Abstract: For detecting movements of a sample with respect to an objective, the sample is imaged onto an image sensor comprising an array of pixels by means of the objective. Images of the sample are recorded in that light coming from the sample is registered at the pixels. Variations of intensities of the light coming from the sample and registered at the pixels are determined during a set-up period in that a temporal course of the intensity of the light, which has been registered at a respective one of the pixels over the set-up period, is analyzed. Using these variations as a criterion, a subset of not more than 90% of the pixels of the image sensor is selected. Parts of the images that each correspond to the selected subset are compared to parts of at least one reference image that also correspond to the subset.

Abstract: A bandpass filter for light has variable lower and upper cut-off wavelengths. The bandpass filter comprises an areal long-pass filter defining the variable lower cut-off wavelength and an areal short-pass filter defining the variable upper cut-off wavelength. The long-pass filter has different lower cut-off wavelengths in different first area regions which follow to one another in a first direction, and the short-pass filter has different upper cut-off wavelengths in different second area regions which follow to one another in a second direction. The long-pass filter and the short-pass filter are connected in series and spatially fixed relative to one another. The first direction and the second direction are oriented crosswise to one another.

Abstract: The present invention is related to a method, a computer program, and apparatus for adapting an estimator for use in a microscope for estimating a position of an emitter in a sample based on a method, in which the sample is illuminated with light at one or more sets of probe positions and fluorescence photons are acquired for the sets of probe positions. The invention is further related to a microscope, which makes use of such a method or apparatus. The sample is illuminated with light at one or more sets of probe positions and fluorescence photons are acquired for the sets of probe positions. Photon counts of the acquired photons are then added to vectors of photon counts or sums of photon counts are determined for the sets of probe positions. A value representative of background noise is determined and used for adapting the estimator in real-time.

Abstract: An apparatus is provided for monitoring a focal state of a microscope having an object plane and a main imaging area. The apparatus has an auxiliary light source providing an auxiliary light beam and coupling the auxiliary light beam into the microscope in such a way that the coupled auxiliary light beam runs within a plane which is spanned outside of the main imaging area by a straight line running in the object plane and a normal to the object plane, and that the coupled auxiliary light beam is inclined at an angle to a normal to the object plane. A part of the coupled auxiliary light beam reflected by a reference boundary surface in the microscope impinges on a registration device in an area of incidence. The registration device registers position changes of the area of incidence on the registration device.

Abstract: A fluorescence microscope (10) includes a sample illumination beam path including a source (9) for illumination light, a first wave front modulator (24) for providing the focused illumination light (8) with a central intensity minimum, a beam splitter (26) and a second adjustable wave front modulator (34) arranged in a pupil plane (30) of an objective (20). A first detection beam path section including the second wave front modulator (34) and a telescope (11) and ending at the beam splitter (26) coincides with the sample illumination beam path. A separate second detection beam path section includes a detector (38) for luminescence light from a sample. The telescope (11) images a first pupil (31) formed in the pupil plane (30) in a smaller second pupil (32), and transfers a beam of the illumination light (8) collimated in the second pupil (32) into an expanded beam collimated in the first pupil (31).

Abstract: For checking the confocality of a scanning and descanning microscope assembly comprising a light source providing illumination light focused into a focal area in a focal plane, a detector detecting light coming out of the focal area and having a detection aperture to be arranged in a confocal fashion with respect to the focal area, and a scanner, an auxiliary detection aperture of an auxiliary detector arranged in the focal plane is scanned with the focal area of the illumination light to record a first comparison intensity distribution of the illumination light registered by the auxiliary detector, and the detection aperture of the detector is scanned with auxiliary light that exits out of an auxiliary emission aperture of an auxiliary light source concentrically arranged with respect to the auxiliary detection aperture in the focal plane to record a second comparison intensity distribution of the auxiliary light registered by the detector.

Abstract: An apparatus for selectively shaping phase fronts of a first light beam that is incident along an optical axis and that has a first linear input polarization direction running orthogonal to the optical axis comprises birefringent optical material arranged in all or all bar one of at least three different partial areas that follow to one another in a direction around the optical axis. The optical material is arranged such that a phase of the first light beam is delayed differently in the different partial areas to an extent that increases from partial area to partial area over a round around the optical axis, whereas a phase of a second light beam that is incident along the optical axis and that has a second linear input polarization direction orthogonal to the first linear input polarization direction and to the optical axis is not delayed differently in the different partial areas.

Abstract: For setting a laser-scanning fluorescence microscope to a correct alignment in which an intensity maximum of excitation light and an intensity minimum of fluorescence inhibition light coincide in a focal area of an objective lens, a structure in a sample marked with a fluorescent dye is scanned with the intensity maximum of the excitation light to generate first and second pictures of the sample, the first picture corresponding to a higher and the second picture corresponding to a lower intensity of the fluorescence inhibition light. A spatial offset of a first image of the structure in the first picture with regard to a second image of the structure in the second picture is calculated; and the intensity maximum of the excitation light is shifted with regard to the intensity minimum of the fluorescence inhibition light in the direction of the offset calculated to set the microscope to the correct alignment.

Abstract: In order to generate rasterized images of a sample, a pixel size of image points of a rasterized image is set and photons emitted out of the sample which were detected, and for each of which a position of an effective local excitation of the sample for emitting the respective detected photon has been recorded are assigned to that image point of the rasterized image into which the position of the effective local excitation recorded for the respective detected photon falls. To set the pixel size of the image points to an optimized pixel size, the positions of the effective local excitation of the sample for emitting the detected photons are evaluated.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: A high resolution laser scanning microscope has beam shaping elements configured to shape a beam of fluorescence inhibiting light which is directed into a back aperture of an objective connected to form an intensity minimum delimited by intensity maxima of the fluorescence inhibiting light in a focus of the objective. A plurality of optical elements including the objective and the beam shaping elements are arranged in a beam path of the beam to the focus. Using the microscope includes removing or exchanging or altering or adding at least one of the optical elements arranged in the beam path of the beam of fluorescence inhibiting light, and compensating a variation of polarization varying properties of the plurality of the optical elements, that is caused by removing or exchanging or altering or adding the at least one optical element, by adapting the beam shaping elements to the variation.

Abstract: A scanner head for high-resolution scanning fluorescence microscopy comprises a first connector to connect the scanner head to a light microscope, second connectors to connect the scanner head to a light source and a fluorescence light detector, a beam shaper to shape a first part of the light from the light source into a first light intensity distribution in the focus of the light microscope comprising an intensity minimum surrounded by intensity maxima and a second part of the light into a second light intensity distribution in the focus of the light microscope comprising an intensity maximum at the location of the intensity minimum of the first light intensity distribution four tilting mirrors configured scan a sample with the light beam, and a deflector to deflect the fluorescence light to the second optical waveguide port.

Abstract: A device comprises two polarization-selective optical elements for separately modulating wave fronts of two components of a collimated light beam, which are transversally polarized in orthogonal directions. The two polarization-selective optical elements are first and second partial areas of one spatial light modulator (SLM) diffracting the light beam in backward direction. A mirror arranged between the first and second partial areas of the SLM reflects the light beam coming from the first partial area towards the second partial area. A wave plate arranged between the first partial area and the second partial area of the SLM rotates the polarization directions of both components of the light beam by 90°. The mirror reflects the first and second components of the light beam as parallel bundles of light rays resulting in a lateral offset between the first and second components of the light beam behind the second partial area of the SLM.