EP3359997A1

EP3359997A1 - Method and apparatus for examining a sample using structured light-sheet illumination

Info

Publication number: EP3359997A1
Application number: EP16778396.8A
Authority: EP
Inventors: Florian Fahrbach; Werner Knebel
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2015-10-09
Filing date: 2016-10-10
Publication date: 2018-08-15
Also published as: WO2017060506A1; US20190025563A1; US11092792B2; CN108292034A; CN108292034B

Abstract

The invention relates to a method and an apparatus for microscopic examination of a sample (24). The method contains the step of producing an illumination light beam (2) using a light source, the step of spatially splitting the illumination light beam (2) into at least two partial illumination light beams (3, 4) using a splitting means (1), the step of guiding the partial illumination light beams (3, 4) through an illumination objective (7) common to both partial illumination light beams (3, 4), the step of deflecting at least one of the partial illumination light beams (3, 4) after these have passed through the illumination objective (7) using at least one deflection means (29) in such a way that the partial illumination light beams (3, 4) interfere with one another in an illumination plane (11) such that an illumination pattern (12) arises in the illumination plane (11), and the step of producing an image of a sample region illuminated by the illumination pattern (12), wherein the detection light emanating from the sample region reaches a spatially resolving detector through a detection objective (8).

Description

METHOD AND DEVICE FOR EXAMINING A SAMPLE WITH A STRUCTURED SAMPLE

LIGHT SHEET LIGHTING

The invention relates to a method and a device for examining a sample, in particular in light-sheet microscopy.

The Single Plane Illumination Microscopy (SPIM) technique, which provides layer-by-layer illumination of the sample, allows for faster and more accurate acquisition of image data than, for example, point sampling of a sample. A well-known field of application of the SPIM technology is the field of fluorescence microscopy, wherein fluorophores are excited in the sample with laser light. In the case of the SPIM technology, excitation takes place here only in one of an illumination light sheet (also called "light stripe") .This avoids damage to the sample by illumination light in other planes A device operating according to the SPIM method is described in DE 102 57 423 AI In this microscope, a specimen is illuminated with a thin light sheet while the observation is made from a direction perpendicular to the plane of the illuminating light sheet, whereby the illumination and the detection are effected via two separate optical paths with separate optics The specimen is moved through the light sheet which is fixed relative to the detector in order to record fluorescence and / or scattered light layer by layer with a two-dimensional detector, which can then be converted into a three-dimensional image d he assemble sample corresponding record.

From DE 10 2007 015 063 AI an optical arrangement with a light source for emitting a light beam and with optical elements for converting this light beam in the form of a light sheet is known. The optical arrangement is particularly suitable for illuminating individual planes of a three-dimensional sample in the case of selective plane illumination microscopy (SPIM). There are means for varying the cross section of the light sheet, for varying the length of the light sheet and / or for influencing the direction of the radiation within the light sheet extending portions of radiation to the geometry of the light sheet to the lighting requirements in the observation of one and the same sample plane with to enable several different types of lenses.

As an alternative to direct sample illumination, it is also possible to illuminate the sample evanescently. In this case, the excitation light is totally reflected at an interface to the sample, wherein the excitation of the sample is carried out by evanescent electromagnetic field decaying with the penetration depth. For this type of sample examination, the term TIRF Microscopy or TIRFM (Total Internal Reflection Fluorescence Microscopy) has become established.

It is the object of the present invention to provide a method for examining a sample, which allows good accessibility of the sample with improved imaging quality.

The object is achieved by a method which is characterized by the following steps:

a. Generating an illumination light beam with a light source, b. spatially splitting the illumination light beam with a splitting means into at least two partial illumination light bundles, c. Directing the partial illumination light beams through an illumination objective common to both partial illumination light beams, d. Deflecting at least one of the partial illumination light bundles, after they have passed through the illumination objective, with at least one deflection means in such a way that the partial illumination light bundles interfere with one another in an illumination plane, so that a lighting pattern is produced in the illumination plane, and e. Generating an image of a sample area illuminated by the illumination pattern, wherein detection light emanating from the sample area passes through a detection objective to a spatially resolving detector.

It is a further object of the present invention to provide a device for Examine a sample that allows good accessibility of the sample with improved imaging quality.

The object is achieved by a device that

a. a light source that generates an illuminating light beam, b. a splitting means which spatially splits the illuminating light beam into at least two partial illuminating light beams, c. an illumination lens that is traversed by the sub-illumination beams, d. at least one deflecting means, which deflects at least one of the partial illumination light bundles, after they have passed through the illumination objective, in such a way that the partial illumination light bundles interfere with one another in an illumination plane, so that a lighting pattern is produced in the illumination plane, and e. a spatially resolving detector for generating an image of a sample area illuminated by the illumination pattern, wherein detection light emanating from the sample area passes through a detection objective to a spatially resolving detector.

The invention has the very special advantage that an improvement of the imaging quality can be achieved by illumination with a lighting pattern, in light-sheet microscopy and / or in TIRF microscopy. This on the one hand by the fact that at least one background suppression is made possible by the illumination with an illumination pattern generated by interference of at least two partial illumination light bundles. In particular, however, which is described in detail below, advantageously even an increase in resolution can be achieved. Of particular advantage here is that the partial illumination light beams, which are brought to generate the illumination pattern to the interference, the same illumination lens through, so that in the sample only a single illumination lens needs to be present, allowing a particularly good accessibility of the sample. Although it is generally known to increase the lateral resolution and the image contrast in a microscope by structured illumination in the wide-field microscopy or TIRF microscopy. However, illumination by the detection objective has the disadvantage that the possible angles of incidence and thus the possible periods of the interference pattern are very limited by the numerical aperture of the detection objective. An isotropic increase in resolution in the image plane or along all three spatial directions by structured illumination in SPIM microscopy is hitherto unknown.

In general, the increase in resolution depends on the period of the lattice. When an illumination with a grating takes place through the detection objective, this usually takes place in such a way that the illuminating light is irradiated at a steep angle with respect to the optical axis into the sample volume in two opposite directions, so that a standing wave arises as an interference pattern whose period of the angle 2a between the two beams depends. The angle a = arcsin (NA / n) depends in particular on the numerical aperture (NA) of the detection objective and the refractive index of the medium. Typically, NA <1, 2, and thus a <65 degrees in water (n = 1, 33). In any case, the angle a is clearly less than 90 degrees, with an angle of 90 degrees being optimal to achieve the minimum period of the interference pattern. Disadvantageously, the interfering beams before and after they reach the focal plane of the detection lens reach large parts of the sample and there, ie above and below the focal plane of the detection lens (except for TIRF) fluorescence, which causes a strong background signal in the image, the fluorophores of the There sample unnecessarily bleaches and damage the sample by phototoxic effects.

These disadvantages are according to the invention, in particular in a SPIM examination of a sample, advantageously avoided or reduced. In this case, it is even possible, in particular to achieve an increase in resolution, to generate the illumination pattern by superimposing 2, 3 or an even higher number of partial illumination light bundles without requiring an equal number of illumination objectives. The use of a variety of illumination lenses would be expensive, complicated and prone to failure. apart Of these, it is hardly possible to spatially combine a detection objective necessary for achieving high resolutions with a plurality of, for example, four illumination objectives so that the illumination objectives can focus the required illumination sub-beams into the focal plane of the detection objective because high-aperture objectives have a large diameter and require a short working distance , so that there is a spatial collision of the lenses, unless special and therefore expensive illumination lenses with a particularly large working distance and typically low numerical aperture are used .. Another difficulty would be in the use of a variety of illumination lenses that high-aperture lenses also manipulate the polarization of the light so that it leads to incomplete interference of the partial illumination light bundles, thereby, for example, zeroing the illumination pattern are less pronounced and / or the contrast of the illumination pattern decreases overall, which would be detrimental to the image quality. These disadvantages avoid the present invention.

In particular, in order to generate an isotropically over-resolved image in the image plane, different and / or differently positioned and / or differently oriented illumination patterns can be generated in the illumination plane one after another, with which the same sample area is illuminated in each case. The data of the images generated in this way can then be offset against each other in order to achieve an overall increase in resolution. For this purpose, a lighting pattern, for example with regard to a lattice constant and / or with regard to its position and / or with regard to its orientation, can be changed in different ways, in order to generate a changed illumination pattern in each case.

For example, it is possible to change the relative phase of the partial illumination light beams. This can be achieved, for example, by changing the optical light path of a subset of the interfering partial illumination light bundles. Alternatively or additionally, it is also possible to change the direction of incidence of the interfering partial illumination light bundles. This can be achieved, for example, by diverting the partial illumination light bundles in each case with differently arranged deflecting means or differently arranged deflecting elements of a deflecting means before they interfere that the interfering partial illumination light beams each fall from different directions, which will be explained in more detail below.

Alternatively or additionally, it is also possible that the angle of incidence of the interfering partial illumination light beam is changed to the illumination plane, in particular, for example, to change a lattice constant.

In a particularly advantageous embodiment of the method according to the invention, a further illumination pattern is generated in addition to the illumination pattern, which is superimposed coherently or incoherently on the illumination pattern.

For example, a further illumination pattern can be generated by splitting off at least two further partial illumination light bundles from the illumination light bundle which interfere with one another in the illumination plane. Alternatively, it is also possible that at least two further partial illumination light beams are generated by means of a further light source, which interfere with one another in the illumination plane, so that the further illumination pattern arises in the illumination plane.

In this way, for example, an over-resolved image can be generated. As a result of the superimposition of a plurality of illumination patterns, a high-frequency illumination pattern results overall in the illumination plane. If the relative phase of the partial illumination light bundles belonging to each illumination pattern is then changed, the phase of the high-frequency grating is also changed so that the respective resulting image data are raw data for generating or calculating an over-resolved and / or contrast-enhanced imaging of the illuminated in the illumination plane sample area can be used. In this case, it can be provided, in particular, advantageously that the sample in the illumination plane is illuminated with a plurality of crossed illumination gratings which are coherently superimposed.

Apart from a coherent superimposition of illumination patterns, in particular a superimposition of crossed illumination grids, there is, as already mentioned, also the possibility of an incoherent superimposition of the illumination pattern at least one further illumination pattern. (The main difference between the two types of superimpositions is that in one case isolated maxima are generated, for example for excitation, in the other case isolated minima / zeros are generated.The incoherent overlay can be understood as a simple sum of the two grids: the lattices overlap without any deviation due to interference.) The resulting illumination pattern then has an annular character on the one hand. In particular, real zeros are then only in the center or at the intersections of the (linear) zeros of the two crossed lattices. In an incoherent superimposition of a lighting pattern with a single further, rotated by 90 degrees in the lighting level, further lighting patterns are the real zeros only in the center of the lighting level within a square formed of four adjacent points (intensity maxima). In addition, on the other hand, a total illumination pattern generated by incoherent superposition has a smaller period. In particular, the illumination pattern generated by incoherent superimposition can also be exploited for parallelized STED or RESOLFT experiments when parallel or alternating excitation light of a different, suitable wavelength is irradiated (alternating with switchable fluorophores, with STED / RESOLFT-like techniques simultaneously) ,

The superimposition of two gratings with different wavelengths is particularly attractive. With light of a first wavelength, a point grid is generated by coherent superposition, whose maxima lie at the zeros of a grid, which was generated with light of a second wavelength and is generated by incoherent superimposition. In this case, the first wavelength is suitable for exciting the fluorophores, the second wavelength for stimulated excitation by means of the STED process or for switching the fluorophores by means of RESOLFT is explained below.

Compared with far-field realizations for RESOLFT experiments (reversible saturable optical fluorescence transitions), the present invention offers the very special advantage in a higher spatial frequency of the resulting illumination pattern and the resulting higher resolution. In addition, RESOLFT experiments and STED (Stimulated Emission Depletion) due to the higher grid frequency, a higher resolution at lower illumination light achievable achievable. In addition, the present invention also provides more efficient use of the light because it illuminates only in a narrow area outside the illumination plane.

In a particularly advantageous embodiment, the interfering partial illumination light bundles and / or the further interfering partial illumination light bundles are designed as Bessel beams. In a particularly advantageous manner, it can be provided that the interfering partial illumination light bundles and / or the further interfering partial illumination light bundles are designed as sectioned-Bessel beams. In particular, it can initially be provided that the interfering Bessel beams, for example, using one or more with respect to the deflection angle adjustable beam deflecting device, are moved in the lighting plane synchronously back and forth.

The background of illumination using Bessel beams or Sectioned Bessel beams is as follows:

Focusing the light passing through a ring aperture causes intensity maxima along the optical axis in the region of the focus due to constructive interference. A disturbance in a small partial area of the annular illumination light beam has almost no effect in the area of the focus on the optical axis because the entire illumination light there interferes. Hiding in a small portion outside the optical axis thus has almost no influence on the constructive interference. In the areas of focus with an axial distance to the optical axis, a disturbance in the area of the incident light is "healed" in that the remaining light behind the disturbance avoids a shadow cast by the disturbance.

A Bessel beam generated with an annular illumination is rotationally symmetric with respect to the optical axis. In addition, such a Bessel beam has intensity maxima in the form of concentric rings in a cross section perpendicular to the optical axis, the largest intensity being located on the optical axis. If one now disregards segments of the annular illumination light, in particular symmetrically, one speaks of a "sectioned Bessel beam." For example, one could use an annular diaphragm which additionally covers two peripheral areas lying opposite one another with mirror symmetry The light then passes only through the two intermediate circumferential regions of the annular diaphragm The focus of such a "sectioned Bessel beam" is not rotationally symmetrical in cross-section perpendicular to the optical axis. Rather, in such a "sectioned Bessel beam" parts of the originally annular intensity maxima are hidden, so that adjacent curved, aligned to the optical axis curved intensity maxima in the form of ring segments or "sections" remain.

By laterally displacing, for example using a deflection angle adjustable beam deflector, by a fraction of the period of the grated sectioned beam (eg, one third of the grating period), multiple images (second three images) can be successively generated and image data respectively can be obtained from which a total image can be calculated with a higher contrast. As an alternative to the superimposition of two or more illumination patterns oriented perpendicular to one another, a similar structure can be produced by interfering with two mutually running sectioned-Bessel beams. In that regard, such an embodiment of the method according to the invention for achieving an increase in resolution and / or to achieve a contrast enhancement is particularly advantageous. The main advantage of the Sectioned-Bessel rays is that a grid can be created by superimposing only two rays from opposite directions. So you only need two mirror caps or lenses. The possibility of moving the grating also laterally by means of a scan mirror is only necessary if the width of the image field is greater than that of the sectioned-beam beam.

If the illumination pattern and the at least one further illumination pattern are to coherently interfere, it is imperative to ensure that the partial illumination light beams that are brought into interference have the same polarization. When using deflecting mirrors as deflecting here is to be noted that the reflection Linearpolarisationsrichtung the partial illumination light beam is changed when the incident on the mirror Teilbeleuchfungsfundbündel has a parallel to the plane of incidence aligned linear polarization. In contrast, the linear polarization direction is not changed when the respective incident partial illumination light beam has a linear polarization direction which is oriented perpendicular to the plane of incidence.

If the illumination pattern and the at least one further illumination pattern are to be superimposed incoherently, this can be achieved, for example, by having different polarizations at the location of the superposition in the illumination plane. For example, two perpendicularly aligned illumination patterns, which are generated by four partial illumination light beams (two each), are incoherently superimposed, the partial illumination light beams being equally polarized in the entrance pupil of the illumination objective, and after passing through the illumination objective each being guided by one of four deflection mirrors aligned in a square, in particular by 90 degrees, are deflected into the illumination plane, since they have, because of the described effects with respect to the linear polarization in the reflection, after deflecting in the illumination plane mutually perpendicular linear polarization directions.

If an incoherent superimposition of the illumination pattern is desired, it is also possible to illuminate in a temporally pulsed manner, wherein the pulse length and the repetition rate and the temporal offset of the light pulses of the illumination pattern relative to the light pulses of the further illumination pattern are selected such that the illumination pattern and the further Illumination patterns arise alternately at different times in the illumination plane.

Alternatively, to produce an incoherent superposition, it is also possible to generate the light for the illumination pattern and for the further illumination pattern respectively with different light sources, because the light of independent light sources per se has no coherence.

Another possibility for generating an incoherent superimposition is the light path of the partial illumination light beam and the light path of the other Design partial illumination light beam such that the optical path lengths differ from each other by more than the coherence length. For example, the illumination light for the illumination pattern could be coupled directly into the sample volume, while the light for the further illumination pattern is guided by a long glass fiber that is longer than the coherence length, or vice versa.

Alternatively, it is also possible to couple the light for generating the illumination pattern and the light for generating the further illumination pattern in each case in a polarization-maintaining glass fiber, which are rotated against each other or not rotated against each other. The light of the light source is coupled into four fibers, which in turn couple the light into the pupil of the illumination objective. The fibers are arranged in pairs opposite each other and thereby rotated so that the emitted light is polarized in the same direction. With this arrangement, it is easily possible to switch between coherent superimposition of the crossed gratings and incoherent superposition by rotating the individual fibers of one pair of opposing fibers 90 degrees about their own axis (and only one end opposite to the other) Fiber).

As a further alternative, to generate an incoherent overlay, it is also possible to use a modulator, for example an AOM (Acusto Optical Mudulator) or an AOD (Acusto Optical Deflector), in order to manipulate the frequency of the illumination light of the sub-illuminating light bundle so that it stops interfere with the unmanipulated light of the further partial illumination light beams. The modulator should be able to shift the frequency of the light, for example by photon-photon interaction.

If, by contrast, a coherent superimposition is desired, the linear polarization direction of the two partial illumination light beams of one of the illumination patterns can be rotated, for example by means of a lambda / 2 plate. In a particularly advantageous embodiment, a phase plate with four quadrants is used for this purpose, wherein two diagonally opposite quadrants do not influence the linear polarization direction, while the other two diagonally opposite quadrants each as lambda / 2 phase plates are executed. For example, the illuminating pattern-generating partial illuminating light bundles can each extend through one of two diagonally opposite quadrants, while the further illuminating pattern-generating further partial illuminating light bundles each extend through one of the two other diagonally opposite quadrants.

The phase plate can advantageously be arranged in a plane conjugate to the focal plane of the illumination objective. As an alternative to a phase plate, it is also possible to influence the polarization by means of active optical components, such as an SLM (spatial light modulator), in particular a ferroelectric SLM, or, for example, by means of an LcoS component ("liquid crystal on silicon" component) The use of components of this kind has the particular advantage that the polarization can be changed in a location-dependent and individually controlled manner In this way, a change in the illumination pattern and / or the at least one further illumination pattern can advantageously be achieved simply and quickly and reliably and / or quickly be switched from a coherent superimposition of the illumination pattern to an incoherent superposition of the illumination pattern.

As already explained above, the image acquisition of several images can each take place with structured illumination with the illumination pattern and / or the further illumination pattern by successive shifting of the respective illumination pattern in the illumination plane. For this purpose, the relative phase of the illumination pattern generating partial illumination light beam or the further illumination pattern generating further

Part illumination light beam to be changed. This is possible, for example, by changing the length of the light path of a subset of the respectively interfering partial illumination light bundles or further partial illumination light bundles. A change in the length of the light path can be achieved, for example, by means of an active mirror, which can be displaced, for example, with a piezoelectric element. Alternatively or additionally, the use of an acousto-optical element is possible. As a further alternative, it is also possible to change the light path, optionally different optical elements with different thickness and / or different optical density in to introduce the beam path. It is also possible to use spatial light modulators (SLM) or digital mirror devices (DMD) or deformable mirrors (DM) to change the light path. For example, the splitting means may comprise a simple beam splitter or a polarization beam splitter. In a particular embodiment, the splitting agent has a phase grating. Such a design is particularly advantageous because a largely symmetrical beam path can be achieved for the different partial illumination light bundles.

The deflection means may for example comprise one or more mirrors (deflecting mirrors) as deflection elements. In particular, the mirrors may be flat. However, it is also possible that one or more mirrors are curved. It is, alternatively or additionally, also possible that the deflection means comprises at least one prism.

The device according to the invention can advantageously have a deflecting means which deflects the illuminating light bundle after it has passed through the illuminating objective in such a way that the illuminating light bundle extends in the illumination plane. In particular, the deflection means may be arranged such that the deflected illumination light beam has an angle different from zero degrees to the optical axis of the illumination objective and / or the detection objective. In particular, the illumination plane can be aligned perpendicular to the optical axis of the illumination objective.

In particular, it can advantageously be provided that the illumination light bundle, in particular formed into a light sheet, initially runs in the vertical direction through the illumination objective and is then deflected in the horizontal direction with the deflection device in order to illuminate the sample in the sample plane along the illumination strip. The detection light emanating from the illumination strip, in particular fluorescent light, preferably runs in the vertical direction through a detection objective. Such a construction allows the use of upright or inverse standard microscope stands for the production of the optical device according to the invention. In a particularly advantageous embodiment, it is provided that the illumination objective and the deflection device, which may, for example, have one or more deflection mirrors, are arranged so as to be movable relative to one another in order to be able to move the illumination light bundle relative to the sample. Alternatively or additionally, it can also be provided that the deflection device, in particular movably, is fastened and / or arranged on the detection objective or the illumination objective.

The illumination pattern and / or the further illumination pattern for fluorescence excitation of the sample in the sample plane is preferably used. Alternatively or additionally, the sample plane is simultaneously subjected to homogeneous de-excitation light.

Most preferably, the illumination pattern is used to switch fluorescent dyes.

In principle, the lattice illumination can also be used in a particularly favorable manner for RESOLFT-like effects (for example STED, stimulated emission depletion or for GSD, ground-state depletion). The difference to structured illumination for fluorescence excitation is that a structured or unstructured illumination for fluorescence excitation is superimposed more or less at the same time (e.g., offset in GatedSTED) with further structured illumination (at a different wavelength) in the sample. For example, a "common" eg blue light sheet could be irradiated into the sample and superimposed with eg a red "grid", where the red grid would cause the fluorophores to be either stimulated to be de-excited or held in a dark state or by conformational change into one can not be transferred to the stimulable state of the blue light sheet, whereby the emission of fluorescence in the direction of the detector is always effectively suppressed by the 'red' illumination.

According to a variant, the excitation light may also have a gifter form for this purpose. The maxima of the excitation would then have to be in the minima of the excitement. The problem here is that the light beams have different wavelengths and therefore does not adjust the same grating period when the grid be generated in the same way. As a solution, therefore, it is appropriate to extend the period of the lattice having the shorter period in order to match the period of the longer lattice. This is possible by tilting the two opposing light beams which are to produce the grating against one another within the illuminated plane (ie not counter-rotating at an angle of 180 ° but, for example, counter-rotating at an angle of 160 °).

It should be pointed out at this point that the concepts described here with respect to the switchability of the fluorescent dyes and / or the concepts around the RESOLFT, STED, GatedSTED and / or GSD on the one hand in connection with the present invention and on the other hand also detached from the concrete combination of features present invention according to the originally formulated independent claims can be realized. In particular, in the latter case, it is not necessary for the partial illumination light beam to be deflected by an illumination objective common to both partial illumination light bundles and / or for at least one of the partial illumination light bundles to be deflected with at least one deflection means such that the partial illumination light bundles are deflected after passing through the illumination objective interfere with each other in an illumination plane, so that a lighting pattern is created in the illumination plane.

A device which is suitable for carrying out the method according to the invention can advantageously be constructed on the basis of a scanning microscope, in particular a confocal scanning microscope. In particular, the use of an inverted microscope stand is suitable. Of particular advantage in this respect is the use of a (possibly-present in a laboratory anyway) grid microscope for carrying out the method according to the invention.

In the drawing, the subject invention is exemplified and shown schematically and will be described with reference to the figures below, wherein the same or equivalent elements are usually provided with the same reference numerals. Showing:

1 shows an embodiment of a detail of a device according to the invention, 2 schematically shows the generation of an illumination pattern in a device according to the invention designed as a SPIM microscope,

3 schematically shows the generation of the superimposition of an illumination pattern and a further illumination pattern in a device according to the invention designed as a SPIM microscope,

4 schematically shows the generation of the superimposition of an illumination pattern with three further illumination patterns in a device according to the invention designed as a SPIM microscope,

5 shows the light distribution in the superimposition of an illumination pattern and a further illumination pattern in a device according to the invention designed as a SPIM microscope,

6 shows the light distribution in the coherent superposition of a lighting pattern and a further illumination pattern,

7 shows the light distribution in the case of the incoherent superimposition of a lighting pattern and of a further illumination pattern,

8 shows the interference pattern of two of the counter-propagating Sectioned Bessel beams, FIG. 9 shows schematically the generation of an illumination pattern in a device according to the invention designed as a TIRF microscope, and FIG

Fig. 10 is a detail view of the device shown in Fig. 9. Fig. 1 shows an embodiment of a detail of a device according to the invention. The device includes a splitting means 1, which spatially splits an illuminating light beam 2 into two partial illuminating light bundles 3, 4. The partial illumination light beams 3, 4 pass through a lens 5 which focuses the partial illumination light beams 3, 4 and a tube lens 6 which collimates the partial illumination light beams 3, 4 and in the opposite direction tilted the optical axis in the entrance pupil of a lighting objective 7 couples.

After passing through the illumination objective 7, the partial illumination light bundles 3, 4 respectively strike one of two deflecting mirrors 9, 10 of a deflecting means arranged opposite one another on a detection objective 8, which deflects the partial illumination light bundles 3, 4 in such a way that the partial illumination light bundles 3, 4 in an illumination plane 1 1, which is also the focal plane of the detection lens 8, interfere with each other, so that in the illumination plane 1 1 creates a lighting pattern.

2 schematically shows the generation of an illumination pattern 12 in a sample volume 34 in a device according to the invention designed as a SPIM microscope. The illumination pattern 12 is formed in the illumination plane 1 1 by interference of two partial illumination light beams, which are deflected by opposite deflection elements, namely deflecting mirrors 9, 10, after passing through an illumination objective 7, so that they propagate in the illumination plane with opposite propagation directions. The vertical lines between the deflecting mirrors show, by way of example, the positions of the maxima / minima of a forming standing wave for two counter-rotating beam bundles with a planar phase front, as they are known for SPIM microscopes usual illumination beams, also called light sheets, to a good approximation.

3 schematically shows the generation of the superimposition of an illumination pattern 12 and of a further illumination pattern 13 in a device according to the invention designed as a SPIM microscope. The illumination pattern 12 is formed in the illumination plane 1 1 by interference of two partial illumination light bundles, which are deflected by mutually opposite deflection elements, namely deflecting mirrors 9, 10, after passing through an illumination objective. The further illumination pattern 13 is formed in the illumination plane 1 1 by interference of two further partial illumination light beams, which are deflected by opposing further deflection elements, namely further deflection mirrors 14, 15, after passing through an illumination objective. Due to the superimposition and interference of the illumination pattern 12 with the further illumination pattern 13, a dot grid 16 arises in the center. 4 schematically shows the generation of the superimposition of an illumination pattern 12 with three further illumination patterns 13, 17, 18 in a device according to the invention designed as a SPIM microscope. It should be emphasized at this point that Fig. 4 is not necessarily an illustration of a real deflection means, but rather also shows the possibility of temporal integration, which can be implemented, for example, by using a rotatable deflection means: The in the figure shown deflecting mirror can be permanently installed, but they must not all be present in the structure. In particular, a movable pair of deflecting mirrors 9, 10 may be provided rotatable so that it can successively occupy the positions of the deflecting mirrors 9, 10 and then 21, 22 and then 14, 15 and then 19, 20 or any number of any other positions. Ideally, a strip-shaped illumination pattern 12 is thereby rotated twice by angles of 60 degrees in order to achieve an isotropic increase in resolution in the plane 11. This happens during the exposure of a single image of the camera.

If several mirrors are permanently installed and their grids overlap, this leads to complex lighting patterns. These would then enable structured illumination of higher orders. If the illumination pattern is not a regular sine, then you have to move the grid more than two times and take more than three pictures, but also get a higher resolution.

The illumination pattern 12 is formed in the illumination plane 1 1 by interference of two partial illumination light bundles, which are deflected by mutually opposite deflection elements, namely deflecting mirrors 9, 10, after passing through an illumination objective. The further illumination pattern 13 is formed in the illumination plane 11 by interference between two further partial illumination light beams, which are deflected by opposing (further) deflection elements, namely further deflection mirrors 14, 15, after passing through an illumination objective

The further illumination pattern 17 is formed in the illumination plane 1 1 by interference of two partial illumination light bundle, the opposing deflection elements, namely deflecting mirrors 19, 20 (or Deflection mirrors 9, 1 0 which were rotated to the position of 19, 20) are deflected after passing through a lighting objective. The further illumination pattern 18 is formed in the illumination plane 11 by interference between two further partial illumination light beams, which are deflected by opposing further deflection elements, namely further deflection mirrors 21, 22, after passing through an illumination objective.

Due to the superimposition of the illumination pattern 12 and with the further illumination patterns 1 3, 1 7, 18, a dot grid 23 is formed in the center. A subgroup, e.g. of 12 and 1 3 or 1 7 and 18 are brought to overlay.

5 shows the light distribution in the superimposition of an illumination pattern and a further illumination pattern in a device according to the invention designed as a SPIM microscope.

Concretely, FIG. 5 shows in the upper line a lighting pattern just produced in the illumination in three different views. In the left-hand illustration, a view of the illumination plane 11 is seen looking along the optical axis of the detection objective (Z-direction). The other two diagrams show the illumination pattern with a viewing direction along the X direction (propagation direction of the rays of the first illumination pattern) and with a viewing direction along the Y direction perpendicular to X and Z (propagation direction of the rays of the second illumination pattern).

FIG. 5 shows in the middle row a further illumination pattern just generated in the illumination in the three different views as in the upper row. In the left-hand illustration, a view with a viewing direction along the optical axis of the detection objective (Z-direction) can be seen. The two other illustrations show the illumination pattern with a viewing direction along the X direction and with a viewing direction along the Y direction. The further illumination pattern is just rotated by 90 degrees in the illumination just relative to the illumination pattern shown in the upper line. Fig. 5 shows in the bottom line the result of the coherent superimposition of the Illumination pattern with the further illumination pattern in the three different views as in the upper line. In the left-hand illustration, a view is seen along the optical axis of the directional objective (Z-direction). The two other illustrations show the illumination pattern with a viewing direction along the X direction and with a viewing direction along the Y direction.

FIG. 6 shows an enlarged view of the light distribution of the coherent superposition of the illumination pattern with the further illumination pattern, as already illustrated in the lower line of FIG.

FIG. 7 shows the light distribution which arises during the incoherent superimposition of the illumination pattern and the further illumination pattern. Fig. 8 shows the interference pattern of two mutually opposed Sectioned Bessel beams. For example, by changing the phase and lateral position of the overlapping Sectioned Bessel beams, the interference pattern can be successively changed to generate multiple images, from which image data can be computed to yield a higher contrast, higher resolution image.

9 shows schematically the generation of an illumination pattern in a device according to the invention designed as a TIRF microscope. The device has an illumination objective 7 and a detection objective 8, between which a sample 24 to be examined is arranged in an examination position. The sample 24 is in contact with an optically transparent medium 25, which has a higher refractive index than the sample 24. The sample may also be prepared on a cover glass, which is in contact with the optically transparent medium 25, wherein in the space an immersion medium can be inserted to minimize jumps in the refractive index.

Two partial illumination light bundles 3, 4 pass over a beam deflection device 26, which can be adjusted with respect to the deflection angle, a scan lens 27 and a Tubus lens 28 to the illumination lens 7, pass through this and are then deflected by a deflection means 29 which has two deflecting elements 30, 31 and is attached to the detection objective 8. The deflection means 29 has a first deflection element 30 and a second deflection element 31, wherein the optically transparent medium 25 (in addition to its function of providing a flat boundary surface to the sample 24) also forms a further deflection element. The deflection elements 30, 31 and the entrance windows of the optically transparent medium 25 redirect the partial illumination light bundles 3, 4 successively with deflection angles of different sign and different amounts.

At the interface 32 between the optically transparent medium 25 and the sample 24, the interfering Teilbeleuchtungslichtbundel 3, 4 for evanescent illumination of the sample 24 are totally reflected. The evanescent field 33 protrudes into the sample 24 and leads there to an optical excitation of fluorescent dyes.

The fluorescent light emanating from the sample 24 passes through the detection objective 8 and is focused on the active surface of a spatially resolving detector (not shown), which is embodied as an area detector, for example.

The beam deflection device 26 has a gimbal-mounted mirror, which makes it possible for the partial illumination light bundles 3, 4 to be able to deflect in two mutually perpendicular planes with any desired deflection angle. In this way, the spatial position and the propagation direction of the emerging from the illumination lens 7 Teillichtuchtungslichtbundel 3, 4 can be adjusted largely freely. In this way, it is possible, for example, to direct the partial illumination light bundles 3, 4, instead of the deflection elements 30, 31, to another pair of deflection means, for example to change the orientation of the illumination pattern.

As an alternative to a gimbal-mounted mirror 26, the beam deflection device could, for example, at least two optically in series switched baffles, in particular deflecting mirror, which can divert the partial illumination light beams 3, 4 in mutually perpendicular Ablenkebenen. The partial illumination light beams 3, 4 are generated from an illumination light beam 2 by means of a splitting means 1, which may be formed, for example, as a grating.

Alternatively or additionally, it is also conceivable, for example, for the splitting means 1 to be rotatable or gimbal-mounted about an axis and for the rotatability of the beam deflecting device 26 to be restricted to only one axis, as long as the free alignability of the partial illuminating light bundle remains ensured.

FIG. 10 shows a detail view of the sample area of the device illustrated in FIG. 9 and in particular the light paths of the interfering partial illumination light bundles 3, 4 in the region of the sample 24.

Finally, it should be particularly noted that the embodiments discussed above are merely for the purpose of describing the claimed teaching, but do not limit it to the exemplary embodiments. In particular, all features contained in this description and / or their functions, effects and properties are to be considered in isolation and / or in combination with each other as disclosed herein, which one skilled in the art may, individually or in combination thereof, possibly taking into account his or her expertise to solve the objective task or related problems.

LIST OF REFERENCES:

1 break-up m † el

2 illumination light bundles

3 partial illumination light bundle 4 partial illumination light bundle

5 lenses

6 tube lens

7 illumination lens

8 detection objective

9 deflection mirror

10 deflecting mirrors

1 1 lighting level

12 lighting patterns

13 further illumination pattern 14 further deflecting mirror

15 further deflection mirrors

1 6 point grid

1 7 further lighting pattern

18 further illumination pattern 19 further deflecting mirror

20 further deflection mirrors

21 further deflection mirrors

22 further deflection mirrors

23 point grid

24 sample

25 optically transparent medium

26 beam deflector

27 scan lens

28 tube lens

29 deflection

30 deflecting element

31 deflecting element

32 interface

33 evanescent field

34 sample volumes

Claims

claims

1 . Method for examining a sample (24), in particular in light-sheet microscopy, characterized by the following steps: a. Generating an illumination light beam (2) with a light source, b. spatial splitting of the illumination light beam (2) with a splitting means (1) into at least two partial illumination light bundles (3, 4), c. Directing the partial illumination light bundles (3, 4) through an illumination objective (7) common to both partial illumination light bundles (3, 4), d. Redirecting at least one of the partial illumination light bundles (3, 4), after having passed through the illumination objective (7), with at least one deflecting means (27) such that the partial illumination light bundles (3, 4) in an illumination plane (1 1) interfere with each other, so that in the illumination plane (1 1) creates a lighting pattern (12), and e. Generating an image of a sample area illuminated by the illumination pattern (12), wherein detection light emanating from the sample area passes through a detection objective (8) to a spatially resolving detector.

2. The method according to claim 1, characterized in that in the illumination plane (1 1) successively different and / or differently positioned and / or differently oriented illumination pattern (12), 13) are generated and thereby in each case at least one image of the illumination pattern ( 12,13) illuminated sample area is generated.

3. The method according to claim 2, characterized in that the successively generated images are offset against each other and / or that the successively generated images are offset against each other to an over-resolved and / or contrast enhanced image.

4. The method according to claim 2 or 3, characterized in that different and / or differently positioned and / or differently oriented illumination pattern (12, 13) by a. Changing the phase of the partial illumination light beams (3, 4) are generated and / or by b. Changing the direction of incidence of the interfering partial illumination light bundles (3, 4) and / or by c. Changing the angle of incidence of the interfering partial illumination light beams (3, 4) on the illumination plane (1 1) are generated.

5. The method according to any one of claims 1 to 4, characterized in that a. at least two further partial illumination light beams are generated from the illumination light bundle (2), which interfere with one another in the illumination plane, so that a further illumination pattern (13) arises in the illumination plane (1 1), or in that b. at least two further partial illumination light beams are generated by means of a further light source, which interfere with one another in the illumination plane (1 1), so that a further illumination pattern (13) is produced in the illumination plane (11).

6. The method according to claim 5, characterized in that a. the illumination pattern (12) and the further illumination pattern (13) in the illumination plane (1 1) are coherently superimposed or that b. the illumination pattern (12) and the further illumination pattern (13) in the illumination plane (1 1) are superimposed incoherently.

7. The method according to any one of claims 1 to 6, characterized in that a. the interfering partial illumination light bundles (3, 4) and / or the further interfering partial illumination light beams are designed as Bessel beams and / or that b. the interfering partial illumination light beams (3, 4) and / or the further interfering partial illumination light beams are designed as sectioned Bessel beams.

8. The method according to any one of claims 1 to 7, characterized in that a. each partial illumination light beam (3, 4) and / or each further partial illumination light beam is designed as a light sheet, and / or that b. each partial illumination light beam (3) and / or each further partial illumination light beam (4) is focused into the entrance pupil of the illumination objective (7), and / or that c. the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles are focused at different angles of incidence and / or at different positions into the entrance pupil of the illumination objective (7).

9. The method according to any one of claims 1 to 8, characterized in that a. propagate the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles after deflection within the illumination plane (1 1) with different propagation directions, and / or that b. propagate two partial illumination light bundles (3, 4) and / or two further partial illumination light bundles after deflecting within the illumination plane (1 1) with mutually opposite directions of propagation, and / or that c. the illumination plane (1 1) has an angle different from zero degrees relative to the optical axis of the illumination objective (7), and / or that d. the illumination plane (1 1) is aligned perpendicular to the optical axis of the illumination objective (7), and / or that e. the illumination plane (1 1) has an angle different from zero degrees relative to the optical axis of the detection objective (8), and / or f. the illumination plane (1 1) is aligned perpendicular to the optical axis of the detection objective (8).

Method according to one of claims 1 to 7, characterized in that the sample (24) is in contact with an optically transparent medium (25) which has a higher refractive index than the sample (24) and that the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles interfere on the interface between the optically transparent medium (25) and the sample (24) for generating the illumination pattern (12) and are totally reflected there for evanescent illumination of the sample (24).

Method according to one of claims 1 to 10, characterized in that a. the deflecting means (29) or at least one of a plurality of deflecting elements of the deflecting means (29) are arranged and / or fastened to the detection objective (8) or to the illumination objective (7) - in particular rotatable, and / or that b. the deflection means (29) has a plurality of deflection elements which are oriented differently, and / or that c. and that each partial illumination light beam (3, 4) and / or further partial illumination light beam is deflected in each case by means of its own deflection element.

Method according to one of claims 1 to 1 1, characterized in that the deflection means (29) has at least one deflection mirror (9) and / or that at least one of a plurality of deflection elements of the deflection means (29) as a deflection mirror (9, 10) is formed.

Method according to one of claims 1 to 12, characterized in that the optical axis of the illumination objective (7) and the optical axis of the detection objective (8) are mutually aligned (anti) parallel or coaxial and / or that the detection objective (8) and the illumination lens (7) are opposed to each other and aligned opposite each other.

14. The method according to any one of claims 1 to 13, characterized in that the illumination pattern (12) and / or the further illumination pattern (13) for fluorescence excitation of the sample (24) are used in the sample plane and that the sample plane simultaneously applied uniformly with de-excitation light becomes.

15. The method according to any one of claims 1 to 13, characterized in that the illumination pattern (12) is used for switching of fluorescent dyes.

16. A device for examining a sample (24), in particular in light-sheet microscopy, comprising: a. a light source that generates an illumination light beam (2), b. a splitting means which spatially splits the illuminating light beam (2) into at least two partial illuminating light bundles (3, 4), c. an illumination objective (7) which is traversed by the sub-illumination light beams (3, 4), d. at least one deflection means (29) which deflects at least one of the partial illumination light bundles (3, 4) after they have passed through the illumination objective (7) in such a way that the partial illumination light bundles (3, 4) interfere with one another in an illumination plane (11) that in the illumination plane (1 1) a lighting pattern (12) is formed, and e. a spatially resolving detector for generating an image of a sample area illuminated by the illumination pattern (12), wherein detection light emanating from the sample area passes through a detection objective (8) to a spatially resolving detector.

17. The device according to claim 16, characterized in that in the illumination plane (1 1) successively different and / or differently positioned and / or differently oriented illumination patterns (12, 13) can be generated, and in each case at least one image of the illumination pattern (12 , 13) lit sample area can be generated, wherein a computational the successively generated mappings offset with each other and / or wherein a calculation device, the consecutively generated images offset each other to an over-resolved and / or contrast enhanced image.

18. Device according to claim 17 or 18, characterized by a. a phase changing means for changing the phase of the partial illumination light beams (3, 4), and / or by b. at least one beam deflecting device (26) adjustable with regard to the deflection angle for changing the position and / or the alignment of at least one of the partial illuminating light bundles (3, 4).

19. Device according to one of claims 16 to 18, characterized in that a. the splitting means (1) splits off two further partial illumination light bundles which interfere with one another in the illumination plane (1 1), so that a further illumination pattern (13) arises in an illumination plane (1 1), or in that b. a further light source generates two further partial illumination light bundles, which interfere with one another in the illumination plane (1 1), so that a further illumination pattern (13) is formed in an illumination plane (1 1).

20. The apparatus according to claim 19, characterized in that a. the illumination pattern (12) and the further illumination pattern (13) in the illumination plane (1 1) are coherently superimposed or that b. the illumination pattern (12) and the further illumination pattern (13) overlap incoherently in the illumination plane (1 1).

21. Device according to one of claims 16 to 20, characterized in that α. the interfering Teillichtuchtungslichtbundel (3, 4) and / or the other interfering Teillichtuchtungslichtbundel are designed as Bessel beams and / or that b. the interfering Teilbeleuchtungslichtbundel (3, 4) and / or the other interfering Teillichtuchtungslichtbundel are formed as Sectioned Bessel beams.

22. Device according to one of claims 16 to 21, characterized in that a. at least one astigmatic optic for forming the illumination light beam (2) and / or the partial illumination light beam (3, 4) is present in each case as a light sheet, and / or that b. an optic is present, which focuses each partial illuminating light bundle (3, 4) and / or each further partial illuminating light bundle into the entrance pupil of the illumination objective (7), and / or that c. at least one optical system is present, which focuses the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles at different angles of incidence and / or at different positions into the entrance pupil of the illumination objective (7).

23. Device according to one of claims 1 6 to 22, characterized in that a. propagate the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles after deflection within the illumination plane (1 1) with different propagation directions, and / or that b. propagate two partial illumination light bundles (3, 4) and / or two further partial illumination light bundles after deflecting within the illumination plane (1 1) with mutually opposite directions of propagation, and / or that c. the illumination plane (1 1) has an angle different from zero degrees relative to the optical axis of the illumination objective (7), and / or d. the illumination plane (1 1) is aligned perpendicular to the optical axis of the illumination objective (7), and / or that e. the illumination plane (1 1) has an angle different from zero degrees relative to the optical axis of the detection objective (8), and / or that f. the illumination plane (1 1) is aligned perpendicular to the optical axis of the detection objective (8).

24. Device according to one of claims 16 to 23, characterized in that the sample (24) is in contact with an optically transparent medium (25) which has a higher refractive index than the sample (24) and that the partial illumination light bundles (3, 4) and / or the further partial illumination light bundles on the interface between the optically transparent medium (25) and the sample (24) for generating the illumination pattern (12) interfere and are totally reflected there for evanescent illumination of the sample (24).

25. Device according to one of claims 16 to 24, characterized in that a. the deflection means (29) or at least one of a plurality of deflection elements of the deflection means (29) are arranged and / or fixed to the detection objective (8) or to the illumination objective (7), and / or that b. the deflection means (29) has a plurality of deflection elements which are oriented differently, and / or that c. and that each partial illumination light beam (3, 4) and / or further partial illumination light beam is deflected in each case by means of its own deflection element.

26. The device according to one of claims 16 to 25, characterized in that the deflection means (29) has at least one deflection mirror (9) and / or that at least one of a plurality of deflection elements of the deflection means (29) as a deflection mirror (9, 10) is formed , Device according to one of claims 16 to 26, characterized in that the optical axis of the illumination objective (7) and the optical axis of the detection objective (8) are aligned parallel or coaxial with each other and / or that the detection objective (8) and the illumination objective (7 ) are opposed to each other and aligned opposite each other.