Leica Microsystems Cms Gmbh

Jonas Foelling

Lars Friedrich

Marcus Dyba

Arnold Giske

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 system for coupling at least one optical beam includes at least one optical beam entry port, an optical beam exit port, the optical beam exit port including a lens performing a Fourier Transform, at least one optical beam deflector, and an optical base element, wherein the at least one directly fixed optical beam deflector is allowed to rotate around a rotation axis, wherein the corresponding at least one optical beam and/or its assigned optical beam entry port is configured such that the semi-major axis of the elliptical cross section of the optical beam on a deflection surface of a respective optical beam deflector is oriented parallel to the rotation axis, and wherein, after having passed the optical beam exit port, the elliptical cross section of the at least one optical beam overlaps the circular cross section of the light receiver.

System and method for coupling an optical beam into a light receiver

A laser scanning microscope includes a light source configured to emit an illumination light beam. The illumination light beam has a transverse light intensity profile comprising an intensity minimum. The laser scanning microscope further includes a scanning device configured to scan the illumination light beam along a closed trajectory in a target area of a specimen, and a detector configured to detect fluorescence light emitted by a fluorophore within the target area of the specimen. The fluorophore is excited by the illumination light beam. The laser scanning microscope further includes a processor configured to determine an intensity distribution of the fluorescence light as a function of time and to determine a position of the fluorophore within the target area based on the intensity distribution of the fluorescence light.

Laser scanning microscope and method for determining a position of a fluorophore

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

A method for imaging a sample using a fluorescence microscope with stimulated emission depletion includes controlling the fluorescence microscope and an imaging process of the fluorescence microscope by a microscope controller. An overview image of a target region is generated with a second spatial resolution prior to the imaging process, the second spatial resolution being lower than a first spatial resolution used for scanning sample segments in the imaging process and higher than a third spatial resolution that has been adapted to an extent of an excitation light distribution. The overview image is analyzed to identify image regions without relevant image information. A radiant flux of the depletion light distribution is reduced within a scope of the imaging process when scanning sample segments which are assigned to the image regions without relevant image information.

Method for imaging a sample using a fluorescence microscope with stimulated emission depletion

A scanning microscope includes an objective and a scanning element that is adjustable for a time-variable deflection to guide a focused illumination beam across the sample in a scanning movement. A detection beam is guided across sensor elements of an image sensor in a movement which corresponds to the scanning movement of the focused illumination beam. A dispersive element of a predetermined dispersive effect arranged upstream of the image sensor spatially separates different spectral components of the detection beam from one another on the image sensor. A controller detects the time-variable adjustment of the scanning element, assigns the spatially separated spectral components of the detection beam to the sensor elements of the image sensor based on the detected time-variable adjustment, while taking into account the predetermined dispersive effect of the dispersive element, and individually reads out the sensor elements assigned to the spectral components.

Scanning microscope

A method for setting an evaluation parameter for a fluorescence microscope includes exciting dye particles in a sample to fluoresce and detecting fluorescent light from the particles. A graphical representation of a distribution of the fluorescent light is determined and a signal is generated for use in displaying the graphical representation on a display unit. Each subregion of the graphical representation is associated with a comparison value that is representative of a light quantity in the subregion. A predefined threshold is used as an evaluation parameter and compared to the comparison values. The subregions having a comparison value that is greater than the threshold value are marked on the display unit with predefined markings. The threshold value is changed and the comparison values are compared to the changed threshold value. The marked regions are defined as events and a complete image of the sample is obtained based on the events.

Method and device for setting a suitable evaluation parameter for a fluorescence microscope

A light-microscopic method of localization microscopy for localizing point objects in a sample arranged in an object space includes imaging, by an imaging optical unit having a depth of field range of predetermined axial z-extension along its optical axis in the object space, the sample onto a detector; localizing the point objects in the sample within the depth of field range in that, on the basis of a sample image, lateral x/y-positions of the point objects in a direction perpendicular to the optical axis are ascertained; displacing, in the object space relative to the sample the depth of field range within which the point objects are localized in the object space relative to the sample along the optical axis at least once by a predetermined axial z-travel distance; and imaging, by the imaging optical unit in the event of an axially displaced depth of field range.

Light-microscopic method of localization microscopy for localizing point objects

A method for illumination and detection in RESOLFT microscopy using a pulsed or continuous light source for excitation light and switching light is characterized in that the excitation light () is irradiated in pulses and in that the pulse of the excitation light () is longer than 150 picoseconds, preferably up to a few hundred picoseconds, and even up to a few nanoseconds. A corresponding apparatus uses the method according to the present invention.

Method and apparatus for illumination and detection in RESOLFT microscopy

A device and a method for acquiring a microscopic image of a sample structure are described. An optic for imaging the sample structure and a reference structure is provided, as well as a drift sensing unit for sensing a drift of the sample structure relative to the optic on the basis of the imaged reference structure. The optic comprises a first sharpness plane for imaging the sample structure and at the same time a second sharpness plane, modifiable in location relative to the first sharpness plane, for imaging the reference structure.

Device and method for acquiring a microscopic image of a sample structure

A widefield microscope illumination system and method with a microscope objective and an illumination light source that sends widefield illumination light along illumination beam paths. Illumination light penetrates into the microscope objective through illumination light entry sites located within a predetermined annular or annular-segment-shaped illumination light entry area having a large offset to an optical objective axis of the objective. A spatially resolving light detector detects light sent from an illuminated sample through the microscope objective along a detected light beam path. An automatic illumination light beam path manipulation device is controlled by a control system, which is arranged in front of the microscope objective in relation to the direction of the illumination light beam path, and by means of which illumination light beam path manipulation device the illumination axes are automatically movable at time intervals to a plurality of different illumination light entry sites.

Wide-field microscope illumination system and wide-field illumination method

A light-microscopy method for locating point objects in a sample arranged in an object space includes imaging the sample onto a detector by an imaging optical unit having a depth of field of predetermined axial extent along an optical axis in the object space, onto which the detector is imaged. The point objects in the sample are located within the depth of field. The first sample image generated by the imaging of the sample onto the detector is evaluated. For locating a respective first point object in a direction of the optical axis, a parameter of a first light spot of one or more light spots of the first sample image representing the first point object is determined, and a rough axial z position related to the first point object is assigned to the parameter based on predetermined association information.

Light-microscopy method for locating point objects

A widefield microscope illumination system and a method for illumination, the system having a microscope objective with an optical objective axis, an illumination light source sending widefield illumination light along illumination beam paths having corresponding illumination axes along which the illumination light penetrates into the microscope objective through illumination light entry sites located within a predetermined illumination light entry area, a spatially resolving light detector detecting detected light sent from an illuminated sample through the microscope objective along a detected light beam path, and an automatic illumination light beam path manipulation device, controlled by a control system, which is arranged in front of the microscope objective in relation to the direction of the illumination light beam path, and by which illumination light beam path manipulation device the illumination axes are automatically movable at time intervals to a plurality of illumination light entry sites.

Widefield microscope illumination system and widefield illumination method

A microscopic device for three-dimensional localization of point-like objects, encompassing a detection optical system that images point-like objects, each in the form of a three-dimensional focus light distribution, into an image space; a color separation apparatus that divides the light into at least two separate light bundles of different wavelength regions; at least two image space detector units , one receiving one light bundle and the other receiving the other light bundle, each detector unit comprising a light-spot-sensing detection surface ; an evaluation unit that ascertains a lateral X-Y position and an axial Z position relative to the sharpness plane in a direction perpendicular to the sharpness plane; at least one Z-position correction value for at least one of the wavelength regions being stored in the evaluation unit, which value indicates a detection optical system longitudinal chromatic aberration in that wavelength region; and the evaluation unit correcting the Z position.

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Heidelberg, Germany

Jonas Foelling