DE102017103252B3 - Method for microscopic grid illumination - Google Patents

Abstract

Described is a method of microscopic raster illumination using a scanning illumination device (10) for a microscope, comprising a light source (12), a raster system (14), an intermediate imaging optics (16) and an illumination objective (18) arranged in an illumination beam path in that order (20) are arranged. The raster system (14) has two raster elements (26, 28) which are designed to divert an illumination light bundle (34) emitted by the light source (12) one after the other in two different raster directions. The intermediate imaging optical system (16) is designed to generate an image of an objective pupil (36) of the illumination objective (18) within the raster system (14). A pupil displacement module (38) is designed to selectively displace the image of the objective pupil (36) within the grid system (14) along the illumination beam path (20).

Description

The invention relates to a method for microscopic grid illumination according to the preamble of claim 1.

From the prior art grid illumination devices are known, in particular for microscopic applications such as confocal fluorescence microscopy, which serve to scan the sample in two different scanning directions, which are generally perpendicular to one another, with an illuminating light bundle. For this purpose, such a raster illumination device has a raster system which deflects the illumination light beam in the above-mentioned raster directions in order to realize illumination scanning the sample in two dimensions.

Usually, a raster system is used for the two-dimensional deflection of the illumination light beam, which either a single biaxial grid element which is tiltable about two mutually orthogonal scanning axes, or two separate uniaxial grid elements which are tiltable only about a scan axis. In the latter embodiment, the scanning axes of the two uniaxial grid elements are again arranged orthogonal to each other. The raster elements may, for example, be tilt mirrors, but also Risley rotary prisms, electro-optical or acousto-optical deflectors.

Particularly in microscopic applications, telecentric positioning of the raster system within the illumination beam path is desirable. In the case of a telecentric positioning, the raster system is located along the illumination beam path at a location at which an image of the pupil of the illumination objective, which is generated for example via the intermediate imaging optics, is located. By a telecentric arrangement of the grid system namely an axis-parallel exit of the deflected by the grid system illumination light beam is guaranteed for all grid positions.

If the grid system is formed from a single biaxial grid mirror, the aforementioned telecentric positioning can be realized comparatively easily by arranging the single raster mirror at the location of the pupil image. However, a raster system formed from a two-axis raster mirror is technically more complex and therefore more expensive than a system that operates with two separate uniaxial raster mirrors. In a system with two separate uniaxial raster elements, however, it is not possible to simultaneously position both raster mirrors at the location of the pupil image and thus bring them into a telecentric arrangement. Also, it comes in realizations from the prior art, such as gimbal suspensions of tilting mirrors, to speed losses of the raster process.

Solutions are known from the prior art, even telecentric raster systems that operate with an arrangement of two single-axis raster mirrors. For example, it is proposed in J. Pawley, Handbook of biological confocal microscopy, Springer 2006, ISBN 978-0387259215 (esp., Chapter 9, pp. 207ff), to image the first raster mirror onto the second raster mirror by means of relay optics. In the DE 4 026 130 C2 On the other hand, a system consisting of three uniaxial scanning mirrors is described, in which two of the three scanning mirrors are provided for one of the two orthogonal scanning directions at the same time.

These two raster mirrors are arranged such that they define a virtual tilting point which lies on the raster mirror associated with the other raster direction.

Grid systems operating with relay optics are technically complex and expensive. They also need a lot of space. Systems consisting of three grid mirrors are also expensive in terms of their mechanics and their electronic control. This applies in particular to that of the two scanning axes, which are assigned to two of the three raster mirrors. Consequently, this scanning axis is also referred to as a slow axis, while the scanning axis realized by a single scanning mirror is called a fast axis.

To keep the technical effort as low as possible, grid systems are therefore often preferred, which consist of two uniaxial grid mirrors. In order to keep the vignetting in such a grid system in particular low, the two raster mirrors are usually arranged symmetrically to the image of the objective pupil. Such a symmetrical arrangement can be used, for example, in a confocal microscopic application. However, this does not apply to applications in which a telecentric positioning of at least one of the two grid elements is absolutely necessary in order to enable an axially parallel light emission. An example of this is a light sheet microscopic application, as described in the document US 9 104 020 B2 is described.

If a fixed telecentric positioning is selected for one of the two mirrors in the case of one of the two mirror mirrors, the result is an asymmetrical arrangement which causes correspondingly asymmetrical and often particularly strong vignetting. This is especially true when imaging the objective pupil is used on the telecentrically positioned raster mirror Kepler telescope as Zwischenabbildungsoptik, which usually has a lateral magnification in a range of 3 to 4 and thus an axial magnification in a range of 9-16. The distance then the other, ie not telecentrically positioned, raster mirror relative to the image of the objective pupil, results in this example, based on the product of the distance of this raster mirror from the telecentrically positioned raster mirror and a factor between 9 and 16.

Thus, it should be noted that the known from the prior art solutions are limited only to specific applications. In any case, it has not been readily possible to flexibly use a grid system consisting of two uniaxial grid elements in various applications.

The publication US 2002/0 048 025 A1 discloses a lighting device that is optionally usable for optical coherence tomography and optical coherence microscopy. This illumination device comprises a light source, a raster system, an intermediate imaging optics and an illumination objective, which are arranged in this sequence in an illumination beam path. To compensate for different pupil positions as a result of using different lenses, the illumination device has a plane-parallel plate, which can be introduced in the illumination beam path. The introduction of the plane-parallel plate ensures that the intermediate imaging optics always image the pupil of the respectively used objective onto a galvanometer mirror contained in the grid system.

From the publication WO 95/06 895 A2 For example, a scanning illuminator for a microscope is known that allows both confocal and non-confocal imaging. This raster illumination device comprises a raster system with two raster mirrors which deflect an illumination light beam emitted by the light source in different raster directions. In order to avoid nonlinearities in the raster process, it is proposed to image the objective pupil onto the raster system.

The publication US 2004/0 263 959 A1 discloses a confocal scanning microscope with a raster mirror which deflects an illuminating light beam in one direction while moving a stage in a direction perpendicular thereto to make a two-dimensional scan of the sample. Alternatively, it is proposed to provide two raster mirrors arranged at equal intervals on opposite sides of an entrance pupil position.

The object of the invention is to provide a method for scanning sample illumination, which makes it possible with little technical effort to use a grid system formed from two separate grid elements in different microscopy applications.

The invention solves this problem by the subject matter of claim 1.

The scanning illumination device according to the invention for a microscope comprises a light source, a raster system, an intermediate imaging optics and an illumination objective, which are arranged in this sequence in an illumination beam path, wherein the grid system comprises two raster elements which are formed, one emitted from the light source illuminating light beam successively in two different Deflecting raster directions, and wherein the intermediate imaging optics is designed to generate an image of an objective pupil of the illumination objective within the raster system. The invention provides a pupil displacement module which is designed to selectively displace the image of the objective pupil within the grid system along the illumination beam path.

The pupil displacement module makes it possible to adapt the grid illumination device according to the invention to different microscopy applications with little technical effort. For this purpose, it is only necessary to optionally position the image of the objective pupil generated by the intermediate imaging optics by means of the pupil displacement module within the grid system, as is advantageous for the selected microscopy application.

Thus, it is possible, in particular, to optimize the louver illumination device for a light-sheet microscopic application. In such an application, the raster system serves, on the one hand, to generate a light sheet from the illumination light beam emitted by the light source and, on the other hand, to deflect this light sheet as a whole along a predetermined raster direction in order to scan the sample in this direction with the light sheet. In this case, a first of the two raster elements can be used to generate the light sheet by deflecting the illumination light bundle along a first raster direction, while a second raster element deflects the illumination light bundle along a second raster direction which is preferably orthogonal to the first raster direction and thus through the first Raster element generated light sheet along this second scanning direction as it were moved through the sample.

In such a light sheet microscopy application, the pupil displacement module can now be used to position the image of the objective pupil within the raster system to lie on the aforesaid second raster element, i. the element that scans the sample in a raster motion with the light sheet. Thus, it is important in this raster movement to the axis-parallel exit of the illumination light beam from the illumination objective, which is ensured by the inventive positioning of the second raster element at the location of the pupil image. In other words, in the aforementioned application, the second raster element is positioned telecentrically to the disadvantage of the first raster element. The fact that the first raster element is not arranged in a telecentric manner, however, is not a significant disadvantage in the light-sheet microscopic application explained above. Thus, the raster movement of the illuminating light bundle caused by the first raster element and intended for generating the light sheet is, as it were, averaged over the exposure time with which a detector detects the fluorescence radiation generated by the light sheet from the sample.

If, on the other hand, the raster illumination device according to the invention is to be optimized, for example, for a confocal microscopic application, it is possible to arrange the image of the objective pupil in a symmetrical arrangement exactly between the two raster elements by means of the pupil displacement module. In this case, although neither of the two raster elements is precisely telecentric, this symmetrical arrangement allows a substantial reduction in vignetting.

The pupil displacement module comprises at least two displacement elements which can be introduced into the illumination beam path and which can be removed from the illumination beam path. In a first operating state, in which neither of the two displacement elements is introduced into the illumination beam path, the image of the objective pupil is located along the illumination beam path in a first position. In a second operating state, in which one of the two displacement elements is introduced into the illumination beam path, the image of the objective pupil is located along the illumination beam path in a second position. In a third operating state, in which the other of the two displacement elements is introduced in the illumination beam path, the image of the objective pupil is located along the illumination beam path in a third position. One of the three named positions lies on one of the two grid elements. Another of the three positions mentioned lies on the other grid element. The remaining of the three positions lies between the two locking elements. In this embodiment, the raster illumination device can operate in three different operating states, namely in two operating states, in which either one or the other raster element is arranged telecentrically, and in another operating state, in which neither of the two raster elements is telecentrically positioned, but the image of Lens pupil between the two grid elements is located. The two first-mentioned operating states can, for example, again be used for a light-sheet microscopic application, while the last-mentioned operating state can be provided for a confocal microscopic application.

The position between the two latching elements, in which the image of the objective pupil is generated, preferably has the same distances from the two latching elements along the illumination beam path. In this non-telecentric, symmetrical to the pupil image arrangement of the two grid elements, the vignetting can be minimized.

Preferably, the respective displacement element is a transparent, in particular plane-parallel plate of predetermined thickness. In this case, the thickness of the plate is selected so that the illuminating light beam undergoes an optical Weglängenänderung when passing through the plate, which leads to the desired displacement of the pupil image. If the pupil displacement module comprises a plurality of plates, they have different thicknesses in order to allow pupil displacements of different sizes.

In a preferred embodiment, the respective transparent plate has a light entry surface and a light exit surface, which are tilted relative to one another to avoid interference effects. The surface tilt is only slight, e.g. in a range of a few tens of arc seconds, so that the plate is still to be described as essentially plane-parallel despite this surface tilting. By tilting the light entry surface and the light exit surface of the plate against each other, interfering interference effects that otherwise occur within the plate due to multiple reflections between the light entry surface and the light exit surface can be avoided.

Disturbing interference effects are largely avoided by using a transparent, plane-parallel plate but already that the thickness of such a plate to provide the desired optical path length change in the rule anyway much larger than that usual coherence length of a diode laser commonly used as a light source. In addition, it should be noted that in the event that the grid system, for example, in contrast to a confocal Auflicht scanning microscope only for lighting purposes (such as in the aforementioned document US 9 104 020 B2 ) is used, multiple reflections within the transparent plate are tolerable anyway.

In a particularly preferred embodiment, the pupil displacement module comprises a changing device. This changing device can be operated manually or by motor. In the case of a motor-driven changing device, a control unit may be provided which controls the pupil displacement module as a function of the currently set operating state. The changing device is designed for example in the form of a revolver, a linear sliding device or a folding device.

Instead of a transparent, plane-parallel plate, for example, a Galilean telescope or another relay system for influencing the optical path length can be used. However, the preferred embodiment in the form of a plane-parallel plate over the latter systems formed of spherical optical elements has the advantage that the positioning of the plate is insensitive to centering errors.

In a preferred embodiment, one of the two raster elements is a mirror which can be tilted about a first tilting axis and the other raster element is a mirror which can be tilted about a second tilting axis. The two tilt axes are preferably orthogonal to each other.

The mirrors are designed, for example, as galvanometer mirrors or as microelectromechanical mirrors. However, they are not limited to the aforementioned embodiments. Thus, single-axis mirror actuators of other types of construction can also be used with which the desired deflection of the illumination light beam can be realized.

Preferably, the pupil displacement module is arranged in a part of the illumination beam path formed by an infinity beam path. The pupil displacement module can then be placed anywhere within the infinity beam path.

In a further advantageous embodiment, the pupil displacement module is arranged between the grid system and the intermediate imaging optics. The intermediate imaging optics in this embodiment are formed, for example, from a tube lens system facing the illumination objective and an eyepiece lens system facing the pupil displacement module.

As already indicated above, the grid system is designed in a particularly preferred embodiment on a scanning sample illumination by means of a light sheet-like illumination light distribution by the light sheet-like illumination light distribution can be generated by the means of one of the two raster elements effected deflection of the illumination light beam in one of the two raster directions the deflection of the illuminating light bundle in the other scanning direction, the light-sheet-like illuminating light distribution, is movable in this other scanning direction.

In this embodiment, the pupil displacement module preferably displaces the image of the objective pupil on the aforementioned other raster element.

• 1 eine schematische Darstellung einer erfindungsgemäßen Rasterbeleuchtungseinrichtung, die ein Pupillenverlagerungsmodul mit zwei verschiedenen Verlagerungselementen umfasst, in einem ersten Betriebszustand, in dem keines der beiden Verlagerungselemente in einen Beleuchtungsstrahlengang eingebracht ist;
• 2 eine schematische Darstellung der Rasterbeleuchtungseinrichtung in einem zweiten Betriebszustand, in dem eines der beiden Verlagerungselemente in den Beleuchtungsstrahlgang eingebracht ist; und
• 3 eine schematische Darstellung der Rasterbeleuchtungseinrichtung in einem dritten Betriebszustand, in dem das andere der beiden Verlagerungselemente in den Beleuchtungsstrahlengang eingebracht ist.

The invention will be explained in more detail below with reference to an embodiment with reference to the figures. Show:

• 1 a schematic representation of a grid illumination device according to the invention, which comprises a Pupillenverlagerungsmodul with two different displacement elements, in a first operating state in which neither of the two displacement elements is introduced into an illumination beam path;
• 2 a schematic representation of the raster illumination device in a second operating state, in which one of the two displacement elements is introduced into the illumination beam path; and
• 3 a schematic representation of the raster illumination device in a third operating state, in which the other of the two displacement elements is introduced into the illumination beam path.

1 shows a purely schematic representation of an embodiment of a raster illumination device 10 , which is part of a microscope that can be used in various applications. Thus, the aforementioned microscope can be operated, for example, in the manner of a light-beam microscope and, alternatively, in the manner of a confocal microscope. It should be noted that in 1 only those components of the grid illumination device 10 are shown, which are necessary for the understanding of the invention.

The scanning microscope 10 includes a light source 12 , a grid system 14 , an intermediate imaging optics 16 and a lighting lens 18 , in the this order in a lighting beam path 20 are arranged. The intermediate imaging optics 16 contains an eyepiece lens system 22 and a tube lens system 24 ,

The grid system 14 is from a first grid element 26 and a second raster element 28 educated. The two grid elements 26 . 28 , in the 1 are shown enlarged in a partial view, in each case, for example, in the form of a Galvanometerspiegels or a MEMS mirror are executed. The first grid element 26 is a first tilt axis 30 tiltable, with reference to the in 1 shown xyz coordinate system is parallel to the x-axis. The second grid element 28 is about a second tilt axis 32 tiltable, parallel to the y-axis and thus orthogonal to the first tilt axis 30 lies.

The light source 12 emits an illumination light beam 34 on the first grid element 26 the lighting light bundle 34 on the second grid element 28 reflected. At the second grid element 28 becomes the illumination light beam 34 in the direction of the intermediate imaging optics 16 reflected. After passing through the intermediate imaging optics 16 the illumination light beam arrives 34 into a lens pupil 36 of the illumination lens 18 , The illumination lens 18 directs the illumination light beam 34 on an in 1 not shown sample to illuminate this.

The two the grid system 14 forming raster elements 26 and 28 have the function, the lighting beam 34 deflect in succession in two orthogonal raster directions that are parallel to the y-axis or to the x-axis, in order to realize an illumination that scans the sample two-dimensionally. For this purpose, the two Abtastspiegel 26 . 28 under the control of an in 1 not shown control unit coordinated with each other about the first tilt axis 30 or the second tilt axis 32 tilted.

The objective pupil 36 is through the Zwischenabbildungsoptik 16 in the way on the grid system 14 imaged that within the grid system 14 a picture of the objective pupil 36 is produced. The position in which the intermediate imaging optics 16 in the grid system 14 the image of the objective pupil 36 is generated in the present embodiment along the optical axis of the illumination beam path 20 displaceable. For this purpose, the grid illumination device 10 a pupil relocation module 38 on, one in 1 purely schematically illustrated changing device 40 as well as two plane parallel glass plates 42 . 44 (see. 2 and 3 ), which by means of the changing device 40 optionally in a part of the illumination beam path formed by an infinity beam path 20 which is between the eyepiece lens system 22 the intermediate imaging optics 16 and the grid system 14 defined, can be brought in. The glass plates 42 . 44 each have a light entry surface 42a . 44a and a light exit surface 42b . 44b on.

Like a synopsis of 1 to 3 can be illustrated by the pupil displacement module according to the invention 38 that came from the changing device 40 as well as the two plane-parallel glass plates 42 . 44 is formed, three operating states of the grid lighting device 10 realize, in terms of location, in which the image of the objective pupil 36 in the grid system 14 is generated, differ from each other.

In 1 a first operating state is shown in which neither the plane-parallel glass plate 42 still the second plane-parallel glass plate 44 in the illumination beam path 20 is introduced. The optical components of the raster illumination device 10 , in particular the intermediate imaging optics 16 and the grid system 14 , are in the present embodiment so coordinated that the Zwischenabbildungsoptik 16 the objective pupil 36 in the first operating state on the second locking element 28 maps. To illustrate this fact, is in the presentation after 1 (and accordingly in the 2 and 3 ) the illumination light beam 34 in two sub-bundles 34a and 34b split, which come from different field points. In this representation lies the place where the image of the objective pupil 36 along the illumination beam path 34 within the grid system 14 is generated, in the intersection of the central rays of the two sub-beams 34a and 34b , In 1 are the central rays of the partial bundles 34a and 34b with Za or Zb and the aforementioned intersection with SP.

2 shows the grid illumination device 10 in a second operating state, in which the first plane-parallel plate 42 in the illumination beam path 20 is introduced. Like the grid system 14 enlarged showing partial view after 2 illustrated, causes the in the illumination beam path 20 introduced plane-parallel plate 42 an optical path length change, which is such that the central rays Za . zb the two subbundles 34a . 34b exactly in the middle between the two raster elements 26 . 28 cut, so that exactly at this point by the Zwischenabbildungsoptik 16 generated image of the objective pupil 36 lies. This central position of the image of the objective pupil is in 2 defined by a plane labeled E. For comparison, this level e also in the 1 and 3 specified.

3 shows a third operating state of the raster illumination device 10 in which instead of the first plane-parallel plate 42 the second plane-parallel plate 44 in the illumination beam path 20 is introduced. The second plate 44 has one along the optical axis of the illumination beam path 20 measured thickness, which is greater than the thickness of the first plane-parallel plate 42 is. The thickness of the second plane-parallel plate is 44 so chosen that the image of the lens pupil 36 exactly on the first grid element 26 is produced. In the illustration after 3 this in turn is through the intersection SP the two central rays Za . zb the sub-bundle 34a . 34b illustrated.

In the present embodiment, the first and the third operating state after the 1 or 3, in which the objective pupil 36 on one of the two grid elements 26 . 28 is mapped and thus a telecentric positioning of this grid element 26 . 28 is given to be used, the grid illumination device 10 to operate in a light sheet microscopic application. In contrast, the non-telecentric second operating state is suitable 2 in which the two grid elements 36 . 28 are spaced symmetrically from the pupil image located centrally between them, for a confocal microscopic application.

It goes without saying that the present invention is not limited to the embodiment described above. For example, instead of the glass plates 42 . 44 one or more afocal Galilean telescope systems can be used for the desired influencing of the optical path length of the illumination light bundle.

LIST OF REFERENCE NUMBERS

10

Grid lighting device

12

light source

14

grid system

16

Between imaging optics

18

lighting lens

20

Illumination beam path

20a

Infinite beam

22

eyepiece

24

tube lens

26

first grid element

28

second grid element

30

first tilt axis

32

second tilt axis

34

Illumination light beam

34a, 34b

partial bundle

36

objective pupil

38

Pupils shift module

40

changing device

42, 44

glass plates

42a, 42b

Light entry surfaces

44a, 44b

Illuminating surfaces

Zb, Za

central beams

SP

intersection

e

level

Claims (13)

Method for microscopic raster illumination using a raster illumination device (10) comprising a light source (12), a raster system (14), intermediate imaging optics (16) and an illumination objective (18) arranged in an illumination beam path (20) in this order, and a pupil displacement module (38), wherein an illuminating light bundle (34) emitted by the light source (12) is deflected successively in two different raster directions by means of two raster elements (26, 28) contained in the raster system (14), by means of the intermediate imaging optics (16). an image of an objective pupil (36) of the illumination objective (18) is generated within the raster system (14), and the image of the objective pupil (36) within the raster system (14) is selectively displaced along the illumination beam path (20) by means of the pupil displacement module (38) , characterized in that the pupil displacement module (38) used at least ns two displacement elements (42, 44), which are selectively introduced into the illumination beam path (20) or removed from the illumination beam path (20), wherein a first operating state of the raster illumination device (10) is set, in which neither of the two displacement elements (42 , 44) are introduced into the illumination beam path (20) whereby the image of the objective pupil (36) is arranged along the illumination beam path (20) in a first position, wherein a second operating state of the raster illumination device (10) is set, in which one of the two displacement elements (42, 44) into the illumination beam path ( 20), whereby the image of the objective pupil (36) is arranged along the illumination beam path (20) in a second position, and wherein a third operating state of the scanning illumination device (10) is set, in which the other of the two displacement elements (42, 44 ) is introduced into the illumination beam path (20), whereby the image of the objective pupil (36) along the illumination beam path (20) is arranged in a third position, wherein one of the three positions on one of the two raster elements (26, 28) is located other of the three positions mentioned lies on the other grid element (26, 28) and the remaining of the dre i positions between the two raster elements (26, 28) is located. Method according to Claim 1 , characterized in that for the telecentric positioning of one of the two raster elements (26, 28) the image of the objective pupil (36) by means of the pupil displacement module (38) on this raster element (26, 28) is displaced. Method according to Claim 1 or 2 , characterized in that between the two raster elements (26, 28) lying position in which the image of the objective pupil (36) is generated, along the illumination beam path (20) equal distances from the two raster elements (26, 28). Method according to one of the preceding claims, characterized in that the respectively used displacement element (42, 44) is a transparent plate of predetermined thickness. Method according to Claim 4 , characterized in that a plane-parallel plate is used as the transparent plate. Method according to Claim 4 , characterized in that as a transparent plate (42, 44) a plate is used, which has a light entry surface (42a, 44b) and a light exit surface (42b, 44b), which are tilted against each other to avoid interference effects. Method according to one of the preceding claims, characterized in that the pupil displacement module (38) used comprises a changing device (40). Method according to one of the preceding claims, characterized in that one of the two grid elements (26, 28) used is a mirror tiltable about a first tilting axis (30) and the other grid element is a tiltable mirror about a second tilting axis (32). Method according to Claim 8 , characterized in that the mirrors used are each formed as Galvanometerpiegel or as a micro-electro-mechanical mirror. Method according to one of the preceding claims, characterized in that the pupil displacement module (38) used is arranged in a part of the illumination beam path (20) formed by an infinity beam path (20a). Method according to Claim 10 , characterized in that the pupil displacement module (38) used is arranged between the grid system (14) and the intermediate imaging optics (16). Method according to one of the preceding claims, characterized in that using the raster system (14) a scanning sample illumination by means of a light sheet-like illumination light distribution is performed by the by means of one of the two raster elements (26, 28) caused distraction of the illumination light beam (34) in one the two raster directions, the light sheet-like illumination light distribution is generated and by the means of the other raster element (26, 28) effected deflection of the illumination light beam (34) in the other raster direction, the light sheet-like illumination light distribution is moved in this other raster direction. Method according to Claim 12 , characterized in that by means of the Pupillenverlagerungsmodul (38), the image of the objective pupil (36) is displaced on said other grid element (26, 28).

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