WO2007131602A1 - Dispositif et procédé de microscopie en transmission confocale, en particulier également pour la mesure d'objets de phase mobiles - Google Patents

Dispositif et procédé de microscopie en transmission confocale, en particulier également pour la mesure d'objets de phase mobiles Download PDF

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Publication number
WO2007131602A1
WO2007131602A1 PCT/EP2007/003644 EP2007003644W WO2007131602A1 WO 2007131602 A1 WO2007131602 A1 WO 2007131602A1 EP 2007003644 W EP2007003644 W EP 2007003644W WO 2007131602 A1 WO2007131602 A1 WO 2007131602A1
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beam path
phase
chromatic
light microscopy
optical
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PCT/EP2007/003644
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German (de)
English (en)
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Klaus KÖRNER
Evangelos Papastathopoulos
Wolfgang Osten
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Universität Stuttgart
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    • 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
    • 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
    • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
    • 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
    • G02B21/0068—Optical details of the image generation arrangements using polarisation

Definitions

  • No. 6,091,496 does not disclose any approach for spectral interferometric transmitted light microscopy, but there is a first indication of the same in WO2006 / 042696, page 118, using a Mach-Zehnder interferometer with longitudinal chromatic aberration and compensation thereof.
  • E. Papastathopoulos, K. Körner and W. Osten "Chromatically dispersed interferometry with wavelet analysis”, in OPTICS LETTERS, Vol. 31, No. 5, pp. 589-591, 2006 the spectral Interferometry with chromatic depth splitting of the detection field in the object space with the analysis of the resulting wavelet is shown, but this approach has not been described for transmitted light microscopy.
  • the invention for transmitted light microscopy can be used for a variety of tasks in medicine and biology, in particular for the analysis of individual, unstained, untreated, living animal or human cells and culture cells by determining their optical thickness and depth position.
  • the invention can also be used for the analysis of chromosomes in human and cytogenetics.
  • the quantitative determination of the distribution, the position in space and the lateral dimensions of opaque microparticles can be carried out in a transparent medium. This must be "introduced” between the condenser and the lens It is possible with the invention to determine the distribution and the lateral dimensions of microfibers in a transparent or even partially cloudy medium, such as special plastics, ie to use the invention for technical applications.
  • the analysis of Si structures in transmission can be carried out, whereby the structures should have an approximately constant thickness because of the spherical aberration.
  • the determination of refractive index changes can also be carried out in the inventive solution.
  • the above-mentioned applications can be carried out in the DUV, UV, VIS or even in the NIR range, whereby immersion techniques in oil, water or glycerine can also be used used tuned lenses become.
  • the inventive approach also makes it possible to read volume data memories that operate in transmission by detecting and evaluating changes in the refractive index in the irradiated medium.
  • the object of the present invention for transmitted-light microscopy, in particular for measuring the optical thicknesses of transparent and partially opaque objects, also known as phase objects, is to provide new items for commercial use.
  • the goal here is in particular the quantitative detection of thin phase objects, e.g. single, also undyed, living cells.
  • the technical aim of the invention is to increase the speed when measuring the optical thicknesses and increasing the usable depth measuring range, in particular when using an imaging system with a comparatively high numerical aperture, for example in the range from 0.4 to 1.25 (water immersion). or 1.5 (oil grade) to values equal to a multiple of the depth of field given by wave optics.
  • optical signals obtained with the inventive sensor during measurement should be detected by means of a radiation detector or a screened radiation detector, such as a line camera or an area camera.
  • Another goal is to provide a lesson for reading volumetric data memories that operate in transmission.
  • optical signals from these depths without the mechanical movement of components - in particular in the object space - are obtained, in particular the optical thickness or To be able to determine changes in the optical thickness of an object or its components or also of several objects.
  • an at least approximately diffraction-limited lateral imaging of the object is to be effected by an imaging system with a comparatively high numerical aperture, in particular also in immersion technology.
  • the inventive task is to be solved for the Nomarski phase contrast method, the differential interference contrast (D ⁇ C) method, the Hoffmann modulation contrast (HMC) method or by means of microscopes based on the two-beam interferometry, especially by means of a Mach-Zehnder interferometer, highly accurate, quantitative measurements of optical thickness or thicknesses, including variations same, of transparent objects with very high resolution.
  • this involves a resolution in the depth direction in the size of a few nanometers.
  • the inventive task is to be achieved that electromagnetic interference modulations are generated over time, which have a sufficiently small and therefore technically good evaluable frequency from this information in particular the optical To be able to determine the thickness or the change in the optical thickness.
  • This inventive task is intended in particular for a confocal differential interference microscope, in particular for the Normarski DIC microscope, that is to say on the basis of polarization-optically generated lateral shear, and also a confocal two-beam interference microscope, in particular also for a confocal Mach - Zehnder interference microscope, to be solved.
  • Main feature in a method for transmitted-light microscopy, in particular also for quantitative testing of one or more objects or a part or parts thereof or the same in the object space with a polychromatic light source in the illumination beam path and with a
  • these images are sequential in the light propagation direction separated, so that object points from different depths can be detected by means of the micro-apertures or the microlenses.
  • a value for the depth position of a sharply imaged object point of a very thin phase object as well as its change in the optical thickness in relation to the surrounding medium can be calculated. This can be done in a range z_c, which can be a multiple of the optical depth of field.
  • the wavelength of the shading can be determined, from which the contour of microparticles can be determined.
  • the source of electromagnetic radiation broadband for example, in the DUV UV, in the VIS or in the IR range, be formed and short-pulse, for example, a short pulse laser, and its pulses with a camera which serves as a detector, for example in the 100th Hz range, to be synchronized.
  • a wavelength-tunable laser can also be used.
  • a spectrally broadband source electromagnetic radiation can be used, for example, a fiber-coupled, broadband white light laser, in which case a controllable monochromator is arranged downstream to tune the wavelength.
  • the light is spatially structured in the object space, in which a focus pattern or a focus line pattern are formed in the object space predetermined by means of multiple micro-apertures or multiple by means of multiple microlenses macroblende, and in the illumination beam path a chromatic depth splitting of these foci or focus lines is performed by means of at least partially chromatic refractive power, so that these foci or focus lines in the light propagation direction, ie in depth, are separated.
  • the at least partially chromatic refractive power in the illumination and the at least partially chromatic refractive power in the detection beam path are predetermined so determined that in the object space the amount and the direction of the chromatic depth splitting of the foci or focus lines for illumination and in the detection beam path, the amount and the direction of the chromatic Depth splitting of the redrawn micro-apertures or the multiple mapped macro-aperture are at least approximately equalized.
  • the images A 'of the centers A of the apertures BA of the illumination beam path or the images A' of the foci A and the images B 'of the centers B of the apertures BB of the detection beam path which respectively correspond laterally and spectrally, namely for example, A'o_ ⁇ min-B'o_ ⁇ min and A'o_ ⁇ max-B'o_ ⁇ max, optically conjugated.
  • their planes of sharpness in object space at least approximately coincide.
  • the inventive method for increasing the depth measuring range over the wavelength optical depth of field also only two monochromatic sources of electromagnetic radiation of different wavelengths can be used with frequency modulation, so that the known heterodyne method can be used. With the radiation of each wavelength, the object is scanned at a slightly different depth.
  • the process of dark field microscopy is preferably used in the inventive method for transmitted-light microscopy.
  • the camera which also scatter or diffract the light and are also at least approximately in the focal plane of a reflected diaphragm for confocal discrimination, the focal plane being one of light of a predetermined wavelength of the spectrum polychromatic light source or the wavelength tunable light source.
  • Objects that do not scatter or diffract the light remain dark, since the directly transmitted light is not detected.
  • the position of an object point can be determined by determining the wavelength position of the maximum of the intensity and from this the depth position.
  • an object-related adaptation of the distance between the condenser and the objective that is to say an axial displacement of the same, can also remedy this problem, which compensates for the optical difference in thickness between the object and the surrounding medium and thus again enables sharp object matching.
  • phase objects here "thick" on the wave-optical depth of field of the microscopic image is assumed and this is usually a multiple of the same, the axial displacement of components to adapt to the optical thickness, only the scattered light from the thick Object overcome the confocal discrimination, so that the object brings a higher intensity than the object-free environment for imaging.
  • the method of phase microscopy based on the interference of electromagnetic waves is preferably used in the inventive method for transmitted-light microscopy. This allows a particularly high resolution and photometric sensitivity in the measurement of objects.
  • Feature 5 Furthermore, preferably in the inventive method for transmitted-light microscopy with chromatic depth splitting, the phase contrast method, in particular also that according to F. Zernike, the differential phase contrast method, especially according to G. Nomarski, as DIC method (DIC: differential interference contrast), ie methods based on polarization-optical shear interferometry, the Hoffmann HMC method (HMC: Hoffmann modulation contrast) or else a phase-microscopic method according to the two-beam interference method, in particular also based on a Mach-Zehnder interferometer.
  • DIC method DIC: differential interference contrast
  • HMC Hoffmann modulation contrast
  • a phase-microscopic method according to the two-beam interference method in particular also based on a Mach-Zehnder interferometer.
  • an optical path difference OPD OPD: optical path difference
  • Beam path of a phase microscope which is at least one wavelength of the maximum detected wavelength, but preferably up to 100 wavelengths of the maximum detected wavelength.
  • interference fringes in particular also in the form of a wavelet, are also known as Müllersche strips or Tolansky strips, ie intensity profiles over the wavelength ⁇ or the wavenumber k are formed in the detected spectral range in a predetermined manner.
  • a thin and laterally small phase object for example of the order of magnitude of the extension of the diffraction-limited point image of the microscope-produces a local phase change in the interference fringes, ie in particular also in a wavelet.
  • This local phase change is evaluated.
  • a laterally extended phase object which completely covers the measuring field, generates a change in the frequency in the interference pattern, ie in particular also in a wavelet, which leads to an altered rise of the phase, ie the phase gradient dtydk, over the wave number k.
  • an extremum can be determined if preferably a sufficiently large optical path difference in the DIC microscope is given with polarization-optical Shear.
  • a comparatively thick retardation plate in the DIC microscope can be used, which preferably has an optical path difference between the two polarization directions in the order of 100 wavelengths of the largest wavelength used in order to increase the accuracy of the Phasenaustician- strategies.
  • phase shifting LCD in the DIC microscope in order to vary the optical path difference in sublambda steps for the phase shift method predetermined.
  • the optical path difference can be adjusted in the interferometer so that the interference fringes over the wavelength, in particular in the form of a wavelet, can be detected by means of a spectrometer.
  • an optical path difference of several tens to several hundred wavelengths of the largest wavelength used can be set, in which case a wavelength range of approximately 150 nm is preferably evaluated in the case of application in the near infrared range.
  • a referencing of the wave number over the depth is necessary for accurate measurements.
  • in a microscope on the basis of the phase contrast method according to F.
  • a comparatively thick phase ring with respect to the light wavelength can be introduced, which thus introduces an optical path difference of several wavelengths, preferably of the order of 100 wavelengths of the largest wavelength used.
  • additional information can be obtained by using light-diffusing objects to evaluate a phase variation over the wavenumber-possibly also in conjunction with an intensity variation-in an interference pattern.
  • laterally small and thin phase objects are marked by local phase changes in the interference fringes over the wavelength ⁇ or the wavenumber k, in particular also in the form of a wavelet, depending on the optical thickness of the phase object (s) and also depending on their depth position also depending on their lateral extent.
  • the usable depth range ⁇ z_c is also given by the size of the chromatic depth splitting in the imaging beam path for the object bundles.
  • the acquisition of the measured variable for example the optical thickness of the object in a specific object depth, can take place by means of the evaluation of the interference fringes, which are also referred to as Müllersche strips.
  • the interference fringes which are also referred to as Müllersche strips.
  • the zero crossing of the first derivative d ⁇ / d & phase curve ⁇ ()) can be evaluated with high accuracy.
  • monochromatic radiation source electromagnetic interference modulations over time can be generated, which have a technically good evaluable frequency by the size of the set path difference in the interferometer and the speed of the wavelength scan.
  • the phase of the interference is preferably evaluated in the inventive method for transmitted-light microscopy.
  • a resolution in the axial direction in the size of a few nanometers can be achieved, and this strongly depends on the shape of the interference pattern, that is to say in particular also of a wavelet, and also on the evaluation strategy.
  • a multiple phase shift is performed in the interferometer in order to detect very small phase deviations even very small deviations from the at least approximately straight phase curve, in which case a small local deviations of the phase ⁇ - in the sense of a local disturbance of the phase curve ⁇ (£), preferably with a local extremum - yes indicates the existence of a phase object.
  • Feature 8 Furthermore, the optical path difference in the infinity beam path or in the chromatically weakly focused beam path of the microscopic image is preferably introduced in the inventive method for transmitted light microscopy. Thus aberrations such as spherical aberration are minimized or largely avoided.
  • the method of chromatic depth splitting and compensation thereof in conjunction with confocal discrimination is applicable to all interferometric methods, including digital microscopic scale holography.
  • Feature 9 In an inventive arrangement for transmitted-light microscopy, especially with condenser and lens and in particular for the quantitative examination of a micro-object in the object space, with a polychromatic light source in the illumination beam path and with a spectrometer in the detection beam path or a wavelength-tunable light source in the illumination beam path with means for confocal discrimination in the detection beam path, such as a plurality of micro-apertures, preferably a micro-aperture array, or a multi-microlenses multiply mapped macro-aperture and a camera in the detection beam means are arranged with at least partially chromatic power for chromatic depth splitting in the detection beam path.
  • a polychromatic light source in the illumination beam path and with a spectrometer in the detection beam path or a wavelength-tunable light source in the illumination beam path with means for confocal discrimination in the detection beam path, such as a plurality of micro-apertures, preferably a micro-aperture array, or a multi-
  • the confocal Discrimination greatly reduces the scattered light in optical imaging, which occurs in light scattering samples such as biological tissues or partially cloudy media.
  • the means are preferably arranged with at least partially chromatic refractive power for chromatic depth splitting in the Fourier plane, ie the rear focal plane of the objective, or in a plane optically conjugate to this. This ensures that the imaging scale in the detection is independent of the wavelength.
  • Feature 11 Furthermore, in the inventive arrangement for transmitted-light microscopy, means with at least partially chromatic refractive power for chromatic depth splitting in the illumination beam path are preferably arranged. Furthermore, means for structured illumination of the object space are preferably arranged. With these structured light, for example in the form of a Foki pattern with equidistant focal distances, ie an array of light spots generated, whereby the scattered light in the imaging of the object can significantly reduce, since only the area of the object is focused focused, the is detected. In this case, the array of light points can be flat, for example, as shown in DE 10 2006 007 172 Al, formed, with a harnesspixlige camera is used for spectral detection in this arrangement. For example, 64 x 64 object points can be spectrally analyzed simultaneously.
  • Feature 12 Furthermore, in the inventive arrangement for transmitted-light microscopy, means with at least partially chromatic refractive power for chromatic depth splitting in the Fourier plane, ie the rear focal plane of the condenser, or in an optically conjugate plane to the latter are preferably arranged. This ensures that the magnification of the illumination is independent of the wavelength.
  • Feature 13 Furthermore, preferably in the inventive arrangement for transmitted light microscopy when using a Nomarski DIC microscope with two Wollaston- prisms an optical, birefringent retarder (retarder) with an optical path difference between the two polarization directions to produce interference, ie Interference strip over the wavelength ⁇ or the wave number k, in particular with interference fringes in the form of a wavelet, arranged.
  • the optical path difference of the birefringent retarder is at least one wavelength of the maximum, detected wavelength, wherein the retarder is preferably arranged in the space between the two Wollaston prisms of the Nomarski DIC microscope.
  • the design of the optical retarder allows the spatial frequency of the interference fringes, especially in the form of a wavelet, to be adjusted so that it is optimally adapted to the spectral resolution of the spectrometer, so that an evaluation of the strip is still technically possible.
  • the optical, birefringent retarder is designed in the form of a calcite plate. This material has a particularly high birefringence.
  • the means with chromatic refractive power are preferably designed as diffractive-optical elements (DOEs). These have a high chromatic resolution.
  • DOEs diffractive-optical elements
  • the means for confocal discrimination in the detection beam path such as a micro-aperture array or else a microlens array, are preferably arranged with high precision laterally displaceable.
  • a gapless lateral scanning of an object which is at least partially transparent can take place.
  • Feature 17 Furthermore, preferably in the inventive arrangement for transmitted light microscopy, the means for structuring the light in the illumination beam path, such as a micro-aperture array or a microlens array, arranged with high accuracy laterally displaceable. These means are shifted synchronously with the means for confocal discrimination in the detection beam path, so that the lateral assignment of the images of the same in the object space in the displacement and in the image acquisition by means of a camera is always maintained.
  • the means for structuring the light in the illumination beam path such as a micro-aperture array or a microlens array, arranged with high accuracy laterally displaceable.
  • the means for structuring the light in the illumination beam path and the means for confocal discrimination in the detection beam path are rigidly coupled by a convolution of the optical beam path, wherein the magnification in the illumination and in the detection beam path is made equal, so that Only a single mechanical scanning system for lateral displacement of the same means is necessary.
  • the optical coupling of the inventive arrangement for transmitted light microscopy can be performed with an optical, computer-controlled tweezers.
  • living cells in the object space of a phase microscope can be predetermined and moved with high precision.
  • a cell can be measured in terms of their optical thickness or their optical thicknesses by means of phase microscopy.
  • the lateral variation of the optical thickness of a Cell to be determined.
  • a position determination and microforming of cells by means of optical tweezers is possible by, for example, selected cells are transported into the measuring volume for surveying.
  • a phase shift can be performed by mechanical scanning in the reference arm.
  • an improvement of the signal-to-noise ratio can be achieved due to a high and strongly light-weakening object density.
  • This phase shift can be performed by means of a mechanical scan on an angle mirror, but also by means of a phase-mostly-LCD.
  • This LCD is then preferably located in an intermediate image plane ZBE in the reference beam path.
  • phase objects to be measured should rather be arranged singly in the measuring volume in the inventive arrangement, but may also be superimposed if the partial transmission of a spherical wave to an object point in the measuring volume is still present without the illumination aperture being substantially reduced. Otherwise, the evaluation becomes much more difficult or even impossible.
  • a numerical aperture of over 1 can be realized. Even for technical objects in oil immersion technology, a numerical aperture of more than 1 can be realized in the optical system.
  • axial readjustment may be required on either of the two distance adjustment microscope objectives, so that the confocality in the optical system is maintained. It can already be one Opening error in the image occur, which can be remedied by an adaptive optical system.
  • the condenser is preferably formed by means of a GRIN lens.
  • the objective is preferably formed by means of a GRIN lens. This leads to a very compact arrangement.
  • a camera with a logarithmic characteristic is preferably used in a transmitted-light microscope according to the invention with chromatic depth splitting. With this usually a much larger range of brightness on the object compared to cameras with linear characteristic is detected.
  • a system of an electrically controllable, diffractive optical element and an electrically controllable Elektrowetting lens can be arranged, which at least partially compensates the average power of the diffractive optical element.
  • the degree of chromatic depth splitting can be changed by means of a diffractive optical element, wherein the readjustment of the refractive power of the electrowetting lens keeps the planes of sharpness and thus the position of the depth measuring range in the object space at the same depth.
  • the detector or the camera of the transmitted-light microscope according to the invention may preferably be preceded by a further two-beam interferometer with which a spectral analysis of the interfering radiation of the two-beam interferometer can take place by scanning the optical path difference therein.
  • FIG. 1 illustrates an application for a Quantitative 3D Transmitted Light Microscope for Thin Phase Objects based on a Mach-Zehnder Interferometer (MZI) to determine the optical thickness or variations of the optical thickness of an object in a spatially resolved and highly accurate manner.
  • MZI Mach-Zehnder Interferometer
  • the arrangement is constructed as a chromatic-confocal spectral interferometer.
  • the application can be used in biological research, for accurate determination of the optical thicknesses of cell components in cancer research, in medical diagnostics, but also for technical applications.
  • the light source Ia is designed as a fiber-coupled white light laser.
  • the light emerging from the fiber passes through a collimator 2 and illuminates a microlens array 3, which is followed by a micro-aperture array 4, wherein the micro-diaphragms are each arranged in the focus position of the microlenses.
  • FIG. 1a shows this situation for a micro-diaphragm BA with the diaphragm center A, which also corresponds to a focus and also represents the origin of a spherical wave K.
  • the microlens array 3 and the micro-aperture array 4 are fixedly connected to each other, this assembly is associated with a not shown here fast Rezisons xy scanner, which also has a highly accurate lateral start position.
  • the spherical wave K which is shown here representative of a plurality of spherical waves, is split at the beam splitter 5 into an object wave and a reference wave.
  • the spherical wave emitted by a micro diaphragm BA from the center A thereof is collimated by the objective 6, ie converted into a plane wave, ie the point A is imaged infinity, thereby passing a diffractive-optical element in the form of a Fresnel zone lens 7 with a focusing effect.
  • This zone lens 7 is at least approximately in a plane PE '10, which is optically conjugate to the pupil plane PElO and to the Fourier plane of a downstream condenser 10.
  • this Fresnel zone lens 7 There is a different focus by means of this Fresnel zone lens 7 as a function of the wavelength.
  • the refractive power of the Fresnel zone lens 7 is compensated by means of a directly downstream, weakly refractive diverging lens 8 to allow only slight deviations from the ideal collimation state, so that for the central wavelength ⁇ O only one plane or very weak one is always used curved wavefront arises.
  • the following first imaging stage 9 images the Fresnel zone lens 7 into the Fourier plane FlO of the condenser 10, which is identical to the pupil plane PE10, so that there are weakly diverging, at least approximately plane, as well as weakly converging waves as a function of the wavelength available.
  • the condenser 10 can be readjusted laterally in the depth and possibly also laterally in order to precisely ensure the confocality of the transmitted-light microscope.
  • a thin phase object 11 present in the object space alters the optical path difference, so the phase also shifts in the MZI.
  • the phase object 11 is usually optically so thin that the confocality of the transmitted-light microscope still exists, ie the object-caused optical path length change is still below the wavelength of the optical depth of field.
  • a water immersion objective 12 forms the object 11.
  • the spherical waves detected by the objective 12 are converted into approximately plane or weakly focused or divergent waves and strike the second imaging stage 13, which images the diffractive optical element in the form of a Fresnel Zone lens 14 with divergent effect allows.
  • This Fresnel zone lens 14 is at least approximately in a plane of the objective 12 conjugate to the plane of the pupil and Fourier plane F '12, ie in the plane F "12.
  • the amount of refractive power of this Fresnel zone lens 14 is at least approximately equal to the Fresnel zone lens for each wavelength
  • the chromatic splitting of the illumination beam path ie the depth splitting by the Fresnel zone lens 7
  • Subsequent angular mirrors 16 cause the diversion to the tube objective 17 with a wavefront inversion
  • the beam splitter 18 which here serves to combine the beam with the light from the reference beam path, the foci of all wavelengths are produced by the compensating effect of the Fresnel zonal lens 14 at least approximately again in a plane.
  • the second micro-aperture array 25 for confocal discrimination.
  • FIG. 1b shows the micro-aperture array 25 with the micro-aperture BB.
  • the imaging in the reference arm takes place via the long-focal-length objective 19, the angular mirror 20, which enables a setting of the optical path difference in the MZI which is suitable for the detection of Müller's stripes.
  • the angle mirror 20 an optical path difference of 80 ⁇ m is set for the MZI.
  • the image in the reference arm is still on the wedge plate pair 21, which is used for dispersion compensation, the wedge angle are chosen sufficiently small, and the lenses 22, 23 and 24, wherein an intermediate image in the plane ZBE takes place.
  • the picture A'r of BA as well as the redrawn image B'r of the center B of the corresponding micro-aperture BB.
  • an amplitude matching ie an adjustment of the intensity for the reference radiation, can be carried out for optically dense samples.
  • the plane ZBE represents a possible position for an LCD for amplitude matching and phase matching.
  • the coherent foci of the object and the reference beam A "o_l A" r_l coincide, for example, in the point B of the micro-aperture BB to the interfering focal point A 'Or I.
  • Confocal filtering takes place through the micro-aperture array 25, in this case, for example, in the micro-aperture BB with the center B.
  • the micro-aperture array 25 is likewise assigned a fast precision xy scanner, not shown here, which synchronously and with high precision with respect to the fast precision xy scanner is moved by the assembly microlens array 3 and micro-aperture array 4.
  • a micro-aperture of the micro-aperture array 4 is imaged onto one and the same, respectively corresponding micro-aperture of the micro-aperture array 25, for example the micro-aperture BA onto the corresponding micro-aperture BB.
  • the confocality of the system necessarily requires an axial readjustment on the condenser 10, which may also be accompanied by a lateral readjustment.
  • the interfering spherical waves emerging from the micro-apertures of the micro-aperture array 25 pass through a spectrometer, which consists of the refractive-diffractive assembly 27 and the imaging system with the lenses 26 and 28.
  • the spectrally split, interfering bundles reach a camera 29 and are detected there.
  • the spectrum of intensity over the wavelength the Müller interference fringes, in the form of a wavelet.
  • the evaluation can take place by means of the evaluation of the phase variation over the wavelength, since the intensity over the wavelength is dependent on the optical path difference in the MZI.
  • the evaluation of the interference fringes is not shown in this document.
  • An MZI in FIG. 1 has a second interferometer output at the beam splitter 18.
  • a visual observation can be made via a fixed micro-aperture array 30 and the microlens array 31 and a microscopic imaging system 32 with an optical narrow-band filter, not shown, when the fast precision xy scanner of FIG the microlens array 3 assembly and the micro-aperture array 4 are in the start position because the fixed micro-aperture array 30 is laterally aligned with this position.
  • the optical narrowband filter By changing the optical narrowband filter, another depth of the object space can be seen sharply. This possibility of visual observation can be used for research purposes as well as for object positioning.
  • the lateral scan is performed, so that integration takes place over all positions.
  • microlens array 3 and coupled micro-aperture array 4 and the micro-aperture array 25 are, for example, scanned together in 10 ⁇ 10 positions, since this should be the ratio of the focus spot diameter to the focal distance of 1:10.
  • a camera image is taken in each position by means of the camera 29, which is best designed as a high-speed camera.
  • the spectrometer is designed as a surface spectrometer, as shown in DE 10 2006 007 172 A1, with a 4 mega-pixel camera.
  • FIG. 2 shows the bundles in the transmitted-light microscope with chromatic splitting of the bundles during illumination on the basis of a Mach-Zehnder interferometer in the object beam path of the MZI of FIG. 1.
  • the object 11 is a thin phase object.
  • the range of the depth splitting z_ ⁇ and the range z_c in which thin phase objects can be detected are shown.
  • FIG. 3 illustrates the beam path in the reference arm of the MZI of FIG. 1. Since there are no chromatic components in the reference arm, the wavefronts of all fall Wavelengths together and the point A provides in the ZBE exactly one pixel A'r for all wavelengths.
  • FIG. 4 shows a calculated wavelet for three superimposed phase objects.
  • the associated spectral phase is indicated over the wavenumber, the optical thickness of the phase difference being determined from the deviation .DELTA..phi._o from the phase straight line, ie the spectral phase .phi Calculates phase object.
  • FIG. 6 shows the first derivative of the spectral phase ⁇ over the wavenumber k. From the zero crossing of the differentiated spectral phase d ⁇ / dA: the position k_o of the phase object can be determined with high precision.
  • FIG. 7 represents an economically and technically very interesting embodiment.
  • the light source Ib is designed as a fiber-coupled wavelength-tunable laser.
  • the light emerging from the fiber passes through a collimator 2 and illuminates a microlens array 3, which is followed by a micro-aperture array 4, the micro-diaphragms being arranged in each case in the focus position of the microlenses.
  • the microlens array 3 and the micro-aperture array 4 are fixedly connected to each other, this assembly is associated with a not shown here fast Rezisons x-y scanner, which also has a high-precision start position.
  • the spherical wave emanating from a micro-diaphragm from the center A thereof is collimated by the objective 6, that is to say converted into a plane wave, that is to say in FIG. H.
  • the point A is imaged to infinity and passes in the region of the Fourier plane of the long focal length lens 6, a polarizer 33, a Wollaston prism 34 and a diffractive-optical element in the form of a Fresnel zone lens 7 with focusing effect.
  • Wollaston prism 34 there is a splitting into ordinary and extraordinary bundles that are polarized perpendicular to each other and have a very small angle in the propagation to each other.
  • Fresnel zone lens 7 It is carried out by means of Fresnel zone lens 7 depending on the wavelength a different focus.
  • the refractive power of the Fresnel zone lens 7 is compensated by means of a directly downstream, low refractive diverging lens 8, to allow only slight deviations from the state of ideal collimation.
  • the following first imaging stage 9 images the Fresnel zone lens 7 into the Fourier plane of the condenser 10, so that there are weakly diverging as well as at least approximately plane as well as weakly converging waves as a function of the wavelength.
  • this imaging stage 9 is an optical retarder 35 with a thick Kalkspatplatte, so that a optical delay of about 80 microns between the vertical and the horizontal Poalarisationscardi, ie the ordinary and the extraordinary bundles.
  • a chromatic depth splitting in the object space whereby two groups of spherical waves of different focussing arise depending on the wavelength whose centers are shifted in depth, so that when imaging the point A here the foci A's_ ⁇ min until A's_ ⁇ min arise and A'p_ ⁇ min through A'p_ ⁇ min which have a lateral-shear 2 ⁇ q to each other. This is shown in FIGS. 8 and 9.
  • the water-immersion condenser 10 in Figure 7 can be readjusted in depth to ensure the confocality of the transmitted-light microscope.
  • An existing in the object space thin phase object 11 changes the optical path difference, so shifts the phase in the interferometer between the bundles with polarization-optical lateral shear.
  • the phase object 11 is usually optically so thin that the confocality of the transmitted-light microscope still exists, ie the object-induced optical path length change is still smaller than the range of the wavelength-optical depth of field.
  • a water immersion objective 12 images the object 11 as well as the light of the differently focused spherical waves, so that here a bright field arrangement is given.
  • the spherical waves detected by the lens are converted into approximately plane or weakly focused or divergent waves and meet the second imaging stage 13, which allows as an afocal stage an image on the diffractive-op table element in the form of a Fresnel zone lens 14 with divergent effect.
  • This Fresnel zone lens 14 is at least approximately in the pupil plane.
  • the amount of refractive power of this Fresnel zone lens 14 is at least approximately equal to the Fresnel zone lens 14 for each wavelength.
  • the subsequent, directly downstream, low refractive converging lens 15 compensates again for the average refractive power of the Fresnel zone lens 14.
  • the polarizer 33, the Wollaston prism 34 and the Wollaston prism 36 and the analyzer 37 form with the microscope components a polarization-optical shear interferometer.
  • FIG. 8 shows the beam path of the bundles in the object space in the case of the transmitted light microscope with differential interference contrast (DIC) according to Nomarski for thin phase objects.
  • the lateral shear here amounts to approximately 0.5 ⁇ m.
  • FIG. 9 illustrates the situation for a single wavelength ⁇ i, wherein the comparatively large optical path difference OPD is symbolically indicated here.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

La présente invention concerne un procédé et un dispositif pour la microscopie en transmission confocale avec une source de lumière polychromatique et un spectromètre ou avec une source de lumière réglable en longueur d'onde ainsi qu'une caméra, en particulier également pour la microscopie d'interférence à saut de phase, en particulier également pour la mesure ou la localisation tridimensionnelle d'un ou plusieurs objets de phase à l'échelle microscopique comme des cellules vivantes mobiles et incolores ou pour la lecture d'enregistreurs de données par transmission optique. Une résolution chromatique en profondeur est réalisée selon l'invention dans le chemin optique de détection au moyen d'une réfringence chromatique. Les microscopes en transmission peuvent se fonder sur le principe d'interférence en contraste de phase de Zemike, Normaski DIC, Hoffman HMC, ou également sur le principe d'interféromètre de Mach-Zender. La différence de chemin optique dans un microscope en transmission à saut de phase est ici réglée de telle sorte que les franges d'interférence, également en particulier sous forme d'ondelettes, peuvent être calculées, les objets de phase minces se caractérisant par des changements de phase dans les ondelettes en fonction de leur position en profondeur. Le chemin optique d'éclairage comporte une deuxième réfringence chromatique agissant à l'inverse de telle sorte que pour les objets de phase minces dans l'espace objet du microscope en transmission, la confocalité est égale à (A'λi-B'λi).
PCT/EP2007/003644 2006-05-16 2007-04-25 Dispositif et procédé de microscopie en transmission confocale, en particulier également pour la mesure d'objets de phase mobiles WO2007131602A1 (fr)

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GB2589327B (en) * 2019-11-26 2023-09-13 Andor Tech Limited Differential phase contrast microscope

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