Leica Microsystems Cms Gmbh

Lars Friedrich

Jonas Foelling

Werner Knebel

Holger Birk

Florian Fahrbach

Bernd Widzgowski

Dirk-Oliver Fehrer

Kai Walter

Wernher Fouquet

Jonas Fölling

Manuel Kremer

Arnold Giske

Lioba Kuschel

Juergen Schneider

Jochen Sieber

A fluorescence microscope includes excitation and de-excitation light sources designed to generate excitation and de-excitation light distributions, which excite and de-excite fluorophores present in a sample, respectively. An illumination unit is designed to combine the light distributions such that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed on one another in an illumination target point. A detector is designed to detect the fluorescence photons as a function of their arrival times. The processor is designed to evaluate the detected fluorescence photons with respect to their arrival times and, based thereon, to control a delay which a light pulse or a light modulation of the de-excitation light distribution has at a position of the illumination target point in relation to a light pulse or a light modulation of the excitation light distribution.

Fluorescence microscope and method for imaging a sample

A method for scanning a sample in microscopy includes generating at least three illumination spots in order to form a spot pattern that contains at least two illumination spots having a first wavelength and an illumination spot having a second wavelength that differs from the first wavelength. At least one specified region of the sample is scanned by moving the spot pattern formed by the illumination spots along a first direction for generating scan lines, which are each associated with the illumination spots of the spot pattern, and by moving the spot pattern formed by the illumination spots along a second direction for generating scan lines respectively after the scan lines.

Method and device for scanning a sample

A method for estimating a stimulated emission depletion microscopy (STED) resolution includes generating a first frame representing a reference image from a field-of-view, the reference image having a predetermined reference resolution, and generating at least one second frame representing a STED image from the same field-of-view, the STED image having the STED resolution to be estimated. The at least one second frame is blurred by applying a convolution kernel with at least one fit parameter to the second frame. An optimal value of the at least one fit parameter of the convolution kernel is determined for which a difference between the first frame and the blurred at least one second frame is minimized. The STED resolution is estimated based on the optimal value of the at least one fit parameter and the predetermined reference resolution.

Method and device for estimating a STED resolution

A method for scanning a sample includes generating at least two illumination points in order to form a point pattern, wherein the point pattern has a settable number of illumination points. At least one freely selectable parameter for defining the point pattern is preset or is set. At least one predefined region of the sample is scanned by moving the point pattern defined by the freely selectable parameter along a first direction such that scan lines assigned to the illumination points of the point pattern are generated, and along a second direction such that further scan lines are generated in each case following the scan lines. The movement of the point pattern in the second direction is carried out in scan steps of identical size or at a constant speed. The illumination points of the point pattern are arranged on a line along the second direction.

A fluorescence scanning microscope includes excitation and de-excitation light sources, which are designed to generate an excitation and a de-excitation light distribution, respectively. An illumination unit combines the light distributions to form a light distribution scanning over multiple illumination target points of a sample in such a way that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed on one another. A detector detects fluorescence photons emitted from the respective illumination target point as a function of their arrival times. A processor evaluates the fluorescence photons with respect to the arrival times, generates a first pixel and a second pixel based thereon, assembles the first and second pixels to form first and second sample images, respectively, and, by means of the two sample images, determines a spatial offset between the intensity maximum and the intensity minimum.

Fluorescence scanning microscope and method for imaging a sample

An optical assembly includes a volume grating configured to influence a beam direction of at least one light beam, and a switching device arranged in a beam path upstream of the volume grating. The switching device is configured to switch the beam direction and/or beam position of the at least one light beam from a first beam direction and/or beam position, in which the at least one light beam does not impinge on the volume grating at an acceptance angle of the volume grating, to a second beam direction and/or beam position, in which the at least one light beam impinges on the volume grating at the acceptance angle, and/or vice versa.

Optical assembly and method for influencing the beam direction of at least one light beam

An illumination apparatus for a microscope, for producing a de-excitation or switching light distribution, includes a light source configured to produce a primary illumination light beam and a beam splitter configured to divide the primary illumination light beam into two partial illumination light beams. An illumination objective is configured to focus the partial illumination light beams onto and/or into a sample such that the partial illumination light beams extend, spatially separated from one another, through an entry pupil of the illumination objective and are spatially superposed on and/or in the sample after passing through the illumination objective. A phase influencer is configured to cause a relative phase offset of the partial illumination light beams with respect to one another in such a way that the partial illumination light beams in the entry pupil of the illumination objective have a phase offset of &#x3c0;.

Illumination apparatus for a microscope

The invention relates to a method for investigating a sample with regard to the lifetime of an excited state, in particular a fluorescence lifetime, and/or with regard to a property of a sample which is correlated with a lifetime of an excited state, in particular with a fluorescence lifetime, a sample region being illuminated with a sequence of excitation light pulses. The method is characterized in that the light quantity and/or number of photons of the detected light, in particular fluorescent light, proceeding from the sample region is measured temporally between the excitation light pulses exclusively within a detection time window in each case, at least two detection time windows having different temporal lengths.

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Weinheim, Germany

Lars Friedrich