DEVICE FOR IMAGING A SAMPLE

Abstract

The invention relates to a device and a method for imaging a sample (2) arranged in an object plane (1). Such a device comprises an optical relay system (3) which images an area of the sample (2) from the object plane (1) into an intermediate image plane (4). Here, the object plane (1) and the intermediate image plane (4) with an optical axis (5) of the relay system (3) include an angle different from 90°. The optical relay system (3) is composed of several lenses. The device also comprises an optical imaging system (6) with an objective, the optical axis (7) of which lies perpendicularly on the intermediate image plane (4) and which is focused on the intermediate image plane (4), with the result that the object plane (1) can be imaged undistorted onto a detector (8). Finally, the device also comprises an illumination apparatus (10) for illuminating the sample (2) with a light sheet (11), wherein the light sheet (11) lies essentially in the object plane (1) and defines an illumination direction, and wherein the normal of the object plane (1) defines a detection direction.

In such a device, the object plane (1) with the optical axis (5) of the relay system (3) includes an angle, the value of which is smaller than the aperture angle of an object-side detection aperture cone (12) of the relay system (3), and the object plane (1) lies at least partially within the object-side detection aperture cone (12). The intermediate image plane (4) with the optical axis (5) of the relay system (3) also includes an angle, the value of which is smaller than the aperture angle of an intermediate image-side detection aperture cone (13) of the relay system (3), and the intermediate image plane (4) lies at least partially within the intermediate image-side detection aperture cone (13).

Claims

1. Device for imaging a sample (2) arranged in an object plane (1), comprising an optical relay system (3) which images an area of the sample (2) from the object plane (1) into an intermediate image plane (4), wherein the object plane (1) and the intermediate image plane (4) with an optical axis (5) of the relay system (3) include an angle different from 90° and the optical relay system (3) is composed of several lenses, an optical imaging system (6) with an objective, the optical axis (7) of which lies perpendicularly on the intermediate image plane (4) and which is focused on the intermediate image plane (4), with the result that the object plane (1) can be imaged undistorted onto a detector (8), an illumination apparatus (10) for illuminating the sample (2) with a light sheet (11), wherein the light sheet (11) lies essentially in the object plane (1) and defines an illumination direction, and the normal of the object plane (1) defines a detection direction, characterized in that the optical imaging system (6) is formed so as to detect a detection angular range distributed asymmetrically around the optical axis (7) of the objective and to image it onto a detector (8).

2. Device according to claim 1, characterized in that the object plane (1) and the intermediate image plane (4) with the optical axis (5) of the relay system (3) each include an angle, the value of which is smaller than the aperture angle of an object-side detection aperture cone (12) or of an intermediate image-side detection aperture cone (13) respectively of the relay system (3), and the object plane (1) and the intermediate image plane (4) lie at least partially within the object-side detection aperture cone (12) or the intermediate image-side detection aperture cone (13) respectively.

3. Device according to claim 1 or 2, comprising an evaluation unit (9) connected to the detector (8) for image processing while taking into account an asymmetrical point spread function on the basis of the asymmetrical detection angular spectrum and/or a compressed point spread function.

4. Device according to one of claims 1 to 3, characterized in that the numerical aperture of the optical imaging system (6) is larger than the numerical aperture of the relay system (3).

5. Device according to one of claims 1 to 4, characterized in that a first optical medium is arranged between the optical relay system (3) and the intermediate image plane (4) and a second optical medium is arranged between the intermediate image plane (4) and the optical imaging system, wherein the intermediate image plane (4) lies in the boundary surface between first and second optical medium and the second optical medium has a higher refractive index than the first optical medium.

6. Device according to one of claims 1 to 5, characterized in that the relay system (3) is constructed symmetrically in relation to a plane of symmetry between the subsystems perpendicular to the optical axis (5) of the relay system (3), with the result that the imaging by the relay system (3) takes place on an image scale of 1:1, and/or in that the relay system (3) is constructed catadioptrically.

7. Method for imaging a sample (2) arranged in an object plane (1), in which the sample is illuminated with a light sheet (11), wherein the light sheet (11) lies essentially in the object plane (1) and defines an illumination direction, and the normal of the object plane (1) defines a detection direction, an area of the sample (2) is imaged from the object plane (1) by means of an optical relay system (3) into an intermediate image plane (4), wherein the object plane (1) and the intermediate image plane (4) with an optical axis (5) of the relay system (3) include an angle different from 90°, the intermediate image plane (4) is imaged by means of an optical imaging system (6) with an objective, the optical axis (7) of which lies perpendicularly on the intermediate image plane (4) and which is focused on the intermediate image plane (4), undistorted onto a detector (8) and is recorded there as an image, wherein the optical imaging system (6) detects a detection angular range distributed asymmetrically around the optical axis (7), the recorded image is processed in an evaluation unit connected to the detector (8) while taking into account an asymmetrical point spread function on the basis of the asymmetrical detection angular spectrum and/or a compressed point spread function.

8. Method according to claim 8, characterized in that the object plane (1) and the intermediate image plane (4) lie at least partially within the object-side detection aperture cone (12) or the intermediate image-side detection aperture cone (13) respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention is explained in yet more detail below by way of example with reference to the attached drawings, which also disclose features essential to the invention. There are shown in:

[0029] FIG. 1 the structure of a device for imaging a sample,

[0030] FIG. 2 the detectable angular distribution of the detection light according to the state of the art,

[0031] FIG. 3 detection angular spectra for a first embodiment of such a device according to FIG. 1,

[0032] FIG. 4 detection angular spectra for a second embodiment of such a device,

[0033] FIG. 5 detection angular spectra for a third embodiment of such a device.

DETAILED DESCRIPTION OF THE DRAWINGS

[0034] With reference to FIG. 1, firstly the basic mode of operation of the device for imaging a sample 2 arranged in an object plane 1 will be explained. The device comprises an optical relay system 3 which images an area of the sample 2 from the object plane 1 into an intermediate image plane 4. The object plane 1 and the intermediate image plane 4 with an optical axis 5 of the optical relay system 3 include an angle different from 90°. The optical relay system 3 here is composed of several lenses. For example, it can be constructed symmetrically in relation to a plane of symmetry between the subsystems perpendicular to the optical axis 5 of the relay system, with the result that the imaging by the relay system 3 takes place on an image scale of 1:1. In this case, it can for example also be constructed as a so-called 4f system, wherein each subsystem comprises an objective and a tube lens. It can also be constructed catadioptrically, i.e. the one or more lenses are at least partially reflecting, whereby the structural size and number of lenses can be reduced. The relay system 3 can also be constructed non-symmetrically in order to produce a correspondingly magnified image in the intermediate image plane. This can also be achieved through the choice of suitable media—in particular immersion media—on the object or intermediate image side which differ in their refractive indices.

[0035] The device also comprises an optical imaging system 6 with an objective, the optical axis 7 of which lies perpendicularly on the intermediate image plane 4 and which is focused on the intermediate image plane 4, with the result that the object plane 1 as a whole can be imaged undistorted onto a detector 8. An evaluation unit 9 for image processing is connected to the detector 8.

[0036] Finally, the device for imaging the sample 2 also comprises an illumination apparatus 10 for illuminating the sample 2 with a light sheet 11. In the device shown in FIG. 1, illumination light in the intermediate image plane 4 is coupled into the beam path of the relay system 3 and directed onto the sample 2 by the relay system 3. Instead of the coupling-in in the intermediate image plane 4, a pupil plane of the relay system 3 can also be used for the coupling-in. It is furthermore conceivable for the illumination to be carried out independently of the relay system directly through irradiation in the sample space. The light sheet 11 is directed onto the sample 2 by the relay system 3 and lies essentially in the object plane 1; the illumination direction is defined in this way. The normal of the object plane 1 corresponds to the detection direction. The illumination is thus carried out at an angle, which is different from zero, to the detection direction. The light sheet 11 lies essentially in the object plane 1, wherein the term “essentially” means that the light sheet 11, as indicated in FIG. 1, has in the xz plane shown here a thickness which is different from zero and increases with increasing distance from the focal point. Here, the thickness of the light sheet 11 is represented by the two envelopes to the left and right of the object plane 1 or the intermediate image plane 4. Perpendicular to the plane of drawing, the light sheet 11 has a considerably greater extent.

[0037] The detection direction here lies perpendicularly on the object plane 1 or the intermediate image plane 4. The aperture of the sample-side objective of the optical relay system 3 in combination with the focus limits the maximum possible angular range in which emission light—for example fluorescent light which was excited by the light sheet—can be detected. This maximum possible angular range is, for the xz plane on the object side and intermediate image side, denoted by the short-dashed lines and when considered three-dimensionally the aperture defines an object-side detection aperture cone 12 and an intermediate image-side detection aperture cone 13, the section of which is represented in the xz plane here. The relay system 3 here is constructed symmetrically with the result that the intermediate image-side detection aperture cone 13 corresponds to a reflected object-side detection aperture cone 12.

[0038] In the state of the art the detection angular range is limited to a section of the object-side aperture cone 12 that is located symmetrically around the detection axis, which section is represented here by the hatched area within the object-side detection aperture cone 12 and correspondingly by a hatched area on the intermediate image side. In the state of the art the reasons for this are that a part of the beam path is reserved for the illumination and/or that the detection beam path is limited in the optical imaging system and/or that during the image evaluation no additional actions have to be carried out as a result of an asymmetrical detection angular spectrum, meaning that the evaluation is considerably simpler.

[0039] The associated detection angular spectrum, which can be detected by the optical imaging system 6 in this case, is represented in FIG. 2 by the dot-dashed line. The continuous line represents the maximum theoretically possible angular spectrum which is dependent solely on the aperture of the relay system 3 and/or of the optical imaging system 6. The normalized amplitude is shown in arbitrary units, and on the x-axis the relative angle in relation to the optical axis 7 of the optical imaging system 6 is shown in rad. Here, the detection angular spectrum that can actually be detected is highly cropped compared with the theoretically possible detection angular spectrum because of the requirement that it has to be located symmetrically with respect to the optical axis 7 of the optical imaging system 6.

[0040] In the device shown in FIG. 1, however, the situation is such that the object-side detection aperture cone 12 corresponds to the maximum aperture cone of the relay system 3 that is actually possible, the dimensions of which are specified solely by the corresponding data from an object-side objective of the relay system 3. The detection aperture cone 13 may also be slightly smaller than the maximum possible aperture cone of the relay system 3. In the example shown here, the object plane 1 with the optical axis of the relay system 3 includes an angle, the value of which is smaller than the aperture angle of the object-side detection aperture cone 12 of the relay system 3, and the object plane 1—and thus also the plane of the light sheet 11—lies at least partially within the object-side detection aperture cone 12. On the intermediate image side, the situation is comparable, that is to say the intermediate image plane 4 with the optical axis 5 of the relay system 3 includes an angle, the value of which is smaller than the aperture angle of the intermediate image-side detection aperture cone 13 of the relay system 3, and the intermediate image plane lies at least partially within the intermediate image-side detection aperture cone 13. This can clearly be seen in FIG. 1.

[0041] The intermediate image plane 4 is thus located within the reflected detection aperture cone 13. The optical imaging system 6 is aligned with its optical axis 7 perpendicular to the intermediate image plane 4 and collects the maximum fluorescent light in an imaging-side detection aperture cone 14, which is denoted here by the long-dashed lines. Since fluorescent light or generally emission light, which propagates at an angle of greater than 90° relative to the optical axis 7 of the optical imaging system 6, can in principle not be detected, the maximum theoretically possible partial area of the detection angular spectrum in the xz plane that can be detected with the setup shown in FIG. 1 is designated by the triangle with the corner points A, B and C. Since the intermediate image plane 4 or the plane of the imaged light sheet 11 is located in the potentially possible intermediate image-side detection aperture cone 13, the actually possible partial area is cropped and is smaller than the potentially possible partial area. In addition, this maximum theoretically possible partial area which is defined by the triangle ABC is asymmetrical in relation to the optical axis 5 of the relay system 3.

[0042] The partial area that can actually be detected by the optical imaging system 6 is sometimes restricted to an even greater extent than the maximum theoretically possible partial area. This is dependent on the aperture of the optical imaging system 6. In each case, the detection angular range that can actually be detected by the optical imaging system 6 and is designated by the triangle CDE covers a considerably larger angular range than the symmetrical hatched cone in accordance with the state of the art, but is distributed asymmetrically around the optical axis 7 of the optical imaging system 6. This detection angular range which is larger compared with the state of the art results in a higher resolution of the device, even though the detection angular spectrum is distributed asymmetrically around the optical axis 7 of the optical imaging system 6.

[0043] Here, the imaging-side detection aperture cone 14 can be further enlarged if a larger numerical aperture of the optical imaging system 6 is also chosen. The numerical aperture of the optical imaging system 6 is therefore preferably larger than the numerical aperture of the relay system 3. Additionally or alternatively, a boundary surface which causes a jump in the refractive index can also be introduced in the intermediate image plane. For this purpose, a first optical medium is arranged between the optical relay system 3 and the intermediate image plane 4 and a second optical medium is arranged between the intermediate image plane 4 and the optical imaging system 6. The intermediate image plane 4 lies on the boundary between first and second optical medium, i.e. in the boundary surface. The second optical medium has a higher refractive index than the first optical medium. The optical media in each case cover the beam paths up to the relay system 3 or optical imaging system 6 and they can be for example immersion media or gelatinous substances; glasses are also possible embodiments. In this way, the partial area that can actually be detected can come as close as possible to the theoretically possible partial area. In each case, an asymmetry remains which, as regards imaging, produces an asymmetrical or not point-symmetrical point spread function for the entire device. The evaluation unit 9 is preferably suitable for image processing while taking into account an asymmetrical point spread function on the basis of the asymmetrical detection angular spectrum, and in this way the point spread function can be used for the image evaluation and can contribute to an increase in sharpness in the context of a deconvolution. Alternatively or additionally, the evaluation unit is also suitable for taking into account or offsetting a compressed point spread function, as occurs at the described boundary surface in the intermediate image plane due to Snell's law of refraction.

[0044] In FIGS. 3-5, the detection angular spectra are shown for various configurations of the device. The figures in each case show a section in the xz plane through the amplitude portion of the light emitted by the sample, i.e. of the detection angular spectrum, by means of a continuous line. The phase portion is in this case disregarded, which is appropriate when considering fluorescence emission. The form of the detection angular spectrum here is randomly selected and serves only for illustration. The abscissa shows the angle relative to the optical axis 7 of the optical imaging system 6.

[0045] In the case represented in FIG. 3, the numerical aperture of the optical relay system 3 is 1.2 and is identical to the numerical aperture of the optical imaging system 6. Only one medium was used here, namely water with a refractive index of 1.33. The relay system 3 is constructed symmetrically and the optical imaging system 6 is at an angle of 48° to the relay system 3 in relation to the optical axes. The maximum theoretically possible partial area of the detection angular spectrum that can be detected and corresponds to the triangle ABC in FIG. 1, which can be detected downstream of the relay system 3, is shown as a dashed line. Compared with the original angular spectrum, this partial area is cropped because of the aperture of the relay system 3 and due to the fact that the intermediate image plane 4 lies within the detection aperture cone 13, in relation to which the optical imaging system must detect perpendicularly. The partial area that can actually be detected and corresponds to the triangle CDE in FIG. 1 is shown as a dotted line in FIGS. 3-5. Here, there is further cropping due to the fact that in turn only a certain partial area can be covered with the aperture of the optical imaging system 6, and the point spread function of the system is asymmetrical in relation to the optical axis 7 of the optical imaging system 6.

[0046] While the area that can actually be detected is thus already enlarged compared with the state of the art, it may be attempted in order to increase the resolution to detect an even larger part of the theoretically possible spectrum shown as a dashed line. This case is shown in a simulation in FIG. 4. The numerical aperture of the optical imaging system 6 here is 1.329, which however cannot be achieved when water is used as immersion medium with a refractive index of 1.33 since the entire half-space would have to be detected and the point E of the triangle would be shifted into the intermediate image plane.

[0047] While there are therefore limits to the increase in the numerical aperture of the optical imaging system 6, an enlargement of the area that can actually be detected can also be effected by introducing a jump in the refractive index in the intermediate image plane 4. This is shown in FIG. 5; here water with a refractive index of 1.33 is used as first medium between relay system 3 and intermediate image plane 4 and BK7 glass with a refractive index of 1.52 is used as second medium between the intermediate image plane 4 and the optical imaging system 6. The numerical aperture of the optical imaging system 6 is then 1.329 and lies in the achievable range as a result of the choice of media. The theoretically possible spectrum is then completely detected, which is shown by the dotted line in FIG. 5. It can also be seen that it is compressed due to Snell's law of refraction, but not distorted. Information is not lost in this way, however the compression, like the asymmetry which is still present, equally has to be taken into account during image processing, during which the point spread function is also taken into account. Compression and point spread function can be taken into account within the scope of a deconvolution.

[0048] If an asymmetrical, possibly compressed detection angular spectrum and associated therewith an asymmetrical and likewise possibly compressed point spread function are allowed in the detection, the resolution of the system as a whole can be increased.

LIST OF REFERENCE NUMBERS

[0049] 1 object plane

[0050] 2 sample

[0051] 3 optical relay system

[0052] 4 intermediate image plane

[0053] 5 optical axis

[0054] 6 optical imaging system

[0055] 7 optical axis

[0056] 8 detector

[0057] 9 evaluation unit

[0058] 10 illumination apparatus

[0059] 11 light sheet

[0060] 12 object-side detection aperture cone

[0061] 13 intermediate image-side detection aperture cone

[0062] 14 imaging-side detection aperture cone

[0063] A, B, C,

[0064] D, E, F corner points of triangles