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

Deutsches Krebsforschungszentrum

Evotec Oai Ag

Christian Eggeling

Stefan W Hell

Mariano Bossi

Jonas Fölling

Alexander Egner

Andreas Schönle

Volker Westphal

Christian Wurm

Vladimir Belov

Stefan Jakobs

Gael Moneron

Johann Engelhardt

Gerald Donnert

Rolf Guenther

Karsten Gall

Giuseppe Vicidomini

Katrin Willig

Kirill Kolmakov

Gyuzel Mitronova

Matthias Reuss

Marcel Lauterbach

Vladimir N Belov

Claus Seidel

Leif Brand

Christian Ringemann

Lars Meyer

Jakob Bierwagen

Kyu-Young Han

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 present invention relates to novel fluorinated 3,6-diaminoxanthene compounds derived from the basic structural formula (I) and to their uses as photostable fluorescent dyes, e.g. for immunostainings and spectroscopic and microscopic applications, in particular in conventional microscopy, stimulated emission depletion (STED) reversible saturable optically linear fluorescent transitions (RESOLFT) microscopy, and fluorescence correlation spectroscopy. The claimed compounds are also useful as molecular probes in various spectroscopic applications.

Fluorinated rhodamines as photostable fluorescent dyes for labelling and imaging techniques

The invention relates to novel and improved photostable rhodamine dyes of the general structural formulae I or II and their uses as fluorescent markers, e.g. for immunostainings and spectroscopic and microscopic applications, in particular in conventional and stimulated emission depletion (STED) microscopy and fluorescence correlation spectroscopy. The partially deuterated analogues are useful as molecular mass distribution tags in mass spectroscopic applications, wherein R=an unsubstituted or substituted alkyl group, including a cycloalkyl group, or heterocycloalkyl group; R=H, an unsubstituted or substituted alkyl group, including a cycloalkyl group, or heterocycloalkyl group, or an unsubstituted or substituted aryl group or heteroaryl group, or any combination of such groups; X=CH, C&#x2550;O, C&#x2550;NOR, C&#x2550;NNRNR, CH(OR), O, S, SO, SO, or any other derivatives of these groups, with Ra and Rb independently being H or an organic residue, in particular an unsubstituted or substituted (cyclo)alkyl group or heterocycloalkyl group, an unsubstituted or substituted aryl group or heteroaryl group; Z=a negatively charged group with 1, 2, 3, 4 or 5 charges per anion. 

Hydrophilic and lipophilic rhodamines for labelling and imaging

In high spatial resolution imaging, a structure in a specimen is marked with a substance which, in a first electronic state, is excited by light of one wavelength to emit fluorescent light, which is also converted from its first into a second electronic state by that light, and which returns from its second into its first electronic state. The specimen is imaged onto a sensor at a spatial resolution not resolving an average spacing between neighboring molecules of the substance, and exposed to the light at such an intensity that the molecules in the first state are alternately excited to emit fluorescent light and converted into their second state, and that at least 10% of the molecules presently in their first state lie at a distance from their closest neighboring molecules in their first state which is greater than the spatial resolution of the imaging onto the sensor.

High spatial resolution imaging of a structure of interest in a specimen

For imaging of a structure, the structure is marked with a substance which can be converted by a switching signal from a first into a second state, and which provides an optical measurement signal in one of its states, only. The switching signal is applied such that at least 10% of the molecules of the substance being in the measurement signal providing state are at a distance from their closest neighbors, which is greater than the spatial resolution limit of imaging the specimen onto a sensor array, which in turn is greater than an average distance between the molecules of the substance. From an intensity distribution of the measurement signal recorded with the sensor array, the position is only determined for those molecules of the substance which are at a distance from their closest neighboring molecules in the measurement signal providing state, which is greater than the spatial resolution limit.

For the high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have a fluorescent first state and a nonfluorescent second state; which can be converted fractionally from their first state into their second state by light which excites them into fluorescence, and which return from their second state into their first state; the specimen's structure of interest is imaged onto a sensor array, a spatial resolution limit of the imaging being greater (i.e. worse) than an average spacing between closest neighboring molecules of the substance in the specimen; the specimen is exposed to light in a region which has dimensions larger than the spatial resolution limit, fractions of the substance alternately being excited by the light to emit fluorescent light and converted into their second state, and at least 10% of the molecules of the substance that are respectively in the first state lying at a distance from their closest neighboring molecules in the first state which is greater than the spatial resolution limit; and the fluorescent light, which is spontaneously emitted by the substance from the region, is registered in a plurality of images recorded by the sensor array during continued exposure of the specimen to the light.

The inventive method for optically measuring a sample consists in temporarily repeatedly transmitting an electromagnetic signal () to the sample in such a way that a substance contained in the sample is transferred from a first electronic state () into a second electronic state (), wherein at least one part of said substance in the second state () emits photons which are used for carrying out the optical measurement of the sample, the signal () is transmitted to the same sample area at a certain repetition interval and said repetition interval of the signal () is adjusted with a lifetime of the second state () of the substance having an order of magnitude of 1 ns on a value of at least 0.1 &#x3bc;s which is optimized with respect to photon yield from the substance.

Method and device for optically measuring a sample

A device for the measurement of chemical and/or biological samples, in particular by means of luminescence spectroscopy, comprises an irradiation unit, a sample receiver, at least one optical unit and a detector unit. The color marker in the sample, which contains at least one color marker, is stimulated by irradiation into producing luminescence and gives off light. The light emitted by the color markers is detected by detectors in the detector unit. According to the invention, the measurement results may be improved by the irradiation unit generating pulsed irradiation. The irradiation unit is thus preferably controlled by a control unit in such a way that the irradiation pulses impinge on the sample in a temporal sequence.

Method and device for the measurement of chemical and/or biological samples

Growing community of inventors

Goettingen, Germany

Christian Eggeling