Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B11/00—Measuring arrangements characterised by the use of optical techniques
  • G01B11/24—Measuring arrangements characterised by the use of optical techniques for measuring contours or curvatures
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
  • G01B2210/00—Aspects not specifically covered by any group under G01B, e.g. of wheel alignment, caliper-like sensors
  • G01B2210/50—Using chromatic effects to achieve wavelength-dependent depth resolution

Definitions

• the invention relates to a method for determining a spatially resolved height information of a sample with a
• optical section 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
• 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.
• 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
• 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.
• PMT x s photomultiplier tubes
• the transmission of the filter and thus the detected wavelength and, finally, a height information is determined.
• confocal wide-field systems which are based on structured illumination.
• Illumination light is obtained height information from the sample.
• DE 10 2007 018 048 A1 describes such a system in which two illumination patterns are projected onto the sample.
• optical sections can be generated.
• 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.
• the object is achieved by a method according to claim 1 and by a wide field microscope according to claim 7.
• the chromatic confocal principle is applied to a wide-field optical cross-sectional imaging method and
• Wavelength-dependent filters in the detection beam path are Wavelength-dependent filters in the detection beam path
• the illumination beam path becomes chromatic
• At least one far field image is detected by detecting sample light reflected or emitted by the sample in a detection beam path.
• Sample light (eg when using a Nipkow disc), but also be composed of confocal and non-focal portions of the sample light.
• 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
• the intensity signal is in the invention
• Device may also depend on x, y
• Measuring process or chromatic modulation also includes beam splitters, etc.
• the ratio can be formed to:
• a wavelength-dependent filter 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
• 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
• An example of the parallel arrangement is the use of a Bayer pattern color camera with two color channels each.
• 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.
• 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.
• Deflection unit 3 for example, provides a fast
• switchable mirrors e.g., galvo mirrors
• AOD Acoustic-optic deflector
• polarization rotation e.g., polarization rotation
• a structured element 4 is arranged in a plane A conjugate to a sample plane P.
• 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.
• 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.
• Optical fibers 9 can optionally be made a polarization filtering.
• 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).
• 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.
• a beam splitter 12 is used to combine the transmission and reflection beam path.
• 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.
• a polarization filter 18 can furthermore be arranged in front of the detector unit 17.
• Detection unit 17 may be a simple camera with
• FIG. 2 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.
• 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).
• a switching element 27 serves for the sequential switching of filter functions.
• 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.
• 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
• At least two measuring processes are evaluated and from this directly on the wavelength is closed.
• 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.
• Wavelength may then be determined not directly, but using an iterative method.
• Measuring range exceeds, so if necessary, a z-stitching is required, in which similar measurements at
• the filter functions are usefully like that
• Beam paths can be realized, including, for example, an electro-optical modulator (EOM) or an acousto-optic modulator (AOD) can be used.
• EOM electro-optical modulator
• AOD acousto-optic modulator
• Modulation in the pupil plane of the lens 7 causes.
• One Grid is here according to the Fourier transform in
• 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
• the evaluation with regard to the wavelength takes place as described above.
• the structure 4 is completely eliminated and a sharpening function over the wavelength is determined in each case only for each local image area.
• 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.
• 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
• polarization filters 18 can also be arranged in the detection beam path here.
• both a wide-field image and a confocal image can be calculated.
• the intensity information as a function of the wavelength yields the sought height information for each detection pixel.
• the resulting color image can also be used directly to represent a color image with extended depth of field information.
• 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,
• 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.
• 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.
• filters 46 and 47 are now different filters 46 and 47.
• 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.
• FIG. 10 A further advantageous embodiment variant is shown in FIG. 10 again.
• 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

Landscapes

• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Optics & Photonics (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Microscoopes, Condenser (AREA)
• Length Measuring Devices By Optical Means (AREA)
• Instruments For Measurement Of Length By Optical Means (AREA)

Priority Applications (2)

Applications Claiming Priority (2)

Publications (1)

Family

ID=56092898

Family Applications (1)

Country Status (4)

Cited By (2)

Families Citing this family (4)

Citations (2)

Family Cites Families (4)

• 2015
  • 2015-06-01 DE DE102015210016.2A patent/DE102015210016A1/de active Pending
• 2016
  • 2016-05-23 CN CN201680029710.4A patent/CN107710046B/zh active Active
  • 2016-05-23 JP JP2017556932A patent/JP6595618B2/ja active Active
  • 2016-05-23 WO PCT/EP2016/061581 patent/WO2016193037A1/de active Application Filing

Patent Citations (2)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events