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.

The present disclosure is directed to a method of disturbance correction and to a laser scanning microscope carrying out this method. Specifically, it is directed to an image recording method according to the MINFLUX principle, in which a spatially isolated fluorescence dye molecule is illuminated at a sequence of scan positions by an intensity distribution with a local intensity minimum, and the number of fluorescence photons emitted by the fluorescence dye molecule is detected at each of the scan positions. The location of the molecule is determined with a high spatial resolution from the scan positions and the numbers of fluorescence photons. A disturbance is captured when illuminating the fluorescence dye molecule and detecting the fluorescence light, said disturbance being considered in corrective fashion when determining the location of the fluorescence dye molecule.

A method, an arrangement for microscopy and a microscope for three-dimensional imaging in microscopy, in which aberrations of a specimen detection radiation coming from a specimen are corrected in a detection beam path by means of a correction element and the corrected specimen detection radiation is captured in a spatially resolved form. The inventions are distinguished by the fact that a best-possible correction setting of the correction element, with which aberrations occurring at the time are reduced as much as possible, is determined; and, on the basis of the best-possible correction setting, a flawed correction setting is determined, a setting with which aberrations occurring lead to an asymmetric point spread function of the specimen detection radiation.

Apparatus and method for capturing an image having a detection beam path for guiding detection radiation from a sample to a detector having a plurality of detector elements. The detector has no more than ten and, preferably, four or five detector elements; and an evaluation unit, which is configured to carry out an evaluation in accordance with the Airyscan method on the image data captured by means of the detector and which generates a high-resolution image.

A method for measuring a position of a fluorophore includes configuring a set of compact light distributions, the set having at least one member, each light distribution characterized by a center, so that there is substantially zero intensity at the center of the set of compact light distributions. The method additionally includes moving the set of compact light distributions in relation to a set of hypothesized positions of the fluorophore, detecting, in a plurality of locations corresponding to the hypothesized set of positions, a set of images; and estimating the position of the fluorophore, by determining from the set of images a set of parameters describing the position of the fluorophore using an inverse problem method.

An immersion objective includes a correction group or correcting a spherical aberration. The displacement of the correction group along the optical axis leads to a substantially negligible defocus aberration.

A confocal microscope device for scanning a two-dimensional array of illumination beams over a target surface and scanning a corresponding two-dimensional array of emission beams stimulated by the array of illumination beams on to a sensor of an imaging device. The device comprises first scanning optics operable to scan the array of illumination beams over the target surface along a first axis and scan the array of emission beams over the sensor along the first axis. The device further comprises second scanning optics operable to deflect, on a second axis, the array of illumination beams as they are scanned over the target surface along the first axis, such that uneven stimulation of the target surface by the array of illumination beams due to interference of the illumination beams is reduced, and deflect, on the second axis, the array of emission beams as they are scanned over the sensor of the imaging device along the first axis such that uneven stimulation of the sensor by the array of emission beams due to interference of the emission beams is reduced.

A microscope includes an illumination unit for illuminating a region of a specimen to generate an illuminated region, an imaging optical unit for magnified imaging of the illuminated region, an image sensor disposed downstream of the imaging optical unit for capturing the magnified image of the illuminated region, a camera for recording an overview region of the specimen without using the imaging optical unit and a control unit for controlling the image sensor and the camera. The overview region includes a part of the illuminated region and a non-illuminated region of the specimen. The control unit actuates the camera to make a recording of the overview region. The control unit actuates the image sensor to cause a recordation of the magnified image of the illuminated region. The control unit generates an overview image based on the recording of the overview region and the recording of the magnified image.

An arrangement for increasing resolution of a laser scanning microscope has a simplified adjustment and lower susceptibility to errors. The pupil beam from the laser scanning microscope is coupled into a shortened common path interferometer, to make wavefronts of a pupil image mirrored at at least one axis and wavefronts of an unchanged pupil image interfere. The area of a pupil from the pupil beam is split into two complementary portions P and Q producing two partial beams separately supplied to at least one beam deflection means by total-internal reflection along the common path interferometer. The light of the interferometer branches from transmitted light of the one interferometer branch and reflected light of the other interferometer branch is made to interfere at a partly transmissive beam splitter layer to cause constructive interference C and destructive interference D of the wavefronts from the two different portions P and Q of the pupil.

Disclosed is a diffractive optical element includes a substrate (BS) having a first surface and a second surface opposite the first surface, being transparent to light in at least one spectral range and having, in the spectral range, a refractive index that is greater than that of water, at least one metasurface able to diffract light radiation of wavelength λ within the spectral range, incident with an angle of incidence, according to a diffracted radiation, so that the diffracted radiation propagates in the substrate and reaches the second surface of the substrate at a diffracted angle θ.sub.d that is greater than or equal to a limit angle (θ.sub.c) of total internal reflection between the substrate and water, the metasurface being designed to have, for the angle of incidence, a transmission with a 0 order of diffraction below 5% and a transmission of the diffracted radiation corresponding to a −1 or +1 order of diffraction above 50%.