DE102007017598A1 - Method and arrangement for positioning a light sheet in the focal plane of a detection optical system - Google Patents

Abstract

The invention relates to a method and an arrangement for positioning a light sheet (10) in the focal plane (7) in the examination of a sample by the method of selective plane illumination microscopy (SPIM). The following method steps are provided according to the invention: imaging a second focal plane (17) enclosing an angle 0 ° <alpha <90 ° with the light sheet (10) on a spatially resolving detector (15), determining the actual position of the maximum contrast or the maximum Intensity in the image of the second focal plane (17), wherein the actual position corresponds to the intersection of the light sheet (10) with the second focal plane (17), - Determining the distance a 'between the actual position and the desired position of the maximum Contrast or the maximum intensity in this figure, wherein the desired position corresponds to the intersection of the two focal planes (7, 17), and - moving the light sheet (10) or the detection optics (1) until the distance a 'between the actual Position and the target position is zero.

Description

The The invention relates to a method for positioning the a light beam shaped beam of light in the investigation a sample according to the method of selective-plane illumination microscopy (SPIM). The invention further relates to an arrangement to practice this procedure.

in the Difference to Confocal Laser Scanning Microscopy (LSM), at a three-dimensional sample in single, different depths Levels is scanned point by point and the gained Image information following a three-dimensional image the sample is assembled, the SPIM technology is based on the wide-field microscopy and allows the visual representation the sample based on optical sections by individual Levels of the sample.

The Advantages of SPIM technology exist among others in the larger one The speed with which the image information is captured, the lower risk of fading of biological samples as well in the possibility of the lateral resolution of the Set the image independently of the depth resolution to be able to.

in principle become in the SPIM technology fluorophores, which in itself in the Sample are included or were introduced for contrasting in the sample, excited with laser light, wherein the laser radiation to a so-called "light sheet" is shaped. With the light sheet each becomes a selected level illuminated in the depth of the sample and with an imaging optics obtained an image of this sample plane in the form of an optical section. The light sheet can also by fast back and forth of a thin, rotationally symmetrical laser beam in the focal plane The imaging optics are modeled, which essentially to Excitation with a static light sheet is equivalent.

An example of the SPIM method is off WO 2004/053558 A1 known. In this case, the sample to be examined, for example a medaka embryo, is embedded in an aqueous gel in which it continues to live for a certain time, but is virtually unable to move.

Around To illustrate the geometry of the light sheet is related assumed with the present invention that the light sheet is a vertical to the optical axis of the detection optics in the coordinates X and Y extended length and width adapted to the object field and one in the direction of the optical axis of the detection optics or in the coordinate direction Z has extending thickness in the Range of 0.5 microns to 20 microns. It is usually the thickness does not exceed the entire length constant, but has a central, called beam waist Constriction on.

One recurrent practical problem in the investigation of Samples according to the SPIM method is that the light sheet as possible must be brought exactly into the focal plane of the detection optics, for optimal results when imaging the illuminated sample plane and to achieve the subsequent evaluation of this sample level. A key reason for the resulting difficulties is that the samples are typically enclosed by a gel and together with this gel from an aqueous medium are surrounded.

there the space between the detection optics and the sample can be complete filled with an aqueous medium and / or with gel be, or it remains an air-filled subspace between the detection optics and located within a sample chamber Sample. Depending on the use of the same detection optics arise due to the different refractive indices of gel, aqueous Medium and air position deviations for the focal plane.

in the In the prior art, the positioning of the light sheet in the focal plane of the detection optics so far by visual, subjective Assessment based on the contrast in the sample image. Whether there the optimal setting is achieved depends largely from the observer's perception. In addition, the handling time consuming and the process hardly reproducible.

From Based on this prior art, the invention has the object to provide a method and an arrangement with which the light sheet in an uncomplicated and time-saving manner reproducible and largely free from subjective influences in the Focusing plane of the detection optics can be brought.

• – eine objektseitige, mit der optischen Achse und dem Lichtblatt jeweils einen Winkel α im Bereich 0°< α < 90° einschließende zweite Fokusebene auf einen ortsauflösenden Detektor mit einer zur zweiten Fokusebene konjugierten Empfangsebene abgebildet wird,
• – der Kontrast oder die Intensität in dieser Abbildung bewertet wird, und
• – das Lichtblatt oder die Detektionsoptik in der Richtung der optischen Achse verschoben wird, bis die Bewertung ergibt, dass das Lichtblatt die erste Fokusebene überdeckt.

In a method for positioning the light sheet in the focal plane of the detection optics of the type described at the beginning, according to the invention, it is provided that:

• An object-side second focal plane enclosing in each case an angle α in the region 0 ° <α <90 ° with the optical axis and the light sheet is imaged on a spatially resolving detector having a reception plane conjugate to the second focal plane,
• - the contrast or intensity is rated in this figure, and
• - The light sheet or the detection optics is displaced in the direction of the optical axis until the evaluation shows that the light sheet the first focal plane covered.

• a) Ermitteln der Ist-Position des maximalen Kontrastes oder der maximalen Intensität in der Abbildung der zweiten Fokusebene, wobei die Ist-Position dem Schnittpunkt des Lichtblattes mit der zweiten Fokusebene entspricht,
• b) Bestimmen des Abstandes zwischen der Ist-Position und der Soll-Position des maximalen Kontrastes oder der maximalen Intensität in dieser Abbildung, wobei die Soll-Position dem Schnittpunkt der beiden Fokusebenen mit der optischen Achse entspricht, und
• c) Verschieben des Lichtblattes oder der Detektionsoptik in der Richtung der optischen Achse, bis der Abstand zwischen der Ist-Position und der Soll-Position gleich Null ist.

In a first embodiment of the method according to the invention, the following method steps are provided with regard to the evaluation of the contrast:

• a) determining the actual position of the maximum contrast or the maximum intensity in the image of the second focal plane, wherein the actual position corresponds to the intersection of the light sheet with the second focal plane,
• b) determining the distance between the actual position and the desired position of the maximum contrast or the maximum intensity in this figure, wherein the desired position corresponds to the intersection of the two focal planes with the optical axis, and
• c) shifting the light sheet or the detection optics in the direction of the optical axis until the distance between the actual position and the desired position is equal to zero.

In a second embodiment of the invention Method is provided, the light sheet a structure of a perpendicular to its propagation direction periodic intensity profile impose, and the thus structured light sheet in the Move the direction of the optical axis until the contrast in the image of the second focal plane has reached its maximum. The structuring of the light sheet is then again picked up to take pictures with a unstructured lighting to get from the sample.

at Both aforementioned embodiments may be provided that initially using a first spatially resolving detector whose Receiving plane is conjugated to the second focal plane, the light sheet is positioned in the first focal plane. Is that then Light sheet in the first focal plane, can the illuminated there level the sample for the purpose of observation and evaluation to another Spatial detector can be imaged, the one with has a receiving plane conjugate to the first focal plane.

There the first detector excluding the positioning or the alignment of the light sheet in the first focal plane, he does not have to take a high-quality picture. It is sufficient, For this purpose, a low-cost CMOS camera with relatively low Sensitivity or high noise factor. Possibly the intensity of the fluorescence radiation can be raised to compensate for disadvantages resulting from the low sensitivity can result.

When second detector used to image the illuminated sample plane for the purpose of observation and evaluation, should be a high Photosensitive CCD or EMCC camera can be used.

in the The scope of the method lies it, for positioning the light sheet emanating from the sample Fluorescence radiation of a first wavelength / a first Wavelength range to use, and to evaluate the with the light sheet illuminated plane of the sample one of the sample outgoing fluorescence radiation of a second wavelength / a second wavelength range to use.

The For example, it is achieved by adding a quantity of fluorescent beads to the sample from which, upon excitation with the light sheet, a first Subset the fluorescence radiation of the first wavelength / first wavelength range and a second subset the Fluorescence radiation of the second wavelength / of the second Wavelength range radiates.

In order to it is possible to use the method steps for positioning of the light sheet in the first focal plane using the first detector and the observation of the illuminated with the light sheet sample plane make simultaneous with the second detector. It's just to make sure that the first detector for the Fluorescence radiation of the one wavelength / the one wavelength range is sensitive and the second detector for fluorescence radiation the other wavelength / the first wavelength range.

at a particularly advantageous embodiment variant of the invention Procedure, only one detector is provided, both for positioning the light sheet in the first focal plane and for imaging the illuminated with the light sheet level of the sample is used.

there becomes the receiving level of this one detector first for positioning the light sheet aligned so that second focal plane is conjugate. Following the positioning process the receiving plane of the detector is aligned so that it is now is conjugated to the first focal plane. The orientation of the reception level can be advantageously changed by this pivoted about its intersection with the optical axis and so on their inclination to the optical axis changed becomes. Alternatively, the light sheet instead of the receiving level of the detector for the focusing operation to be tilted.

The inventive method further comprises an embodiment in which for the positioning of the light sheet alternatively fluorescent light is used, which emanates from the sample to be examined, or fluorescent light is used that of a reference sample starts, wherein after positioning of the light sheet in the first focal plane, the sample to be examined is placed at the location of the reference sample. The reference sample may be a homogeneously fluorescent aqueous solution, a gel interspersed with fluorescent substances or a plastic interspersed with fluorescent substances.

• – eine optische Einrichtung zum Abbilden einer objektseitigen, mit der optischen Achse und dem Lichtblatt jeweils einen Winkel α im Bereich 0° < α < 90° einschließenden zweiten Fokusebene auf die Empfangsebene eines ortsauflösenden Detektors,
• – eine Auswerteeinrichtung zur Bewertung des Kontrastes oder der Intensität in dieser Abbildung, und
• – eine Einrichtung zur Positionsänderung des Lichtblattes relativ zur ersten Fokusebene in Abhängigkeit von dieser Bewertung.

The invention furthermore relates to an arrangement for positioning the illumination beam shaped into a light sheet in an object-side first focal plane intersecting the optical axis of a detection optical system when investigating a sample according to the method of selective plane illumination microscopy (SPIM), comprising:

• An optical device for imaging an object-side, with the optical axis and the light sheet in each case an angle α in the range 0 ° <α <90 ° enclosing the second focal plane to the receiving plane of a spatially resolving detector,
• An evaluation device for evaluating the contrast or intensity in this figure, and
• - A device for changing the position of the light sheet relative to the first focal plane in dependence on this evaluation.

• – zum Ermitteln der Ist-Position des maximalen Kontrastes oder der maximalen Intensität in dieser Abbildung, wobei die Ist-Position dem Schnittpunkt des Lichtblattes mit der zweiten Fokusebene entspricht, und
• – zum Bestimmen des Abstandes zwischen der Ist-Position und der Soll-Position des maximalen Kontrastes oder der maximalen Intensität in dieser Abbildung, wobei die Soll-Position dem Schnittpunkt der beiden Fokusebenen mit der optischen Achse entspricht, und
• – zum Generieren von Stellbefehlen zum Verschieben des Lichtblattes in Abhängigkeit von diesem Abstand.

In a first embodiment of the arrangement according to the invention, the evaluation device is formed

• - For determining the actual position of the maximum contrast or the maximum intensity in this figure, wherein the actual position corresponds to the intersection of the light sheet with the second focal plane, and
• - For determining the distance between the actual position and the desired position of the maximum contrast or the maximum intensity in this figure, wherein the desired position corresponds to the intersection of the two focal planes with the optical axis, and
• - To generate setting commands for moving the light sheet as a function of this distance.

These first embodiment of the arrangement according to the invention is particularly to the execution of those described above first embodiment of the method according to the invention suitable.

The Device for changing the position of the light sheet is for its displacement in the direction of the optical axis of the detection optics, d. H. in the direction of the coordinate Z, provided, assuming is that the light sheet parallel to the first focal plane and thus is oriented perpendicular to the optical axis of the detection optics, However, the first focal plane not yet covered, so the sample plane in the first focus plane is not or insufficient for optimal observation is illuminated.

insofar becomes with the shift of the light sheet in the direction of the optical Axis that is a parallel shift of the light sheet, the position of the Light sheet continuously changed until the light sheet superimposed on the first focal plane.

optional may be the device for changing the position of the light sheet also for its displacement perpendicular to the optical axis of the detection optics, d. H. in the direction of the coordinate X and / or the coordinate Y, designed be. With this option the possibility arises, for example following the positioning in the first focal plane the light sheet to shift so that his beam waist in the image field center lies. This allows the illumination in the optimize the first focal plane located sample level so far, as the contrast or intensity maximum is now also in the center of the image.

The inventive arrangement can over have two detectors. One of the detectors has it a reception plane conjugate to the first focal plane and is for Imaging of illuminated by the light sheet sample plane provided. The other detector has a conjugate to the second focal plane Receiving level and serves as an aid to the positioning of the light sheet in the first focal plane. The detection beam path is in this case so branched that one of the branches to the receiving level of the one Detector and the second branch to the receiving level of the other Detector are directed.

The Method steps for positioning the light sheet in the first Focal plane, on the one hand, and the image of the one located in the first focal plane Sample level for the purpose of observation and evaluation be with this embodiment variant of the invention Arrangement made with independent detectors.

In In this context it can be provided that the sample when excited with the light sheet fluorescent light with wavelengths / wavelength ranges radiates for which the first detector is sensitive, and at the same time fluorescent light with wavelengths / wavelength ranges radiates, for which the second detector is sensitive, whereby It is possible to use the method steps for positioning of the light sheet and the observation of the illuminated with the light sheet To carry out the sample plane simultaneously.

Alternatively, but also an out design variant of the inventive arrangement proposed in which only one detector is present and this is coupled to a device for tilting its receiving plane at its intersection with the optical axis of the imaging beam path. This ensures that in an end position of the tilting of the receiving plane is conjugate to the second focal plane and the detector is available as an aid for positioning the light sheet to the second focal plane available, and in another end position of tilting the receiving plane to the first focal plane conjugate and the detector is usable for imaging and evaluation of illuminated with the light sheet sample plane.

Here Although only one detector is required, however, the use is this detector for positioning the light sheet on the one hand and for sample observation, on the other hand, only in temporal succession possible.

component The inventive arrangement can also be a Reference sample, which serves as an aid to positioning the light sheet in the first focal plane. It should be at suggestion with the Light sheet from the reference sample fluorescence radiation of the same Wavelength / same wavelength range go out, as with excitation of the sample to be examined with the light sheet. A The exception to this is of course for the design variant conceivable, in the case of two to different fluorescence radiation calibrated detectors are used, as described above.

When Reference sample can be a homogeneously fluorescent aqueous Solution, a interspersed with fluorescent substances Gel provided or interspersed with fluorescent substances plastic be.

at the version with plastic is for positioning the light sheet and thus for calibrating the inventive Arrangement of a reusable reference sample with large Shelf life available.

To the Purpose of spectral suppression of scattered excitation light and thus to increase the accuracy of the evaluation the contrast or the intensity of the positioning and also to increase the image quality of illuminated sample plane can in the detection beam path Before the reception levels fluorescence filters can be arranged, which only the fluorescent light intended for the respective detector let pass.

The spatially resolving detectors advantageously consist of individual, lying in the receiving level and in an array of lines and Columns arranged optoelectronic sensors whose signal outputs communicate with the evaluation device. In this regard, come into consideration for positioning the light sheet detectors, those with a number from 2 to 2000 sensor lines, preferably with a number of 20 to 200 sensor lines, two-dimensional images be able to record.

Of the Angle α, the image-side focal plane with the optical Axis of detection optics should include in the field between 20 ° and 70 °, preferably at 40 °.

Of the Structure of a second embodiment of the invention Arrangement, in particular for the implementation of the above described second embodiment of the invention Method is suitable, partially corresponds to the structure of the first Configuration of the arrangement. However, here is the use a reference sample provided in the form of a calibration chamber, the with a homogeneously fluorescent aqueous solution is filled, and in the illumination beam path are optical means provided, with which the light sheet a structure from a periodic Intensity profile is impressed. The periodic Intensity profile can be determined by introducing an amplitude or phase grating can be achieved in the illumination beam path.

In In this case, the evaluation unit for evaluation of the lattice contrast formed in the image of the second focal plane, and in dependence From the result of this evaluation, the light sheet is so far in the coordinate direction Z shifted until the grid contrast has reached its maximum. The maximum grid contrast arises when the structured one is Light sheet is located in the focal plane. The structured light sheet can also be simulated by a thinner, rotationally symmetric laser beam reciprocated and thereby periodically switched on and off at an even higher frequency becomes.

To The positioning is replaced by the calibration chamber observing sample. At the same time the optical means removed from the illumination beam path, with which the light sheet the periodic intensity profile was imprinted, so that this structure is lifted and the light sheet now a has homogeneous intensity distribution. Now the Level of the sample to be taken, located in the first focal plane located.

The Invention will be described below with reference to embodiments be explained in more detail. In the associated Drawings show

1 the principle of the arrangement according to the invention in an embodiment with two detectors,

2 a representation of the recorded with one of the detectors intensity distribution in the image of the second focal plane,

3 the principle of the arrangement according to the invention in an embodiment with a detector,

4 the principle of the arrangement according to the invention in an embodiment with a structured light sheet,

5 a representation of the recorded with one of the detectors intensity using the embodiment according to 4 ,

Out 1 First, the basic structure of a microscopic device for observing a sample by the method of selective plane illumination microscopy (SPIM) can be seen. These include as essential components a detection lens 1 , a fluorescence filter 2 , a tube optic 3 and a camera 4 , For example, a CCD camera. The camera 4 has a detector 5 on, consisting of individual, arranged in an array of rows and columns and a receiving level 6 forming photoelectric sensors, so the camera 4 is suitable for recording two-dimensional images. The array has, by way of example, 1000 to 5000 rows and as many columns, wherein advantageously only a portion of the rows or columns is used for the positioning process in order to speed up the process.

The focal plane 7 cuts the optical axis 8th of the detection lens 1 at a right angle. The reception level 6 is located in a focal plane 7 of the detection lens 1 optically conjugate position.

Is there a fluorescent substance in the focal plane? 7 and this is excited with an excitation radiation, for example, the wavelength of 488 nm, for fluorescence, is from the focal plane 7 outgoing fluorescence radiation from the detection objective 1 picked up and in the imaging beam path through the fluorescence filter 2 and the tube optics 3 through to the reception level 6 of the detector 5 directed.

The signal outputs of the optoelectronic sensors of the detector 5 are connected to an electronic image processing and evaluation device (not shown in the drawing).

With this arrangement, a two-dimensional image of itself in the focal plane 7 generated fluorescent substance and thus the observation of one in the focal plane 7 lying level of a particular biological sample allows.

The prerequisite for this, however, is exactly that in the focal plane 7 lying and to be imaged plane of the sample is illuminated with the excitation radiation. Typically, this excitation occurs in the SPIM technology with one to a light sheet 10 shaped illumination beam path.

This in 1 indicated light sheet 10 has one in the direction of the optical axis 8th or the coordinate Z measured thickness in the range between 0.5 .mu.m to 20 .mu.m. Its extension in the coordinate X, which lies in the plane of the drawing, and in the coordinate Y, which extends perpendicular to the plane of the drawing, corresponds to the extent of the field to be observed microscopically in the focal plane 7 ,

The problem insufficiently solved in the prior art to date is the light sheet 10 To bring as exactly as possible into a position in which the focal plane 7 from the light sheet 10 is completely covered.

As in 1 represented is the light sheet 10 although parallel to the focal plane 7 aligned, but does not cover them.

In this regard, the invention provides, in the imaging beam path between the detection lens 1 and the fluorescence filter 2 a beam splitter 11 , For example, a dichroic, which decouples a part of the fluorescent light forming the imaging radiation path and through a second fluorescence filter 12 and a second tube optic 13 through to a second camera 14 , here for example a CMOS camera, which directs a detector 15 which in turn consists of individual optoelectronic sensors arranged in rows and columns, which together form a receiving plane 16 form. The reception level 16 closes with the optical axis 8th the detection optics 1 an angle α, which is for example between 20 ° and 70 ° and should be 40 ° in the described embodiment.

The reception level 16 is located in a to a focal plane 17 optically conjugate position.

How out 1 can be seen cuts the focal plane in this constellation 17 also the light sheet 10 , this intersection being an amount a from the optical axis 8th the detection optics 1 is removed.

Will now be the focal plane 17 to the reception level 16 of the detector 15 the camera 14 shown, the results in 2a illustrated In intensity distribution on an exemplary taken out sensor line of the receiving level 16 , In the 2a to be recognized actual position of the intensity maximum 18 corresponds to the position of the intersection of the light sheet 10 with the focal plane 17 ,

In 2a is still the intersection of the two focal plane 7 and 17 with each other and with the optical axis 8th defined insofar as this position is assigned to specific optoelectronic sensors on this sensor line. This position is the nominal position of the intensity maximum. In this respect, the distance a 'in 2a proportional to the distance a in 1 and thus a measure of the filing of the light sheet 10 from the focal plane 7 ,

The signal outputs of the optoelectronic sensors of the detector 15 are located at an evaluation unit 19 in which the actual position of the intensity maximum is compared with its nominal position. The output of the evaluation unit 19 is with an adjusting device 20 coupled, which has control elements that move the light sheet 10 in the direction of the optical axis 8th or in the coordinate direction Z are formed (not shown in the drawing).

Returns the evaluation of the intensity distribution in 2a for example, the in 1 Drawn filing of the light sheet 10 to the focal plane 7 , are in the evaluation unit 19 Control commands generated and to the actuator 18 forwarded a parallel shift of the light sheet 10 to the focal plane 7 effect. Alternatively, the detection optics 1 be moved in the direction of its optical axis.

The continuous evaluation of this intensity distribution during this shift makes it possible to track the reduction of the distance a 'until, finally, the intensity maximum 18 is at its desired position, as in 2 B shown. This is the case when the light sheet 10 the focal plane 7 covered.

Thus, the object of the invention is achieved, namely the light sheet 10 independent of subjective influences of an observer to coincide with the focal plane 7 bring to.

In the arrangement according to 1 can be used to image the focal plane 17 to the reception level 16 the camera 15 the fluorescence radiation emanating from the sample to be examined.

It can be provided that the sample upon excitation with the light sheet 10 Fluorescent light emits at wavelengths or wavelength ranges for which the first detector is sensitive, and at the same time emits fluorescent light with wavelengths or wavelength ranges, for which the second detector is sensitive. So it is possible both cameras 5 . 15 to use simultaneously and the process steps for positioning the light sheet 10 and the observation of the light leaf 10 lighted sample plane simultaneously.

In an alternative variant of the invention, fluorescence radiation which starts from a reference sample can also be used for this purpose. In the illustration after 1 however, the use of a reference sample is provided which is in a calibration chamber 21 located. For this purpose, the calibration chamber 21 filled with a fluorescent substance, for example with a gel interspersed with fluorescence beads or with a plastic interspersed with fluorescent beads.

Such a reference sample is advantageously reusable, it has a long life, and the process steps in the positioning of the light sheet 10 in the focal plane 7 are highly reproducible with such a reference sample.

After positioning the light sheet 10 with the help of the calibration chamber 21 the sample to be examined is placed in its place, and it is possible to start the examination of the sample plane which is located in the focal plane 7 located.

3 shows an embodiment of the inventive arrangement, in which, in contrast to the arrangement according to 1 only the camera 4 present and these both for the positioning of the light sheet 10 in the focal plane 7 as well as for mapping and evaluation of each in the focal plane 7 provided sample level is provided.

For the sake of clarity, in 3 for assemblies already in 1 are shown, also the same reference numerals as in 1 used.

Unlike the arrangement after 1 is in the arrangement after 3 the camera 4 , For example, in turn, the CCD camera, pivotally arranged, in such a way that the angle, the receiving plane 6 the camera 4 with the optical axis 9 of the imaging beam path, is changeable in two end positions.

In a first end position is the reception level 6 so inclined that they have optical axis 9 encloses the angle α, so that it to the focal plane 17 is optically conjugated.

The camera is used in this constellation 4 for positioning the light sheet 10 in the focus bene 7 as based on 1 described. For this purpose, it is at least during the course of the process steps for positioning the light sheet 10 with the evaluation device 19 connected. The case by means of the camera 4 The resulting image again gives the intensity distribution in the focal plane 17 as in 2a shown. This intensity distribution is in the evaluation unit 19 evaluated, and it will be generated and commands to the actuator 20 transmitted by means of (not graphically illustrated) control elements, the displacement of the light sheet 10 in the direction of the optical axis 8th cause.

In an alternative structural embodiment of the arrangement according to the invention, the control commands can be used instead of the displacement of the light sheet 10 a shift in the detection optics 1 in the direction of the optical axis 8th to cause until the distance a '= 0 and is light sheet 10 and focal plane 7 cover.

After the positioning of the light sheet 10 is completed, is either manually, but preferably also in the evaluation 19 Generated positioning commands cause the camera 4 so tilted that their receiving level 6 the optical axis 9 the imaging beam path intersects at right angles, so that these now to the focal plane 7 is optically conjugated.

The camera is used in this orientation 4 for mapping each in the focal plane 7 level of a sample to be examined. As already described above, the positioning of the light sheet is also here 10 in the focal plane 7 alternatively based on the use of a reference sample in the calibration chamber 21 outgoing fluorescence radiation or based on the use of emanating from the sample to be examined fluorescence radiation possible.

In 4 the principle of an embodiment of the arrangement according to the invention is shown, the structure of which essentially according to the structure 1 equivalent. However, here is a calibration chamber 21 used, which is filled with a homogeneous fluorescent aqueous solution as a reference sample. In addition, optical means are provided in the illumination beam path with which the light sheet 10 a structure of a perpendicular to its propagation direction, which corresponds to the coordinate direction Y, a periodic intensity profile is impressed.

The means for producing such a structure are well known in the art. Thus, for example with a phase grating or an amplitude grating can be achieved that the light sheet 10 has alternating light and dark areas perpendicular to its propagation direction. The grating can be designed to be electronically controllable (eg LCD, DMD) or mechanically pivotable in and out.

Due to the inclination of the focal plane 17 becomes the structured light sheet 10 with the help of the camera 15 partially reproduced, as exemplified in 5a shown. By means of the evaluation unit 19 the lattice contrast is evaluated, and depending on the result of this evaluation, the light sheet 10 moved so far in the coordinate direction Z until the grid contrast has reached its maximum. The maximum lattice contrast arises exactly when the structured light sheet 10 in the focal plane 7 is located, bringing the positioning of the light sheet 10 is completed.

Now the calibration chamber 21 removed and placed in their place the sample to be observed. With the removal of the calibration chamber 21 At the same time, the phase or amplitude grating is swiveled out of the illumination beam path, so that the structure in the light sheet 10 is lifted and the light sheet 10 has a homogeneous intensity distribution. With the camera 4 Now the level of the sample can be recorded, which is in the focal plane 7 located.

In this embodiment of the arrangement according to the invention is advantageous that in the calibration chamber 21 to be filled homogeneously fluorescent aqueous solution is easy to prepare. In particular, the calibration chamber 21 be equipped with an inflow and outflow for the aqueous solution, which makes it possible, the calibration chamber 21 to empty and then to equip with the sample to be examined.

This is the calibration chamber 21 at the same time usable as a receiving chamber for samples, so that a separate sample chamber is unnecessary. Optical errors, which could occur due to a change of chamber, are thus avoided.

All Variants of the method according to the invention can be done in an equivalent way, though for the positioning process in addition or instead of the respective detector or the receiving plane to the light sheet an angle not equal to 90 ° with respect to the optical Axis of the imaging optics is tilted.

1

detection objective

2

fluorescence filters

3

tube optical system

4

camera

5

detector

6

reception plane

7

focal plane

8th

optical axis

9

Imaging beam path

10

light sheet

11

beamsplitter

12

fluorescence filters

13

tube optical system

14

camera

15

detector

16

reception plane

17

focal plane

18

intensity maximum

19

evaluation

20

setting device

21

calibration

a, a '

distance

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• WO 2004/053558 A1 [0005]

Claims (19)

Method for positioning the light sheet ( 10 ) shaped illumination beam in an object-side, the optical axis ( 8th ) of a detection optics ( 1 ) intersecting first focal plane ( 7 ) in the examination of a sample by the method of selective plane illumination microscopy (SPIM), in which - an object-side, with the optical axis ( 8th ) and the light sheet ( 10 ) an angle α in the range 0 ° <α <90 ° enclosing second focal plane ( 17 ) to one to the second focal plane ( 17 ) conjugate reception level ( 16 ) of a spatially resolving detector ( 15 ), - the contrast or the intensity is evaluated in this figure, and - the light sheet ( 10 ) or the detection optics ( 1 ) in the direction of the optical axis ( 8th ) is moved until the evaluation shows that the light sheet ( 10 ) the first focal plane ( 7 ) covered. Method according to Claim 1, characterized by the following method steps: d) Determining the actual position of the maximum contrast or the maximum intensity in the image of the second focal plane ( 17 ), the actual position being the point of intersection of the light sheet ( 10 ) with the second focal plane ( 17 ), e) determining the distance between the actual position and the desired position of the maximum contrast or the maximum intensity in this figure, wherein the desired position is the intersection of the two focal planes ( 7 . 17 ) with the optical axis ( 8th ) of the detection ( 1 ), and f) shifting the light sheet ( 10 ) or the detection optics ( 1 ) in the direction of the optical axis ( 8th ), until the distance between the actual position and the desired position is zero and thus the light sheet ( 10 ) in the first focal plane ( 7 ) is positioned. Method according to claim 1, characterized in that: - the light sheet ( 10 ) a structure of a perpendicular to the propagation direction periodic intensity profile is impressed, and - the thus structured light sheet ( 10 ) in the direction of the optical axis ( 8th ) is shifted until the contrast in the image of the second focal plane ( 17 ) has reached its maximum, whereby the light sheet ( 10 ) in the first focal plane ( 7 ) is positioned. Method according to one of the preceding claims, wherein first - the light sheet ( 10 ) with the aid of the spatially resolving detector ( 15 ) with one to the second focal plane ( 17 ) conjugate reception level ( 16 ) and then - the one with the light sheet ( 10 ) illuminated plane of the sample on a second spatially resolving detector ( 5 ) with one to the first focal plane ( 7 ) conjugate reception level ( 6 ) is displayed. Method according to Claim 4, in which - for positioning the light sheet ( 10 ) a fluorescence radiation emanating from the sample of a first wavelength / a first wavelength range is used, and - for the evaluation of the light sheet ( 10 ) illuminated level of the sample from the sample outgoing fluorescence radiation of a second wavelength / a second wavelength range is used. Method according to one of claims 1 to 3, in which - a spatially resolving detector ( 5 ) first for positioning the light sheet ( 10 ) in the first focal plane ( 7 ) and after that with the light sheet ( 10 ) illuminated level of the sample is used, wherein - for positioning the light sheet ( 10 ) the receiving level ( 6 ) of the detector ( 5 ) is aligned so that it to the second focal plane ( 17 ) is conjugated, and after positioning the receiving plane ( 6 ) of the detector ( 5 ) is aligned in such a way that it reaches the first focal plane ( 7 ) is conjugated. Method according to Claim 6, in which the orientation of the reception plane ( 6 ) is changed by being at its intersection with the optical axis ( 9 ) of the imaging beam path is pivoted. Method according to one of the preceding claims, in which for positioning the light sheet ( 10 ) - Fluorescence light is used, which starts from the sample to be examined, or - Fluorescence light is used, which starts from a reference sample, wherein after positioning of the light sheet ( 10 ) in the first focal plane ( 7 ) the sample to be examined is placed in the place of the reference sample. The method of claim 8, wherein as a reference sample a homogeneously fluorescent aqueous solution, a permeated with fluorescent substances gel or with Fluorescent substances interspersed plastic is used. Arrangement for positioning the to a light sheet ( 10 ) shaped illumination beam in an object-side, the optical axis ( 8th ) of a detection optics ( 1 ) intersecting first focal plane ( 7 in the examination of a sample by the method of selective plane illumination microscopy (SPIM), comprising: an optical device for imaging an object-side, with the optical axis ( 8th ) and the light sheet ( 10 ) each have an angle α in the range 0 ° < α <90 ° enclosing second focal plane ( 17 ) to the reception level ( 16 ) of a spatially resolving detector ( 15 ), - an evaluation device ( 19 ) - to determine the actual position of the maximum contrast or the maximum intensity in this figure, the actual position being the intersection of the light sheet ( 10 ) with the second focal plane ( 17 ), and - for determining the distance between the actual position and the desired position of the maximum contrast or the maximum intensity in this figure, the desired position being the intersection of the two focal planes ( 7 . 17 ) with the optical axis ( 8th ), and - a device for changing the position of the light sheet ( 10 ) in relation to the first focal plane ( 7 ). Arrangement according to Claim 10, in which the device for changing the position of the light sheet ( 10 ) is formed - for shifting the light sheet ( 10 ) in the direction of the optical axis ( 8th ) of the detection optics ( 1 ), and optionally - for shifting the light sheet ( 10 ) perpendicular to the optical axis ( 8th ) of the detection optics ( 1 ). Arrangement according to one of claims 10 or 11, in which - two spatially resolving detectors ( 5 . 15 ), of which - a first detector ( 5 ) one to the first focal plane ( 7 ) conjugate reception level ( 6 ) and for imaging the light sheet ( 10 ) illuminated sample plane is provided, and - the second detector ( 15 ) one to the second focal plane ( 17 ) conjugate reception level ( 16 ) and as an aid for positioning the light sheet ( 10 ) in the first focal plane ( 7 ) is provided, and - the detection beam path is branched so that one of the branches on the receiving plane ( 6 ) of the first detector ( 5 ) and the second branch to the receiving level ( 16 ) of the second detector ( 15 ) are directed. Arrangement according to Claim 12, in which the sample is excited when it is excited with the light sheet ( 10 ) - emits fluorescent light at wavelengths for which the first detector ( 5 ) in cooperation with a fluorescence filter ( 2 ) is sensitive, and at the same time - emits fluorescent light at wavelengths for which the second detector ( 15 ) in cooperation with a fluorescence filter ( 12 ) is sensitive. Arrangement according to Claim 10 or 11, in which a detector ( 5 ) and this with a device for tilting its reception plane ( 6 ) about their intersection with the optical axis ( 9 ) of the imaging beam path is coupled, wherein - in a first end position of the tilt, the reception plane ( 6 ) to the first focal plane ( 7 ) and the detector ( 5 ) for imaging and evaluation of the light sheet ( 10 ) illuminated sample plane is provided, and - in a second end position of the tilting the receiving plane ( 6 ) to the second focal plane ( 17 ) and the detector ( 5 ) as an aid for positioning the light sheet ( 10 ) to the second focal plane ( 17 ) is provided. Arrangement according to one of claims 10 to 14, in which as an aid for positioning the light sheet ( 10 ) in the first focal plane ( 7 ) a reference sample is provided, from which upon excitation with the light sheet ( 10 ) Emanating fluorescence radiation of the same wavelength / the same wavelength range as from the sample to be examined in their excitation with the light sheet ( 10 ) An assembly according to claim 15, wherein - the reference sample is a fluorescently permeated gel or a plastic interspersed with fluorescent substances, and - the reference sample is in a calibration chamber ( 21 ) is located. Arrangement according to one of Claims 10 to 15, in which there are optical means in the illumination beam path with which the light sheet ( 10 ) is impressed on a structure of a perpendicular to its propagation direction periodic intensity profile and in which a homogeneously fluorescent aqueous solution is provided as a reference sample. Arrangement according to one of Claims 10 to 17, with spatially resolving detectors ( 5 . 15 ), consisting of individual, in the respective receiving level ( 6 . 16 ) lying optoelectronic sensors are formed whose signal outputs to the evaluation ( 19 ) keep in touch. Arrangement according to one of claims 10 to 18, with an angle α in the range 20 ° <α <70 °.