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

Deutsches Krebsforschungszentrum

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Leica Microsystems Heidelberg Gmbh

Stefan W Hell

Christian Eggeling

Vladimir Belov

Volker Westphal

Christian Wurm

Francisco Balzarotti

Yvan Eilers

Klaus Gwosch

Johann Engelhardt

Mariano Bossi

Katrin Willig

Gerald Donnert

Jonas Fölling

Alexander Egner

Stefan Jakobs

Andreas Schönle

Kirill Kolmakov

Lars Kastrup

Norbert Quaas

Fabian Goettfert

Gyuzel Mitronova

Alexey Butkevich

Gael Moneron

Rainer Pick

Shamil Nizamov

Heiko Schill

Johanna Wildanger

Hilmar Gugel

Joerg Bewersdorf

Erdmann Rapp

Giuseppe Vicidomini

Pekka Hanninen

Michael Hilbert

Matthias Bischoff

Matthias Reuss

Maksim Sednev

Heydar Shojaei

Jakob Bierwagen

Lars Meyer

Vadim P Boyarskiy

Claudia Geisler

Marcel Lauterbach

Dirk Kamin

Christian Ringemann

Jan Keller-Findeisen

Brian Rankin

Andriy Chmyrov

Robert Kellner

Jaydev Jethwa

Thorsten Staudt

Marion Lang

Marcus Dyba

Vladimir N Belov

Kyu-Young Han

J&ouml;rg Bewersdorf

Jan Wichmann

Elizaveta Savicheva

Laura Thomas

Marvin J Boehm

René Hennig

Michael Weber

Dirk Meineke

Maxim Sednev

Jale Schneider

Fabian GÖTTFERT

Volker Dr Westphal

Jan Keller

Martin Andresen

Tanja Brakemann

Andre Stiel

Christian A Wurm

Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis The invention relates to fluorescent dyes with multiple negatively charged groups in their ionized form which are aminoacridone sulfonamides or 1-aminopyrenes having of one of the following general formulae A-D: Formula (A), Formula (B), Formula (C), Formula (D), wherein the ionizable groups X are typically selected from the following: SH, COOH, SOH, OSOH, OP(O)(OH), OP(O)(OH)R, P(O)(OH), P(O)(OH)R, where R═C-Calkyl or substituted C-Calkyl. The invention further relates to the use of these dyes as fluorescent tags, in particular for reducing sugars and glycans.

Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis

Disclosed are photoactivable fluorescent dye compounds of formula I:

Photoactivatable fluorescent dyes with hydrophilic caging groups and their use

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

A method of spatially measuring a plurality of nano-scale structures in a sample comprises the steps of: marking the individual structures at different locations with fluorescent markers, coupling the individual structures to individual positioning aids whose positions in the sample are known, exciting the fluorescent markers with excitation light for emission of fluorescence light, wherein an intensity distribution of the excitation light has a local minimum, arranging the local minimum at different positions in a close-up range around the position of respective positioning aid whose dimensions are not larger than the diffraction limit at the wavelength of the excitation light, registering the fluorescence light emitted out of the sample separately for the individual fluorescent markers and for the different positions of the minimum, and determining positions of the individual fluorescent markers in the sample from the intensities of the fluorescence light registered.

Method of and apparatus for spatially measuring nano-scale structures

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in n spatial dimensions in a sample, the excitation light is directed onto the sample with an intensity distribution, which has a zero point and intensity increasing regions adjoining the zero point on both sides in each of the n spatial dimensions. The zero point is arranged at not more than n&#xd7;3 different positions. The luminescence light emitted by the singularized molecule is separately registering for each of the different positions of the zero point. The position of the singularized molecule in the n spatial dimensions in the sample is deduced from intensities of the luminescence light separately registered for the not more than n&#xd7;3 different positions of the zero point.

Method of high spatial resolution determining a position of a singularized molecule which is excitable for emission of luminescence light

For high spatial resolution imaging a structure marked with luminescence markers, light that has an effect on the emission of luminescence light by the luminescence markers is directed onto a sample with an intensity distribution having a central zero point. Scan areas of the sample are scanned with the zero point. Luminescence light emitted out of a local area including the zero point is registered and assigned to the respective location of the zero point in the sample. Several copies of an object of interest are arranged in the scan areas and subjected to varying surrounding conditions. The individual scan areas are scanned with the respective zero point at least two times at two different stages of reactions to the varying surrounding conditions. Dimensions of the scan areas are limited such that they are not larger than 75% of a distance of intensity maxima delimiting the zero point.

Method of locally imaging a structure in a sample at high spatial resolution in order to detect reactions of an object of interest to altered environmental conditions

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in a sample, the excitation light is provided with an intensity distribution comprising an intensity increasing region with a known strictly monotonic course of an intensity of the luminescence light over a distance of the singularized molecule to a model point of the intensity distribution. The model point is arranged at different preliminary positions such that the intensity increasing region extends over a preliminary local area of the sample including the singularized molecule. From intensity values including intensities of the luminescence light separately registered for the preliminary positions of the model point, a further local area is determined which includes the singularized molecule and which is smaller than the preliminary local area. These steps are repeated using the last further local area as the next preliminary local area.

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in n spatial dimensions in a sample, a preliminary local area including the singularized molecule is determined The excitation light is directed onto the sample with an intensity distribution, which has a zero point and intensity increasing regions adjoining the zero point on both sides in each of the n spatial dimensions. At first, the zero point is arranged at preliminary positions on known sides of the preliminary local area. Then, present positions of the zero point are successively shifted into the preliminary local area in each of the n spatial dimensions depending on photons of the luminescence light which is quasi-simultaneously separately registered for the present positions of the zero point in that the zero point is repeatedly shifted between the present positions of the zero point.

Compounds of formula I are disclosed: wherein X, X, X, Xare independently H, F, Cl, Br, I, CN, NO, OR, SR, NRR, COR, COOR, CONRR, PORR, SOR, SORor R; Rand Rare, e.g., H, alkyl or aryl or optionally a ring; Ris, e.g., alkyl, alkenyl, alkynyl, aryl or cycloalkyl; Y is OR, NRR, or NRR; Q is O, S, SO, NR, C(R), Si(R), Ge(R), P(&#x2550;O)Ror P(&#x2550;O)OR; Q and Xcan optionally form part of a ring; L and M are independently OR, SR, NRRand R; L and M can optionally form part of a ring; Z is O, S, NR, CRRor aryl; and Z and Xcan optionally form part of a ring.

Dyes with phosphinic acid, phosphinate, phosphonate and phosphonamidate substituents as auxochromic groups and methods for preparing the same

For high spatial resolution imaging a structure in a sample, the structure being marked with luminescence markers, light that has an effect on the emission of luminescence light by the luminescence markers is directed onto the sample with an intensity distribution having a zero point and intensity maxima neighboring the zero point in at least one direction. A scan area which is a part of the sample is scanned with the zero point. Luminescence light emitted out of a local area including the zero point is registered and assigned to the respective location of the zero point in the sample. Dimensions of the scan area, in at least one direction in which the intensity maxima are neighboring the zero point, are limited such that they are not larger than 75% of a distance of the intensity maxima in the at least one direction.

Locally imaging a structure in a sample at high spatial resolution

A scanning luminescence light microscope for spatial high resolution imaging a structure marked with a luminescent marker comprises a light source for luminescence inhibition light and for further light; a light shaping and aligning device; and a detector registering luminescence light emitted by the luminescent marker. The device, by means of two optical gratings and an objective lens, forms two crossing line gratings of the luminescence inhibition light, and two crossing line gratings of the further light so that local intensity minima of an overall intensity distribution of the luminescence inhibition light are delimited in at least two directions, and that local intensity maxima or local intensity minima of an overall intensity distribution of the further light coincide with the local intensity minima of the luminescence inhibition light. Further, the device moves the overall intensity distributions of the further light and the luminescence inhibition light to scan the structure.

Scanning luminescence light microscope with gratings of luminescence inhibition light and further light

In order to determine the locations of individual fluorescent molecules in a sample, which keep a minimum distance with regard to each other, the individual molecules are excited for emission of fluorescence light by means of excitation light. The fluorescence light is registered for different positions of a zero point of an intensity distribution of the excitation light. The distance between these positions is at least half the minimum distance of the fluorescent molecules. The locations of the fluorescent molecules are derived from the course of the intensity of the fluorescence light over the positions of the zero point of the excitation light.

High-resolution fluorescence microscopy using a structured beam of excitation light

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

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)YYor P(O)YY, where Yand Yindependently denote any of the following residues: OH, O(&#x2212;), ORand OR, NHRand NHR, NRRand NRR, ORand NHR, ORand NRR, NHRand NRR; and any salt thereof.

Fluorescent dyes with phosphorylated hydroxymethyl groups and their use in light microscopy and imaging techniques

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Goettingen, Germany

Stefan W Hell