Leica Microsystems Heidelberg Gmbh

Deutsches Krebsforschungszentrum

Max-planck-gesellschaft Zur F&#xf6;rderung Der Wissenschaften E. V.

Leica Microsystems Cms Gmbh

Leica Lasertechnik Gmbh

Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts

Leica Lasertechink Gmbh

Lecia Microsystems Heidelberg Gmbh

Johann Engelhardt

Juergen Hoffmann

Heinrich Ulrich

Holger Birk

Rafael Storz

Werner Knebel

Joachim Bradl

Kyra Moellmann

Stefan W Hell

William C Hay

Thomas Zapf

Bernd Widzgowski

Klaus Kierschke

Hilmar Gugel

Matthias Reuss

William F Hug

Volker Westphal

Christian Eggeling

Joerg Bewersdorf

Giuseppe Vicidomini

Katrin Willig

Jörg Bewersdorf

Martin Hoppe

Gael Moneron

Jan Keller-Findeisen

Marion Lang

Jonas Marquard

Kyu-Young Han

Christiane Ihrig

Joachim Jehle

Roelof W Wijnaendts Van Resandt

Jale Schneider

Kyra Möllmann

Jan Keller

Jürgen Hoffmann

In a method of detecting changes in direction of a collimated coherent light beam, the light beam is split into partial light beams which are superimposed on a camera to form an interference pattern displaying light intensity minima and maxima alternatingly following to one another in a transverse direction oriented transversely to an average propagation direction of the partial beams. The light beam is focused into at least one focus located in front of the camera. Pictures of the interference pattern including a plurality of the light intensity maxima are registered with the camera. An average shift of the plurality of light intensity maxima with regard to the camera in the at least one transverse direction is determined from the pictures. A change in angular orientation of the collimated coherent light beam in the at least one transvers direction is deduced from the average shift.

Method and apparatus for detecting changes in direction of a light beam

For forming and shifting a light intensity distribution in a focal area of an objective lens, portions of coherent input light are one by one directed into non-identical two-dimensional pupil areas of a pupil of the objective lens. Each of the portions of coherent input light is collimated in the pupil. The pupil areas include a pair of two pupil areas which are axially symmetrically arranged on opposite sides of an optical axis of the objective lens. At least one of the two discrete portions of coherent input light that are directed into the pair of pupil areas is separately modulated with regard to its phase by means of an electro optical modulator such as to form the light intensity distribution in the focal area with a local intensity minimum delimited by intensity maxima and to shift the local intensity minimum laterally with regard to the optical axis.

Method of and apparatus for forming and shifting a light intensity distribution in a focal area of an objective lens

Upstream a microscope objective lens, a polarization direction of a light beam is tilted with a first electro-optical deflector between a first polarization direction with which the light beam is deflected by a first polarization beam splitter by a first angle and a second polarization direction with which it is deflected by a second angle. With a second electro-optical deflector, the polarization direction of the light beam is tilted between a third polarization direction with which the light beam is deflected by a second polarization beam splitter by a third angle and a fourth polarization direction with which it is deflected by a fourth angle. By rotating the polarization direction of the light beam by means of the first and second electro-optical deflectors in a coordinated way the light beam is tilted about a fixed point in a pupil of the objective lens.

Method of scanning a sample with a light beam focused by a microscope objective lens and scanning light microscope

A segmented birefringent chromatic beam shaping device comprises at least three birefringent chromatic segments arranged side by side in a pupil of the beam shaping device. The birefringent chromatic segments are essentially n&#x3bb; waveplates at a first design wavelength. At a second design wavelength, the birefringent chromatic segments are essentially (m+1/2)&#x3bb; waveplates. Each of the birefringent chromatic segments comprises a stack of birefringent elements including at least three chromatic birefringent elements. Orientations of fast axes of each pair of directly consecutive chromatic birefringent elements of each of the birefringent chromatic segments differ by at least 5 deg. The at least three birefringent chromatic segments comprise same sequences of materials, thicknesses and orientations of their birefringent elements so that they only differ is their orientations in the pupil.

Segmented birefringent chromatic beam shaping device

A fluorescence microscope instrument includes a light source; an objective focusing light from the light source into a sample and collecting fluorescence light emitted out of the sample; a detector detecting the fluorescence light; and a beam path separator arranged in a first beam path between the light source and the objective and in a second beam path between the objective and the detector. Wavelengths of the light to be directed to the sample and of the fluorescence light to be detected by the detector fall into an extended range of wavelengths. The beam path separator separates the two beam paths in that it is transferable between being transparent for any wavelength of the range of wavelengths and coming along the first beam path, and a second state in which it is transparent for any wavelength of the range of wavelengths and coming along the second beam path.

Fluorescence microscope instrument comprising an actively switched beam path separator

A segmented chromatic phase plate includes at least three stacks. The three stacks are arranged around and stacked along a main axis, and have same overall heights. Each stack includes at least two plane-parallel optical flats of different materials. The materials and the thicknesses of the optical flats are selected such that optical path lengths of light of a first wavelength passing through the different stacks along the main axis differ by integer multiples of the first wavelength, whereas optical path lengths of light of a second wavelength differ by integer multiples plus defined fractions of the second wavelength. The materials of the at least two optical flats have different refractive indices for both the first and second wavelengths; and derivatives of the differences between the optical path lengths with respect to wavelength are zero at at least one of the first and second wavelengths.

Chromatic phase plate

In a method of monitoring a relative position of a microscope objective with regard to a sample a test beam of light is directed onto at least one at least partially reflective surface connected to the sample, and light of the test beam reflected at the at least one at least partially reflective surface is registered and evaluated. Additionally, the test beam is directed onto a reflective surface of the microscope objective facing the sample, and light of the test beam reflected at the reflective surface of the microscope objective is also registered and evaluated.

Method of and microscope with installation for focus stabilization

An apparatus forming an area of minimum light intensity enclosed by high light intensity includes two objectives focusing light of opposite directions into a common focal area. Light intensities of a first pair of light beams extinguish each other in a first partial area of the focal area. Beam paths of the first pair of beams pass through the objectives in a first pair of partial areas of the pupils of the objectives. Light intensities of a second pair of light beams extinguish each other in a second partial area of the focal area. Beam paths of the second pair of light beams pass through the objectives in a second pair of partial areas which are offset with regard to the first pair of partial areas of the pupils; and the light of the second pair does not interfere with the light of the first pair of light beams.

4Pi STED fluorescence light microscope with high three-dimensional spatial resolution

In a STED fluorescence light microscope pulses of excitation light () are applied to a sample, which excite fluorescent entities contained in the sample for fluorescence, and which are focused on at least one focal area. Further, de-excitation light () is applied to the sample, which de-excites the excited fluorescent entities and which comprises an intensity zero point in the at least one focal area, as a continuous wave. Fluorescence light emitted by the excited fluorescent entities in the sample is registered after each pulse of the excitation light () and overlapping with applying the de-excitation light () with high temporal resolution between consecutive pulses of the excitation light ().

STED microscopy with pulsed excitation, continuous stimulation, and gated registration of spontaneously emitted fluorescence light

A fluorescence light scanning microscope () comprises a light source providing excitation light () for exciting a fluorophore in a sample to be imaged for spontaneous emission of fluorescence light, and suppression light () for suppressing spontaneous emission of fluorescence light by the fluorophore on a common optical axis (), the suppression wavelength differing from the excitation wavelength; an objective () focusing both the excitation () and the suppression () light to a focus point; a detector () detecting fluorescence light () spontaneously emitted by the fluorophore; and a chromatic beam shaping device () arranged on the common optical axis (), and including a birefringent chromatic optical element () adapted to shape a polarization distribution of the suppression light () such as to produce an intensity zero at the focus point, and to leave the excitation light such as to produce a maximum at the focus point.

Fluorescence light scanning microscope having a birefringent chromatic beam shaping device

To the end of three-dimensionally localizing light emitting marker entities of unknown orientation and unknown position in a sample, the light emitted by each single marker entity is imaged in at least two different ways onto at least one detection plane which corresponds to a focal plane () in the sample resulting in at least two images of the marker entity. Virtual x- and y-positions of the marker entity in parallel to the focal plane () are separately determined from the emitted light intensity distribution over each image of the marker entity. Further, the z-position of the marker entity normal to the focal plane is determined from the emitted light intensity distributions over the images of the marker entity. The real x- and y-positions of the marker entity in parallel to the focal plane () are determined based on its virtual x- and y-positions and on its z-position.

Three-dimensionally localizing light emitting molecules of unknown orientation and unknown Z-position

For dynamically shifting a light beam () with regard to an optic focussing the light beam to scan an object in a two-dimensional scanning range with the focussed light beam, at least two beam deflectors () are connected in series per each direction in which the light beam is to be deflected with regard to the optical axis of the focussing optic. The two beam deflectors deflect the light beam by two deflection angles (and, respectively) which are dynamically variable independently on each other. The deflection angles (to) of all beam deflectors () are predetermined for each point of the scanning range () in such a way that the light beam (), in scanning the whole two-dimensional scanning range, always runs through the pupil of the optic () at essentially the same point.

Method and apparatus for dynamically shifting a light beam with regard to an optic focussing the light beam

For imaging a structure in a sample with spatial resolution, the structure is labeled with a fluorophore which is transferable by an optical transfer signal out of a first into a second photochromic state. Via a common objective, the sample is subjected to both the focussed optical transfer signal and a focussed optical excitation signal only exciting a portion of the fluorophore being in its second photochromic state for fluorescence. The transfer and the excitation signal have a common centre of maximum intensity; and a decrease of intensity of the transfer signal with the distance to this common centre is substantially stronger than any decrease of the effective return rate of the fluorophore back into the first photochromic state. Fluorescence light emitted by the excited fluorophore is detected. Then, the common centre is shifted with regard to the sample; and the steps of subjecting and detecting are repeated.

Method of fluorescence-microscopically imaging a structure in a sample with high three-dimensional spatial resolution

The present invention relates to an optical scanning device that comprises a light source to emit a beam of light, and a beam splitter to split that beam into several beamlets, and further a first objective lens to direct said beamlets onto a focal plane wherein each of said beamlets impinges on the focal plane spacially separated from each other, wherein the beam splitter comprises several birefringent elements for splitting said beam, preferably a stack of Wollaston prisms.

Optical scanning device and method of deriving same

An optical device for combining a first light beam and at least one second light beam includes a first beam splitting device, a second beam splitting device and a position detector. The first beam splitting device splits a first reference beam from the first light beam and a second reference beam from the second light beam. The second beam splitting device splits a third reference beam from the first light beam and a fourth reference beam from the second light beam. The position detector detects respective positions of the reference beams so as to enable a respective propagation direction and/or a respective position of the first and/or second light beams to be adjusted as a function of the detected positions of the reference beams.

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Bad Schoenborn, Germany

Johann Engelhardt