WO2016193037A1 - Method for determining spatially resolved height information of a sample by means of a wide-field microscope, and wide-field microscope - Google Patents

Abstract

The invention relates to a wide-field microscope and to a method for determining spatially resolved height information of a sample (14) by means of 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 capturing a wide-field image in an observation beam path of a sample (14) illuminated in a sample plane (P); a modulator for chromatically modulating the illumination beam path or the observation beam path in a direction perpendicular to the sample plane (P); an evaluating unit for determining chromatic confocal height information in each pixel of the wide-field image. The method comprises the following steps: illuminating the sample (14) by means of a wide-band illumination source (1) in an illumination beam path; chromatically modulating the illumination beam path or a detection beam path; capturing at least one wide-field image from sample light reflected or emitted by the sample in the detection beam path, said sample light having chromatic confocal components; determining, pixel by pixel, height information of the sample from the wide-field image by evaluating chromatic confocal components of the detection beam path in accordance with the chromatic modulation.

Description

Method for determining a spatially resolved height information of a sample with a wide field microscope and a wide field microscope

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 method today

used. In most cases, the sample is sampled in all three spatial directions, i. it is a matter of

Point-scanning systems in which an optical beam is guided in the x / y direction over the sample. To derive the

Height information requires movement of the sample relative to the detector unit (in the z-direction). 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 spent by the raster scan for a 3D topography

is needed. Furthermore, during the xy scan, where there is a fixed geometric arrangement between

Sample body and optical sensor is present, by external shocks or vibrations to uncontrolled movements of the

Sensor head relative to the sample body come, whereby the measurement result can be falsified. To avoid z-rastering, the chromatic confocal principle is used. This is usually a

used polychromatic light source, which the

sample of interest via a chromatic-acting

illuminated 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 whereby also a spectrum is obtained.

Kim et al in "Chromatic confocal microscopy with a novel wavelength detection method using transmittance", OPTICS EXPRESS 6286, Vol. 21, No. 5, describes a point-scanning chromatic confocal arrangement with 50/50 beam splitting in the

Detection beam path behind the pinhole detection. The

Sample light is detected correspondingly with two photomultiplier tubes (PMT ^x s), 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 xy raster scan, long-established 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 every Value a confocal sectional image calculated from images that are structured with eg a grid

Lighting was recorded. As a rule, the wide field image can also be obtained. Taking advantage of the

Polarization or the color properties of the

 Illumination light is obtained height information from the sample. For example, DE 10 2007 018 048 A1 describes such a system in which two illumination patterns 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 is a poorly confocal image with non-focal

Shares and one as a pure wide field image or as a picture with predominantly non-focal shares. 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 have a disturbing effect on the

Can take measurement result.

There are other wide field methods with which

optical sections can be generated. 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

To provide a method for generating a spatially resolved height information of a sample, in which disturbing movements can be avoided on the microscope.

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

customized. This is achieved in particular by encoding the wavelength in the illumination or detection beam path. In a preferred embodiment

Wavelength-dependent filters in the detection beam path

used. In a method according to the invention, a sample with a broadband illumination source in one

Illuminated beam path illuminated.

 The illumination beam path becomes chromatic

confocal principle modulated chromatically.

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 wide field image can be both purely confocal parts of the

Sample light (eg when using a Nipkow disc), but also be composed of confocal and non-focal portions of the sample light. In the observation beam path and / or in the illumination or excitation beam path is at least one

Wavelength-dependent filter function or spectral distribution are used and for each pixel in the x-y direction, at least two measuring operations are carried out with the different filters or spectral distributions. These measurements can take place in parallel (when using several image sensors) or sequentially. For example, if the ratio of the intensities of the at least two measurements in each pixel of the

Farfieldbildes formed, it is from this the wavelength with maximum intensity of the sample light and thus the height value of the sample at each pixel can be determined.

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 is in the invention

Method and microscope in the i-th measuring procedure given by: h {x, y, z) = jάλ 'Ρ {χ, γ, λ') R {x, y, ') Τ ^ χ, γ, λ') _{λ [ ζχ, γ)]} (λ [ζ (χ, γ)] - λ ')

With :

P (x, y, ^r ): spectral characteristics of the light source and the

 Device may also depend on x, y

R {x, y, '): spectral reflectivity of the sample

 Τι {χ, γ, λ '): filter function or spectral distribution in the i-th

 Measuring process or chromatic modulation (also includes beam splitters, etc.)

gXmax (h _max -X): spectral device response function with X _max as

 parameter

max ⁼ [z (x, y)]: maximum reflected wavelength at x, y according to the height 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 gx _max ( _max -X). The

 Parameterization with regard to the wavelength indicates that this function varies spectrally depending on the nature of the chromatic deposition. For further consideration will be

simplifying assumed that the parameterization

is negligible and the function g represents a delta function located at 0. The above formula then simplifies to: li (x, y, z) = P (x, y, [z (x, y)]) A (x, y, A [z (x, y)]) 7Kx, y, A [z (x, y)]) If P, R, and T are sufficiently well known, we can conclude from this

in principle already z (x, y) are derived, which, however, entails enormous calibration effort, also because it is an absolute measurement. However, if at least two detection processes i = l, 2

used, the ratio can be formed to:

/ jfa, z) _7 \ (x, y, l [z (x, y)])

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 can be based on the known filter functions or

Spectral distributions relatively simple determine the value associated with a given intensity ratio λ and thus the value z (x, y). In a special case T _{2 has} no wavelength dependence (T _S (X) = constant). This case can be realized

for example, with the aid of two similar detectors and a beam splitter, wherein only in one beam path, a wavelength-dependent filter is used.

Another possibility is to use only one channel and two sequential measurements with and without or with two different wavelength-dependent filters

but in quick succession

be carried out that can be said to speak of a bullet measurement (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 is also here next to one

sequential arrangement a parallel arrangement possible. 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 = 1T]. Preferred embodiments of the invention will be explained in more detail with reference to FIGS.

Show it: a first preferred embodiment of a wide field microscope according to the invention; an embodiment variant with parallel detectors; an embodiment variant with parallel detectors and filters in the detection beam path; an embodiment variant with a switching element in the detection beam path; an embodiment variant with a chip splitter detector; a second preferred embodiment of a microscope according to the invention; a third preferred embodiment of a microscope according to the invention; an advantageous embodiment variant of the illumination beam path with a switching element; an advantageous embodiment variant of the illumination beam path with two equal moistening light sources; 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 apply

Reference numerals for like elements. Their functional description also apply in the figures or embodiments in which they are not expressly mentioned.

FIG. 1 shows a first preferred embodiment of a wide field microscope according to the invention. A polychromatic light source 1 (z. Ex. Broadband laser, halogen lamp, superluminescent diode, ...), wherein in this embodiment, different spectral distributions can be selected by a selection element 2 ^¬. This selection element 2 can be, for example, an AOTF (acousto-optical tunable filter), a prism, a grating or also a filter selection unit. The illumination light can then be deflected by a deflection unit 3 in different directions. The

Deflection unit 3, for example, provides a fast

switchable mirrors (e.g., galvo mirrors), AOD (Acousto-optic deflector) or 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 4 represents a transmissive 1D or 2D lattice structure. The structure is imaged into the sample space via refractive and / or diffractive longitudinal chromatic aberration-inducing elements 6, 7 (objective), so that here a chromatic splitting 8 is generated in z-direction, ie the focus shifts in dependence on the wavelength in the z-direction.

In the observation beam are advantageously

Optical fiber 9 is arranged. But it can be in others

 Embodiments for this purpose, a simple free beam guidance based on mirrors are used. With the

Optical fibers 9 can optionally be made a polarization filtering. By means of the deflection unit 3, it is possible to sequentially illuminate the structured element 4 by means of a respective collimating lens 11 from two sides (dashed representation). For this purpose, the structured element 4 is executed mirror-coated, it can accordingly be imaged two grid phases in the sample space or the sample plane P. Is this

structured element 4 is not mirrored, so eliminates the deflection unit 3 and the dashed lines shown optics.

To combine the transmission and reflection beam path, a beam splitter 12 is used. The

Illumination light is then passed through a beam splitter 13 on to a sample 14 positioned in the sample space P, the beam splitter 13 advantageously being referred to as

 Polarization beam splitter is executed. Namely, it can continue to be a lambda / 4 plate 16 is arranged in the beam path, so that the sample 14 going to the illumination light and the sample 14 reflected or emitted to be detected sample light having a 90 ° to each other rotated polarization and so well at the beam splitter 13 from each other can be separated.

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, to suppress. For this purpose, a polarization filter 18 can furthermore be arranged in front of the detector unit 17. The

Detection unit 17 may be a simple camera with

corresponding imaging optics, if the spectral

Distributions over the selection element 2 alone in the

Lighting or excitation can be selected. In addition or instead, one of the embodiments according to FIGS. 2 to 5 is also possible. FIG. 2, for example, describes an arrangement in which the sample light is first guided through a color splitter 19, so that two detection channels are operated, each comprising an imaging optic 21 and a camera 22.

In essence, this arrangement is the same as above

In FIG. 3, in comparison to FIG. 2, the color divider is replaced by a beam splitter 23, which initially has no

Wavelength-dependent filtering causes. This is coming

however, in channel I through a filter 24. Optionally, a filter 26 may also be arranged in channel II (T2 would then not be constant, if there is no filter 26, T2 would be constant).

With Figure 4 is already described above

sequential detection mapped. A switching element 27 serves for the sequential switching of filter functions. The

 Switching element 27 can in this case e.g. a fast filter wheel or an AOTF or a suitable beam splitter arrangement with

Switching mirror arrangement be. A particular arrangement, which functions similar to the arrangements described in FIG. 2 or 3, is shown in FIG. Here comes a so-called chip splitter 28

Use, as for example by the company Optosplit

is offered, i. One and the same camera chip of the camera 22 is used for the two measuring operations, which can therefore also take place in parallel.

It is thus with a device according to the above

described figures initially possible, different To image and detect lattice phases or structures on the sample, and it can in this way as in the usual methods of structured illumination an "optical

If the surface of the sample is in focus at this location for a given pixel in xy, it is usually possible to find a wavelength for which this case is given, provided the surface topography in their height dynamics does not go much beyond the associated with the chromatic storage measuring range addition.

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

Evaluation of the filter function as described above, wherein in the simplest case, the intensity ratios of the

at least two measuring processes are evaluated and from this directly on the wavelength is closed.

 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 then

advantageous if the sample is strongly colored and in

shows different spectral ranges a different reflection behavior. 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. Sometimes a calibration is sufficient regardless of the function g as well as the function P is not enough, so these two functions as device properties still have to be included. The

Wavelength may then be determined not directly, but using an iterative method.

Is the surface topography such that its

Height dynamics associated with chromatic storage

Measuring range exceeds, so if necessary, a z-stitching is required, in which similar measurements at

different distances between sensor and sample are performed and then linked together.

The filter functions are usefully like that

adapted chromatic color storage that over the entire wavelength range, a similarly sensitive height determination is possible.

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 Figure 1, the switching of different grid phases can also by a simple switching of

 Beam paths can be realized, including, 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

Illustration of the structured element 4 in transmission and

Reflection. In addition, arrangements are conceivable 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. One Grid is here according to the Fourier transform in

Essentially represented as a dot pattern, which the

corresponds to individual diffraction orders of the grid. 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 may also be formed by a 2D pinhole arrangement, is moved to different positions, and corresponding images are taken with the detector unit 17, but in this case only one light channel of the illumination is used , The detector unit 17 can also be used as a digital PH, so that a truly confocal image

Billing and composition of the images taken at each position is obtained.

The evaluation with regard to the wavelength takes place as described above. In a further variant, the structure 4 is completely eliminated and a sharpening function over the wavelength is determined in each case only for each local image area. This

corresponds to the principle of focus variation, as they are

for example in M. Rahlves, J. Seewig, "Optical Measurements of Technical Surfaces", Beuth Verlag GmbH, Berlin, 2009. With sufficiently structured samples, this is also sufficient to obtain height information.

Similarly, a structure that works on the principle of HiLo method designed. The structured element 4 can also represent an element for targeted introduction of a speckle pattern, which can be completely removed from the beam path. FIG. 6 shows a further exemplary embodiment of a

Chromatic wide field microscope, which corresponds to the combination of the aperture correlation principle with the chromatic confocal technology. In an intermediate image plane Z, a rotatable disk 31 with a mirrored structure 32 is arranged here. The sample light (detection beam path) reflected or emitted back from the sample 14 is detected in two illustrated camera channels in the example shown, a first detector 33 passing through the disc 31

transmitted sample light (confocal components) and a second detector 34 the sample light reflected from the mirrored structure 32 (wide-field image with non-focal portions)

to capture. Optionally, polarization filters 18 can also be arranged in the detection beam path here.

From the two images of the detectors 33, 34 can in

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.

FIG. 7 shows a further exemplary embodiment in which, for example, a pinhole array 41 or a Nipkow disk is used. A detection of the entire sample surface is achieved by a movement (rotation,

Shift) of the pinhole array 41 and / or a

optional scanner unit 42 reached. If the pinhole array 41 is a Nipkow disk and designed as such with structured and unstructured sectors, this embodiment also provides a special case of the aperture correlation in which the confocality evaluation is carried out by offsetting sequentially or parallel recorded structured and non-structured illuminated images. An interferometer element 43 is optionally provided to increase the measurement accuracy. This may also be present in all other embodiments.

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

Thus, FIG. 8 shows the polychromatic light source 1, whose light can be conducted with a fast switching element 44 into different channels, which in turn is the same as the two measuring processes in analogy to the one discussed above. In both channels now different filters 46 and 47

be arranged. In principle, only one of the two filters 46, 47 is sufficient. In each case, a beam combination takes place at the beam combiner element 48, wherein in a skilful variant this is also polarization-sensitive, e.g. can be designed as a polarization beam splitter. If a color splitter is used instead of the beam combiner element 48, the filters 46, 47 may possibly be dispensed with, which corresponds in its overall effect to the case described in FIG. 2 in a sequential design.

Fig. 9 shows an embodiment variant of the invention, in which two similar light sources 1 with, respectively

downstream filters 46, 47 are used, which are connected in quick succession.

A further advantageous embodiment variant is shown in FIG. 10 again. Here are two different sources of illumination 51 and 52 are used, which differ in terms of their spectral characteristics. The beam combining element 53 is now designed as a pure beam combiner. The spectral characteristics already yield the desired filter functions. For example, the spectra of the

 Illumination sources 51, 52 are slightly shifted from each other and gaussförmig. From the intensity ratio of the two with each one of the illumination sources 51, 52 coupled

Measurements is then immediately derived the wavelength.

Reference sign list

01 illumination source 32 mirrored structure

02 selection element 33 first detector unit

03 deflecting unit 34 second detector unit

04 structured element 35

05 41 pinhole array

 06 Color longitudinal error- 42 Scanner unit

 inducing element 43 interferometer

 07 Lens 44 switching element

 08 chromatic decomposition 45

 09 optical fiber 46 filter

10 47 filters

 11 collimating lens 48 beam combining element

12 beam splitter 49

 13 beam splitter 50

 14 sample 51 illumination source

15 52 Lighting source

 16 λ / 4 plate 53 beam union element

17 detector unit

 18 polarization filters A, Z - field level

 19 color dividers

20

 21 imaging optics

 22 camera

 23 beam splitters

 24 filters

25

 26 filters

 27 switching element

 28 chip splitter

29

 30

 31 disc

Claims

claims Method for determining a spatially resolved  Height information of a sample (14) with a  Wide field microscope, comprising the following steps:  Illuminate the sample (14) with a broadband Lighting source (1) in one  Beieuchtungsstrahlengang;  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 evaluation of chromatic confocal components of the  Detection beam path as a function of the chromatic modulation. The method of claim 1, further comprising the steps of:  Capture a first image with a first one  chromatic modulation adjustment;  Acquiring a second image with a second chromatic modulation setting at the same time or with a time delay to acquire 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 li (x, y, z) = / dX P (x, y, X ') R (x, y, X') Τ ^ χ, γ, λ ') gx _{[z (x, y)]} ([z (_x, y)] - λ '), where: P (x, y, X ^r ) a spectral characteristic of the light source and the device;  R {x, y, X ') has a spectral reflectivity of the sample; Ti {x, y, ') the chromatic modulation; gAmax (max-) a spectral device response function with X _max as parameter;  max a maximum reflected wavelength at one Place x, y according to a height 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 Pictures for each pixel according to the instructions l {x, y, z) _ T _x {_x, y, X \ z {_x, y) l ₂ (x, y, z) r ₂ (x, y, A [z (x, y)]) is formed. Method according to one of claims 1 to 4, characterized in that the chromatic modulation of the illumination beam path by a sequential Switching a filter in the illumination beam path or in the observation beam path takes place. 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 takes place. Wide field microscope comprising  an illumination source (1, 52, 53), which in one  Illuminating beam path is arranged;  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 Wide field microscope according to claim 7, characterized 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 Observation beam paths as a modulator 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 Observation beam is arranged. Wide field microscope according to claim 7, characterized characterized in that a switching element in Illuminating beam path is arranged. Wide field microscope according to one of claims 1 to 11, characterized in that the chromatic modulator is a filter.