DE102015210016A1 - Method for determining a spatially resolved height information of a sample with a wide field microscope and a wide field microscope - Google Patents

Abstract

The invention relates to a wide field microscope and a method for determining a spatially resolved height information of a sample (14) with a wide field microscope. The wide field microscope comprises an illumination source (1, 52, 53) which is arranged in an illumination beam path; a first detector unit (17, 33) for detecting a wide-field image in an observation beam path of a sample (14) illuminated in a sample plane (P); a modulator for chromatic modulation of the illumination beam path or the observation beam path in a direction perpendicular to the sample plane (P); an evaluation unit for determining a chromatically confocal height information in each pixel of the wide field image. The method comprises the following steps: illuminating the sample (14) with a broadband illumination source (1) in an illumination beam path; chromatic modulation of the illumination beam path or a detection beam path; Detecting at least one wide field image of specimen light having chromatic confocal portions reflected or emitted from the specimen in the detection beam path; Pixel-wise determination of height information of the sample from the wide-field image by evaluating chromatically confocal components of the detection beam path as a function of the chromatic modulation.

Description

The invention relates to a method for determining a spatially resolved height information of a sample with a wide-field microscope and a wide-field microscope.

The determination of a spatially resolved height information of a sample is also referred to as an optical section. Such optical sections are used in particular in microsopy to determine topographies of a sample or surface properties of a sample such as e.g. To measure roughness.

For the characterization of technical surfaces, confocal microscopy is the standard procedure used today. In most cases, the sample is sampled in all three spatial directions, i. These are point-scanning systems in which an optical beam is guided in the x / y direction over the sample. To derive the height information, a movement of the sample relative to the detector unit (in the z-direction) is required. From the intensity maximum as a function of the z-position, the height information and thus the topography can be derived for each x-y location.

A disadvantage of this method is, among other things, the long time required by the raster scan for a 3D topography. Furthermore, during the xy scan, in which there is a fixed geometric arrangement between the specimen and the optical sensor, external shocks or oscillations can lead to uncontrolled movements of the sensor head relative to the specimen, which can falsify the measurement result.

To avoid z-rastering, the chromatic confocal principle is used. Here, a polychromatic light source is usually used, which illuminates the sample of interest via a chromatically acting refractive and / or diffractive element, whereby the z-information is spectrally encoded. If now the spectrum is measured behind a confocal pinhole in the detection, then the height information can be derived therefrom. Also possible, but time consuming, is the use of a tunable light source with sequential confocal detection which also gives a spectrum.

Kim et al. Describe in "Chromatic confocal microscopy with a novel wavelength detection method using transmittance", OPTICS EXPRESS 6286, Vol. 21, no. 5 a point-scanning chromatic confocal arrangement with 50/50 beam splitting in the detection beam path behind the pinhole detection. The sample light is correspondingly detected with two photomultiplier tubes (PMTs), wherein one filter is connected upstream of the one PMT. From the intensity ratio of the two PMTs, the transmission of the filter and thus the detected wavelength and, finally, a height information is determined.

To circumvent the disadvantage of the x-y raster scan, long-range confocal wide-field systems already exist, in which surface-area cameras are generally used. An example of this is the spinning-disc process with Nipkow disc. Here several points are detected almost simultaneously according to the confocal principle. The approach of different z-positions to determine a sectional image is also required here.

Furthermore, confocal wide-field systems are known which are based on structured illumination. Here, for each z-value, a confocal sectional image is calculated from images taken with a structured illumination given, for example, by a grid. As a rule, the wide field image can also be obtained. Taking advantage of the polarization or the color characteristics of the illumination light, height information is obtained from the sample. In the DE 10 2007 018 048 A1 For example, such a system is described in which two patterns of illumination are projected onto the sample.

Also related is the process of aperture correlation. Here, a continuously changing structured illumination is used and the optical sectional image is calculated from two parallel or sequentially recorded images, one of which can be regarded as a poorly confocal image with non-focal portions and one as a pure wide field image or as an image with predominantly non-focal proportions. An advantage of this method based on structured illumination is that parallel to the confocal image, a wide field image can also be obtained virtually in one shot.

In the end, all systems based on structured illumination have in common that during the change of the phase pattern and / or during the movement of the sample or the sensor in the z direction, vibrations can have a disturbing influence on the measurement result.

There are other wide-field methods that can be used to create optical sections. Here, for example, the focus variation should be mentioned, in which the image sharpness is evaluated as a function of z in order to calculate a maximum similar to the confocal case. It also spatial information of the system are used.

With regard to susceptibility to vibration, the same problems exist as in the aforementioned methods.

It is the object of the invention to provide a microscope and a method for generating a spatially resolved height information of a sample, in which disturbing movements on the microscope can be avoided.

The object is achieved by a method according to claim 1 and by a wide field microscope according to claim 7.

According to the invention, the chromatic confocal principle is applied to a wide-field optical cross-sectional imaging method and adapted. This is achieved in particular by encoding the wavelength in the illumination or detection beam path. In a preferred embodiment, wavelength-dependent filters are used in the detection beam path.

In a method according to the invention, a sample is illuminated with a broadband illumination source in an illumination beam path. The illumination beam path is chromatically modulated according to the chromatic confocal principle. Furthermore, at least one far field image is detected by detecting sample light reflected or emitted by the sample in a detection beam path.

The far-field image may comprise both purely confocal portions of the sample light (e.g., when using a Nipkow disc), but may also be composed of confocal and extra-focal portions of the sample light.

At least one wavelength-dependent filter function or spectral distribution is used in the observation beam path and / or in the illumination or excitation beam path, and at least two measurement operations with the different filters or spectral distributions are performed for each pixel in the x-y direction. These measurements can take place in parallel (when using several image sensors) or sequentially.

If, for example, the ratio of the intensities of the at least two measurement processes is formed in each pixel of the wide-field image, then the wavelength with maximum intensity of the sample light and thus the height value of the sample at the respective pixel can be determined therefrom.

In particular, this is possible in principle independently of the spectral reflectivity of the sample and independently of the spectral properties of the light source and or of the device.

In general, the intensity signal in the method and microscope according to the invention in the ith measuring process is given by: I _i (x, y, z) = ∫ dλ'P (x, y, λ ') R (x, y, λ') T _i (x, y, λ ') g _{λ [z (x, y) ]} (λ [z (x, y)] - λ '),

P(x, y, λ'):

spektrale Charakteristik der Lichtquelle und des Geräts eventuell auch abhängig von x, y

R(x, y, λ'):

spektrale Reflektivität der Probe

T_i(x, y, λ'):

Filterfunktion oder Spektralverteilung im i-ten Messvorgang bzw. chromatische Modulation (beinhaltet auch Strahlteiler etc.)

g_λmax(λ_max – λ):

spektrale Geräte-Response-Funktion mit λ_max als Parameter

λ_max = λ[z(x, y)]:

maximal reflektierte Wellenlänge an der Stelle x, y entsprechend der Höhenfunktion

z(x, y):

Höhenfunktion der Probe

With:

P (x, y, λ '):

Spectral characteristics of the light source and the device may also depend on x, y

R (x, y, λ '):

spectral reflectivity of the sample

T _i (x, y, λ '):

Filter function or spectral distribution in the i-th measuring process or chromatic modulation (also includes beam splitters, etc.)

g _max (λ _max - λ):

spectral device response function with λ _max as parameter

λ _max = λ [z (x, y)]:

maximum reflected wavelength at the location x, y according to the altitude function

z (x, y):

Height function of the sample

We are now looking for the height function z (x, y) for the sample to be examined. The confocal or quasi-confocal detection manifests itself in particular in the function g _λmax (λ _max -λ). The parameterization with respect to the wavelength indicates that this function varies spectrally depending on the nature of the chromatic deposition. For the sake of further consideration, it is assumed for the sake of simplification that the parameterization is negligible and the function g represents a delta function located at 0. The above formula simplifies to: I _i (x, y, z) = P (x, y, λ) [z (x, y)]) R (x, y, λ [z (x, y)]) T _i (x, y, λ [z (x, y)])

If P, R and T are sufficiently well known, z (x, y) can in principle already be deduced therefrom, which, however, entails an enormous calibration effort, also because it is an absolute measurement.

However, if at least two detection processes i = 1,2 are used, the ratio can be formed to:

P and R no longer matter. As an approximation, this also applies if P and R are constant over the integration range given by the form of g. From the right side, it is relatively easy to determine the value associated with a given intensity ratio, and thus also the value z (x, y), on the basis of the known filter functions or spectral distributions.

In a special case T _{2 has} no wavelength dependence (T _s (λ) = constant). This case can be realized, for example, with the aid of two similar detectors and a beam splitter, wherein a wavelength-dependent filter is used only in one beam path.

Another possibility is to use only one channel and to carry out two sequential measurements with and without or with two different wavelength-dependent filters, which, however, are carried out in such a rapid sequence that quasi a shot measurement can be spoken (total measurement <100ms).

Also possible is the use of a filter in the excitation, so that is measured with the same light source alternately with and without filter.

Another special case is the use of two spectrally offset bandpass filters in excitation and / or detection. In the detection, a parallel arrangement is also possible here in addition to a sequential arrangement. An example of the parallel arrangement is the use of a Bayer pattern color camera with two color channels each.

Another special case is, for example, the use of two detection channels and a dichroic beam splitter such that T ₂ = 1 - T ₁ .

Preferred embodiments of the invention will be explained in more detail with reference to FIGS.

Show it:

1 a first preferred embodiment of a wide field microscope according to the invention;

2 : an embodiment variant with parallel detectors;

3 : an embodiment variant with parallel detectors and filters in the detection beam path;

4 an embodiment variant with a switching element in the detection beam path;

5 an embodiment variant with a chip splitter detector;

6 a second preferred embodiment of a microscope according to the invention;

7 a third preferred embodiment of a microscope according to the invention;

8th : An advantageous embodiment variant of the illumination beam path with a switching element;

9 : An advantageous embodiment variant of the illumination beam path with two identical illumination light sources;

10 : An advantageous embodiment variant of the illumination beam path with two spectrally different illumination light sources.

In the following description of the figures, the same reference numbers apply to the same elements. Their functional descriptions also apply in the figures or embodiments in which they are not expressly mentioned.

1 shows a first preferred embodiment of a wide field microscope according to the invention. A polychromatic illumination source 1 (For example, broadband laser, halogen lamp, super luminescence diode, ...), in this embodiment, different spectral distributions by a selection element 2 are selectable. This selection element 2 For example, it may be an AOTF (Acousto-Optical Tunable Filter), a prism, a grid, or a filter selection unit. The illumination light can then pass through a deflection unit 3 be diverted in different directions. The deflection unit 3 represents, for example, a fast-switching mirror (eg galvo mirror), an AOD (Acousto-optical deflector) or a polarization rotation based switching unit.

A structured element 4 is arranged in a plane A conjugate to a sample plane P. In the simplest case, the structured element represents 4 a transmissive 1D or 2D lattice structure. The structure is via refractive and / or diffractive longitudinal chromatic aberration inducing elements 6 . 7 (Lens) imaged in the sample space, so here is a chromatic splitting 8th in the z direction is generated, ie the focus shifts depending on the wavelength in the z-direction.

In the observation beam path are advantageously optical fiber 9 arranged. However, in other embodiments, a simple free beam guidance based on mirrors can also be used for this purpose. With the optical fibers 9 Optionally, polarization filtering can be performed.

Through the deflection unit 3 is it possible the structured element 4 with the help of a collimating lens 11 from two sides to illuminate sequentially (dashed line). This is the structured element 4 executed mirrored, it can be mapped accordingly two grating phases in the sample space or the sample plane P. Is the structured element 4 not mirrored, so eliminates the deflection 3 and the dashed lines shown optics.

To combine the transmission and reflection beam path is a beam splitter 12 used. The illumination light is then passed through a beam splitter 13 to a sample positioned in the sample space P 14 led, wherein the beam splitter 13 is advantageously designed as a polarization beam splitter. It can still continue a lambda / 4-plate 16 be arranged in the beam path, so that the sample 14 going illumination light and from the sample 14 reflected or emitted to be detected sample light having a 90 ° to each other twisted polarization and so on the beam splitter 13 can be separated well from each other.

Furthermore, it is possible with such a configuration, disturbing reflections from the optical elements of a detection unit 17 and not from the sample 14 stem, suppress. For this purpose, further in front of the detector unit 17 a polarizing filter 18 be arranged. The detection unit 17 can be a simple camera with appropriate imaging optics, if the spectral distributions on the selection element 2 be chosen alone in the lighting or stimulation. Additionally or alternatively, one of the embodiments according to the 2 to 5 possible.

2 describes, for example, an arrangement in which the sample light is first passed through a color splitter 19 is guided, so that two detection channels are served, each having an imaging optics 21 and a camera 22 include.

In essence, this arrangement corresponds to the special case described above with T2 = 1 - T1.

In 3 is compared to 2 the color divider through a beam splitter 23 replaced, which initially causes no wavelength-dependent filtering. However, this comes in channel I through a filter 24 conditions. Optionally, also in channel II a filter 26 be arranged (T2 would not be constant then there is no filter 26 , T2 would be constant).

With 4 the sequential detection already described above is mapped. A switching element 27 serves the sequential switching of filter functions. The switching element 27 can be eg a fast filter wheel or an AOTF or a suitable beam splitter arrangement with switching mirror arrangement.

A special arrangement similar to the one in 2 or 3 works described in is 5 shown. Here comes a so-called chip splitter 28 used, as it is offered for example by the company Optosplit, ie it is one and the same Kamerachip the camera 22 used for the two measuring processes, which can thus also run in parallel.

It is thus initially possible with a device according to the figures described above to image and detect different grating phases or structures on the sample, and it is thus possible to produce an "optical cross-sectional image" as in the conventional structured illumination methods for a given pixel in xy only has a significant signal if the sample surface is in focus at that location. As a rule, one can always find a wavelength for which this case is given, provided that the surface topography in its height dynamics does not significantly exceed the measuring range associated with the chromatic deposition.

The evaluation of the image data takes place in such a way that the wavelength at which the optical sectional image signal becomes maximum is determined for each pixel. From this, the function z (x, y) or the surface topography can be directly deduced. This is done, for example, by evaluating the filter function as described above, wherein in the simplest case the intensity ratios are evaluated from the at least two measurement processes and from this is directly deduced to the wavelength. Of course, multiple measurements are useful here in order to obtain a better signal-to-noise ratio. In addition, it can also be useful to carry out the multiple measurements at different absolute heights by moving the sample relative to the sensor. This is advantageous if the sample is strongly colored and shows a different reflection behavior in different spectral ranges.

Depending on the sample, HDR imaging, for example, by multiple measurements with different exposure times is also useful, so that the noise for each pixel is essentially shot-noise limited. Occasionally, a calibration is not sufficient regardless of the function g and the function P, so that these two functions as device properties still have to be involved. The wavelength may then be determined not directly, but using an iterative method.

If the surface topography is such that its height dynamic exceeds the measurement range associated with chromatic storage, z-stitching may be required where similar measurements are made at different distances between the sensor and the sample and then linked together.

The filter functions are usefully adapted to the chromatic color storage so that a similar sensitive height determination is possible over the entire wavelength range.

Sometimes it may be useful to apply a filter function in both lighting and detection, as well as to perform multiple measurements with different filter functions to achieve better sensitivity in terms of altitude determination.

With regard to 1 the switching of different grating phases can also be realized by a simple switching of optical paths, for which purpose, for example, an electro-optical modulator (EOM) or an acousto-optic modulator (AOD) can be used. In the case shown here results, for example, a phase-shifted mapping of the structured element 4 in transmission and reflection. Arrangements are also conceivable, for example, where, for example, an EOM or an AOD or even a galvo mirror a fast grid switching by a direct modulation in the pupil plane of the lens 7 causes. A grating is represented here in accordance with the Fourier transformation substantially as a dot pattern, which corresponds to the individual diffraction orders of the grating. By means of an angle modulation, it would be possible, for example, to switch quickly between different grid positions.

In another variant, the structured element 4 , which can also be formed by a 2D pinhole arrangement, moved to different positions, and it is with the detector unit 17 taken corresponding images, but here only one light channel of the illumination is used. The detector unit 17 can be used as a digital PH, so that a truly confocal image is obtained by calculating and composing the images taken at each position.

The evaluation with regard to the wavelength takes place as described above.

In another variant, the structure is eliminated 4 completely and it is only determined for each local image area a sharpness function on the wavelength. This corresponds to the principle of focus variation, such as in M. Rahlves, J. Seewig, "Optical Measurement of Technical Surfaces", Beuth Verlag GmbH, Berlin, 2009 , is described. With sufficiently structured samples, this is sufficient to obtain height information.

Similarly, a structure that works on the principle of HiLo method designed. The structured element 4 can also be an element for specific introduction of a speckle pattern, which can be completely removed from the beam path.

6 shows another embodiment of a wide-field chromatic microscope, which corresponds to the combination of the aperture correlation principle with the chromatic confocal technology. In an intermediate image plane Z here is a rotatable disc 31 with a mirrored structure 32 arranged. That from the sample 14 sample light reflected back or emitted (detection beam path) is detected in the example shown in two camera channels, wherein a first detector 33 that through the glass 31 transmitted sample light (confocal portions) and a second detector 34 from the mirrored structure 32 detect reflected sample light (wide-field image with off-focus portions). Optionally, polarization filters can also be used here 18 be arranged in the detection beam path.

From the two pictures of the detectors 33 . 34 Depending on the wavelength, both a wide-field image and a confocal image can be calculated. Again, the intensity information as a function of the wavelength yields the sought height information for each detection pixel. Of course, the resulting color image can also be used directly to represent a color image with extended depth of field information. Likewise, structures are possible in which the two channels are arranged on only one camera chip.

In 7 a further embodiment is shown in which, for example, a pinhole array 41 or a Nipkow disc is used. A detection of the entire sample surface is achieved by a movement (rotation, displacement) of the pinhole array 41 and / or one optional scanner unit 42 reached. Is the pinhole array 41 a Nipkow disk and as such with structured and unstructured sectors, this embodiment also represents a special case of the aperture correlation, in which the confocality evaluation takes place by offsetting sequentially or parallel recorded structured and non-structured illuminated images.

An interferometer element 43 is optional for increasing the measuring accuracy. This may also be present in all other embodiments.

8th to 10 show possible design variants for the use of filter functions or different spectral distributions in the illumination beam path.

So shows 8th the polychromatic light source 1 whose light comes with a fast switching element 44 can be routed into different channels, which in turn, in analogy to the above discussed equal to the two measurement processes. Both channels now have different filters 46 and 47 be arranged. In principle, only one of the two filters is sufficient 46 . 47 , In any case, a beam combination takes place at the beam combiner element 48 instead, this can be designed in a clever variant polarization-sensitive, for example, as a polarization beam splitter. Will instead of Strahlvereinigerelementes 48 a color divider can be used on the filters 46 . 47 be omitted if necessary, which in the overall effect of the basis of 2 described case in sequential execution corresponds.

9 shows an embodiment variant of the invention, in which two similar light sources 1 each with downstream filters 46 . 47 used, which are switched quickly in a row.

A further advantageous embodiment variant is 10 again. Here are two different sources of illumination 51 and 52 used, which differ in terms of their spectral characteristics. The beam combining element 53 is now designed as a pure Strahlvereiniger. The spectral characteristics already yield the desired filter functions. For example, the spectra of the illumination sources 51 . 52 be slightly shifted to each other and gaussförmig. From the intensity ratio of the two with each one of the illumination sources 51 . 52 Coupled measuring processes, then the wavelength can be derived immediately.

LIST OF REFERENCE NUMBERS

01

 lighting source

02

 selection element

03

 Deflector

04

 structured element

05

06

 Color longitudinal error inducing element

07

 lens

08

 chromatic decomposition

09

 optical fiber

10

11

 collimating lens

12

 beamsplitter

13

 beamsplitter

14

 sample

15

16

 λ / 4 plate

17

 detector unit

18

 polarizing filter

19

 color splitter

20

21

 imaging optics

22

 camera

23

 beamsplitter

24

 filter

25

26

 filter

27

 switching element

28

 Chip Splitter

29

30

31

 disc

32

 mirrored structure

33

 first detector unit

34

 second detector unit

35

41

 Pinhole array

42

 scanner unit

43

 interferometer

44

 switching element

45

46

 filter

47

 filter

48

 Beam combining element

49

50

51

 lighting source

52

 lighting source

53

 Beam combining element

A, Z

 field level

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102007018048 A1 [0008]

Cited non-patent literature

• Kim et al. Describe in "Chromatic confocal microscopy with a novel wavelength detection method using transmittance", OPTICS EXPRESS 6286, Vol. 21, no. 5 [0006]
• M. Rahlves, J. Seewig, "Optical Measurement of Technical Surfaces", Beuth Verlag GmbH, Berlin, 2009 [0065]

Claims (12)

Method for determining a spatially resolved height information of a sample ( 14 ) with a wide field microscope, comprising the following steps: illuminating the sample ( 14 ) with a broadband illumination source ( 1 ) in a illumination beam path; - Chromatic modulation of the illumination beam path or a detection beam path; Detecting at least one wide-field image of sample light with chromatic confocal components reflected or emitted by the sample in the detection beam path; Pixel-wise determination of height information of the sample from the wide-field image by evaluating chromatically confocal components of the detection beam path as a function of the chromatic modulation. The method of claim 1, further comprising the steps of: - acquiring a first image having a first chromatic modulation setting; Capturing a second image with a second chromatic modulation setting at the same time or with a time delay for capturing the first image; Determining a ratio of the intensity signals of the two images for each pixel; Determining a height value z (x, y) of the sample ( 14 ) for each pixel. A method according to claim 2, characterized in that the intensity signal is defined by I _i (x, y, z) = ∫ dλ'P (x, y, λ ') R (x, y, λ') T _i (x, y, λ ') g _{λ [z (x, y) ]} (λ [z (x, y)] - λ '), where: P (x, y, λ ') is a spectral characteristic of the light source and the device; R (x, y, λ ') has a spectral reflectivity of the sample; T _i (x, y, λ ') the chromatic modulation; g _λmax (λ _max - λ) a spectral device response function with λ _max as a parameter; λ _max = λ [z (x, y)] a maximum reflected wavelength at a location x, y corresponding to an altitude function; and z (x, y) represents the height function of the sample. A method according to claim 2 or 3, characterized in that the ratio of the intensity signals of the two images for each pixel according to the rule

is formed. Method according to one of claims 1 to 4, characterized in that the chromatic modulation of the illumination beam path is effected by a sequential circuit of a filter in the illumination beam path or in the observation beam path . Method according to one of claims 2 to 5, characterized in that the detection of a first wide field image and a second wide field image in two separate detection channels. Wide field microscope comprising - a source of illumination ( 1 . 52 . 53 ) disposed in an illumination beam path; A first detector unit ( 17 . 33 ) for detecting a wide-field image in an observation beam path of a sample illuminated in a sample plane (P) ( 14 ); - A modulator for chromatic modulation of the illumination beam path or the observation beam path in a direction perpendicular to the sample plane (P); - An evaluation unit for determining a chromatically confocal height information in each pixel of the wide field image Wide field microscope according to claim 7, characterized in that it comprises a second detector, which is similar to the first detector. Weifeldmikroskop according to claim 8, characterized in that between the first and the second detector, a beam splitter is arranged and in one of the observation beam paths as a modulator, a wavelength-dependent filter is arranged. Weifeldmikroskop according to claim 8, characterized in that the first detector in the observation beam path of a device for detecting chromatic confocal components is arranged downstream and the second detector for detecting non-focal components in the observation beam path is arranged. Wide field microscope according to claim 7, characterized in that a switching element is arranged in the illumination beam path . Wide field microscope according to one of claims 1 to 11, characterized in that the chromatic modulator is a filter.

Images