Microscope device

There is provided a method for a microscope device for optionally examining a sample by at least a first light beam bundle from a first direction or a second light beam bundle from a second direction different to the first direction, having a microscope objective and a beam deflection element, wherein the beam deflection element is operable by a drive in order to optionally couple the first light beam bundle or the second light beam bundle into the microscope objective, and wherein the beam deflection element is rotatable by the drive in order to optionally change at least an exit direction of the first light beam bundle from the beam deflection element if the first light beam bundle is coupled into the microscope objective.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a microscope device which allows for examination of a sample in at least two different illumination modes.

2. Description of Related Art

Recent microscopic examination methods frequently require the use of different illumination methods and illumination modes and their combined utilization in a single device. According to WO 2004/077121 A1, for example, a “beam hub” concept is proposed, wherein beam multiplex elements which can be actuated allow for the selection of certain illumination beams from a plurality of possible illumination beams.

DE 102 33 549 A1 relates to a scanning microscope having a light source that generates an illuminating beam for illumination of a sample, which can be directed through or over the sample by a beam deflection device. A further light source generates a manipulation light beam which likewise can be directed through or over the sample by the beam deflection device.

It is a first object of the invention to provide for a microscope device for optional examination of a sample by at least a first light beam bundle and a second light beam bundle, wherein it is possible to switch in a simple manner between the first and the second light beam bundle for illumination and wherein further at least one geometric property of the selected light beam bundle should be adjustable in a simple manner in the object plane.

It is a further object of the invention to provide for a microscope device for examination of a sample, wherein at least two different geometric properties of a light beam bundle are adjustable in a simple manner in the object plane.

SUMMARY OF THE INVENTION

According to the invention, the first object is achieved by a microscope device in which a beam deflection element is operable by means of a drive in order to couple a selected one of a first light beam bundle and a second light beam bundle into the microscope objective, and in which the beam deflection element is rotatable by means of the drive in order to change at least an exit direction of the first light beam bundle from the beam deflection element when said first light beam bundle is coupled into the microscope objective. This solution is beneficial in that by actuating the drive of the beam deflection element one cannot only switch between the first and the second light beam bundle as the light source, i.e. the beam deflection element serves to selectively deflect one of the two beam bundles into the microscope objective, but simultaneously one also can change the exit direction of the selected light beam bundle from the beam deflection element in a selective manner in order to selectively adjust at least one geometric property of the light beam bundle in the object plane. Since for both functions only a single element, namely the beam deflection element, is required, a particularly simple construction of the microscope device can be achieved.

According to a preferred embodiment the beam deflection element is arranged in or close to a plane which is conjugate to the plane of the objective pupil, wherein then a rotation of the beam deflection element causes a shift of the position of the first light beam bundle in one dimension in the object plane without substantially changing the impingement direction of the first light beam bundle in the object plane and without resulting in a vignetting effect caused by shading effects in the objective pupil. Thus, by actuation of the beam deflection element an illumination pattern can be moved in a simple and selective manner in one direction over the specimen, and the sample can be scanned at least in one dimension. This is necessary if a FRAP (Fluorescence Recovery After Photo Bleaching) illumination—which is preferably realized by laser light—is used or if confocal layer images are to be taken by structured illumination or by scanning by a dot pattern or a bar pattern.

According to another embodiment the beam deflection element is arranged in or close to a plane conjugate to the object plane, wherein rotation of the beam deflection element causes a change of the angle of incidence of the first light beam bundle in the object plane, without essentially changing the point of impingement of the first light beam bundle in the object plane. Thus, by rotation of the beam deflection element, the angle of the excitation beam and hence the illumination angle on the sample can be changed in a selective manner, what is particularly beneficial in case that the light beam bundle is laser light for TIRF (Total Internal Reflection Fluorescence) illumination of the sample.

According to the invention, the second object is achieved by a microscope device in which a scanning device, via a first path and a second path, respectively, is imaged differently in such a manner that, upon coupling of a light beam bundle via the first path, deflection of the light beam bundle by means of a scanning device causes a shift of a position of the light beam bundle in one of an object plane and a plane close to the object plane, whereas, upon coupling of the light bean bundle via the second path, deflection of the light beam bundle by means of the scanning device causes a shift of a position of the light beam bundle in a plane of one of an objective pupil and a plane close to the plane of the objective pupil. According to this solution, the operable beam deflection element does not only serve to select a light beam bundle from a plurality of at least two light beam bundles and to adjust the exit angle of the selected beam, but it also allows to couple the selected beam via a first path or a second path into the microscope objective. The optical elements of these two paths differ in that in the first case the beam deflection element is imaged into the objective pupil whereas in the other case it is imaged into the object plane. Thus, actuation of the scanning device in the first case may cause a shift of the position of the light beam bundle in the object plane, without the beam moving in the objective pupil, whereas in the second case actuation of the scanning device causes a shift of the position of the light beam bundle in the plane of the objective pupil (what enables to change the illumination angle), without the illuminated field being changed thereby. According to the invention only a single light source and only a single scanning device is necessary for optionally adjusting different geometric properties of the light beam bundle in the objective plane in a selective manner, namely on the one hand the position in the object plane and on the other hand the angle of incidence in the object plane. Thereby a very flexible microscope device can be provided for in a particularly simple manner.

According to a preferred embodiment the light beam bundle serves as a FRAP illumination when it is coupled in via the first path, whereas the light beam bundle servers as a TIRF illumination when it is coupled in via the second path.

According to a further preferred embodiment the second light source may be provided for supplying a second light beam bundle which arrives from a direction different to that of the first light beam bundle on the beam deflection element, wherein the beam deflection element is operable for optionally deflecting the second light beam bundle towards the objective. Preferably the second light beam bundle is light for wide-field incident light illumination. A mask, through which the second light beam bundle passes, is located in a plane conjugate to the object plane between the second light source and the beam deflection element, with the image of the mask being shiftable in the sample plane to some extent by selective rotation of the beam deflection element. Thereby movable structured illumination of the sample can be realized.

These and further objects, features and advantages of the present invention will become apparent from the following description when taking in connection with the accompanying drawings which, for purposes of illustration only, show several embodiments in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FRAP measurements require the selective activation and deactivation, respectively, of proteins or other cell-active substances by a focused laser beam, and they require the capability to observe the course of the reaction of the cell in incident light fluorescence. FRAP measurements, like, for example, laser micro dissection methods or optical pincers, nowadays are included in the standard repertoire of advanced microscopy. The combining of the beam paths which serve for laser bleaching or laser excitation with those which are necessary for observation in incident light fluorescence usually occurs by means of beam splitters, which, however, either result in loss of light (if they are, for example, designed as semi-transparent mirrors) or, if dichroitic beam splitters are used, which restrict the free choice of the respective wave length.

In the embodiment of a microscope device according to the invention shown inFIG. 1to this end a quickly rotating beam deflection element10is provided which is driven by a drive12which is preferably a galvanometer scanner in order to optionally couple either 100% of a laser beam14or 100% of an illumination beam16necessary for incident light observation into the microscope18which comprises an object plane20including a sample22, a microscope objective24and a tube lens26, wherein the plane of the objective pupil is designated by28. InFIG. 1the beam deflection element10is designed as a planar mirror which is rotatable by the drive12around an axis30which is perpendicular to the paper plane between a position in which the laser beam14is deflected into the microscope18and a position in which the illumination beam16is deflected into the microscope18, with both positions shown inFIG. 1. The switching between the two positions can be effected by means of a galvanometer scanner in a few 100 msec, so that, for example, first the laser beam14is brought onto the sample22for FRAP excitation and immediately thereafter the sample22can be illuminated by the illumination beam16.

The beam deflection element10is located in a plane conjugate to the objective pupil plane28or close to such a plane, since in this case the rotation of the beam deflection element10around the rotation axis30can be utilized in order to selectively vary the exit direction of the laser beam14(and/or of the illumination beam16), whereby the position of the (focussed) laser beam14(or the illumination beam16, respectively) in the object plane20—and hence on the sample22—can be shifted selectively at least in one dimension. Thereby the position of the illumination beam in the object field can be influenced in a selective manner so that, for example, a field having a defined size and shape can be moved in one dimension as a “light carpet” across the object field. This can be utilized, for example, for the so-called “structured illumination” in order to construct three-dimensional layer images from planar images or for multi-focal confocal images, wherein layer images are composed of a plurality of partial images recorded by means of bar masks or dot masks. To this end a corresponding mask is placed in the beam14or the beam16at an appropriate location.

The imaging of the beam deflection element10in the objective pupil plane28in the example shown inFIG. 1is achieved with the help of a lens32which is located between the microscope18and the beam deflection element10. By means of this imaging it is ensured that the illumination angle, i.e. the angle of incidence of the illumination light in the object plane20, does not or at least not essentially change upon the one-dimensional shifting of the beam bundle in the object plane20achieved by selective rotation of the beam deflection element10around the axis30.

A two-dimensional shift of the beam bundle in the object plane20may be achieved by locating an additional scan element, which provides for beam deflection perpendicular to the beam deflection achieved by the beam deflection element10, in a plane conjugate to the objective pupil plane28, whereby any desired space-time beam profile may be generated in the object plane20by combining the actuation of the beam deflection element10and of the additional scan element accordingly.

InFIG. 2a modified embodiment is shown which differs from the embodiment ofFIG. 1essentially in that the beam deflection element10is not located in or close to a plane conjugate to the objective pupil plane28but rather in or close to a plane conjugate to the object plane20. In this case the lens32is eliminated and the beam deflection element10is imaged by means of the tube lens26to infinity and then it is imaged by the microscope objective24into the object plane20. In contrast to the embodiment ofFIG. 1in the embodiment ofFIG. 2consequently rotation of the beam deflection element10around the axis30causes a one-dimensional shift of the position of the beam bundle in the pupil plane28so that the illumination angle (angle of incidence) of the beam bundle in the object plane20changes correspondingly, whereas no or no essential shift of the impingement position of the beam bundle in the object plane20takes place (in the embodiment ofFIG. 1the beam bundle does not move in the pupil plane28upon rotation of the beam deflection element10, but rather the angle of incidence of the beam bundle with regard to the pupil plane28changes; the angle of incidence of the beam bundle, however, remains constant in the embodiment ofFIG. 2).

The embodiment ofFIG. 2is particularly suited for TIRF measurements. In general, TIRF measurements become increasingly popular, since thereby observation is selectively restricted to layers located close to a glass-liquid boundary layer. Most of these TIRF measurements nowadays are performed by means of special objectives in incident light configuration. Different methods are described, see, for example, DE 103 09 269 A1 or US 2004/0174523 A1, in order to combine the beam path of lasers usually used for TIRF measurements and the beam path for the usual wide-field incident light image.

With regard to these known systems the solution according toFIG. 2is significantly simplified, since on the one hand by rotation of the beam deflection element10around the axis30it is possible to switch between a TIRF laser beam14and an incident light illumination beam16in a simple manner, and on the other hand by rotating the beam deflection element10around the axis30the illumination angle of the light beam14(or16) deflected into the microscope18with regard to the object plane20(inFIG. 2this angle is designated by α) can be adjusted in a selective manner, i.e. the change of the beam exit angle with regard to the intermediate image plane34, in which the beam deflection element10and the rotation axis30thereof, respectively, are arranged, causes a corresponding adjustment of the TIRF angle α in the object plane20. If the latter angle becomes larger than the angle of total reflection between the object carrier and the sample, total internal reflection and the creation of a spatially limited evanescent field in the sample occur. The penetration depth of the evanescent field in the sample depends on the selected TIRF angle α, which can be varied, as already mentioned, in turn via the adjusted deflection angle of the deflection element10in one dimension.

An arrangement as shown inFIG. 2also may be modified in such a manner that it serves to combine three or more beam paths one after the other and to simultaneously influence the geometric beam properties in the object plane20. A corresponding example is shown inFIG. 3, wherein the beam deflection element10is rotatable around the axis30in order to switch between a wide-field incident light illumination beam16, a FRAP laser15and a TIRF laser14, respectively, as the illumination source. Also in this case, the beam deflection element10does not only serve for beam selection but in addition it allows influencing of at least one geometric property of one of the selected beams in the object plane20. In the embodiment ofFIG. 3the beam deflection element10is located in or close to a plane34conjugate to the object plane20, i.e. in or close to an intermediate image plane.

Thereby it is possible to switch not only between the three illumination beams14,15and16by means of the beam deflection element10, but in addition the illumination angle α in the object plane20can be adjusted as in the embodiment ofFIG. 2by rotating the beam deflection element around the axis30, i.e. the TIRF angle α can be adjusted for the TIRF beam14by means of the beam deflection element.

Shifting of the FRAP beam14in the object plane20, i.e. selective bleaching of the sample22by means of the FRAP beam15, can be realized by using an additional scanning device (not shown) which correspondingly deflects the FRAP beam15in one or two dimensions before it impinges on the beam deflection element10. In this case the beam deflection element10only has the function to select the FRAP beam15for illumination of the sample.

Corresponding two-dimensional scanning devices are described, for example, in U.S. Pat. No. 6,433,908 B2 and DE 103 28 308 A1. Such scanning devices have in common that two adjustable mirrors provide for independent beam deflection in two directions in space, which is translated by a scanning lens into a corresponding scanning movement of the beam focus in the intermediate image plane34, and hence—after passing through the beam path of incident light of the microscope18—also in the object plane20.

According to a modification of the embodiment ofFIG. 3the beam deflection element10may be located, as in the embodiment ofFIG. 1, in a plane conjugate to the pupil plane, wherein in this case in addition to the switching between the three incident light beams14,15and16a shifting of the beam position in the object plane20by rotating the beam reflection element10around the axis30is enabled. In order to adjust the TIRF angle α then a corresponding scanning device would be necessary, whose imaging plane should lie in a plane conjugate to the pupil plane28.

InFIG. 4an embodiment is shown wherein a light beam41, which preferably consists of laser light, is focused by means of a subsequent scanning device42in a scanning plane50and can be freely positioned therein. By actuating a beam deflection element10, depending on the path taken by the selected light beam before it is deflected into the microscope18, optionally illumination including shifting of the beam position in the object plane20with essentially constant illumination angle, or a variation of the illumination angle in the object plane20with essentially constant beam position in the object plane20, can be realized. To this end, by rotating the beam deflection element10around the axis30, one can select a first optical path wherein the light beam bundle44leaving the scanning device42can be deflected directly into the tube lens26of the microscope18(seeFIG. 4a) and a second optical path, wherein the incident beam bundle44first is passed to a mirror46a, from there to a mirror46b, from there through a lens48to a mirror46cand from there via two mirrors46dand46eagain onto the beam deflection element10in such a manner that the beam from there is deflected into the tube lens26of the microscope18.

The beam deflection element10preferably is located in or close to an intermediate image plane, i.e. a plane conjugate to the object plane20.

The scanning device42focuses the light beam bundle44into a plane50which is located between the scanning device42and the beam deflection element10. If the beam deflection element10is switched to the first optical path (seeFIG. 4a), the vision plane50is imaged to the object plane20, i.e. it lies in an intermediate image plane, so that in this configuration the position of the light beam in the sample plane20may be shifted by actuating the scanning element42, as it is beneficial for FRAP measurements, i.e. in the illumination mode ofFIG. 4athe sample22may be scanned by the light from the light source40by means of the scanning device42.

In the illumination mode ofFIG. 4b, wherein the light beam bundle44from the beam deflection element10is sent via the second path, i.e. via the mirrors46ato46eand the lens48, the focal plane50of the scanning element42is not imaged to the object plane20but rather to the pupil plane28, i.e. in this position of the beam deflection element10the plane50lies in a plane conjugate to the pupil plane28. In this case shifting of the position of the light beam bundle44in the plane50, by actuating the scanning device42, causes a corresponding movement of the light beam in the pupil plane28which movement in turn is translated by the microscope objective24into a corresponding variation of the illumination angle in the object plane20. In the illumination mode ofFIG. 4bthe light from the light source40, for example, may be used for TIRF measurements wherein the TIRF angle can be varied by actuating the scanning device42.

According toFIG. 4cthe beam deflection element10may take a third rotational position in which it serves to deflect a second light beam52from second light source54, which has a different direction from that of the light beam bundle44from the first light source40, into the tube lens26of the microscope18in order to provide, for example, for wide-field incident light illumination. Thereby the beam deflection element10can be utilized for switching between three different illumination modes according toFIGS. 4a,4band4c, respectively.

If the rotation axis30of the beam deflection element10is not located directly in an intermediate image plane but rather only close to such an intermediate image plane, by a slight rotation of the deflection element10around the axis30a slight shift of the illuminated object field can be achieved, i.e. the second light beam52moves in one dimension in the object plane20, wherein, however, the second light beam52also moves in the pupil plane28and hence the illumination angle in the object plane20changes accordingly. However, if only a small shift of the illuminated object field, as it is necessary for structured illumination, is realized, the movement of the beam52in the pupil plane28is negligible.

This may be used for a variation of the path of the illumination beam, as it shown in an example inFIG. 4d, wherein in an intermediate image plane, i.e. in a plane56conjugate to the object plane, a mask58is introduced which, for example, may be designed as a bar mask or a dot mask. The mask58is imaged accordingly onto the sample22and into the object plane20, respectively, where it creates a corresponding bar pattern or dot pattern due to the passage of the second light beam52through the mask58. This pattern then may be moved across the sample22by a slight rotation of the deflection element10around the axis30. Thereby a so-called structured illumination and a multifocal confocal illumination, respectively, can be realized, i.e. a method for obtaining layer images with simultaneous suppression of information from planes outside the focal plane.

While various embodiments in accordance with the present invention have been shown and described, it is understood that the invention is not limited thereto, and is susceptible to numerous changes and modifications as known to those skilled in the art. Therefore, this invention is not limited to the details shown and described therein, and includes all such changes and modifications as encompassed by the scope of the appended claims.