METHOD AND APPARATUS FOR DETECTING FLUORESCENCE SIGNALS IN A THREE-DIMENSIONAL REGION OF A SAMPLE

Abstract

The disclosure relates to a detection method for optical signals in a three-dimensional region of a sample, and a detection method for marked antibodies and/or antigens on a biological surface. In the process, signals with a depth of field that is extended in relation to an original depth of field are captured and evaluated. Also, an apparatus includes an optical element in a detection beam path, a depth of field that is extended in relation to an original depth of field being generated by the effect of said optical element.

Claims

1.-10. (canceled)

11. A detection method for optical signals in a three-dimensional region of a sample, the method comprising: capturing image data of the three-dimensional region by means of an optical apparatus in a two-dimensional image plane, wherein signals in the form of fluorescence signals are captured with a depth of field that is extended in relation to an original depth of field of the apparatus and are projected into an image plane; ascertaining a number of signals in the image plane; and storing the number in a manner assigned to the image region and providing the assigned number.

12. The method as claimed in claim 11, wherein the three-dimensional region of the sample includes photoswitchable molecules used as markers, and further comprising: exciting a subset of the photoswitchable molecules used as markers to emit signals; and capturing the emitted signals in a time series.

13. The method as claimed in claim 11, wherein at least two focal planes with an original depth of field or at least two focal regions with an extended depth of field or at least one focal plane with an original depth of field and at least one focal region with an extended depth of field are generated and an extended depth of field is generated as a result.

14. A detection method for an antigen on a biological surface, the method comprising: in a biological surface incubated with at least one antibody which is compatible with the antigen to be detected and which is provided with markers suitable for induced emission of signals in the form of fluorescence signals and/or induced light signals, exciting marked antibodies bound to an antigen to emit signals; capturing the emitted signals by means of an optical apparatus, wherein the apparatus includes: a detection beam path, wherein detection radiation is guided and captured along the detection beam path, an objective with an original depth of field in a direction of a detection axis in the detection beam path for capturing the detection radiation, an optical element in a pupil of the detection beam path, wherein a depth of field which is extended over the original depth of field is generated by means of the optical element, a detector configured for two-dimensionally resolved capture of signals, and an evaluation unit, in which image data read from the detector are evaluated and a number of the signals is ascertained, assigned to an image region, and stored; evaluating the image data in respect of the presence of captured signals and ascertaining a number of the captured signals; and storing and providing the number of the captured signals.

15. The method as claimed in claim 14, wherein photoswitchable molecules are used as the markers, and further comprising: exciting a subset of the number of markers, in each case to emit signals; capturing the emitted signals in a time series.

16. The method as claimed in claim 14, wherein at least two focal planes with an original depth of field or at least two focal regions with an extended depth of field or at least one focal plane with an original depth of field and at least one focal region with an extended depth of field are generated and an extended depth of field is generated as a result.

17. The method of claim 14, further comprising: incubating the biological surface with the at least one antibody which is compatible with the antigen to be detected and which is provided with a marker suitable for the induced emission of signals in the form of fluorescence signals and/or induced light signals.

18. An apparatus for detecting signals in the form of fluorescence signals and/or induced light signals from a three-dimensional region of a sample, the apparatus comprising: a detection beam path, wherein detection radiation can be guided and captured along the detection beam path; an objective with an original depth of field in a direction of a detection axis in the detection beam path for capturing the detection radiation; an optical element in a pupil of the detection beam path, wherein a depth of field which is extended over the original depth of field is generated by means of the optical element; a detector configured for two-dimensionally resolved capture of signals as image data; and an evaluation unit, in which image data read from the detector are evaluated and a number of the signals is ascertained, assigned to an image region, and stored.

19. The apparatus as claimed in claim 18, wherein the optical element includes an axicon or an axicon phase mask and wherein the detection radiation is converted into a Bessel beam by the action of the optical element.

20. The apparatus as claimed in claim 18, wherein the optical element includes a cubic phase mask.

21. The apparatus as claimed in claim 18, wherein the optical element includes a ring phase mask.

22. The apparatus as claimed in claim 18, wherein the optical element includes a birefringent element.

23. The apparatus as claimed in claim 18, wherein the optical element includes at least one liquid lens, an adaptive mirror, or a microlens array.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The invention is explained in more detail below on the basis of exemplary embodiments and figures. In the drawings:

[0066] FIG. 1 shows a schematic illustration of a detection method by means of an optical apparatus and a focal plane corresponding to an original depth of field of an objective in accordance with the prior art;

[0067] FIG. 2 shows a schematic illustration of a detection method by means of a first exemplary embodiment of an optical apparatus and a focal region corresponding to an extended depth of field of an objective;

[0068] FIG. 3 shows a schematic illustration of a second exemplary embodiment of an apparatus;

[0069] FIG. 4 shows a schematic illustration of a third exemplary embodiment of an apparatus;

[0070] FIG. 5 shows a schematic illustration of a fourth exemplary embodiment of an apparatus;

[0071] FIG. 6 shows a schematic illustration of a fifth exemplary embodiment of an apparatus;

[0072] FIG. 7 shows a schematic illustration of a sixth exemplary embodiment of an apparatus;

[0073] FIG. 8 shows a schematic illustration of a seventh exemplary embodiment of an apparatus;

[0074] FIG. 9 shows a schematic illustration of a phase distribution in a pupil of a detection beam path by the effect of a symmetric cubic phase mask, respectively as a) a simplified black-and-white sketch, b) as a grayscale image, and c) a simplified illustration of the associated point spread function;

[0075] FIG. 10 shows a schematic illustration of a phase distribution in a pupil of a detection beam path by the effect of an axicon, respectively as a) a simplified black-and-white sketch, b) as a grayscale image, and c) a simplified illustration of the associated point spread function;

[0076] FIG. 11 shows a schematic illustration of a phase distribution in a pupil of a detection beam path without the effect of an optical element, as a) a simplified black-and-white sketch, b) as a grayscale image, and c) a simplified illustration of the associated point spread function;

[0077] FIG. 12 shows a schematic illustration of a) a phase distribution of a first ring phase mask in a pupil plane of a detection beam path and b) a much-simplified illustration of an associated simulated point spread function;

[0078] FIG. 13 shows a schematic illustration of a) a phase distribution of a second ring phase mask in a pupil plane of a detection beam path and b) a much-simplified illustration of an associated simulated point spread function;

[0079] FIG. 14 shows a schematic illustration of a) a phase distribution of a third ring phase mask in a pupil plane of a detection beam path and b) a much-simplified illustration of an associated simulated point spread function; and

[0080] FIG. 15 shows a flowchart of a configuration of a method.

DETAILED DESCRIPTION

[0081] Implementations disclosed herein are illustrated schematically. Here, the same reference signs denote the same technical elements in each case.

[0082] An example implementation is illustrated in FIG. 2 and is compared to the prior art illustrated in FIG. 1. An optical element 14, the effect of which leads to the depth of field of the objective 5 being extended, is arranged in the detection beam path 3 of an optical apparatus. The point spread function PSF is extended in the direction of the Z-axis Z. This extended depth of field EDOF (elucidated by a double-headed arrow) facilitates the capture of the entire sample 7 in the direction of the Z-axis Z with only a single focus setting of the objective 5 in this exemplary embodiment. Accordingly, all markers 8 bound to the sample 7 can be imaged simultaneously by capturing their respective (fluorescence) signals 12 by means of the detector 13. The information about the position of the location of origin (marker 8) of a respectively captured signal 12 in the direction of the Z-axis Z is lost in the process. A marker 8 can only be localized in the detection plane along the X and Y axes.

[0083] A first exemplary implementation of an apparatus is shown in FIG. 3 as part of a microscope 1 not illustrated in any more detail. Illumination radiation is generated and provided in a laser light source 16. The illumination beam reaches the objective 5 via a beam splitter 18 in reflection along the illumination beam path 2, in which optical lenses 17 are situated. An optical element 14 in the form of a phase element is arranged in or in the vicinity of a Fourier plane of the objective 5. In a Fourier plane, a point is transformed into a plane wave and, conversely, a plane wave is transformed into a point. So that the respectively present phase element has the same action on all object points, it should be located in the Fourier plane or be arranged as close as possible to a Fourier plane.

[0084] An extended depth of field EDOF of the objective 5 is achieved by the action of the optical element 14. In the sample 7, the illumination radiation causes detection radiation 4, which strikes the beam splitter 18 along the detection beam path 3. The detection radiation 4 deviates from the illumination radiation in terms of its optical properties and is transmitted by the beam splitter 18. If the detection radiation 4 is formed by radiation of at least two wavelengths, these can be separated from one another and can be captured separately. In the illustrated exemplary embodiment, such a separation is brought about by means of a further beam splitter 18, which is transparent to one wavelength and reflective for another wavelength. Detection radiation 4 split in this way reaches the detector 13 or a further detector 20 along partial beam paths.

[0085] The detectors 13 and 20 are connected to an evaluation unit 23, by means of which the captured signals 12 are processed as image data and evaluated. Moreover, a control unit 24 is present, by means of which the control commands for controlling, e.g., the laser light source 16, the optical element 14, the tube lens 11, and/or the optical lenses 17 can be generated and output. The image data and information regarding generated control commands and/or the current configuration of the apparatus can be displayed on a display 25.

[0086] The arrangement of the optical element 14 in a pupil-conjugate plane (Fourier plane) (FIG. 4) can be chosen so that the illumination radiation does not pass through the optical element 14. In the illustrated third exemplary embodiment, illumination beam path 2 and detection beam path 3, or the illumination radiation and the detection radiation 4, are split from one another by means of a beam splitter 18. The detection radiation 4 is steered to the optical element 14 and the detector 13 by means of a mirror 19.

[0087] A further possible exemplary embodiment of an apparatus (FIG. 5) is suitable for attaining an extended depth of field EDOF by means of a fast displacement of the respective focal plane DOF. An intermediate image downstream of the tube lens 11 is imaged on the detector 13 by means of two lenses 17.1 and 17.2 illustrated in exemplary fashion in the detection beam path 3. Below, the lenses 17.1 and 17.2 can also represent more complex imaging optical units. A varifocal lens 21, which facilitates a displacement of the current focal plane DOF and the generation of an extended depth of field EDOF, is present between the lenses 17.1 and 17.2. The varifocal lens 21 can additionally include a compensation lens 21.1, as shown schematically in FIG. 5. The varifocal lens 21 is controllable by means of the control unit 24. In particular, control can be implemented so quickly that at least two current focal planes DOF are set within a capture time interval of the detector 13 and consequently a resultant extended depth of field EDOF is generated. The detection radiation 4 is captured with the (resultant) extended depth of field EDOF.

[0088] An axicon is arranged in the detection beam path 3 as an optical element 14 in a further exemplary implementation of the apparatus (FIG. 6). The optical element 14 is arranged in the vicinity of the lens 17.1, the latter in turn being arranged at a distance from an intermediate image plane 22 in accordance with its focal length.

[0089] A further implementation option of the apparatus can facilitate a dynamic generation of the extended depth of field EDOF using an adaptive mirror or a microlens array as an optical element 14 (FIG. 7). The lenses 17.1 and 17.2 in the detection beam path 3 image a first intermediate image on the detector 13. The optical element 14 is present in a pupil 15 (pupil plane).

[0090] One exemplary embodiment for multifocal imaging combined with an extended depth of field EDOF is elucidated in FIG. 8. The lenses 17.1 and 17.2 image the intermediate image on the detector 13 and simultaneously create a further conjugate pupil 15. The optical element 14 is arranged in the latter. The lenses 17.1 and 17.2 provide a so-called relay optical unit.

[0091] The detection radiation 4 is captured with a respective depth of field DOF with a plurality of mutually different focal planes, wherein the effect of the optical element 14 generates an extended depth of field EDOF1 or EDOF2 around each of the focal planes, as shown schematically in the magnified partial illustration. In this case, the extended depth of field EDOF1 allows imaging of signals 12 of markers 8, antigens 9 connected to marked antibodies 10, and/or marked antibodies 10 of the upper cell membrane 7.1 and the extended depth of field EDOF2 allows imaging of signals 12 of markers 8, antigens 9 connected to marked antibodies 10, and/or marked antibodies 10 of the lower cell membrane 7.2.

[0092] In further embodiments of the apparatus and/or configurations of the method, the depths of field DOF or the extended depths of field EDOF advantageously slightly overlap in the direction of the z-axis Z in order to allow interruption-free imaging of the relevant sample volume.

[0093] Furthermore, an image splitter unit 26 is arranged in the detection beam path 3. By means of the latter, the components of the detection radiation 4 from the different focal planes DOF or the different extended depths of field EDOF1, EDOF2 are directed at different detectors 13 or at different regions of at least one detector 13 and are captured separately from one another.

[0094] An extended depth of field EDOF can be generated using phase masks. Phase distributions in a pupil of a detection beam path as a result of the effect of different phase masks are shown in FIG. 9 to FIG. 11. In this case, partial FIGS. 9a, 10a, and 11a each show a simplified black-and-white sketch of the grayscale images illustrated in FIGS. 9b, 10b, and 11b with corresponding phase distributions in an xy-plane. FIGS. 9c, 10c, and 11c, respectively, illustrate the simplified point spread functions PSF as a section in an xz-plane in each case.

[0095] FIGS. 9a to 9c show the sketch, the grayscale image, and the PSF of a symmetric cubic phase mask.

[0096] FIGS. 10a to 10c show the corresponding phase distributions and the PSF of an axicon.

[0097] Phase distribution and PSF without the arrangement of an optical element 14 in the detection beam path 3 are illustrated in exemplary fashion in FIGS. 11a to 11c.

[0098] Various ring phase masks and their respective simulated point spread functions are shown in FIGS. 12 to 14. In this case, FIGS. 12a, 13a, and 14a each schematically show a generated ring phase mask. FIGS. 12b to 14b illustrate the associated point spread functions PSF in simplified fashion.

[0099] FIG. 12 relates to a ring phase mask with a phase shift of π between the differently hatched regions. The resultant PSF shows two separate maxima in the direction of the Z-axis Z.

[0100] A ring phase mask with a plurality of different regions, once again with a phase shift of π in each case, is illustrated schematically in FIG. 13a. The associated point spread function PSF is pulled apart in the direction of the Z-axis Z. At least two separate maxima occur, with these having a clearly identifiable tendency of forming further maxima. Ring phase masks with more than the shown regions and phase shifts allow additional maxima and hence focal planes DOF and/or extended depths of field EDOF. Depending on the design of the phase masks, the point spread function PSF can be matched to the sample 7 to be imaged and/or to the sought-after object of the imaging.

[0101] Configuration options of the method are explained on the basis of the flowchart in FIG. 15. In particular, one of the above-described embodiment options of the apparatus is used to carry out the method.

[0102] A sample 7 that is expected to potentially emit fluorescence signals or light-induced light signals is provided. By way of example, such samples 7 are biological surfaces with at least one antibody 10, which is compatible with an antigen 9 to be detected and which is provided with a marker 8 suitable for emitting light signals. However, a biological surface incubated with such an antibody 10 can also be used as a sample 7.

[0103] An extended depth of field EDOF is chosen, by means of which the relevant sample 7 should be examined for the presence of fluorescence signals 12. If a current setting or configuration of the apparatus, for example, a currently set phase ramp, allows the detection of the signals 12 with the desired extended depth of field EDOF, the measurements are carried out with this configuration. To this end, the sample 7 is illuminated with the illumination radiation, the latter facilitating and/or potentially triggering the emission of the signals 12. If the illumination radiation acts as excitation radiation, markers 8 which are present in the illuminated region of the sample 7 and which are receptive to the illumination radiation are excited to emit signals 12.

[0104] The emitted signals 12 are imaged by means of the apparatus and the currently set extended depth of field EDOF on the at least one detector 13, 20 arranged in the detection beam path 3 and are captured by said detector as image data (measurement). The spatial resolution of the detector 13, 20 facilitates a two-dimensional assignment (2-D localization) of the location of origin of the respective signal 12 in an XY-plane. A localization in the direction of the Z-axis Z is possible, at best, to the effect of the origin of the signal 12 being known as coming from the region of the extended depth of field EDOF and so a correspondingly coarse region in the direction of the Z-axis Z being able to be assigned to a position in the XY-plane. The localization assists the check for possible two-time capture of signals 12 and a possibly required correction of the detection and/or count results of the method.

[0105] The captured image data are merged to form a resultant overall image, for example, by means of the evaluation unit 23; this is also referred to as image synthesis or rendering.

[0106] In an alternative course of the method, it is determined that the chosen extended depth of field EDOF cannot be attained with the current configuration of the apparatus, in particular with the current phase ramp. In the illustrated example, the extended depth of field EDOF should be generated by a plurality of focal planes DOF, which are located sufficiently close to one another in the direction of the Z-axis Z.

[0107] Therefore, the optimum spacings of the focal planes DOF and the overlap thereof are set for the desired extended depth of field EDOF. By means of the apparatus set thus, the measurement and the localization of the respectively captured signal 12 is implemented in the respective focal planes DOF and/or in the region of the extended depth of field EDOF.

[0108] The captured image data of the individual focal planes DOF, and hence of the extended depth of field EDOF, are subsequently combined to form an overall data record. In the process, it is possible to use the known spacings and/or overlaps of the focal planes DOF. In addition, or as an alternative thereto, structures of the sample 7, which are contained in the image data and which have been identified, can be used to verify ascertained localization data by way of correlations of the structure data. The overall data record obtained thus, which may have been verified where necessary, is merged to form a resultant overall image.

[0109] Additionally, a value of the plane spacing can be provided and can be included in the combination method step to form an overall data record. By way of example, this optional value is advantageous if a plurality of objects, e.g., cells, are present above one another or if image data are captured in tissues.

REFERENCE SIGNS

[0110] 1 Microscope [0111] 2 Illumination beam path [0112] 3 Detection beam path [0113] 4 Detection radiation [0114] 5 Objective [0115] 6 Slide [0116] 7 Sample [0117] 7.1 Lower cell membrane [0118] 7.2 Upper cell membrane [0119] 8 Marker [0120] 9 Antigen [0121] 10 Antibody [0122] DOF Focal plane; original depth of field [0123] EDOF Focal region; extended depth of field [0124] PSF Point spread function [0125] 11 Tube lens [0126] 12 Fluorescence signal [0127] 13 Detector [0128] 14 Optical element [0129] 15 Pupil [0130] 16 Laser light source [0131] 17 Lens [0132] 17.1 Lens [0133] 17.2 Lens [0134] 18 Beam splitter [0135] 19 Mirror [0136] 20 Further detector [0137] 21 Varifocal lens [0138] 21.1 Compensation lens [0139] 22 Intermediate image plane [0140] 23 Evaluation unit [0141] 24 Control unit [0142] 25 Display [0143] 26 Image splitter unit