Abstract: Methods and a device for imaging a sample stained with multiple dyes are disclosed. The methods enable nanoscopic imaging with up to molecular resolution to be placed in the spatial context of microscopic images, or nanoscopic tracking of individual molecules to be performed in the spatial context of a microscopic image. The methods combine different light-optical microscopy techniques in a particularly efficient manner. Molecular resolution can be achieved by a localization microscopic method, in particular by localization according to a MINFLUX or STED-MINFLUX method. These methods are characterized by the fact that, on the one hand, they are particularly gentle on the sample during the steps that precede imaging with molecular resolution, and on the other hand, they make optimal use of the fluorescence photons during localization or enable optimal use.

Abstract: The invention relates to a method for determining positions of mutually spaced molecules (M) in a sample (20), having the steps of generating (101) a plurality of light distributions, each light distribution having a local intensity minimum (110, 310) and adjacent regions (120, 320) of increasing intensity, comprising an excitation light distribution (100) and a deactivation light distribution (300); illuminating (102) the sample (20) with the excitation light distribution (100) and the deactivation light distribution (300); detecting (103) photons emitted by the molecule (M) for different positions of the excitation light distribution (100); and deriving (104) the position of the molecule (M) on the basis of the photons detected for the different positions of the excitation light distribution (100), wherein the local minimum (110) of the excitation light distribution (100) is arranged at a plurality of scanning positions (201) one after the other within a scanning region (200), and the light intensity of the

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: A fluorescence microscope (10) comprises 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: 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: 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 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.

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.

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: The invention relates to novel fluorescent dyes with phosphorylated hydroxymethyl groups, a method for preparing the same as well as to their use in imaging techniques. The fluorescent dyes are coumarin, rhodamine or BODIPY dyes having of one of the following general formulae I-III: wherein W=OP(O)Y1Y2 or P(O)Y1Y2, where Y1 and Y2 independently denote any of the following residues: OH, O(?), ORa and ORb, NHRa and NHRb, NRaRb and NRcRd, ORa and NHRb, ORa and NRbRc, NHRa and NRbRc; and any salt thereof.

Abstract: The invention relates to novel fluorescent dyes with phosphorylated hydroxymethyl groups, a method for preparing the same as well as to their use in imaging techniques. The fluorescent dyes are coumarin, rhodamine or BODIPY dyes having of one of the following general formulae I-III: wherein W=OP(O)Y1Y2 or P(O)Y1Y2, where Y1 and Y2 independently denote any of the following residues: OH, O(?), ORa and ORb, NHRa and NHRb, NRaRb and NRcRd, ORa and NHRb, ORa and NRbRc, NHRa and NRbRc; and any salt thereof.

Abstract: The invention relates to novel fluorescent dyes with phosphorylated hydroxymethyl groups, a method for preparing the same as well as to their use in imaging techniques. The fluorescent dyes are coumarin, rhodamine or BODIPY dyes having of one of the following general formulae I-III: wherein W=OP(O)Y1Y2 or P(O)Y1Y2, where Y1 and Y2 independently denote any of the following residues: OH, O(?), ORa and ORb, NHRa and NHRb, NRaRb and NRcRd, ORa and NHRb, ORa and NRbRc, NHRa and NRbRc; and any salt thereof.

Abstract: The invention relates to novel fluorescent dyes with phosphorylated hydroxymethyl groups, a method for preparing the same as well as to their use in imaging techniques. The fluorescent dyes are coumarin, rhodamine or BODIPY dyes having of one of the following general formulae I-III: wherein W=OP(O) Y1Y2 or P(O) Y1Y2, where Y1 and Y2 independently denote any of the following residues: OH, O(?), ORa and ORb, NHRa and NHRb, NRaRb and NRcRd, ORa and NHRb, ORa and NRbRc, NHRa and NRbRc; and any salt thereof.

Abstract: The inventive method for optically measuring a sample consists in temporarily repeatedly transmitting an electromagnetic signal (2) to the sample in such a way that a substance contained in the sample is transferred from a first electronic state (1) into a second electronic state (3), wherein at least one part of said substance in the second state (3) emits photons which are used for carrying out the optical measurement of the sample, the signal (2) is transmitted to the same sample area at a certain repetition interval and said repetition interval of the signal (2) is adjusted with a lifetime of the second state (3) of the substance having an order of magnitude of 1 ns on a value of at least 0.1 ?s which is optimized with respect to photon yield from the substance.

Abstract: The inventive method for optically measuring a sample consists in temporarily repeatedly transmitting an electromagnetic signal (2) to the sample in such a way that a substance contained in the sample is transferred from a first electronic state (1) into a second electronic state (3), wherein at least one part of said substance in the second state (3) emits photons which are used for carrying out the optical measurement of the sample, the signal (2) is transmitted to the same sample area at a certain repetition interval and said repetition interval of the signal (2) is adjusted with a lifetime of the second state (3) of the substance having an order of magnitude of 1 ns on a value of at least 0.1 ?s which is optimized with respect to photon yield from the substance.