METHOD FOR DETERMINING HEIGHT INFORMATION OF A SAMPLE, AND SCANNING MICROSCOPE

Abstract

The invention relates to a method for determining height information of a sample, and to a scanning microscope. The method comprises the following steps: generating an illumination spot; illuminating the sample with the illumination spot; capturing an image of a reflection of the illumination spot at the sample; evaluating the lateral distribution of the image; determining the height information from the lateral distribution; wherein the illumination spot has a three dimensional illumination pattern and/or the image in a detection beam path has a three dimensional detection pattern. The scanning microscope is characterized in that an illumination device (07) and/or a detector device comprise(s) a means for generating a three dimensional pattern with a change in the lateral intensity distribution that is asymmetrical along the optical axis, and an evaluation device is configured to determine height information from the detector signal.

Claims

1. Method for determining height information of a sample comprising the following steps: generating an illumination spot; illuminating the sample with the illumination spot; capturing an image of a reflection of the illumination spot at the sample; evaluating the lateral distribution of the image; determining the height information from the lateral distribution; wherein the illumination spot has a three dimensional illumination pattern and/or the image in a detection beam path has a three dimensional detection pattern.

2. Method according to claim 1, characterized in that the illumination pattern and/or the detection pattern have/has a change in the lateral intensity distribution which is asymmetrical along the optical axis, wherein the optical axis runs parallel to a plane normal of a sample plane in which the sample is positioned.

3. Method according to claim 1, characterized in that a phase profile is used for generating the illumination pattern and/or the detection pattern.

4. Method according to claim 3, characterized in that the phase profile is linear.

5. Method according to claim 1, characterized in that intensity information of the image is furthermore determined.

6. Method according to claim 1, characterized in that the illumination pattern is generated by the use of a laser beam with a rotating intensity distribution.

7. Method according to claim 1, characterized in that a plurality of illumination spots are scanned simultaneously over the sample plane.

8. Scanning microscope, comprising an illumination device for illuminating a sample with an illumination spot; a scanning apparatus for scanning the illumination spot over the sample; a detector device for the spatially resolved detection of the illumination spot reflected by the sample; an evaluation device for evaluating the signal of the detector device; characterized in that the illumination device and/or the detector device comprise(s) a means for generating a three dimensional pattern with a change in the lateral intensity distribution that is asymmetrical along the optical axis; and the evaluation device is configured to determine height information from the detector signal.

9. Scanning microscope according to claim 8, characterized in that the means for generating the three dimensional pattern is a phase element.

10. Scanning microscope according to claim 9, characterized in that the phase element comprises a glass wedge which is arranged in a pupil plane of the scanning microscope, and covers at least part of the illumination spot and/or of the image.

11. Scanning microscope according to claim 10, characterized in that the glass wedge is a first glass wedge and the phase element comprises a second glass wedge, wherein the first glass wedge and the second glass wedge are arranged in opposite directions and each cover half of the illumination pattern.

12. Scanning microscope according to claim 8, characterized in that the means for generating the illumination pattern is a laser beam with a rotating intensity distribution.

13. Scanning microscope according to claim 8, characterized in that the detector device is a quadrant photodiode.

Description

[0035] In the figures:

[0036] FIG. 1: shows various point spread functions;

[0037] FIG. 2: shows an illumination pattern or detection pattern in various z positions;

[0038] FIG. 3: shows one preferred embodiment of the evaluation of an illumination pattern reflected by a sample or of a detection pattern;

[0039] FIG. 4: shows a circuit diagram for the evaluation of the detector signals;

[0040] FIG. 5: shows an intensity signal of a detector device as a function of a topography (Z) of a sample;

[0041] FIG. 6: shows a basic schematic diagram of a laser scanning microscope in a first preferred embodiment with an open control loop;

[0042] FIG. 7: shows a basic schematic diagram of a laser scanning microscope in a second preferred embodiment with a closed control loop.

[0043] FIG. 1 shows various point spread functions (PSF) such as occur or are evaluated for example in the detection of a laser scanning microscope.

[0044] The figure shows at the bottom a standard point spread function such as arises as a result of diffraction at a pinhole. The illustration shows the lateral resolution as a function of the Z position.

[0045] The figure shows at the top a so called double helix point spread function (DH-PSF) such as is described e.g. in [Pavani et al., Optics Express 16, 26 (2008)] for 3-D data generation in super resolution microscopy (PALM). Such a point spread function that is asymmetrical in a lateral (X-Y) and axial (Z) direction is preferably used as illumination pattern. It can likewise be used as detection pattern.

[0046] FIG. 2 shows a different illumination pattern or detection pattern, which was generated by the use of a glass wedge covering one half of the pupil in a laser scanning microscope. A similar detection point spread function is described in [Baddeley et al., Nano Res. 4, 589 (2011)].

[0047] According to the invention, such and similar point spread functions can be generated both in illumination and in detection and can be used as illumination pattern or detection pattern, wherein, with the use of laser sources, if appropriate, it is necessary to take account of the coherence in the sense of a point light source.

[0048] FIGS. 3 and 4 describe the measurement and evaluation principle with the use of an illumination pattern or detection pattern.

[0049] FIG. 3 shows a pattern 01 at two different Z positions represented by 500 nm and 750 nm. A detector is schematically depicted in the pattern. The detector is a position sensitive detector (PSD) and, in the example illustrated, a four quadrant photodiode 02. It can be discerned that the intensities in the respective quadrants in the different Z positions have different magnitudes.

[0050] FIG. 4 shows an exemplary evaluation circuit that forms a differential signal of in each case two quadrants. In the case of the present pattern having two symmetrical spots proceeding in opposite directions, the differential signal of the quadrants {A and B} and {C and D} would be identical and can be averaged in order to increase the sensitivity in one embodiment of the invention.

[0051] The normalized differential signals are formed as follows:

[00001]$\frac{I_A-I_B}{I_A+I_B}\propto z$$\frac{I_C-I_D}{I_C+I_D}\propto z$

[0052] The summation information of all the quadrants would correspond to the intensity signal of the scanned point and be used to determine the overall image in a known manner.

[0053] Alternatively and depending on the point pattern, the sum of two of the four quadrants suffices, that is to say that only one of the two spots is used; the lateral resolution is thus increased.

[0054] FIG. 5 illustrates a signal profile of a pattern evaluated according to the above calculation.

[0055] A central region 03 between maximum 04 and minimum 06 exhibits a strictly monotonic behaviour with very high signal quality, which moreover is linear over a wide range. In this region 03, the z information can thus be unambiguously obtained directly from the detector differential signal. A non-linear behaviour (for example when a different type of illumination pattern is used) can also be mapped by corresponding gauging and calibrating curves which are sufficiently known to the person skilled in the art.

[0056] FIG. 6 shows a laser scanning microscope in a first preferred embodiment with an open control loop (open loop).

[0057] The laser scanning microscope is firstly constructed in a known manner. A laser 07 provides illumination light in an illumination beam path 08, said illumination light being scanned point by point onto a sample plane 11 by a scanner 09.

[0058] Light reflected and/or emitted by a sample positioned in the sample plane 11 is fed in an observation beam path 12 through an objective 13 and a beam splitter 14 to a detector 16. The detector 16 is a position sensitive detector (PSD).

[0059] A phase element 17 is provided at least in one of the two beam paths, in the illumination beam path or in the detection beam path, or in both beam paths, in order to generate an unambiguous intensity distribution, which is greatly variable as a function of z, transversely and parallel with respect to the optical axis. The phase element 17 can be for example a simple glass wedge as in [Baddeley et al., Nano Res. 4, 589 (2011)] or a complex phase mask as in [Grover et al., Biomedical Optics Express 2, 3012 (2011)]. The phase element 17 is situated in or near a conjugate pupil of the system in excitation and/or detection. A phase element 18 is illustrated by dashed lines in the figure; the phase element 18 can be used as an alternative or in addition to the phase element 17.

[0060] Alternatively, the phase element 17, 18 can also be a Spatial Light Modulator (SLM). If the latter acts in transmission (e.g. liquid crystal SLM), there is no change to the construction; if a reflection SLM (e.g. Micromirror-Array, Deformable Mirror) is involved, the beam path must be correspondingly folded.

[0061] A camera or a detector array can also be used instead of the detector 16. The number of pixels can be small (=fast); it suffices to image the reflected illumination pattern. Thus the entire image information of the illumination pattern can then be analysed.

[0062] A detector array as described in DE 10 2013 019 347 A1 is particularly suitable for this purpose. In this regard, e.g. a stored system PSF can be adapted.

[0063] Instead of the scanning movement, the sample can also be moved relative to the measuring head.

[0064] FIG. 7 shows a system which is in principle of the same construction as shown in FIG. 6, but embodied as a closed control loop (closed loop). Identical reference signs have the same meaning as described above.

[0065] The system additionally comprises a piezo actuator 19 for adjusting the sample plane 11 in the z direction and a controller 20.

[0066] The controller 20 receives e.g. the differential signal of the detector 16, which is determined as described above, as controlled variable r. In order to keep this controlled variable at a setpoint value w (e.g. “0”), the controller 20 applies a control signal s to the piezo actuator 19. Said signal is directly proportional to z (topography) and is detected and evaluated by an evaluation unit 21. In this case, the evaluation unit 21 can have a dedicated signal processor for data processing. In addition, an error signal f (deviation from the controlled variable) can be detected and recorded, which error signal, e.g. with the use of a PI controller, to an approximation is proportional to the change in topography (derivation).

[0067] The advantage of this embodiment consists in a high sensitivity (=topography resolution) which results from the great gradient of the signal at the zero crossing (FIG. 5), and in a high operating range in the z direction. The latter is no longer provided by the depth of focus of the illumination pattern (e.g. ˜1-1.5 μm at 100×0.9 NA), but rather by the movement range of the piezo actuator 19, which may be a few 10-100 μm. This mode by its nature is slower than the embodiment described in FIG. 6, owing to the closed loop control and the actuating time of the piezo actuator 19.

[0068] What is ideal is a demand controlled switching between the operating modes “open loop” (fast, limited in z) and “closed loop” (slow, large z operating range, sensitive).

[0069] In this embodiment, too, one phase element 18 is illustrated by dashed lines; it can be used as an alternative or in addition to the phase element 17.

LIST OF REFERENCE SIGNS

[0070] 01 Illumination pattern [0071] 02 Four quadrant photodiode [0072] 03 Central region [0073] 04 Maximum [0074] 05 [0075] 06 Minimum [0076] 07 Laser [0077] 08 Illumination beam path [0078] 09 Scanner [0079] 10 [0080] 11 Sample plane [0081] 12 Observation beam path [0082] 13 Objective [0083] 14 Beam splitter [0084] 15 [0085] 16 Detector [0086] 17 Phase element [0087] 18 Phase element [0088] 19 Piezo actuator [0089] 20 Controller [0090] 21 Evaluation unit