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

Deutsches Krebsforschungszentrum

Katrin Willig

Stefan W Hell

Lars Kastrup

Christian Wurm

Gerald Donnert

Vladimir Belov

Kirill Kolmakov

Shamil Nizamov

Heiko Schill

Johanna Wildanger

Johann Engelhardt

Volker Westphal

Christian Eggeling

Giuseppe Vicidomini

Matthias Reuss

Gael Moneron

Kyu-Young Han

Vladimir N Belov

Christian A Wurm

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

A method of high spatial resolution imaging a structure in a sample comprises: marking the structure with molecules of a fluorescent dye; selecting a first wavelength for excitation light which excites the molecules of the fluorescent dye via a multi photon process for spontaneous emission of fluorescent light; focusing pulses of the excitation light into the sample to excite those molecules of the fluorescent dye present in a focal area of the focused excitation light; selecting a second wavelength shorter than the first wavelength for de-excitation light which de-excites excited molecules of the fluorescent dye prior to their spontaneous emission; during a plurality of the pulses of the excitation light, continuously directing the de-excitation light onto the sample to de-excite excited molecules of the fluorescent dye, which are located outside an measurement area which is a fraction of the focal area; and recording the fluorescent light spontaneously emitted by the molecules of the fluorescent dye in the sample.

STED-fluorescent light microscopy with two-photon excitation

Growing community of inventors

Goettingen, Germany

Katrin Willig