Carl Zeiss Microscopy Gmbh

Ingo Kleppe

Ralf Netz

Yauheni Novikau

Ralf Wolleschensky

Thomas Kalkbrenner

Christoph Nieten

Helmut Lippert

Jörg Siebenmorgen

Michael Kempe

Gerhard Krampert

Michael Gölles

Wolfgang Singer

Matthias Wald

Artur Degen

Joerg Siebenmorgen

Lars-Christian Wittig

Rebecca Elsässer

Philipp Schwesig

Daniel Schwedt

Daniel Haase

Manuel Amthor

Mirko Liedtke

Michael Kieweg

Kai Wicker

Toufic Jabbour

Gunther Lorenz

Lutz Schaefer

Stephan Kuppig

An apparatus and method for structured illumination microscopy, having an illumination beam path for irradiating a sample with excitation light with a two-dimensional illumination pattern at angles greater than the angle for total internal reflection. The illumination beam path has an illumination objective used to irradiate the sample. A first separation device for separating the excitation light in a first linear coordinate direction in a pupil plane and a displacement device for laterally displacing the illumination pattern in a sample plane, having a detection beam path containing at least one microscope objective for guiding emission light to a camera. The emission light is emitted by the sample as a consequence of the irradiation by the excitation light. A camera for recording images of the sample, has a control unit for calculating microscopic images of the sample using partial images of the sample recorded for different positions of the illumination pattern in the sample plane. The apparatus has an adjustable second separation device for variably separating the excitation light in a second linear coordinate direction which is independent of the first linear coordinate direction, this variable separation being independent of the separation in the first linear coordinate direction in the pupil plane.

Apparatus for structured illumination microscopy, method for illuminating a sample and method of structured illumination microscopy

Method for super-resolution evaluation of microscope images illuminated in a structured manner and microscope having structured illumination. The resolution can be improved laterally by a factor of up to two using conventional linear structured Illumination (SIM). If a non-linear iterative method is used for the purpose of deconvolution, the achievable resolution can be improved beyond the theoretical limit. However, the known methods only achieve a small amount of increase. The novel method is intended to make improved resolution or improved contrast possible. If a PSF/OTF that is manipulated (individually for each order) in the same (or in a corresponding) way as the relevant order spatial frequency spectrum is used during the re-weighting in the spatial frequency domain (for the deconvolution), the actually achievable resolution can be nearly doubled in comparison with the conventional SIM, both in one-stage and in two-stage variants.

Method for super-resolution evaluation of microscope images illuminated in a structured manner and microscope having structured illumination

A method of image evaluation when performing SIM microscopy on a sample includes: A) providing n raw images of the sample, which were each generated by illuminating the sample with an individually positioned SIM illumination pattern and imaging the sample in accordance with a point spread function, B) providing (S) n illumination pattern functions, which each describe one of the individually positioned SIM illumination patterns, C) providing (S) the point spread function and D) Carrying out an iteration method, which includes following iteration steps a) to e), as follows: a) providing an estimated image of the sample, b) generating simulated raw images, in each case by image processing of the estimated image using the point spread function and one of the n illumination pattern functions such that n simulated raw images are obtained, c) assigning each of the n simulated raw images to that of the n provided raw images which was generated by the illumination pattern that corresponds to the illumination pattern function used to generate the simulated raw image, and calculating n correction raw images by the comparison of each provided raw image with the simulated raw image assigned thereto, d) generating a correction image by combining image processing of the n correction raw images using the point spread function and the n illumination pattern functions, wherein a filtering step is carried out in each implementation of iteration step d), said filtering step suppressing a spatial fundamental frequency of the illumination pattern, and e) reconstructing the estimated image of the sample by means of the correction image and using the corrected estimated image of the sample as the estimated image of the sample in iteration step a) in the next run through the iteration.

Method of image evaluation for sim microscopy and sim microscopy method

A light beam shaping arrangement for a light microscope has a first and a second liquid crystal region or lifting micromirror region, each of which has a plurality of independently switchable liquid crystal elements or mirrors with which a phase of incident light is changeable in a settable manner, an input-/output-coupling polarization beam splitter, a polarization beam splitter arranged between the input-/output-coupling polarization beam splitter and the liquid crystal regions or lifting micromirror regions such that the polarization beam splitter separates the light coming from the input-/output-coupling polarization beam splitter in a polarization-dependent manner into a first partial beam. The first partial beam is directed to the first liquid crystal region or lifting micromirror region, and into a second partial beam, which is directed to the second liquid crystal region or lifting micromirror region, and the two partial beams returning from the liquid crystal regions and directs them together as an outgoing light beam.

Optical arrangement and method for light beam shaping for a light microscope

A method for performing SIM microscopy on a sample, includes: generating n raw images of the sample, in each case by illuminating the sample using the same SIM illumination pattern albeit with an individual positioning for each raw image, wherein p orders of diffraction are assigned to the SIM illumination pattern, and generating an image of the sample from the n raw images. An image reconstruction is carried out using the orders of diffraction, wherein t highest orders of diffraction are suppressed during the image reconstruction and n=p−t applies.

Method for SIM microscopy

An optics arrangement for flexible multi-color illumination for a light microscope includes an acousto-optical tunable filter ('AOTF'). The AOTF is set up to diffract two light components from incident illumination light into different order-of-diffraction directions. The two light components differ in their wavelengths and polarizations. Alternatively, an electro-optical modulator ('EOM') can be used, with which two temporally successive light components of different wavelengths are set to different polarization directions. A polarization beam splitter separates the two light components of different wavelengths and polarizations into reflection light, which is reflected at the polarization beam splitter, and transmission light, which is transmitted at the polarization beam splitter. A light structuring apparatus imprints different structures onto the transmission light and the reflection light. The structured transmission light and the structured reflection light are then recombined by the polarization beam splitter or a further polarization beam splitter onto a common beam path.

Optics arrangement for flexible multi-color illumination for a light microscope and method to this end

A microscope control method for operating a microscope, includes: capturing an item of acoustic, graphically represented and/or electronically coded voice information; comparing the voice information with stored reference commands and determining a voice command on the basis of a predetermined degree of correspondence between at least one section of the voice information and a reference command; selecting that reference command to which the voice command corresponds at least to a predetermined degree; generating at least one control command suitable for operating the microscope, wherein the control command is either an invariable control command assigned to the selected reference command or the control command is generated on the basis of a rule assigned to the reference command for forming a generated control command, and controlling the microscope by means of the assigned or generated control command. Also, a microscope is designed to carry out the microscope control method.

Microscope control method and microscope

In high-resolution scanning microscopy, a sample is excited by illumination radiation to emit fluorescence radiation in such a way that the illumination radiation is focused at a point in or on the sample to form a diffraction-limited illumination spot. The point is imaged in a diffraction-limited manner into a diffraction image on a spatially resolving surface detector, wherein the surface detector has a spatial resolution that resolves a structure of the diffraction image. The sample is scanned by means of different scanning positions with an increment of less than half the diameter of the illumination spot. An image of the sample is generated from the data of the surface detector and from the scanning positions assigned to said data, said image having a resolution that is increased beyond a resolution limit for imaging. For the purposes of distinguishing between at least two predetermined wavelength regions in the fluorescence radiation from the sample, a corresponding number of diffraction structures are generated on the surface detector for the at least two predetermined wavelength ranges, said diffraction structures differing but having a common center of symmetry. The diffraction structures are evaluated when generating the image of the sample.

High-resolution scanning microscopy with discrimination between at least two wave-length ranges

An optical arrangement for light beam shaping in a light microscope has a first and a second liquid crystal region, each of which has a plurality of independently switchable liquid crystal elements with which a phase of incident light is changeable in a settable manner. A first polarization beam splitter is arranged in such a way that incident light is split in a polarization-dependent manner into reflection light, which is reflected in the direction of the first liquid crystal region, and transmission light, which is transmitted in the direction of the second liquid crystal region. The first or a second polarization beam splitter is arranged such that the reflection light and transmission light are combined onto a common beam path after phase modulation by means of the liquid crystal regions.

A microscopy method for three-dimensionally imaging an object, including imaging the object along a beam path into a first image on a first image plane. A first microlens array is arranged on the first image plane, and a second microlens array with the same pitch is arranged downstream of the first array. The two arrays laterally segment the first image and image same into a second image in which the segments are spaced apart and separated by gaps. On a pupil plane downstream of the microlens array, a provided phase mask generates a spot for each segment of the second image according to a pixel diffusion function. A detector detects the shape and structure of the spot, and a controller ascertains a lateral intensity distribution and depth specification from the shape and/or structure of the spot for each segment and generates a depth-resolved image of the object therefrom.

3D microscopy

One or more images of a sample may be split into image data split according to dyes. The sample has at least two different dyes, in particular fluorescent dyes. The method for splitting the one or more images includes providing the one or more images of the sample and inputting the one or more images into a machine learning system. The method then includes generating the image data split according to dyes from the image or the images, using the machine learning system. The machine learning system removes at least one partial structure of the sample that is present in the image data split according to dyes of more than one dye from the image data of one or more dyes.

Method for splitting one or more images of a sample into image data split according to dyes, computer program, computer-readable medium storing the program and system for performing the method

A microscope and method for high resolution scanning microscopy of a sample, having an illumination device, an imaging device for the purpose of scanning at least one point or linear spot across the sample and of imaging the point or linear spot into a diffraction-limited, static single image below a reproduction scale in a detection plane. A detector device is used for detecting the single image in the detection plane for various scan positions, with a location accuracy which, taking into account the reproduction scale in at least one dimension/measurement, is at least twice as high as a full width at half maximum of the diffraction-limited single image.

High-resolution scanning microscopy

Accelerated methods and apparatuses for three-dimensional microscopy with structured illumination, in which focal planes of the sample are focused and each focal plane is illuminated in a plurality of phases sequentially with structured illumination light and the sample light emitted by the sample is recorded in a respective individual image. A resulting image having a resolution that is increased with respect to the individual images is reconstructed from the individual images to produce a super-resolved image stack, By reconstructing a resulting image from individual images of two different focal planes by approximation methods, said resulting image represents a sample plane that is situated between said focal planes, an image stack can be produced in a shorter period with less stress on the sample.

Accelerated methods and apparatuses for three-dimensional microscopy with structured illumination

A method for high-resolution scanning microscopy of a sample in which a sample is illuminated at a point in or on the sample by means of illumination radiation. The point is imaged along an optical axis and according to a point spread function into a diffraction image on a spatially resolving surface detector that comprises detector pixels in which a diffraction structure of the diffraction image is resolved. The point is displaced relative to the sample in at least two scanning directions and pixel signals are read from the detector pixels in various scanning posi- tions, wherein the pixel signals are respectively assigned to that scanning position at which they were read out and adjacent scanning positions overlap one another and are disposed according to a scanning increment. An image of the sample having a resolution that is increased beyond a resolution limit of the imaging is generated from the read pixel signals and the assigned scanning positions, wherein a deconvolution is carried out. Intermediate positions are generated for at least one of the scanning directions in the deconvolution on the basis of the pixel signals and the image of the sample, which contains more image points than scanning positions, is generated.

A microscopy high-resolution scanning method, including exciting a sample with illumination radiation focused at a point to form a diffraction-limited illumination spot so as to emit fluorescence radiation. The point is imaged in a diffraction image on a spatially resolving two-dimensional detector. The sample is scanned at scanning positions with increments that are smaller than half the diameter of the spot. An image of the sample with a resolution increased beyond a resolution limit of the image is generated from the data of the two-dimensional detector and the scanning positions. To discriminate between at least two predetermined wavelength ranges in the fluorescence radiation of the sample, Airy disks corresponding to the wavelength ranges are generated on the two-dimensional detector, the Airy disks being offset laterally from one another such that the diffraction image consists of the mutually offset Airy disks. The Airy disks are evaluated when generating the sample image.

Growing community of inventors

Jena, Germany

Ingo Kleppe