DE102006023887B3 - Transmitted-light microscopy method involves sequentially separating the pictures in light dispersion direction with poly-chromatic source of light to time point or with wavelength-variable source of light - Google Patents

Abstract

The method involves accomplishing a chromatic depth resolution by partially chromatic refraction power in the detection rays path, during the object representation. The pictures of the centers of the micro screen (B) or pictures of the center of the multi-illustrated screen during re-illustration into the object area experience a chromatically caused depth resolution. These pictures are separated sequentially in the light dispersion direction with a poly-chromatic source of light to a time point or with a wavelength-variable source of light. An independent claim is also included for the transmitted-light microscopy arrangement.

Description

State of technology

The Durchfokussierung plays in confocal microscopy since Marvin Minski, [Patentschrift US 3,013,467 of December 19, 1961] has a functionally important role. Many efforts have already been made to realize the focussing without mechanically moving parts.

in the Review by Christopher J. Mann, Lingfeng Yu, Chun-Min Lo, and Myung K. Kim: "High-resolution quantitative phase-contrast microscopy by digital holography "in OPTICS EXPRESS Vol. 13, no. 22, pp. 8693-8698, 2005 becomes a holographic quantitative phase-contrast microscopy method described in which the object information outside the depth of focus numerically be reconstructed. That's why for the three-dimensional capture an object advantageously no mechanical depth scanning required. However, the problem here is that of scattering Samples not entirely avoidable residual scattered light, its influence here by the use of the information of the angle spectrum though is already reduced, but with very high requirements but still something disturbing can work. Furthermore is the achievable depth resolution not always satisfactory, for example, if one depth resolution or a resolution the optical thickness of 10 nm and less is required.

in the Review by Christopher G. Rylander, Digant P. Dave, Taner Akkin, Thomas E. Milner, Kenneth R. Diller, and Ashley J. Welch: "Quantitative phase-contrast imaging of cells with phase-sensitive optical coherence microscopy ", OPTICS LETTERS Vol. 29, no. 13, p. 1509-1511, In 2004, a fiber-based optical coherence tomographic system will be set up the basis of the differential interference contrast (OCT-DIC) described. This system is used for the quantitative measurement of cell components used by making changes the optical path difference are determined high-resolution. However, that is it is not possible Objects in distinctly different depths simultaneously with high lateral resolution as this is the limitation due to the optical depth of field prevented.

In Scripture US 6,091,496 No approach for spectral interferometric transmitted light microscopy is presented, however, a first indication of this is found in WO2006 / 042696, page 118, using a Mach-Zehnder interferometer with longitudinal chromatic aberration and compensation thereof. However, there is no teaching here to apply transmitted light microscopy to phase objects. In the publication by 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.

field of use the invention

The 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, live animal or human cells as well as cultivated cells, by determining their optical thickness and depth position. Especially The invention can also be used for the analysis of chromosomes in the human and Find cytogenetics application. For example, components should also of cancer cells in terms of optical thicknesses with high accuracy can be measured.

Farther can be the quantitative determination of the distribution, the position in the Space and the lateral dimensions of opaque microparticles, for example also of interstellar micro dust (star dust) in a transparent Medium done. This must be "inserted" between the condenser and the lens the invention, the distribution and the lateral dimensions of microfibers in a transparent or partially cloudy medium, such as special plastics, to determine, so use the invention for technical applications. In the NIR range, the analysis of Si structures in transmission feasible being because of the spherical Aberration the structures an approximately should have constant thickness. It is with the inventive solution also a crystal analysis feasible. Also according to the invention the determination of refractive index changes in the transparent media in the microscopic scale laterally highly accurate and depth resolved. The mentioned applications can in the DUV, UV, VIS or in the NIR range. It can also with immersion techniques in oil, water or glycerine are worked, each one being specially tailored Lenses are used.

Of the innovative approach also the readout of volume data storage, which in transmission work by making changes of the refractive index in the irradiated medium detected and evaluated become.

Goals of invention

The object of the present invention for transmitted-light microscopy, in particular for Vermes Sensing the optical thicknesses of transparent and partially opaque objects, also known as phase objects, is to provide something new for commercial use.

The The aim here is in particular the quantitative recording of thin Phase objects, e.g. single, also undyed, living cells.

The technical aim of the invention is to increase the speed of Measuring the optical thicknesses and increasing the usable depth measuring range, especially 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 immersion) to values that correspond to a multiple of the wave-optical depth of field.

The with the inventive sensor when measuring optical signals obtained should be using radiation detector or rastered radiation detector like a line or area camera become.

One Another goal is to teach a reading of volume data storage to give who work in transmission.

task the invention

It to be used in transmitted-light microscopy, in particular for testing Phase objects when optically probing the objects at different depths of the object space optical signals from these depths without the mechanical Moving components - in particular in object space - won in particular the optical thickness or the change the optical thickness of an object or its components or also be able to determine from several objects. It should be at least one approximately diffraction-limited lateral imaging of the object by an imaging system with a comparatively high numerical aperture, in particular also in immersion technology.

In order to the inventive task is to be solved for the Nomarski phase contrast method, the differential Interference contrast (DIC) method, Hoffinann modulation contrast (HMC) method or by means of microscopes based on two-beam interferometry, especially by means of a Mach-Zehnder interferometer, highly accurate, quantitative measurements of optical thickness or thickness, including variations thereof, transparent objects with very high resolution. in this connection So it's all about a resolution in the depth direction in the size of a few Nanometers.

on the other hand should during the wavelength scan using a time wave tunable, spectral narrowband radiation source solved the inventive task that electromagnetic interference modulations are generated over time, the one sufficiently small and therefore technically well evaluable Have frequency from this information in particular the optical thickness or the change to be able to determine the optical thickness. This innovative task especially for a confocal differential interference microscope, in particular for the Nomarski DIC microscope on the basis of polarization-optically generated lateral-shear, as well as a confocal two-beam interference microscope, in particular also for a confocal Mach-Zehnder interference microscope, solved become.

description the invention based on the features

• – mit einer polychromatischen Lichtquelle im Beleuchtungsstrahlengang und mit einem Spektrometer im Detektionsstrahlengang
• – oder mit einer Wellenlängen-durchstimmbaren Lichtquelle im Beleuchtungsstrahlengang

und mit Mitteln zur konfokalen Diskriminierung im Detektionsstrahlengang

• – wie mehrere Mikroblenden
• – oder eine mittels mehrerer gerasterter Mikrooptiken, wie gerasterte Mikrolinsen, mehrfach abgebildete Blende

sowie einer Kamera im Detektionsstrahlengang bei der Objektabbildung eine chromatische Tiefenaufspaltung mittels einer zumindest partiell chromatischen Brechkraft im Detektionsstrahlengang durchgeführt. So wird erreicht, dass Bilder der Mittelpunkte der Mikroblenden oder Bilder des Mittelpunktes der mehrfach abgebildeten Blende bei der Rückabbildung in den Objektraum dort eine chromatisch verursachte Tiefenaufspaltung erfahren. Damit sind diese Bilder bei einer polychromatischen Lichtquelle zu einem Zeitpunkt oder bei einer Wellenlängen-durchstimmbaren Lichtquelle sequentiell in der Lichtausbreitungsrichtung separiert, so dass Objektpunkte aus unterschiedlichen Tiefen mittels der Mikroblenden oder der Mikrolinsen erfasst werden können.The object of the invention is achieved with the following inventive features:
Main feature: According to the invention, in a method for transmitted-light microscopy, in particular also for the quantitative examination 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 spectrometer in the detection beam path
• - or with a wavelength-tunable light source in the illumination beam path

and with means for confocal discrimination in the detection beam path

• - like several micro-apertures
• - Or one by means of multiple rastered micro-optics, such as screened microlenses, multiple-mapped aperture

and a camera in the detection beam path in the object image, a chromatic depth splitting performed by means of an at least partially chromatic power in the detection beam path. It is thus achieved that images of the centers of the micro-apertures or images of the center of the multiple-imaged diaphragm undergo a chromatically-caused depth split in the back-imaging into the object space there. Thus, in a polychromatic light source, these images are sequentially separated in the light propagation direction at a time or at a wavelength-tunable light source, so that object points from different depths can be detected by the micro-apertures or the microlenses.

Thus, a value for the depth position of a sharply imaged object point of a very thin NEN phase object and its change in the optical thickness in relation to the surrounding medium are calculated. This can be done in a range z_c, which can be a multiple of the optical depth of field. For an opaque thin microparticle in a transparent medium, the wavelength of the shading can be determined, from which the contour of microparticles can be determined.

there For example, the source of electromagnetic radiation can be broadband in the DUV UV, in the VIS or in the IR range to be trained and be short-pulsed, for example, a short-pulse laser, and its Pulse with a camera, which serves as a detector, for example in 100 Hz range, synchronized. This is for moving Micro scenes of advantage. On the other hand, also a wavelength tunable Lasers are used. Furthermore, a spectrally broadband can also Source of electromagnetic radiation are used, for example also a fiber-coupled, broadband white-light laser, with a controllable Monochromator is followed to tune the wavelength.

feature 2. Furthermore, it is preferred in the inventive method the light spatially in the object space structured, in which by means of multiple micro-apertures or a means multiple microlenses multi-mapped macroblende a Foki pattern or a focus line pattern are formed in the object space predetermined, and in the illumination beam path, a chromatic depth splitting these foci or focus lines by means of at least partially chromatic Refractive power performed so that these foci or focus lines in the light propagation direction, so in the depth, are separated. These are at least partially chromatic power in the illumination and the at least partially chromatic power in the detection beam path so predetermined introduced that in the object space the amount and direction of the chromatic depth splitting of foci or focus lines to Illumination and in the detection beam path the amount and the direction the chromatic depth splitting of the redrawn micro-apertures or the macro mapped multiple times at least approximately are equalized. Then the images A 'of the centers are in the object space A of the apertures BA of the illumination beam path or the images A 'the foci A and the pictures B 'the Midpoints B of the aperture BB of the detection beam path, which respectively laterally and spectrally correspond, namely, for example, A'o_λmin-B'o_λmin and A'o_λmax-B'o_λmax, optically conjugated. So fall their levels of sharpness in object space at least approximately together. As a result, confocality exists from lighting to lighting Detection, as with thin Phase objects in the object space, the images A 'and B' of all wavelengths coincide, respectively the picture A '' independent of the wavelength also with the center B of a micro-aperture BB in the detection beam path coincides. Due to the confocal discrimination in the detection beam path is the possibly occurring in the object space, sample-caused Scattered light on the way to the camera greatly reduced.

Farther can in the inventive method for increasing the depth measuring range over the Wave-optical depth of field In addition, only two monochromatic sources electromagnetic Radiation of different wavelengths with frequency modulation be used, so that the known heterodyne method is applied can be. With the radiation of each wavelength is the object is scanned at a slightly different depth.

Feature 3. Furthermore, the method of dark field microscopy is preferably used in the inventive method for transmitted light microscopy. Thus, only thin translucent objects or object details are detected by 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 bend the light remain dark, since the directly transmitted light Known not be detected here. Thus, depending on the intensity in the spectrum, 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. However, this requires wavelength-to-depth referencing of the dark field microscope, where the range of measurement is dictated by the degree of chromatic depth splitting that results from the amount of chromatic refractive power. The optical difference in thickness of a thin, translucent object, which results from the difference of the refractive indices to the surrounding medium, wherein the surrounding medium may be water, for example in biological objects, should only be so great that this does not cause a significantly larger optical path length change than the wave-optical depth of field, as this can lead to a contrast deterioration in the imaging of scattering or diffracting objects. Otherwise, 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 provide a remedy which compensates for the optical difference in thickness between the object and the surrounding medium and thus again allows a sharp object adaptation. However, it is also possible to move the micro-diaphragms in the illumination beam path or micro-diaphragms in the detection beam path axially, that is to say in the depth, in order to restore the confocality disturbed in the case of thicker phase objects. In comparatively thick 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 Although dark field microscopy is mainly used for visual observation used. However, in the inventive method over the Wave number, for example, an extremum can be determined, thereby also quantitative statements about the object possible are.

feature 4. Furthermore, preferably in the inventive method for transmitted light microscopy the method of phase microscopy on the basis of interference of electromagnetic waves for use. This allows a very high resolution and photometric sensitivity the measurement of objects.

feature 5: Furthermore, preferably in the inventive method for phase-contrast microscopy with chromatic depth splitting the phase-contrast method, In particular, according to F. Zernike, the differential phase-contrast method, in particular also according to G. Nomarski, also as DIC method (DIC: Differential interference contrast), ie method on the Basis polarization-optical shear interferometry, the Hoffinann HMC method (HMC: Hoffinann modulation contrast) or a phase microscopic Method according to the two-beam interference method, in particular also based on a Mach-Zehnder interferometer. This are imported microscopic procedures by the application of the invention for one even faster object detection, especially for highly accurate Object measurement, can be used.

feature 6: It is further preferred in the inventive method for transmitted light microscopy an optical path difference OPD (OPD: optical path difference) introduced in the beam path of a phase microscope, the at least one wavelength of the maximum detected wavelength is, preferably however, up to 100 wavelengths the maximum detected wavelength. So become interference fringes, especially in the form of a wavelet, also called Müllersche Strips or Tolansky stripes known, so intensity curves over the Wavelength λ or the Wavelength k, formed in the detected spectral range predetermined. This creates a thin and laterally small phase object - for example of the order of magnitude the extension of the diffraction-limited point image of the microscope - according to the invention a local phase change in the interference fringes, thus especially in a wavelet. This local phase change is evaluated. A laterally extended phase object, which the measuring field completely covered, generates a change the frequency in the interference pattern, so in particular in one Wavelet that over the wave number k changed to one Rise of the phase, ie the phase gradient dφ / dk leads.

Especially also with the Nomarski DIC procedure can be due to the chromatic Depth splitting additional information can be obtained by, for example, over the Wavelength k an extremum can be determined, if preferably a sufficiently large optical path difference in the DIC microscope with polarization-optical Shear is given. Preferably, a comparatively thick retardation plate be used in the DIC microscope, which is preferably an optical Gap difference between the two polarization directions in the Order of magnitude 100 wavelengths the largest used wavelength to increase the accuracy of the phase evaluation strategies.

Farther Is it possible, a different in both polarization directions, the phase Insert sliding LCD in the DIC microscope to the optical path difference in sublambda steps for the phase shift method can be predetermined to vary.

Of the optical retardation can be adjusted in the interferometer so that the interference fringes over the wavelength, especially in the form of a wavelet, by means of a spectrometer can be detected. Preferably, therefore, an optical path difference of several tens up to several hundred wavelengths the largest used wavelength can be adjusted, in which case in an application in the near infrared range preferably a wavelength range is evaluated by about 150 nm. This is a referencing for accurate measurements the wavenumber above the Depth necessary.

Preferably, in a microscope on the basis of the phase contrast method according to F. Zernike, 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 in the order of 100 wavelengths of the largest wavelength used. Thus, 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.

in the Inventive methods laterally mark small and thin phase objects through local phase changes in the interference fringe over the Wavelength λ or the Wavenumber k, in particular in the form of a wavelet, depending on the optical thickness of the phase object (s) and also in dependence from their depth position as well as depending on their lateral extent. The usable depth range Δz_c is also due to the size of the chromatic Depth splitting given in the imaging beam path for the object bundles.

The Obtaining the measurand, for example the optical thickness of the object at a certain object depth, can by means of the evaluation of interference fringes, which also called Mullerian Strips are called. This can, for example, the Zero crossing of the first derivative dφ / dk of the phase curve φ (k) be evaluated with high accuracy.

The Method of evaluation of the intensity of the interference pattern over the Wavelength, respectively the phase φ over the wavenumber k to a measurand - such as an information about a change the optical thickness or the optical thickness of the object - to obtain is not an inventive subject matter in this document.

Farther can in a wavelength scan by means of a wavelength tunable, monochromatic radiation source electromagnetic interference modulations over the Time can be generated by the size of the set in the interferometer Gait difference and the speed of the wavelength scan have a technically good evaluable frequency.

at the inventive method of transmitted light microscopy, it is advantageous in the case of microscopic object imaging the spherical aberration, ie the aperture error, sufficiently well to correct, so that at least approximately Diffraction-limited mapping can be done.

feature 7: Furthermore, it is preferable in the inventive method for transmitted light microscopy the phase of the interference, so in particular also in a wavelet given phase, evaluated. This can be a resolution in axial direction in the size of a few Nanometers, this being largely dependent on the shape of the interference pattern, So in particular a wavelet, as well as the evaluation strategy depends. It is also possible a multiple phase shift is performed in the interferometer to even with very thin ones Phase objects still very small deviations from the at least approximately can detect straight phase curve, with a small local deviations of the phase φ - in the sense of a local disturbance of the Phase curve φ (k), preferably with a local extremum - yes the Existence of a phase object displays.

feature 8: Furthermore, it is preferable in the inventive method for transmitted light microscopy the optical path difference in the infinity beam path or in chromatic weakly focused beam path of the microscopic picture brought in. Thus, aberrations such as the spherical aberration become minimized or largely avoided.

The Method of chromatic depth splitting and compensation of the same confocal discrimination is interferometric for all Method usable, including in digital holography on the microscopic scale.

Farther it may be advantageous to indicate the degree of depth splitting to adapt the measurement object to thicker and thinner phase objects respectively to capture optimally.

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. These means enable a sharp imaging of objects or object details from different depths of the object space at the same time, whereby more information should be obtained by means of a spectral analysis of the light compared to a mechanically performed depth scan. The confocal discrimination greatly reduces the scattered light in optical imaging, which in light scattering samples such as biological Tissues or partially cloudy media occurs.

feature 10: Furthermore, they are preferably in the inventive arrangement for transmitted light microscopy the means with at least partially chromatic refractive power for chromatic Depth splitting in the Fourier plane, ie the rear focal plane of the lens, or in an optical conjugate to this plane arranged. This ensures that the magnification with independent of detection from the wavelength is.

Feature 11: Furthermore, preferably 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 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. The array of light points can be flat, for example as in DE 10 2006 007 172 A1 may be formed, wherein for spectral detection in this arrangement, a hochpixlige camera is used. For example, 64 × 64 object points can be spectrally analyzed simultaneously.

feature 12: Furthermore, they are preferably 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 a plane to this optically conjugate arranged. This ensures that the magnification with the lighting is independent from the wavelength is.

feature 13: Furthermore, it is preferable in the inventive arrangement for transmitted light microscopy using a Nomarski DIC microscope with two Wollaston prisms an optical birefringent retarder with an optical retarder Path difference between the two polarization directions Generation of interference, ie interference fringes over the Wavelength λ or the Wavelength k, in particular with interference fringes in the form of a wavelet, arranged. The optical path difference is of the birefringent retarder at least one wavelength the maximum detected wavelength, the retarder preferably in the space between the two Wollaston prisms of the Nomarski DIC microscope is arranged. The design of the optical retarder allows the spatial frequency the interference fringe, in particular also in the form of a wavelet, adjusted so that this optimally to the spectral resolution of Spectrometer is adjusted so that an evaluation of the strip still technically possible is.

feature 14: Furthermore, it is preferable in the inventive arrangement for transmitted light microscopy the optical, birefringent retarder in the form of a calcite plate (calcite plate) formed. This material has a special high birefringence on.

feature 15: Furthermore, they are preferably in the inventive arrangement for transmitted light microscopy the means with chromatic power as diffractive optical Formed elements (DOEs). These have a high chromatic Splitting up.

feature 16: Furthermore, they are preferably in the inventive arrangement for transmitted light microscopy the means for confocal discrimination in the detection beam path, like a micro-aperture array or even a microlens array, highly accurate arranged laterally displaceable. Thus, by means of lateral scan of the Confocal discrimination means a gapless lateral scan an object that is at least partially transparent.

feature 17: Furthermore, preference is given to the inventive arrangement for transmitted light microscopy the means for structuring the light in the illumination beam path, like a micro-aperture array or even a microlens array, highly accurate arranged laterally displaceable. These funds will be in sync with the means for confocal discrimination in the detection beam path shifted so that the lateral assignment of the images of the same in the object space during the displacement and during image acquisition by means of Camera is always preserved. It is preferably also possible that by a convolution of the optical beam path, the structuring means of the light in the illumination beam path and the means of confocal Discrimination in the detection beam path are rigidly coupled, where also the magnification made equal in the illumination and in the detection beam path, so that only a single mechanical scanning system for lateral displacement same means is necessary.

Feature 18: For applications in biological research, the optical coupling of the inventive arrangement for transmitted light microscopy, ie preferably a quantitative phase microscope, can be performed with an optical, computer-controlled tweezers. Thus, in particular living cells in the object space of a phase microscope can be predetermined and moved with high precision. It can by means of a phase microscope a Cell can be measured in terms of their optical thickness or their optical thicknesses. In particular, the lateral variation of the optical thickness of a cell can also be determined. In addition, 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.

Other inventive Characteristics:

It can with the Mach-Zehnder interferometer on the basis of the inventive Arrangement of a phase shift by means of mechanical scanning in the reference arm carried out become. In particular, very weak signals due to a high and strong light-weakening Object density thereby achieves an improvement of the signal-to-noise ratio. This phase shift can be achieved by means of a mechanical scan on an angle mirror, but also be carried out by means of a phase-mostly LCDs. This LCD is then preferably in an intermediate image plane ZBE in the reference beam path. But it is also possible by amplitude division Interference over the wavelength λ in different Phase record simultaneously on a detector. This can preferably also using the second output of the Mach-Zehnder interferometer respectively.

The to be measured phase objects should in the inventive arrangement in the measurement volume rather isolated, but can also possibly one above the other lie when the partial transmission of a spherical wave to a Object point in the measurement volume is still given without the Illumination aperture is significantly reduced. Otherwise it will the evaluation much more difficult or is no longer possible.

Farther Is it possible, By means of the inventive arrangement additionally also spectral Information, for example, in the IR fluorescent light for metabolism analysis to gain living cells, with an additional Auskoppelstrahlengang is used in the arrangement. In addition, the NIR Fourier transform spectroscopy or dispersive spectroscopy to obtain more information be connected to transmitted light microscopy by adding light is decoupled from the object beam path.

It can for biological objects in water immersion technique in the optical system a numerical aperture of about 1 be realized. Also for technical objects in oil immersion technique can be realized in the optical system, a numerical aperture of about 1 become. For thick samples, where thick describes here, if the axial image shift the order of magnitude the mean wavelength of light or more, therefore, because of the wave-optical depth of field, which is low at high numerical aperture, optionally an axial Readjustment on one of the two microscope objectives for distance adjustment done so the confocality retained in the optical system. It may already be an opening error occur in the image, however, by an adaptive optical System can be fixed.

Farther is according to the invention at a Transmitted light microscope according to the invention with chromatic depth splitting preferably the condenser means a GRIN lens formed. Furthermore, according to the invention in a Transmitted light microscope according to the invention with chromatic depth splitting preferably the lens means a GRIN lens formed. This leads to a very compact one Arrangement.

Farther is according to the invention at a Transmitted light microscope according to the invention with chromatic depth splitting preferably a camera with logarithmic characteristic used. This is usually a much larger brightness range detectable on the object compared to cameras with linear characteristic.

Farther can in a transmitted light microscope according to the invention in the object beam path preferably a system of an electrically-controllable, diffractive optical element and an electrically-controllable electrowetting lens be arranged, which the average refractive power of the diffractive optical Elements at least partially compensated. It can the degree of Chromatic depth splitting by means of diffractive optical element changed be, with the readjustment of the refractive power of Elektrowetting lens the planes of sharpness and thus the position of the depth measuring range in the object space in the same depth holds. Thus, an optimal adaptation of the optical system can be achieved. This is possible with an arrangement with immersion liquid be beneficial in object space.

Farther may preferably the detector or the camera of the transmitted light microscope according to the invention another upstream two-beam interferometer, with which by a scan of the optical path difference in the same a spectral Analysis of the interfering radiation of the two-beam interferometer can be done.

It is possible, too, for the Application of the invention, in particular, an inverted microscope use.

description the figures

The 1 set an application for Quantitative 3D transmitted-light microscope for thin phase objects based on a Mach-Zehnder interferometer (MZI) to determine the optical thickness or the variations of the optical thickness of an object in a spatially resolved and highly accurate manner. Here, the arrangement is constructed as a chromatic-confocal spectral interferometer. The application can be used in biological research, for example for the highly accurate determination of the optical thicknesses of cell components in cancer research, in medical diagnostics, but also for technical applications.

The light source 1a is designed as a fiber-coupled white light laser. The light emerging from the fiber passes through a collimator 2 and illuminate a microlens array 3 which is a micro-aperture array 4 is arranged downstream, wherein the micro-apertures are respectively arranged in the focus position of the microlenses. The 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 firmly connected to each other, this assembly is associated with a not shown here fast Präzisons xy scanner, which also has a high-precision lateral start position. The spherical wave K, which is shown here representative of a plurality of spherical waves, is at the beam splitter 5 divided into an object wave and a reference wave. The spherical wave emanating from a micro-diaphragm BA from the center A thereof is emitted by the objective 6 collimated, that is converted into a plane wave, ie, the point A is imaged to infinity and passes while a diffractive-optical element in the form of a Fresnel zone lens 7 with focusing effect. This zone lens 7 is at least approximately in a plane PE'10, which to the pupil plane PE10 and the Fourier plane of a downstream condenser 10 is optically conjugated. It is done by means of this Fresnel zone lens 7 depending on the wavelength, a different focus. For the center wavelength λ0 of the spectrum, the refractive power is the Fresnel zone lens 7 by means of a directly downstream, low refractive diverging lens 8th compensated, so as to allow only slight deviations from the state of ideal collimation, so that for the central wavelength λ0 always only a plane or very weakly curved wavefront arises. The following first image stage 9 forms the Fresnel zone lens 7 in the Fourier plane F10 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. This is done by means of the Fresnel zone lens 7 and the water immersion condenser 10 a chromatic depth splitting in the object space, whereby spherical waves of different focusing depending on the wavelength arise whose centers are shifted in depth, so that in the imaging of the point A here the foci A'o_λmin to A'o_λmin arise. The redrawn images B'o_λmin to B'o_λmin of the corresponding point B which represents the center of a confocal microslice BB, see also 1b , coincide with the foci A'o_λmin to A'o_λmin. The condenser 10 can be readjusted in depth and possibly also laterally to ensure the confocality of the transmitted light microscope precisely. A thin phase object present in the object space 11 Changes the optical path difference, so shifts the phase in the MZI. The phase object 11 but 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 below the wavelength of the optical fiber depth. A water immersion lens 12 forms the object 11 , The from the lens 12 detected spherical waves are converted into approximately plane or weakly focused or divergent waves and meet the second imaging stage 13 showing an image on the diffractive optical element in the form of a Fresnel zone lens 14 with divergent effect. This Fresnel zone lens 14 is at least approximately in a to the pupil plane and Fourier plane F'12 conjugate plane of the lens 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 14 , The following, directly downstream, weakly refractive collecting lens 15 compensates again for the average refractive power of the Fresnel zone lens 14 , As a result of passing the Fresnel zone lens 14 is the chromatic splitting of the illumination beam path, ie the depth splitting by the Fresnel zone lens 7 compensated. The subordinate angle mirror 16 causes the deflection to the tube lens 17 with a wavefront inversion. After passing the beam splitter 18 , which serves here for beam combination with the light from the reference beam path, the foci of all wavelengths arise through the compensating effect of the Fresnel zone lens 14 at least approximately in one plane again. In this level is the second micro-aperture array 25 to confocal discrimination. The 1b represents the micro-aperture array 25 with the micro-aperture BB dar.

In the 1 at the beam splitter 5 spherical waves reflected in the reference arm of the MZI, or the reference beam, experience no chromatic splitting. The image in the reference arm is made via the long-focal-length lens 19 , the angle mirror 20 , the one suitable for the detection of Müller's stripes setting the optical gear below in the MZI. For example, by means of the angle mirror 20 set an optical path difference of 80 microns for the MZI. The image in the reference arm continues to be via the wedge plate pair 21 , which is used for dispersion compensation, wherein the wedge angle are chosen sufficiently small, as well as the lenses 22 . 23 and 24 , wherein an intermediate image takes place in the plane ZBE. Here arises the image A'r of BA as well as the redrawn image B'r of the center B of the corresponding micro-aperture BB. In the intermediate image plane ZBE, an amplitude matching, ie an adjustment of the intensity for the reference radiation, can be carried out for optically dense samples. Thus, the plane ZBE represents a possible position for an LCD for amplitude matching and phase matching.

The coupling of the light from the reference arm in the detection beam path via the beam splitter 18 , In the plane of the second micro-aperture array 25 fall the coherent foci of the object and the reference beam A''o_1 A''r_1 together, for example, in the point B of the micro-aperture BB to the interfering focal point A''or_1. Through the micro-aperture array 25 Confocal filtering takes place here, for example in the micro-aperture BB with the center B. The micro-aperture array 25 is also associated with a high-speed, high-precision xy scanner, not shown here, which is synchronous and highly accurate with respect to the fast precision xy scanner of the microlens array assembly 3 and micro-aperture array 4 is moved. This ensures that in each position, a micro-aperture of the micro-aperture array 4 to one and the same, respectively corresponding micro-aperture of the micro-aperture array 25 is mapped, for example, the micro-diaphragm BA to the corresponding micro-aperture BB. Thus, there is confocality in the system, which is generally not significantly affected by thin phase objects. For somewhat thicker phase objects, the confocality of the system necessarily requires axial readjustment of 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 Leave pass a spectrometer, which from the refractive-diffractive assembly 27 and the imaging system with the lenses 26 and 28 consists. The spectrally split, interfering bundles reach a camera 29 and are detected there. On the camera 29 the spectrum of the intensity over the wavelength, the Müller interference fringes, arises 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 1 points at the beam splitter 18 a second interferometer output. So can via a fixed micro-aperture array 30 and the microlens array 31 and a microscopic imaging system 32 Visual observation is made with an optical narrowband filter, not shown, when the fast precision xy scanner of the microlens array assembly 3 and the micro-aperture array 4 are in the start position because the fixed micro-aperture array 30 is laterally aligned to this position. 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.

• 1. Innerhalb der Zeit eines Kamera-Frames wird der Lateral-Scan ausgeführt, so dass eine Integration über alle Positionen erfolgt.
• 2. In jeder Position beim Lateral-Scan wird ein Bild aufgenommen, gespeichert und zu einem Datensatz verrechnet. Das ergibt gegenüber Modus 1 mehr laterale Informationen.

Because of the lighting of the object 11 a focus pattern results in a reduced degree of filling, ie an "optical pincushion" in optical scanning, but this can be done by synchronized lateral scanning of micro-aperture array 3 and micro-aperture array 4 and the micro-aperture array 25 be completely balanced, leaving a full screen of the object 11 is won. There are two modes:

• 1. Within the time of a camera frame, the lateral scan is performed, so that integration takes place over all positions.
• 2. In each position during the lateral scan, an image is taken, saved and billed to a data set. This gives you more lateral information than mode 1.

The components microlens array 3 and coupled micro-aperture array 4 as well as the micro-aperture array 25 are scanned, for example, together in 10 × 10 positions, since this is the ratio of the focus spot diameter to focus distance to 1:10. The best thing in any position is a camera image using the camera 29 which is best formed as a high-speed camera.

The spectrometer is called an area spectrometer, as in DE 10 2006 007 172 A1 shown, formed with a 4 mega-pixel camera. For example, 64 × 64 object points become spectral at the same time, eg by means of 2048 spectral elements for each object point of the object 11 , for example, 10 × 10 = 100 scans, each with a detection of an image in each scan position by the camera 29 be executed.

The 2 the bundles in the transmitted-light microscope with chromatic splitting of the bundles in the illumination based on a Mach-Zehnder interferometer in the object beam path of the MZI of 1 dar. The object 11 is here at a thin phase object. The range of the depth splitting z_λ and the range z_c in which thin phase objects can be detected are shown.

The 3 sets the beam path in the reference arm of the MZI 1 Since there are no chromatic components in the reference arm, the wavefronts of all wavelengths coincide and the point A provides exactly one pixel A'r for all wavelengths in the ZBE.

The 4 represents a calculated wavelet for three superimposed phase objects.

In 5 the associated spectral phase is given over the wavenumber, whereby the optical thickness of the phase object can be calculated from the deviation Δφ_o from the phase line, ie the spectral phase φ (k). 6 shows the first derivative of the spectral phase φ over the wavenumber k. From the zero crossing of the differentiated spectral phase dφ / dk, the position k_o of the phase object can be determined with high precision.

The 7 represents an economically and technically very interesting design. The light source 1b is designed as a fiber-coupled wavelength tunable laser. The light emerging from the fiber passes through a collimator 2 and illuminate a microlens array 3 which is a micro-aperture array 4 is arranged downstream, wherein the micro-apertures, each arranged in the focus position of the microlenses. The microlens array 3 and the micro-aperture array 4 are firmly connected to each other, this assembly is associated with a not shown here fast Präzisons xy scanner, which also has a high-precision start position.

The spherical wave emanating from a micro-diaphragm from the center A of the same is emitted by the objective 6 collimated, that is converted into a plane wave, ie the point A is mapped to infinity, passing 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. At the Wollaston prism 34 there is a splitting into ordinary and extraordinary bundles, which are polarized perpendicular to each other and have a very small angle in the propagation to each other.

It is done by Fresnel zone lens 7 depending on the wavelength, a different focus. For the middle wavelength of the spectrum, the refractive power is the Fresnel zone lens 7 by means of a directly downstream, low refractive diverging lens 8th compensated to allow only slight deviations from the state of ideal collimation. The following first image stage 9 forms the Fresnel zone lens 7 in 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. In this illustration stage 9 there is an optical retarder 35 with a thick Kalkspatplatte, so that there is an optical delay of about 80 microns between the vertical and the horizontal Poalarisationsrichtung, ie the ordinary and the extraordinary bundles. Subsequently, by means of the water immersion condenser 10 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 in the imaging of the point A here the foci A's_λmin to A's_λmin arise and A'p_λmin to A'p_λmin having a lateral-Shear 2Δq to each other. That is in the 8th and 9 shown. The water immersion condenser 10 in 7 can be readjusted in depth to ensure the confocality of the transmitted light microscope. A thin phase object present in the object space 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 However, as a rule it is 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 lens 12 forms the object 11 as well as the light of the differently focused spherical waves, so that here is 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 hit the second imaging stage 13 , which as an afocal stage, an image on the diffractive optical 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 for each wavelength 14 , The following, directly downstream, weakly refractive collecting lens 15 compensates again for the average refractive power of the Fresnel zone lens 14 , As a result of passing the Fresnel zone lens 14 is the chromatic splitting of the illumination beam path than the depth splitting by the Fresnel zone lens 7 compensated. The polarizer 33 , the Wollaston prism 34 as well as the Wollaston prism 36 and the analyzer 37 form a polarization-optical with the microscope components Shear interferometer.

The further beam path corresponds to that already in the 1 described. However, the chromatic wavelets arise here as a function of the wavelength tuning by means of the wavelength-tunable light source 1b ,

The 8th represents the optical 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 μm. The 9 represents the situation for a single wavelength λi, wherein the comparatively large optical path difference OPD is symbolically indicated here.

Claims (18)

Process for transmitted light microscopy, in particular also for the quantitative testing of one or more objects ( 11 ) or a part or parts thereof or the same in the object space, - with a polychromatic light source ( 1a ) in the illumination beam path and with a spectrometer ( 26 . 27 . 28 ) in the detection beam path - or a wavelength tunable light source ( 1b ) in the illumination beam path and with means for confocal discrimination ( 25 . 30 ) in the detection beam path, such as a plurality of micro-diaphragms (B) or a diaphragm repeatedly imaged by means of a plurality of screened micro-optics, such as screened microlenses, and a camera ( 29 ) in the detection beam path characterized in that in the object image, a chromatic depth splitting by means of at least partially chromatic power in the detection beam path is performed, so that images of the centers of the micro-apertures ( 25 B) 'or images of the center of the multi-mapped diaphragm undergo a chromatically-caused depth split in the back-imaging in the object space there, so that these images (B'o_λmin, B'o_λmax) in a polychromatic light source at a time or at a wavelength tunable light source are separated sequentially in the light propagation direction. Method for transmitted light microscopy according to claim 1, characterized in that the light is spatially structured in the object space, in which by means of several micro-apertures ( 4 , A) or a multi-microlenses multiply mapped macro-aperture - a focus pattern - or a focus line pattern in the object space are formed predetermined, and in the illumination beam path chromatic depth splitting of these foci or focus lines is performed by means of at least partially chromatic power, so that the Images are separated in the light propagation direction (A'o_λmin, A'o_λmax) and that the at least partially chromatic refractive power in the illumination and 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 magnitude and the direction of the chromatic depth splitting of the remapped micro-diaphragms ( 25 , BB) or the multi-mapped macro diaphragm are at least approximately equalized, so that in the object space the images (A ') of the center points (A) of the diaphragm ( 4 , AB) of the illumination beam path or the images (A ') of the foci (A) and the images (B') of the center points (B) of the diaphragm ( 25 , BB) of the detection beam path, which correspond laterally and spectrally, namely A'o_λmin-B'o_λmin and A'o_λmax-B'o_λmax are optically conjugate. Method for transmitted light microscopy after at least one of the claims 1 and 2, characterized in that the method of dark field microscopy is used. Method for transmitted light microscopy after at least one of the claims 1 and 2, characterized in that the method of phase microscopy based on the interference of electromagnetic waves to Application comes. Process for transmitted light microscopy according to claim 4, characterized in that the phase contrast method, in particular also according to F. Zernike, the differential phase-contrast method, in particular also according to G. Nomarski, also as DIC method (DIC: Differential interference contrast), the Hoffinann HMC method (HMC: Hoffmann modulation contrast) or a phase microscopic method after the two-beam interference method, in particular on the Based on a Mach-Zehnder interferometer Method for transmitted-light microscopy according to at least one of claims 4 and 5, characterized in that an optical path difference is introduced in the beam path of a phase microscope, which is at least one wavelength of the maximum, detected wavelength, so that interference fringes, in particular in the form of a Wavelets, ie intensity curves over the wavelength λ or the wavenumber k, are predetermined in the detected spectral range, wherein a thin and laterally small phase object ( 11 ) generates a local phase change in the interference fringes, in particular also in the form of a wavelet. Method for transmitted-light microscopy after An Claim 6, characterized in that the phase of the interference is evaluated. Method for transmitted light microscopy after at least one of the claims 5 to 7, characterized in that the optical path difference in the infinity beam path or in the chromatically weakly focused Beam path of the microscopic image is introduced. Arrangement for transmitted-light microscopy, in particular also with a condenser ( 10 ) and lens ( 12 ) and in particular also for the quantitative examination of a micro object in the object space, - with a polychromatic light source ( 1a ) in the illumination beam path and with a spectrometer ( 26 . 27 . 28 ) in the detection beam path - or a wavelength tunable light source ( 1b ) in the illumination beam path, with means for confocal discrimination in the detection beam path, such as multiple micro-apertures ( 25 , B) or a multi-microlenses multiply mapped macro-aperture, as well as a camera ( 29 ) in the detection beam path, characterized in that means ( 14 ) are arranged with at least partially chromatic refractive power for chromatic depth splitting in the detection beam path. Arrangement for transmitted-light microscopy according to claim 9, characterized in that the means ( 14 ) with at least partially chromatic refractive power for chromatic depth splitting in the Fourier plane (F'12), ie the rear focal plane of the objective ( 12 ), or in an optical conjugate plane (F''12) are arranged. Arrangement for transmitted-light microscopy according to claim 9, characterized in that means ( 7 ) are arranged with at least partially chromatic refractive power for chromatic depth splitting in the illumination beam path. Arrangement for transmitted-light microscopy according to claim 11, characterized in that means ( 7 ) with at least partially chromatic refractive power for chromatic depth splitting in the Fourier plane, ie the rear focal plane (F10), of the condenser ( 10 ), or in a plane (PE'10) which is optically conjugate to this. Arrangement for transmitted-light microscopy according to at least one of claims 9 to 12, characterized in that when using a Nomarski DIC microscope with two Wollaston prisms ( 34 ) ( 36 ) an optical, birefringent retarder generates an optical path difference between the two polarization directions of at least one wavelength of the maximum, detected wavelength, the retarder in the space between the two Wollaston prisms ( 34 ) ( 36 ) of the Nomarski DIC microscope. Arrangement for transmitted light microscopy according to claim 13, characterized in that the optical, birefringent retarder in the form of a calcite ( 35 ) (calcite plate) is formed. Arrangement for transmitted-light microscopy according to at least one of claims 9 to 14, characterized in that the means with chromatic refractive power as diffractive-optical elements ( 7 ) 14 ) are formed. Arrangement for transmitted-light microscopy according to at least one of Claims 9 to 15, characterized in that the means ( 25 ) are arranged laterally displaceable for confocal discrimination in the detection beam path. Arrangement for transmitted-light microscopy according to at least one of Claims 9 to 15, characterized in that the means ( 4 ) are arranged laterally displaceable for structuring the light in the illumination beam path and synchronously with the means ( 25 ) are shifted to the confocal discrimination in the detection beam path, so that the lateral assignment of the images (A ', B') of the same in the object space during the displacement and the image recording by means of camera ( 29 ) is always preserved. Arrangement for transmitted light microscopy after at least one of the claims 9 to 17, characterized in that an optical coupling of the arrangement for transmitted light microscopy is performed with an optical, computer-controlled tweezers.