direction from simultaneously entering the beam splitter from the first and second channels and the second illumination light beam being formed simultaneously with the first main polarization direction and the second main polarization direction may not be desirable, may be formed in the first channel and / or the second channel respectively suitable optical closures, each optically block either the first channel or the second channel. Also in this way it can be ensured that by means of the second beam splitter of the second

Illuminating light beam always only from light of one of the two

Main polarization directions is formed.

As already mentioned, according to the invention the two said optical channels are arranged to emit the light with the first main polarization direction from the first channel and the light with the second main polarization direction from the second channel with different convergence angles.

For example, the first channel may be configured to receive the light of the first

Main polarization direction in the form of a divergent light beam and the second channel may be adapted to emit the light with the second main polarization direction from the second channel in the form of a collimated light beam. For this purpose, as already mentioned, different optical path lengths and / or optical elements are provided in the two channels. In principle, however, a parallel provision of an orthoscopic and a conoscopic beam path can also be effected in a different way than here and below.

Advantageously, in a corresponding scanning microscope, the light source section is adapted to provide the first illumination light beam in the form of a collimated light beam, ie a light source is imaged infinitely. However, due to the two different channels, a raster unit can later be imaged into the objective pupil either along the orthoscopic beam path and from there through an object-side telecentric objective, with the light source being imaged into the sample (front focal plane of the objective) or the raster unit along the conoscopic beam path into the sample, in which case again the light source remains at infinity.

Advantageously, the first beam splitter is preceded by a first optical element, which is set up to provide the one in the form of the collimated light beam

Illuminating illumination light beam in the form of a convergent light beam in the first beam splitter. Since this advantageously does not have convergence-influencing properties in the context of the present invention, the light irradiated into the first beam splitter in the form of the convergent light beam is also guided convergently into the already-mentioned channels. The first optical element can be formed in the simplest case, for example in the form of a converging lens, optionally with suitable optical correction means. It focuses the collimated light beam of the

Illuminating light beam in an image-side plane of the first optical element. In this way, the focused using the first optical element light diverges beyond the focal point of the first optical element and can be in this way without further optical interference, for example, from the first channel in the form of a divergent light beam and irradiated in the second beam splitter. In the second optical channel, a corresponding light beam diverging beyond the focal point can be deflected, collimated, and collimated out of the second channel and irradiated into the second beam splitter by means of suitable optical elements and deflection devices. In particular, in the second channel, a part of a

Be provided Bertrand lens system, as known from DE 10 2013 222 562 A1. The remainder of the Bertrand lens system is advantageously formed by a second, explained below optical element, which is located beyond the second beam splitter,

for example, a tube lens.

As already mentioned, the first channel is advantageously designed to radiate light at least predominantly divergently into the second beam splitter, and the second channel is advantageously configured to radiate light at least predominantly collimated into the second beam splitter. Because the first channel predominantly or exclusively carries light with the first main polarization direction, this is emitted from the first channel in the form of a divergent light beam and radiated into the second beam splitter. In contrast, the light with the second main polarization direction from the second channel is radiated into the second beam splitter in the form of a collimated light beam. Corresponding light can be optically influenced after passing through the second beam splitter in any desired manner. Advantageously, in particular the second beam splitter is followed by an already mentioned second optical element, which is set up to divergently radiate light emitted from the second beam splitter (with predominantly or exclusively the first beam splitter)

Hauptpolarisationsrichtung) to collimate, and divergent emitted from the second beam splitter light (with predominantly or exclusively the second

Main polarization direction). In particular, this second optical element may be the tube lens of the scanning microscope.

In particular, correspondingly collimated light can be radiated into an objective, wherein the angle at which this collimated light strikes a rear objective pupil can be changed by means of a raster unit. This way a can

Raster illumination and sample manipulation are performed. By focusing the collimated light beam from the second beam splitter in the rear lens pupil, for example, an evanescent lighting can be realized by the

Raster unit the lateral focus position in the rear lens pupil is affected. As a result, the exit angle of the light beam collimated by the objective is influenced, and thus the angle of incidence of the light beam on the sample or the interface between immersion medium or cover glass and sample. Thus, the present invention is particularly advantageous in connection with an optical scanning microscope with an attachable in a lens mount and positionable in a lens position lens with a rear lens pupil, wherein the second optical element is adapted to the light with the second

Main polarization direction to focus in a plane of the rear lens pupil. The light having the first main polarization direction collimated by the second optical element, on the other hand, passes in collimated form through the rear lens pupil.

As also mentioned above, by the light source section, simultaneous provision of light having the first and second main polarization directions or provision of light having the first main polarization direction in a first period and light having the second main polarization direction having a second period

be made. In the latter case, preferably a fast switching between light with the first main polarization direction and light with the second takes place

Hauptpolarisationsrichtung by the light source section used.

For a corresponding fast switching between light with the first

Hauptpolarisationsrichtung and light with the second Hauptpolarisationsrichtung is advantageously used a switchable delay element. This switchable delay element, for example, one in the beam path of the

Light source section einschwenkbare or rotatably arranged in the beam path

Delay plate, for example, a λ / 2 plate or more corresponding plates include. However, electro-optical systems and / or acousto-optical systems and / or systems realized on the basis of liquid crystals may also be used with particular advantage as delay elements. Corresponding elements for providing polarized light are known in principle from the prior art, so that reference may be made to relevant specialist literature.

On the other hand, as is also possible in principle, light is emitted by the light source section simultaneously with two different main polarization directions, for example by using light of two differently polarized lasers and merging in the first illumination light beam or by insufficient polarization of the light along a main polarization direction z. B. by inadequacies of the polarization-optical components, instead of a corresponding

Delay element or in addition to a fast closure may be provided in one or both of the channels. Whenever light comes with a first

Hauptpolarisationsrichtung from the first channel is required, in this case the second channel is blocked with a corresponding shutter and vice versa. Corresponding fast closures may be in the form of, for example, synchronized rotating diaphragms or aperture segments, irises, fast LCD devices, electro-optical

Be formed elements and / or acousto-optic elements. In a scanning optical microscope according to a particularly preferred embodiment of the invention, optical elements are provided along the first channel, which are formed and arranged to form a Galilean telescope, which is one or more real or virtual deflection points of the raster device in or close the lens pupil images. A "real or virtual deflection point" is used to denote a point which, on a corresponding grid unit, deflects light of a light source in a spatially limited manner ("punctiform") and thus provides a scanning light beam. Real are corresponding deflection points when they are formed by actual elements, for example mirrors, "virtual" deflection points are representations of corresponding elements in space.

With particular advantage, the illumination system of the present invention is designed removable, wherein in particular the first and the second beam splitter and the first and the second channel, which is formed between the first and the second beam splitter, are arranged on a withdrawable from the scanning microscope insert.

Advantageously, the optical scanning microscope has means for adaptation as a submodule to a wide field microscope.

The present invention also extends to a method of examining a sample by means of a scanning optical microscope comprising an illumination system comprising a light source section, first and second polarization-dependent beam splitters, and first and second optical channels between the first and second beam splitters.

The method according to the invention provides, using the light source section, a first illumination light beam with light of a first and a second one

Hauptpolarisationsrichtung radiate, using the first beam splitter to guide the light of the first main polarization at least predominantly in the first and the light of the second Hauptpolarisationsrichtung at least predominantly in the second channel, using the second beam splitter of light of the first

Hauptpolarisationsrichtung from the first and from the second light

Hauptpolarisationsrichtung from the second channel to form a second illumination light beam, and to emit the light with the first polarization from using the first channel and the light with the second polarization using the second channel with different convergence angles.

In particular, in a corresponding method, a scanning microscope can be used, as previously described and explained in detail. For features and advantages of a corresponding method, reference is expressly made to the above explanations.

Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawings.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention. The invention is illustrated schematically with reference to an embodiment in the drawing and will be described below with reference to the drawing.

figure description

Figure 1 shows a beam path of a scanning microscope with a

Illumination system according to an embodiment of the invention in a simplified, schematic representation. Figure 2 shows a beam path of a scanning microscope with a

Illumination system according to an embodiment of the invention in a simplified, schematic representation.

Figure 3 shows a beam path of a scanning microscope with a

In the figures, corresponding elements with identical reference numerals are given. A repeated explanation is omitted for clarity.

Detailed description of the drawings

In the figures 1 to 3 are each beam paths of a scanning microscope with a

Lighting system according to an embodiment of the invention in a simplified schematic representation shown. The illustrated in Figures 1 to 3

Embodiments include a plurality of common elements, which will be explained below with reference first to FIG. The explanations also apply to the other figures. A scanning microscope illustrated in FIG. 1, which here illustrates a highly schematic illustration, which is shown in dot-dashed lines, and designated by 1, comprises an objective 12 received in an objective receptacle 11 at a lens position 10.

Lens shots of different types can be provided, for example

Objective revolvers, linear objective change mechanisms and the like. An objective pupil of the objective 12 is designated by 109. An illumination system of a corresponding scanning microscope is denoted overall by 20. An orthoscopic beam path is illustrated by 200, a conoscopic beam path by 201 (dashed). The orthoscopic beam path 200 and the conoscopic beam path 201 run over certain distances of the

Lighting system 20 and a beam path realized therein together, in a first optical channel 21 and a second optical channel 22, however, separated from each other. The first channel 21 and the second channel 22 are each formed between a first polarization-dependent beam splitter 105a and a second polarization-dependent beam splitter 105b.

For switching between the orthoscopic and the conoscopic beam path 201, two, for example mutually orthogonal, main polarization directions of an illumination light beam provided by a light source 100 are used. For this purpose, a corresponding light source 100 can be used, which already provides correspondingly polarized illumination light. The light source 100 may be

For example, be a laser light source, a polarization-maintaining optical fiber or a suitable polarization element, or a light source for unpolarized light with a corresponding polarization element. Switching the Hauptpolarisationsrichtung one by a corresponding

Light source 100 provided illumination light beam can be realized by different arrangements, which are greatly simplified here with 102 illustrated. For example, a corresponding arrangement 102 may comprise a fast mechanically switchable delay element (for example a λ / 2 plate), which is preferably arranged at a location of the beam path or the illumination light beam with a small beam diameter and a constant beam position. For example, a corresponding delay element or a corresponding arrangement 102 between a

Fiber collimator and in front of a beam expansion system, as illustrated here in a simplified manner at 101.

Further possibilities for switching between the main polarization directions by means of a corresponding arrangement 102 are, for example, acousto-optic, electro-optical or liquid-crystal elements, as are known in principle from the prior art. In the embodiment of the present invention illustrated in FIG. 1, a light source section formed from the light source 100, the arrangement 102, the beam expanding system 101 and a scanning device 103 always provides either first or first light a second Hauptpolarisationsrichtung available. Deviating embodiments are explained, for example, with reference to FIG.

A scanning device or scanning unit 103 is formed in a manner known per se, this includes, for example, tiltable mirrors, rotatable prisms and / or

acousto-optical means as they are also known in principle from the prior art. Overall, by the light source section 100 to 103 thus a

Illuminating light beam, here illustrated with 203, is provided which optionally has a first main polarization direction or a second main polarization direction and in the example shown is emitted in collimated form by the grid unit 103.

The illumination light beam 203 then passes through a first optical element 104 of one or more lenses and is focused in this way. Of the

Illuminating light beam 203 having the first or the second main polarization direction focusses into the first polarization-dependent beam splitter 105a. At a boundary layer of the first polarization-dependent beam splitter 105a, the light of the second main polarization direction, as illustrated here in the form of the dashed conoscopic beam path 201, is reflected, while light of the first main polarization direction passes through the boundary layer without being deflected.

In this way, the first polarization-dependent beam splitter 105a guides the light with the first main polarization direction into the first optical channel 21 and the light with the second main polarization direction into the second channel 22. Beyond a respective focal point in the first channel 21 and the second channel 22, respectively the light with the first and the second polarization respectively divergent. In the illustrated example, the light having the first main polarization direction in the first channel 21, as illustrated here with the orthoscopic beam path 200, diverges from the first channel and enters the second polarization-dependent beam splitter 105b. By means of a closure 110 it is possible to prevent false light from entering the second polarization-dependent beam splitter.

In the first channel 21, no further optical elements are provided in the embodiment illustrated in FIG. In the second optical channel 22, however, further optical elements 106a and 106b are provided. The light in the second channel 22 extending with the second polarization is further deflected via deflecting elements 107a and 107b. By means of the optical elements 106a and 106b, the light with the second main polarization direction in the second channel 22 collimated and enters in collimated form in the second polarization-dependent beam splitter 105b.

Accordingly, the light having the first main polarization direction out of the first channel 21 diverges therefrom after passing through the second polarization-dependent beam splitter 105b, the light having the second main polarization direction emerges from the second channel 22 after passing through the second channel and the second

polarization-dependent beam splitter 105b, however, collapses from this. By using a second optical element 108, for example a tube lens, the collimated light with the second main polarization direction can be focused from the second optical channel, whereas the light with the first main polarization direction from the first optical channel can be collimated. The second optical element 108 forms a Bertrand lens with the already explained optical elements 106a and 106b. By the formation of the first optical channel 21 and the second optical channel 22 and the arrangement of the illustrated lenses, the light having the first main polarization direction in collimated form can enter the rear lens pupil 109 of the lens 12, whereas the light having the second main polarization direction can enter the rear lens pupil 109 of the lens 12 are focused. The second optical element 108, together with the first optical element 104, forms a Galilean telescope, which surrounds the one or more through the

Scanning device 103 conditional real or virtual deflection points of the

Illuminating light beam into or near the objective pupil 109.

A detection of fluorescent light of a sample, which is arranged in front of the objective 12, can be carried out by a conventional wide field fluorescence microscope. The

Illumination system 20 of the embodiments discussed herein may be coupled in such a wide field fluorescence microscope in the fluorescence illumination beam path at a suitable location and be formed removable. It is particularly advantageous if the Bertrand lens system formed by the optical elements 106a, 106b and 108 has a variable focus, which allows focusing at different mechanical positions of the objective pupil 109, for example when using different objectives or a turret focusing. It is particularly advantageous that the second optical element 108, which can be formed, for example, in the form of a tube lens, can be kept constant in a corresponding focusable Bertrand lens system and therefore does not have to be removed even when changing the illumination system 20, and the afocalism of the Galileo telescope consisting of elements 104 and 108 is preserved. The embodiment of the scanning microscope 2 illustrated in Figure 2 differs from the embodiment illustrated in Figure 1 by the absence of the shutter 110. As mentioned, in the embodiment of the scanning microscope 1 illustrated in Figure 1, this allows blanking due to non-ideal

Light polarization. If this is not necessary because sufficient light is already provided by a light source section 100 to 103, this additional closure 110 can be dispensed with, which makes possible a simpler mechanical design of the microscope 2. In the embodiment of the scanning microscope 3 illustrated in FIG. 3, no variable delay element or a corresponding arrangement 102, as is present in the embodiments illustrated in FIGS. 1 and 2, is provided. Instead, both the first channel 21 and the second channel 22 are fast

Shutter members 110 and 111 are provided. The light source section 100 to 103 and the light source 100 in this case, for example, illumination light of a fixed

Polarization state ready, the light linear combination of two orthogonal

Represents main polarization directions. Therefore, also by means of the first

polarization-dependent beam splitter 105a illumination light in both channels 21 and 22 directed. As a result of the alternating use of the closures 110 and 111 of the two channels 21 and 22, a beam path can now be selectively blocked and in this way light can pass through the only one channel to the objective 12. Of course, it is also possible to provide light by none or both channels 21 and 22 by appropriate control of the closures 110 and 111.

LIST OF REFERENCE NUMBERS

1, 2, 3 Scanning microscope

10 lens position

11 Lens mount

12 lens

20 lighting system

21 first optical channel

22 second optical channel

100 light source

101 beam expansion system

102 delay element

103 Raster unit

104 first optical element

105a, 105b polarization-dependent beam splitter 106a, 106b further optical elements

107a, 107b beam deflecting elements

108 second optical element

109 entrance pupil lens

110 closure

111 closure

200 orthoscopic beam path

201 conoscopic beam path 203 first illumination light beam 204 second illumination light beam

Claims

claims

An optical scanning microscope (1, 2, 3) comprising an illumination system (20) with a light source section (100-103) emanating from a light source (100), a first polarization-dependent beam splitter (105a) and a second polarization-dependent beam splitter (105a, 105b) and a first and a second optical channel (21, 22) between the first and the second polarization-dependent beam splitter (105a, 105b), wherein

- The light source section (100-103) is adapted to a first

Illuminating light beam (203) with light of a first and a second

To radiate main polarization direction,

- The first beam splitter (105 a) is adapted to the light of the first

Hauptpolarisationsrichtung at least predominantly in the first (21) and the light of the second Hauptpolarisationsrichtung at least predominantly in the second channel (22) to lead

- The second beam splitter (105b) is adapted to light of the first

Hauptpolarisationsrichtung from the first (21) and light of the second

Hauptpolarisationsrichtung from the second channel (22) has a second

Illumination light beam (204) to form, and

- The channels (21, 22) are adapted to the light of the first

Hauptpolarisationsrichtung from the first (21) and the light of the second

Hauptpolarisationsrichtung from the second channel (22) with different convergence angles to emit.

An optical scanning microscope (1, 2, 3) according to claim 1, wherein the

Light source section (100-103) is adapted to radiate collimated the first illumination light beam (203). An optical scanning microscope (1, 2, 3) according to claim 2, wherein the first

A beam splitter (105a) is preceded by a first optical element (104), which is adapted to focus the illumination light beam (203) and to radiate convergently into the first beam splitter (105a).

An optical scanning microscope (1, 2, 3) according to claim 3, wherein the first channel (21) is adapted to irradiate light at least predominantly divergently into the second beam splitter (105b), and wherein the second channel (22) is arranged thereto To irradiate light at least predominantly collimated into the second beam splitter (105b).

An optical scanning microscope (1, 2, 3) according to claim 4, wherein the second beam splitter (105b) is followed by a second optical element (108) arranged to collimate and collimate divergently light emitted from the second beam splitter (105b) to focus light emitted from the second beam splitter (105b).

Optical scanning microscope (1, 2, 3) according to claim 5, with one in one

Lens mount (1 1) attachable and in a lens position (10) positionable lens (12) having a rear Objektivpupille (109), wherein the second optical element (104) is arranged to collimated from the second beam splitter (105b)

Focusing light to focus in a plane of the rear lens pupil (109).

Optical scanning microscope (1, 2, 3) according to one of the preceding claims, in which at least part of a Bertrand lens system (106a, 106b) is provided in the second channel (22).

Optical scanning microscope (1, 2, 3) according to one of the preceding claims, wherein an orthoscopic beam path (200) is formed by the light of the first main polarization direction and a conoscopic beam path (201) is formed in the illumination system (20) by the light of the second main polarization direction ,

An optical scanning microscope (1, 2, 3) according to any one of the preceding claims, wherein the light source section (100-103) is adapted to the first

Illuminating light beam (203) in a first period of time with light of the first

Hauptpolarisationsrichtung and in a second period of time to emit light with the second Hauptpolarisationsrichtung.

10. A scanning optical microscope (1, 2, 3) according to claim 9, wherein for emitting the illumination light beam (203) with light having the first Hauptpolarisationsrichtung in the first period and light with the second Hauptpolarisationsrichtung in a second period, a switchable delay element (102 ).

1 1. An optical scanning microscope (1, 2, 3) according to any one of claims 1 to 8, wherein the light source section (100-103) is adapted to irradiate the first illumination light beam (203) simultaneously with light of the first and second main polarization directions.

12. Scanning optical microscope (1, 2, 3) according to any one of the preceding claims, wherein the illumination unit (20) comprises a raster unit (103).

13. A scanning optical microscope (1, 2, 3) according to any one of the preceding claims, wherein along the first channel (21) optical elements (104, 108) are provided, which are formed and arranged to form a Galilean telescope, which images one or more real or virtual deflection points of the rasterizer (103) into or close to the objective pupil (109). 14. An optical scanning microscope (1, 2, 3) according to any one of the preceding claims, wherein in the first channel (21) and / or in the second channel (22) an optical

Closure is arranged.

Optical scanning microscope (1, 2, 3) according to one of the preceding claims, wherein the first and the second beam splitter (105a, 105b) and the first and the second

Channel (21, 22) are arranged on a withdrawable from the scanning microscope (1, 2, 3) insert.

16. An optical scanning microscope (1, 2, 3) according to any one of the preceding claims, which

Has means for adaptation as a sub-module to a wide-field microscope.

17. A method for examining a sample by means of an optical

Scanning microscope (1, 2, 3) having a lighting system (20) with a

Light source section (100-103), a first and a second

polarization-dependent beam splitter (105a, 105b) and a first and a second optical channel (21, 22) between the first and the second beam splitter (105a, 105b), wherein - Using the light source section (100-103) a first

Illuminating light beam (203) with light of a first and a second

Main polarization direction is emitted,

- Using the first beam splitter (105 a), the light of the first

Hauptpolarisationsrichtung at least predominantly in the first (21) and the light of the second Hauptpolarisationsrichtung at least predominantly in the second channel (22) is guided, using the second beam splitter (105b) of light of the first

Hauptpolarisationsrichtung from the first (21) and light of the second

Hauptpolarisationsrichtung from the second channel (22) a second

Illuminating light beam (204) is formed, and

- The light with the first polarization of using the first channel (21) and the light with the second polarization using the second channel (22) is emitted with different convergence angles. 18. The method according to claim 17, wherein a scanning microscope (1, 2, 3) according to one of claims 1 to 16 is used.