WO2008037346A1

WO2008037346A1 - Laser scanning microscope with element for pupil manipulation

Info

Publication number: WO2008037346A1
Application number: PCT/EP2007/007881
Authority: WO
Inventors: Matthias Wald
Original assignee: Carl Zeiss Microlmaging Gmbh
Priority date: 2006-09-27
Filing date: 2007-09-10
Publication date: 2008-04-03
Also published as: DE102006045839A1; DE102006045839B4

Abstract

Disclosed is a light raster microscope with a lens (16) for test imaging, the lens (16) having a pupillary lens (48),and with a scanning device (12) downstream of the lens (16) in the imaging direction for biaxial beam deflection, wherein the scanning device (12) has a first and a second scanning element, each of which is a uniaxial beam deflection scanner, which are placed one behind the other in the imaging direction, and with an element (43) for pupil manipulation (40, 41) arranged in a position relative to the pupillary lens (48), wherein the first scanning element (40) is positioned in the pupillary lens (48) or a pupil conjugated thereto. A relay optics system (42) is arranged downstream of the first scanning element (40) for imaging the pupil (48) containing the first scanning element (40) onto a second pupil (49). The element (43) for pupil manipulation is positioned in the second pupil (49), generated by the relay optics system (42), and is reflective such that the relay optics system (42) is traversed again in the imaging direction and again images the second pupil (49) onto a third pupil (50). The relay optics system (42) comprises a beam deflector (47) which separates the third pupil (50) from the pupil containing the first scanning element (40), and the second scanning element (41) is arranged in the third pupil (50).

Description

Carl Zeiss Microlmaging GmbH September 10, 2007

Lawyer's file: PAT 4024/009-PCT K / 22 / dw

Laser scannin microscope with element for pupil manipulation

The invention relates to a light-scanning microscope with a lens for sample imaging, wherein the lens has an objective pupil, a scanning device for biaxial beam deflection downstream of the objective in the imaging direction, wherein the scanning device has a first and a second, each uniaxially deflecting beam element which in Imaging direction behind each other, and a pupil manipulation element, which is arranged in a position associated with the objective pupil.

Laser scanning microscopes are known in the art. Reference is made, for example, to DE 197 02753 A1 or DE 10257237 A1, both of which describe a light scanning microscope designed as a laser scanning microscope. In this context, it should be noted that the term "light" here is understood to mean the entire region of the electromagnetic radiation which obeys the laws of optics.

Scanning microscopes or laser scanning microscopes usually acquire an object image by scanning the object with a spot or multispot arrangement. The radiation recorded in the spot or multi-spot areas is detected with the highest possible depth resolution so that no structure of the spot or the multi-spot is resolved, for example by a so-called confocal detection. Moving the spot or multispot area over the object then delivers the picture. Thus, only radiation information for the respective spot or multi-spot area is always present at the detector, and electronic joining of this image information to the individual points of the image (corresponding to the spot / multi-spot areas) taking into account the shift of the spot or multi-spot areas results in the desired image. Confocal detection is a common way to achieve a very high depth resolution. The signal evaluation is then restricted substantially to the focal plane, since areas lying outside the focal plane do not provide any significant signal information in the case of confocal detection; they are imaged in front of or behind the confocal aperture. To capture the image, it is therefore important in a laser scanning microscope to deflect the light beam focused on a spot or point group (multispot) over the object. It is known to use a scanning device that deflects the beam biaxially. Commonly used are scanning devices that have two scanning elements, which deflect the beam uniaxially adjustable. Examples of scanning elements used in scanning microscopes are galvanometer mirrors or acoustically optical modulators. The deflection with the two scanning elements is conveniently carried out around mutually orthogonal axes. Based on the recorded image, it is therefore assumed that one scan element causes the deflection in the line direction, the other element in the image direction perpendicular thereto (the term "column direction" would also be conceivable).

The deflection of the light beam or light beam should ideally take place from one point, but this actually requires a single, then biaxial scan element. However, the use of two scanning elements is much less expensive and also allows a faster adjustment of the deflection. It is therefore common in the art to arrange two scan elements as close to the pupil plane of the microscope objective. This has the further advantage that the two scanning elements can be kept small without fear of shading effects. Further, the problem otherwise encountered with adjustable mirrors is avoided, depending on the deflection angle, i. the mirror position, the light path by the stroke of the mirror deflection is different lengths, which can lead to defocusing effects. In conventional laser scanning microscopes, as marketed for example by Carl Zeiss Microlmaging GmbH, Germany, under the trademark ZEISS, therefore, two galvanometer mirrors are arranged as close as possible to each other before or after the pupil of the microscope objective or a pupil conjugated thereto. The term "before" or "after" here and in the following text refers to the microscope beam path in the imaging direction, i. against the direction of illumination.

In microscopy, various microscopy methods are known in which a pupil manipulation occurs. The best known method is probably the dark field method, in which the main maximum of the Fourier transforms is hidden in the pupil plane. Other known methods in which manipulation of the microscope's current radiation are VAREL methods, methods for extended depth of field (Toraldo principle, logarithmic phase masks, cubic phase masks), methods for aberration measurement (Ronchi test), phase masks for Correction of residual aberrations or for fast focusing. In laser scanning microscopy, however, pupil manipulation is difficult. In the conventional realization of laser scanning microscopes, the two scanning elements are both not exactly in the objective pupil. Therefore, each pupil that represents an image of the scanner pupil travels laterally when scanning against the objective pupil. It is therefore limited in elements for pupil manipulation on designs or manipulation operations that are insensitive to this shift. Theoretically, it would also be conceivable to use a controllable element which is changed in time with the control of the scanning elements in order to take account of the displacement.

The invention is based on the object, a light-scanning microscope of the type mentioned in such a way that the possibilities for pupil manipulation are extended and at the same time a pupil manipulation can be performed easily.

The invention solves this problem by a light-scanning microscope of the type mentioned, in which the first scanning element in the objective pupil or a pupil conjugate thereto, the first scanning element downstream of a relay optics for imaging the pupil contained in the first scan element in a second pupil is, the element for pupil manipulation in the second pupil generated by the relay optics and is formed reflective, so that the relay optics is traversed again in the imaging direction and these images the second pupil again into a third pupil, wherein the relay optics a beam deflector, so that the third pupil is spatially separated from the pupil plane containing the first scanning element, and the second scanning element is arranged in the third pupil.

Thus, according to the invention, a relay optic for pupil imaging is used, which is integrated in the beam path after the objective pupil or a conjugated pupil produced therefrom by intermediate imaging, so that three mutually associated pupils are formed. This is possible only because of the special optical effect of the scanning elements, since each scanning element generates a field in only one dimension due to the uniaxial deflection. In two of the pupils, the scanning elements are positioned so that the third pupil is automatically fixed relative to the scanning elements. In this third pupil, the pupil manipulation element is placed. It is further ensured that one of the three pupils coincides with the objective pupil or a pupil conjugated thereto. Thus, the first scanning element is located in the objective pupil (or a pupil conjugated thereto), the pupil manipulation element is in a second pupil obtained by imaging the pupil with the first scanning element, and this is again imaged into a third pupil in which the pupil second scanning element is arranged. - A -

In order to keep the optical structure as simple as possible, the relay optics is provided with a beam deflector and designed so that it is traversed twice in the imaging direction. From the first scanning element, the relay optics effects an image of the pupil with this scanning element on the second pupil. Since, according to the invention, a reflective element for pupil manipulation is arranged there, the relay optics, after reflection at the pupil manipulation element, produce a further pupil image on the third pupil which, due to the effect of the beam deflector, does not coincide with that of the pupil, in which the first scan element stands. The relay optics thus causes a double pupil image: once in the direction of the element for pupil manipulation and the other time away from it in the direction of the second scanning element.

This design of the relay optics allows a very compact design. Forming the beam deflector as a beam splitter or prism, resulting in a total of a T-shaped beam path between the scanning elements. Such a construction is particularly space-saving and easy to adjust.

The element for pupil manipulation can now be chosen almost arbitrarily according to the desired microscopy method. It is particularly preferred to use an element which is adjustable by an electrical control signal. If one does not want to manipulate the pupil, the element is switched to a mode in which it reflects the beam path without any further manipulation. One possible construction for such an element is a spatial light modulator or adaptive mirror. Also, a DMD array can be used.

The structure according to the invention has the following advantages:

1. By means of the relay optics according to the invention, three pupils are created, which are in fixedly assigned positions. A pupil picks up the first scan element. The second pupil contains the element for pupil manipulation. The third pupil is provided for the second scanning element. The three pupils are conjugate to each other, so that an optimal effect of the element for pupil manipulation is achieved and there is no longer any restriction to certain elements.

2. The arrangement according to the invention makes it possible to arrange the first scanning element in a pupil conjugate to the objective pupil or in the objective pupil itself. As a result of the fixed assignment of the two other pupils mentioned, a fixed position of the objective pupil to the pupil with the pupil manipulation element is secured overall. A lateral Displacement of the beam path during scanning does not take place on the pupil manipulation element.

3. The design of the relay optics with a beam deflector and the use of a reflective element for pupil manipulation causes the relay optics in both

Direction of the element for pupil manipulation as well as from this away makes a pupil image. It will therefore pass twice. That for two pupil images, only one set of optical components is needed. This leads to a compact and otherwise cost-effective design.

4. Regardless of the effect of the element for pupil manipulation, the two scanning elements are always exactly in the objective pupil or a pupil conjugated thereto. Thus, negative effects that may occur in scan elements that are outside the pupil plane, avoided even without pupil manipulation operation in the microscope according to the invention. In particular, the light path is not extended as a function of the deflection by impinging the light beam outside the deflection axis of the respective uniaxially deflecting scanning element. On the contrary, it is now ensured that the illumination and the imaging beam always strike the scanning element exactly on the deflection axis, regardless of the position of the other scanning element.

The invention will be explained in more detail by way of example with reference to the drawings. In the drawings shows:

FIG. 1 shows a schematic representation of a light-scanning microscope,

Figure 2 is an enlarged view of a scanning device of the light scanning microscope and

Figures 3 to 5 the beam path of the scanning device of Figure 2 with the corresponding optical elements in three different views.

FIG. 1 shows schematically a light scanning microscope designed as a laser scanning microscope (LSM) 1. With the LSM 1, an object 2 is measured in a manner yet to be explained. The LSM 1 is essentially subdivided into a microscope module 3, a detection module 4 and a lighting or excitation module 5. The excitation module provides excitation radiation and feeds it into the microscope module 3, so that it is directed to the object 2 as spot-shaped illumination. The spot-shaped illumination is guided by the microscope module 3 raster over the object 2. The spot area illuminated with illumination radiation from the illumination module 5 on the object is transmitted via the microscope module 3 from the Detection module 4 detected confocally. If the illumination is designed as excitation radiation, an image of the fluorescence properties of the object 2 can be obtained.

The illumination or excitation module 5 has for illumination light sources 6 and 7, which may be formed for example as a laser. The radiation from the light sources 6 and 7 is conducted via a deflecting mirror 8 or a beam splitter 9 and an illumination optical unit 10 to a beam splitter 11, referred to as a main color splitter, where it is coupled into the microscope module 3. The specific embodiment of the coupling of the radiation, realized in the present embodiment by deflecting mirror 8, beam splitter 9 and illumination optics 10 is for the following invention without further meaning. Other constructions are also possible, for example by means of fiber optics and suitable fiber optic couplers. Also, deviating from the two light sources shown in Figure 1, of course, only a single light source or a larger number of light sources can be used. Essential to the invention here is only that at the main color divider 11, an illumination beam 17 is coupled.

The main color splitter 11 may be constructed, for example, as in the already mentioned DE 197 02 753 A1. Instead of the dichroic main color splitter described therein, a color-neutral splitter can also be used, as it is e.g. is described in DE 10257237 A1.

The radiation arriving from the illumination or excitation module 5 on the main color splitter 11 in the form of the illumination beam 17 is then focused onto or into the object 2 by means of a scanning device 12 and a scanning optics 13 through a tube lens 15 and an objective 16. The focusing is carried out in the embodiment in a diffraction-limited focus whose position in the object 2 along the optical axis by an adjustable sample table 18 can be adjusted. The scanning device 12 deflects the coming of the main color splitter 11 illumination beam 17 biaxially, so that this falls differently deflected beam 19 through the tube lens 15 and the lens 16 on or in the object 2, thus in the object 2 at different locations transverse to the optical axis is focused.

For observation of the object 2 for a microscope user, a deflecting mirror 14 designed as a beam splitter is optionally provided between the scanning optics 13 and the tube lens 15, which optionally permits a visual inspection.

By means of the scanning device 12, which, like the sample table 18, is also controlled by a control unit 26 via lines not designated or not shown, the focus of the deflected illumination beam 19 is placed at different locations in the object by the objective 16. Overall, there is a three-dimensional positioning. The deflection by the scanning device 12 causes the recording of a two-dimensional image whose Depth position in the object 2 as a third dimension by the setting of the sample table 18, that is, the position of the focal plane in the object 2 is determined. Alternatively, a focus adjustment by adjusting the lens 16 is possible.

The radiation generated in object 2, e.g. Fluorescence radiation is detected by imaging the focus in the object 2 by means of the objective 16 and the tube lens 15 as well as the scanning optics 13 into the detection module 4 for each point of the image to be recorded. For this purpose, the main color splitter 11 guides the beam 21, which, after passing through the scanning device 12, is again stationary in the imaging direction to the detection module 4, which has confocal detector elements. Confocal filtering of the radiation from the focus in the object 2 takes place via an output coupler 22 and a pinhole optics 23 at a pinhole 24. The plane of the confocal diaphragm 24 is conjugate to the focal plane in the object 2. A detector 25 picks up the confocal filtered radiation. He is also connected to the controller 26. The control unit 26 thus generates for each position of the focus in the object 2 a corresponding pixel, which is characterized by the intensity information from the detector 25 and its position in the image by the position of the scanning device 12. Depending on the embodiment, the detection module 4 can have a plurality of spectral channels. There are then the corresponding elements 22 to 25 multiple times. In Figure 1, this is symbolized schematically by a second detection channel, the reference numerals are provided with an apostrophe. A dot-dash line indicates that even more detection channels are possible. The output couplers 22 and 22 'thus act as so-called secondary color splitter. Its spectral characteristic determines which spectral range has the radiation detected by the associated detector. The construction of the detection module 4 is of no further interest for the invention described here, in particular it does not determine whether or how confocal filtering takes place. It is only essential for the invention here that after the scanning device 12, a stationary beam 21 is present, which is referred to in the literature as a de-scanned beam.

The scanning device 12 ensures that the beam is deflected biaxially to the deflected beam 19, whereby the focus of the illumination radiation in the object 2 is perpendicular to the

Direction of incidence of the illumination radiation or to the imaging direction lying field sweeps. This is called scanning. In reverse (imaging) direction provides the

Scanning device for the fact that two-dimensionally distributed pixels are time-sequentially imaged onto a non-spatially resolving detector. This is called de-scanning.

Since dot-shaped illumination is of no further concern to this illustration, and therefore of no further importance to the present invention, which is primarily dedicated to scanning device 12 is (it also comes as a wide-field lighting in question), the scanning device 12 is considered below in the imaging direction.

The scanning device 12 is shown schematically in more detail in FIG. It consists of two scanning mirrors 40 and 41, which in this embodiment are examples of uniaxially deflecting scanning elements. Each scanning mirror deflects about a deflection axis, wherein the deflection axes of the scanning mirrors 40 and 41 are preferably orthogonal to one another. In principle, however, any inclination is sufficient to be able to cover a two-dimensional field with the focus.

In the imaging direction, i. 2 comes on the deflected beam 19 from the left, acts in the scanning device 12, a first scanning mirror 40 and this following a second scanning mirror 41 so that after the second scanning mirror 41 of the stationary beam 21 is given. Between the scan mirrors 40 and 41 there is a pupil manipulation unit 42, which is shown only schematically in FIG.

FIGS. 3-5 show the scanning device 12 with the pupil manipulation unit 42 in detail with its beam path. FIG. 4 shows the structure of FIG. 3 in this case from the direction designated A. Finally, FIG. 5 shows the same beam path from the direction designated B. The description follows the de-scanning of the biaxially deflected beam 19 to the stationary beam 21.

The biaxially deflected beam 19 first falls on the first scanning mirror 40, whose uniaxial motion thus uniaxially de-scans. The first scanning mirror 40 is located in a pupil 48 of the objective 16, which is shown schematically in FIG. 3 by a dashed line.

The originally biaxially deflected, i. a two-dimensional field sweeping deflected beam 19 is after the scanning mirror 40 only in one spatial direction, ie line-shaped, deflected. It then falls on a prism 47, which is part of the pupil manipulation unit 42. From the prism 47, the beam passes through a first optical group 44 and a second optical group 45, which together form the pupil 48, in which the first scanning mirror 40 is located, in a second pupil 49. In this second pupil 49, a reflective element for pupil manipulation is arranged. In the exemplary embodiment, this is an adaptive mirror 43, which is controlled by the control unit 26.

Due to the reflection at the adaptive mirror 43, the second pupil 49 becomes the prism 47 again through the second optical group 45 and the first optical group 44 and from there into a third one Pupil 50 shown. In this is the second scanning mirror 41. The objective pupil 48, the second pupil 49 and the third pupil 50 are thus conjugate to each other, so that the first scanning mirror 40, the adaptive mirror 43 and the second scanning mirror 41 are in mutually conjugate layers. The second scanning mirror 41 then causes a de-scanning about the remaining axis, so that thereafter the stationary beam 21 is present.

In the illumination direction, on the other hand, the stationary beam 21, which is generated by the illumination beam 17 in this approach, is first uniaxially deflected by the second scanning mirror 41, guided by the prism 47 through the first and second optical groups 44, 45 toward the adaptive mirror 43 depending on the setting pupil manipulating reflected so that it passes through the second optical group and the first optical group 45, 44 and the prism 47 to the first scanning mirror 40, which makes a biaxial deflection from the hitherto existing uniaxial deflection and the deflected beam 19 provides.

The pupil manipulation unit 42 comprises the prism 47, the first optical group 44 and the second optical group 45, between which an intermediate image 46 is formed. The prism 47 guides (in the imaging direction) the radiation into the first optical group 44 and the second optical group 45 so at an offset to the optical axis of the optical groups 44 and 45, that after reflection on the adaptive mirror 43 of the received beam from the prism 47 is not in the direction the objective pupil 48 is reflected, but reaches the third pupil 50. The prism 47 is designed here as a roof prism. In principle, however, a single mirror surface is sufficient so that only one deflection by the prism 47 has to be carried out. The beam path would then continue without deflection so that the position of the third pupil 50 would be to the left of the pupil manipulation unit (as viewed in the direction of the adaptive mirror).

The construction shown in Figure 3 and in Figures 4 and 5 has the advantage that the first and second scanning mirrors 41, 41 lie on a common optical axis, i. the optical axis of the deflected beam 19 and the stationary beam 21 can coincide. This facilitates the adjustment of the arrangement.

Claims

Carl Zeiss Microlmaging GmbH September 10, 2007Personal Law: PAT 4024/009-PCT K / 22 / dwAnsprüche

1. Scanning microscope with a lens (16) for sample imaging, the lens (16) having an objective pupil (48),

a scanning device (12) arranged downstream of the objective (16) in the imaging direction for biaxial beam deflection, wherein the scanning device (12) has a first and a second, respectively uniaxially deflecting scanning element, which lie one behind the other in the imaging direction,

- an element (43) for pupil manipulation (40, 41) arranged in a position associated with the objective pupil (48), characterized in that

the first scanning element (40) lies in the objective pupil (48) or a pupil conjugated thereto,

a relay optics (42) for imaging the pupil (48) containing the first scanning element (40) is arranged downstream of the first scanning element (40) in a second pupil (49),

the pupil manipulation element (43) lies in the second pupil (49) produced by the relay optic (42) and is designed to be reflective, so that the relay optics (42) are traversed again in the imaging direction and the second pupil () 49) again into a third pupil (50), the relay optic (42) having a beam deflector (47) which separates the third pupil (50) from the pupil containing the first scanning element (40), and

- The second scanning element (41) in the third pupil (50) is arranged.

2. Laser microscope according to claim 1, characterized in that the beam deflector is designed as a beam splitter or prism (47), between the first and second scanning element (40, 41) and deflects the beam path substantially at right angles, so that a total of T shaped beam path between the scanning elements (40, 41) is formed.

3. Microscope according to one of the above claims, characterized in that the element (43) is adjustable for pupil manipulation by an electrical control signal.

4. A microscope according to claim 3, characterized in that the element (43) for pupil manipulation is a spatial light modulator or an adaptive mirror.