Method and arrangement for light sheet microscopy

Abstract

An arrangement for light sheet microscopy that includes a means for scanning a sample volume with a light sheet, which includes an angle ??90? with the optical axis of an objective. The light sheet passes through the entire sample volume in the propagation direction, and the depth of field S.sub.obj of the objective is less than the optical-axis depth T of this sample volume. An optical device, disposed downstream of the objective, increases the depth of field S.sub.obj to a depth of field S.sub.eff?the depth T of this sample volume. The arrangement also includes a means for positioning the sample volume within the region of the depth of field S.sub.eff. A spatially resolving optoelectronic area sensor is disposed downstream of the optical device, and hardware and software are provided to generate sample-volume images from the electronic image signals output by the area sensor.

Claims

1. A method for light sheet microscopy, comprising the following steps: scanning a sample volume with a light sheet which is formed by illumination light and has an angle ??90? with an optical axis of an objective, wherein: the light sheet is directed to the entire sample volume to be imaged in a propagation direction; and the objective directs both the illumination light to the sample volume and detection light from the sample volume; and the illumination light is shined decentrally into an entrance pupil of the objective and in parallel with the optical axis of the objective; a depth of field S.sub.obj of the objective is less than a depth T of the sample volume in a direction of the optical axis; increasing the depth of field S.sub.obj in the detection beam path to a depth of field S.sub.eff that is greater than or equal to the depth T of the sample volume, and positioning the sample volume within the depth of field S.sub.eff; converting the optical image signals obtained with the increased depth of field S.sub.eff into electronic image signals with assignment to the regions of origin thereof in the sample volume; and generating images of the scanned sample volume from the electronic image signals.

2. The method for light sheet microscopy as claimed in claim 1; wherein, in a manner dependent on the sample volume to be scanned: a position of the light sheet in relation to the objective is varied in the x, y or z-direction; the angle ? is varied; a spatial extent of the light sheet within the sample volume is varied by rotation about the optical axis; or any combination of the above is performed.

3. The method for light sheet microscopy as claimed in claim 1; wherein the sample volume is scanned using a reflected-light method.

4. The method for light sheet microscopy as claimed in claim 1; wherein two-dimensional or three-dimensional images of the scanned sample volume are generated according to an extended depth of field technique or a light-field technique with an insertion of diffractive optical elements into the detection beam path.

5. The method for light sheet microscopy as claimed in claim 4; wherein the two- or three-dimensional images of the scanned sample volume are generated according to the extended depth of field technique or light-field technique with the insertion of diffractive optical elements (DOE) into the detection beam path.

6. An arrangement for light sheet microscopy, comprising: a scanning means configured to scan a sample volume to be imaged with a light sheet which is formed by illumination light and has an angle ??90? with an optical axis of an objective, wherein: the objective directs both the illumination light to the sample volume and detection light from the sample volume, the illumination light being shined to an entrance pupil of the object at a location offset in parallel to the optical axis of the objective; and the light sheet is directed into a sample volume to be imaged in a propagation direction; and a depth of field S.sub.obj of the objective is less than a depth T of the sample volume in a direction of the optical axis; a device configured to vary a direction for shining the illumination light into the entrance pupil; and an optical device, disposed downstream of the objective, configured to increase the depth of field S.sub.obj to a depth of field S.sub.eff that is equal to or greater than the depth T of the sample volume; a positioning means configured to position the sample volume within a region of the depth of field S.sub.eff; a spatially resolving optoelectronic area sensor disposed downstream of the optical device; and hardware and software, which are configured to generate images of the sample volume from the electronic image signals output by the area sensor.

7. The arrangement for light sheet microscopy as claimed in claim 6, further comprising: an optical means configured to: generate a light sheet scanned into the sample volume; and vary at least one of: a position of the light sheet in relation to the objective in the x, y, or z-direction; the angle ?; and a spatial extent of the light sheet by rotation about the optical axis or about an axis parallel to the optical axis.

8. The arrangement for light sheet microscopy as claimed in claim 6; wherein the optical device comprises: a cubic phase mask; or a microlens array.

9. The arrangement for light sheet microscopy as claimed in claim 6; wherein a CCD sensor with a reception area aligned at right angles, at least substantially at right angles, to the detection beam path is provided as an area sensor.

10. The arrangement for light sheet microscopy as claimed in claim 6, further comprising: a means for changing the entrance location for shining illumination light into the entrance pupil of the objective, in a decentralized manner in relation to the optical axis thereof.

11. The arrangement for light sheet microscopy as claimed in claim 6; wherein the hardware comprises: a personal computer (PC); and a means for electronic storage and visually perceivable reproduction of two-dimensional or three-dimensional images; and wherein the software comprises a predetermined algorithm for signal evaluation and for generating the two-dimensional or three-dimensional images.

12. An arrangement for light sheet microscopy, comprising: scanning means configured to scan a sample volume to be imaged with a light sheet which is formed by illumination light and has an angle ??90? with an optical axis of an objective, wherein the light sheet is directed into a sample volume to be imaged in a propagation direction; and wherein a depth of field S.sub.obj of the objective is less than a depth T of the sample volume in a direction of the optical axis; an optical device configured as an axicon, disposed downstream of the objective, and configured to increase the depth of field S.sub.obj to a depth of field S.sub.eff that is equal to or greater than the depth T of the sample volume; positioning means configured to position the sample volume within a region of the depth of field S.sub.eff; a spatially resolving optoelectronic area sensor disposed downstream of the axicon; and generating means configured to generate images of the sample volume from the electronic image signals output by the area sensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a first exemplary embodiment of the arrangement according to the invention, in which the reception device has an area sensor, u-stream of which an axicon is arranged in the detection beam path.

(2) FIG. 2 shows a second exemplary embodiment of the arrangement according to the invention, but in this case with a cubic phase mask arranged upstream of the area sensor in the detection beam path.

(3) FIG. 3 shows a third exemplary embodiment of the arrangement according to the invention, in this case with a microlens array arranged upstream of the area sensor in the detection beam path.

(4) FIG. 4 shows the realization of a first exemplary position and alignment of a light sheet in a sample in relation to the optical axis of an objective.

(5) FIG. 5 shows the realization of a second exemplary position and alignment of a light sheet in a sample in relation to the optical axis of an objective.

(6) FIG. 6 shows a depth of field S.sub.eff obtained according to the invention in comparison with the nominal depth of field S.sub.obj of an objective and in comparison with the depth T of a sample volume, and the extent of the light sheet within the sample volume in comparison with the depth T of a sample volume.

DETAILED DESCRIPTION OF EMBODIMENTS

(7) It is to be understood that the FIGS. and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein.

(8) The present invention will now be described in detail on the basis of exemplary embodiments.

(9) The exemplary embodiment shown in FIG. 1 has an objective 1 with a fixed focal length, imaging toward infinity, said objective being provided for use both as an illumination objective and as a detection objective. Nominally, it can be designed for a depth of field S.sub.obj in the range from 0.5 ?m to 170 ?m.

(10) A laser light source 2, a beam widening optical unit 3 and a scanning device 4 generate an illumination beam path 5, which is deflected toward the objective 1 by means of the splitter surface 6 of a beam splitter 7 and which is shone with lateral offset from the optical axis 8 of the objective 1 into the entrance pupil 24 of the objective 1 (cf. FIG. 4 and FIG. 5). The widened, collimated laser beam, which is e.g. only focused in one direction by means of a cylindrical lens (not depicted in the drawing) and thus formed into a light sheet 9, is projected into the sample volume 10 byway of the objective 1. In alternative embodiments, the light sheet 9 can be generated by means of a punctiform laser focus moved in a scanning manner or by means of any other light-shaping procedure.

(11) The illumination light being shone in a decentral manner in relation to the optical axis 8 causes the propagation direction of the light sheet 9 to include an angle ? with the optical axis 8 which differs from 90?. Here, the size of the angle ? is dependent on the distance between the entrance location 23 of the illumination light into the entrance pupil 24 and the optical axis 8.

(12) A consequence of the light sheet 9 being at an angle relative to the optical axis 8 is that the light sheet 9 also illuminates regions of the sample volume 10 which lie at different depths in front of, and behind, the focal plane 11 in the axial direction, i.e. in the z-direction.

(13) With the scanning movement in the x- and/or y-direction, the scanning device 4 prescribes the current position and alignment of the light sheet 9 within the sample volume 10. Said scanning device 4 is also embodied to rotate the light sheet 9 about an angle about the optical axis 8 or about an axis parallel to the optical axis 8 (cf. FIG. 4 and FIG. 5). Moreover, the scanning unit 4 enables the displacement of the entrance location 23 of the illumination light in the pupil plane along the circumference of a partial circle 25, the radius of which corresponds to the distance of the entrance location 23 from the optical axis 8.

(14) Here, the scanning device 4 is embodied in an exemplary manner to change the distance between the entrance location 23 of the illumination light and the optical axis 8, displace the entrance location 23 in the pupil plane along a partial circle 25, the radius of which corresponds to the distance of the entrance location 23 from the optical axis 8, and to change the direction for shining the illumination light into the entrance pupil 24 of the objective 1.

(15) However, the scope of the invention explicitly also includes each one of these functions being exercised by separate devices embodied therefor.

(16) Reflected, scattered or excited light coming from the illuminated sample regions enters the objective 1 as detection light. The objective 1 forms a detection beam path 12, to which the splitter surface 6 is transparent, as depicted in FIG. 1. A tube optical unit 13 matched to the objective 1 is used to generate an intermediate image 14, which is subsequently imaged on the reception plane 18 of an area sensor 17 by means of two further lens groups 15 and 16a and an axicon 19. The reception plane 18 is fixedly positioned in an image plane of the objective 1 which is conjugate to the focal plane 11.

(17) In the detection beam path, the axicon 19 is positioned between the two lens groups 15, 16a. The axicon 19 transforms the detection light into a Bessel-shaped light beam with a very long focus and therefore, according to the invention, brings about an increase in the nominal depth of field S.sub.obj of the objective 1 by a multiple to a depth of field S.sub.eff, as a result of which the whole sample volume 10 scanned by the light sheet 9 is imaged sharply on the reception plane 18.

(18) A depth-resolved image of each illuminated thin section of the sample volume 10 is established by calculation from the intensity distribution in the detection beam path 12 registered by the area sensor 17 and said image is depicted in a manner that is visually perceptible by an observer. Such restoration methods are known per se. In the case of the so-called wavefront coding by means of phase masks, the data record is initially decoded numerically. This is carried out by a deconvolution with a known point spread function (PSF) determined by the mask, the objective and the microscope system. A projection of the sample volume along the optical axis is obtained after this first step. In a further step, the a priori information about the respectively current illumination geometry is included in the sample restoration in such a way that a unique spatial assignment of each detected event to the scanned sample volume is carried out.

(19) As can be seen from FIG. 1, a computing unit 26 is provided in this respect, for example in the form of a PC with appropriate processing software. The computing unit 26 receives the image signals from the area sensor 17 byway of the signal path depicted in FIG. 1, information about the spatial situation and alignment of the light sheet 9 relative to the illuminated sample volume 10 from the scanning device 4 and information about the position of the sample volume 10 in the z-direction relative to the object 1 from the sample stage 22, and it links this information by means of the processing software with the image signals for the purposes of obtaining two- or three-dimensional images of the scanned sample volume 10. The visually perceptible representation and/or storage of the results is carried out by means of a memory and reproduction unit 27 connected to the computing unit 26.

(20) In a specific embodiment, the distance of the tube optical unit 13 from the position of the intermediate image 14 is f.sub.13=200 mm. Byway of example, the lens group 15 is at a distance of f.sub.15=200 mm from the position of the intermediate image 14 and generates an infinite beam path. The axicon 19 is at a distance of 41 mm from the lens group 15. The cone angle of the axicon 19 is ?2; for example, it is 0.5 in this case. The distance between the axicon 19 and the reception plane 18 varies depending on the embodiment of the arrangement according to the invention; it can be up to 50 cm and is determined by the length of the Bessel beam which is generated by the axicon 19. The reception plane 18 is positioned in the region of the Bessel beam.

(21) The lens groups 15, 16a can both consist of a plurality of lenses and be embodied as individual lenses. In principle, there is no need for a lens group 16a; the scope of the invention also includes an embodiment of the arrangement according to the invention without a lens or lens group 16a. However, a lens group 16a can advantageously be used to set optical parameters such as resolution and lateral magnification.

(22) FIG. 2 shows a second exemplary embodiment of the arrangement according to the invention. To the extent that the components illustrated here are comparable to components from FIG. 1 in respect of the functionality thereof, they are also provided with the same reference signs as in FIG. 1.

(23) The difference to the exemplary embodiment according to FIG. 1 essentially consists of the fact that provision is not made here for an axicon 19 but for a cubic phase mask 20 between two lens groups 15 and 16b, which cubic phase mask is positioned in a pupil embodied between the lens groups 15, 16b.

(24) The phase mask 20 transforms the detection light into an Airy-shaped light beam and likewise, according to the invention, brings about an increase in the nominal depth of field S.sub.obj of the objective 1 by a multiple to a depth of field S.sub.eff, as a result of which the whole sample volume 10 scanned by the light sheet 9 is also imaged sharply on the reception plane 18 in this case.

(25) By applying the extended depth of field (EDoF) technology, a depth-resolved image of each illuminated thin section of the sample volume 10 is established in turn by calculation using the computing unit 26 from the intensity distribution in the detection light beam registered by the area sensor 17 and said depth-resolved image is depicted in a manner visually perceivable by an observer by means of the memory and reproduction unit 27.

(26) FIG. 3 depicts a third exemplary embodiment. Here too, the same reference signs are also used for components, to the extent that these are comparable with components from FIG. 1 and FIG. 2.

(27) The exemplary embodiment according to FIG. 3 differs from the preceding exemplary embodiments according to FIG. 1 and FIG. 2 in that provision is made neither for an axicon 19 nor for a phase mask 20, but for a microlens array 21 arranged upstream of the reception plane 18 instead.

(28) The microlens array 21 increases the depth of field S.sub.obj of the objective 1 by a multiple to a depth of field S.sub.eff by virtue of the sample volume 10 scanned by the light sheet 9 being imaged as a whole on the reception plane 18. Below, a three-dimensional reconstruction of this volume is undertaken by virtue of a depth-resolved image of the illuminated thin section being established by calculation by means of the light-field technology from the registered intensity distribution of the detection light beam and said depth-resolved image being depicted in a visually perceivable manner.

(29) In the case mentioned here, obtaining and using the a priori information about the respectively current illumination geometry, as described further above on the basis of FIG. 1, can be included to improve the results of the three-dimensional sample representation.

(30) Here too, two- or three-dimensional images of the scanned sample volume 10 are obtained by means of a computing unit 26.

(31) FIG. 4 and FIG. 5 depict two of the possible positions and alignments which the light sheet 9 can assume in relation to the optical axis 8 and to a sample stage 22 during the scanning of a sample volume 10 in an exemplary and perspective manner. For reasons of clarity, depicting an objective and a sample which is placed in a sample stage 22 and scanned by the light sheet 9 has been dispensed with in FIG. 4 and FIG. 5.

(32) In a plan view of the entrance pupil 24 of an objective, FIG. 4a shows the entrance location 23 of the illumination beam path 5 into the entrance pupil 24, which is determined by the angle and the distance from the optical axis 8 and which causes the position and alignment of the light sheet 9 in the sample depicted in FIG. 4b.

(33) By contrast, FIG. 5a shows in a plan view of the same entrance pupil 24 the entrance location 23 of the illumination beam path 5 into the entrance pupil 24, which is once again determined by the angle and the distance from the optical axis 8, but which brings about the position and alignment of the light sheet 9 in the sample depicted in FIG. 5b in this case.

(34) In both cases, this pupil illumination shows, in an exemplary manner, the generation of an inclined elliptic Gaussian light sheet.

(35) As can be seen from FIG. 4 and FIG. 5, it is possible to adapt the position and alignment of the light sheet 9 to a sample volume 10 to be scanned by varying the entrance location 23, here, as an example, by changing the angle ?, wherein, however, the angle ? between the optical axis 8 and the light sheet 9 is maintained. The intersection point of the light sheet 9 in respect of a sample plane away from the focal plane of the objective 1 in this case extends along a circular path.

(36) A variation of the distance of the entrance location 23 from the optical axis 8 brings about a change in the angle ? between the light sheet 9 and the optical axis 8. An adaptation of the position of the light sheet 9 to the sample volume 10 which extends in the x- and y-direction is achieved by changing the entrance angle of the illumination light into the pupil plane of the objective 1. A displacement of the focal plane 11 or of the region of the depths of the field S.sub.obj, S.sub.eff in the z-direction relative to the sample can be obtained by displacing the sample stage 22 in the z-direction.

(37) In FIG. 6, the effective depth of field S.sub.eff obtained according to the invention is contrasted for comparison purposes with the nominal depth of field S.sub.obj of an objective 1 and the depth T of the illuminated volume 10 of a sample placed on the sample stage 22.

(38) Moreover, it is possible to identify from FIG. 6 that the light sheet 9 extends over the whole depth T of the sample volume 10. In FIG. 6, the light sheet 9 extends in the z-direction over a range ?z=L, where L includes the depth T of the sample volume 10 such that the light sheet 9 illuminates the sample volume 10 over the entire depth T thereof.

(39) This condition is also satisfied by the case L=T and therefore likewise lies within the scope of the invention.

(40) Within the region L, the spatial extent of the light sheet 9 in the direction of the optical axis 8 does not exceed the value of e.g. 10 ?m and it is therefore suitable for detecting or measuring a sample volume 10 according to the principle of light sheet microscopy.

(41) In conjunction with the change in the angle and the displacement of the light sheet 9 or the sample in the x- or y-direction, the depth of field S.sub.eff?T enables the detection and imaging of the entire sample volume 10.

(42) While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.

LIST OF REFERENCE SIGNS

(43) 1 Objective 2 Laser light source 3 Beam widening optical unit 4 Scanning apparatus 5 Illumination beam path 6 Splitter surface 7 Beam splitter 8 Optical axis 9 Light sheet 10 Sample volume 11 Focal plane 12 Detection beam path 13 Tube optical unit 14 Intermediate image 15 Lens group 16 Lens group 17 Area sensor 18 Reception plane 19 Axicon 20 Phase mask 21 Microlens array 22 Sample stage 23 Entrance location 24 Entrance pupil 25 Partial circle 26 Computing unit 27 Memory and reproduction unit