Spatial resolution imaging of structure of interest in specimen involves marking specimen structure, imaging the specimen, exposing the specimen to light, registering the fluorescent light and determining position of molecules of substance

High spatial resolution imaging of a structure of interest in a specimen (102) involves selecting substance, which can be excited by light (103); marking the specimen's structure of interest with molecules of the substance; imaging the specimen onto a sensor array; exposing the specimen to the light of the one wavelength; registering the fluorescent light which is spontaneously emitted from the region of molecules; and determining the position in the specimen of the molecules of the substance, where the first and the second state are different electronic states of substance. High spatial resolution imaging of a structure of interest in a specimen (102) involves selecting a substance from a group of substances, which have a first state with first fluorescent properties and a second state with second fluorescent properties, which can be excited by light of one wavelength to spontaneously emit fluorescent light (103), which can be converted from the first state into their second state by the light of the one wavelength and which can return from their second state into their first state; marking the specimen's structure of interest with molecules of the substance; imaging the specimen 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; exposing the specimen to the light of the one wavelength in at least one region which has dimensions larger than the spatial resolution limit of the imaging of the specimen onto the sensor array, fractions of the molecules of the substance alternately being excited by the light of the one wavelength to spontaneously emit fluorescent light and being 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 ...

Verfahren und Vorrichtung zum räumlich eng begrenzten Anregen eines optischen Übergangs

Zum räumlichen eng begrenzten Anregen eines optischen Übergangs wird ein sich über einen Fokuspunkt (11) hinweg erstreckender Fokusbereich (12) eines Anregungslichtstrahls (3), dessen Wellenlänge auf den anzuregenden optischen Übergang abgestimmt ist, mit einem räumlichen, ein Intensitätsminimum an dem Fokuspunkt (11) und auf verschiedenen Seiten des Fokuspunkts (11) mehrere Intensitätsmaxima aufweisenden Interferenzmuster eines den optischen Übergang auf irgendeine Weise beeinflussenden, in Teilstrahlen (22, 23) aufgespaltenen und in dem Fokusbereich (12) in Form seiner fokussierten Teilstrahlen (22, 23) aus unterschiedlichen Richtungen mit sich selbst zur Interferenz gebrachten Abregungslichtrate (18) überlagert. Dabei werden die Wellenfronten der aus entgegengesetzten Richtungen auf den Fokuspunkt (11) fokussierten Teilstrahlen (22, 23) vor dem Fokussieren auf den Fokuspunkt (11) aberriert, so dass auf jeder der beiden Seiten des Fokuspunkts (11) die Intensitätsmaxima des Interferenzmusters ...

ADVANCED METHODS FOR AUTOMATED HIGH-PERFORMANCE IDENTIFICATION OF CARBOHYDRATES AND CARBOHYDRATE MIXTURE COMPOSITION PATTERNS AND SYSTEMS THEREFORE AS WELL AS METHODS FOR CALIBRAT ION OF MULTI WAVELENGTH FLUORESCENCE DETECTION SYSTEMS THEREFORE, BASED ON NEW FLUORESCENT DYES

The present invention relates to improved (simplified/easier, more robust and more reproducible) methods for identification of carbohydrates compositions, e.g. out of complex carbohydrate mixtures, as well as the determination of carbohydrate mixture composition patterns (e.g.: of glycosylation patterns) based on advanced internal standards to determine precise and highly reproducible migration and retention time indices using novel fluorescent dyes in combination with high performance separation technologies, like capillary (gel) electrophoresis (C(G)E) or (ultra)high performance liquid chromatography (U)HPLC with a highly sensitive detection like (laser induced) fluorescence detection. In a first aspect, the present invention relates to methods for an automated determination and/or identification of carbohydrates and/ or carbohydrate mixture composition pattern profiling as well as a method for an automated carbohydrate mixture composition pattern profiling based on the use of at least ...

PROCEDURES AND DEVICE FOR SPATIALLY THE CLOSE LIMITED A SUGGESTING OF AN OPTICAL TRANSITION

PROCEDURE FOR THE MICROSCOPIC INVESTIGATION OF A SPATIAL FINE STRUCTURE

Sample`s interesting structure spatial high-resolution imaging method, involves adjusting intensity of signal such that ten percentage of molecules of substance have specific distance to adjacent molecules

The method involves marking an interesting structure of a sample (2) with a substance. Portions of the substance with a switching signal (7) are transferred into a specific condition. An intensity of the switching signal is adjusted in such a manner that 10 percentage of molecules of the substance have a specific distance to the adjacent molecules during transferring the portion of the substance, where the distance is larger than spatial resolution limit of imaging of the sample. An independent claim is also included for a fluorescence light-optical microscope.

SPATIALLY HIGH-DISSOLVED PRODUCING A PERMANENT STRUCTURE

NOVEL HYDROPHILIC AND LIPOPHILIC RHODAMINES FOR LABELLING AND IMAGING

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═O, C═NOR, C═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. 2. The compound according to claim 1 , wherein Ris —[CH]—COY with Y being F claim 1 , Cl or Br claim 1 , and n being an integer from 1 to 21 claim 1 , or —[CH]—COORwith Rbeing H or an organic residue claim 1 , n being an integer from 1 to 21 claim 1 , and Ris H or an unsubstituted or substituted (cyclo)alkyl group or heterocycloalkyl group claim 1 , including Rbeing —[CH]—COY or —[CH]—COORas defined above for R.5. The compound according to claim 4 , wherein Ris —[CH]—COORwith Rbeing H or an organic residue claim 4 , or Ris a —[CH]—Rgroup with Rbeing an N-linked maleimido group or a —S—S—Rgroup with Rbeing an organic residue claim 4 , n is an integer from 1 to 21 claim 4 , and Ris H or an unsubstituted or substituted (cyclo)alkyl group or heterocycloalkyl group claim 4 , or any combination of such groups claim 4 , ...

Probe structure reproducing method, involves directing optical signal with polarization as another polarization of another optical signal to discriminate fluorescent light from color molecule in probe

The method involves stimulating a fluorescence color in a fluorescently stimulated condition. The fluorescence color is selected with an optical signal (12) with polarization (14) so that a spontaneously emitting fluorescence light (9) comes from a color molecule (4) of the fluorescence color. The optical signal (12) with the polarization (14) is directed as a polarization (11) of an optical signal (5) to discriminate the fluorescent light from the color molecule in a probe (3). An independent claim is also included for a fluorescence light microscope for reproducing a structure of a probe, which is marked with a fluorescent eye.

RÄUMLICH HOCHAUFGELÖSTES ERZEUGEN EINER DAUERHAFTEN STRUKTUR

Verfahren und Vorrichtung zur Mehrphotonenanregung einer Probe

Verfahren zur Mehrphotonenanregung einer Probe, wobei ein Laserstrahl in mindestens zwei kohärente Teilstrahlen gleicher Intensitätsverteilung um die jeweilige Strahlachse aufgespalten wird und wobei die Teilstrahlen aus verschiedenen Richtungen auf eine gemeinsame, quer zu den Strahlachsen verlaufende Meßebene ausgerichtet werden, so daß die Teilstrahlen in dem Bereich der Meßebene miteinander interferieren, dadurch gekennzeichnet, daß die Teilstrahlen (11, 12) zueinander unter einem Kippwinkel (14) < 1 ausgerichtet werden und daß die Teilstrahlen (11, 12) durch ein gemeinsames Linsensystem (21) auf die Meßebene ausgerichtet werden, so daß eine durch die Interferenz der Teilstrahlen (11, 12) hervorgerufene Intensitätsverteilung in der Meßebene Bereiche maximaler Intensität (31, 33) neben Bereichen minimaler Intensität aufweist.

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

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 ...

Verfahren und Vorrichtung zum optischen Messen einer Probe

Zum optischen Messen einer Probe wird ein elektromagnetisches Signal (2) wiederholt vorübergehend auf die Probe gerichtet, um eine Substanz in der Probe von einem ersten Zustand (1) in einen zweiten elektronischen Zustand (3) zu überführen, wobei zumindest ein Teil der Substanz aus dem ersten Zustand (1) oder dem zweiten Zustand (3) heraus Photonen aussendet, die zum optischen Messen der Probe verwendet werden. Das Signal (2) wird dabei mit einem zeitlichen Wiederholungsabstand auf dieselben Bereiche der Probe gerichtet. Dieser Wiederholungsabstand und eine damit einhergehende Änderung bei einer Ausbeute von Photonen von der Substanz werden beobachtet, um den Wiederholungsabstand in Bezug auf die Ausbeute an Photonen zu optimieren.

Energieoptimierter Lager- und Fermentationsbehälter für Energieerzeugungs- und Energiespeicheranlagen sowie Verfahren zur Optimierung der Wärmenutzung in einem solchen Behälter

Die vorliegende Erfindung betrifft einen energieoptimierten Lager- und Fermentationsbehälter für Energieerzeugungs- und Energiespeicheranlagen sowie ein Verfahren zur Optimierung der Wärmenutzung in dem Fermentationsbehälter. Der Behälter umfasst einen in einer Kammer (5) angeordneten Gasauslass (17) zum Überleiten des bei der Fermentation oder der Nachgärung in einer Kammer (5) entstehenden Biogases in den Gasspeicherraum (2), wobei nach dem Gasauslass (17) einer Kammer (5) wenigstens eine Wärmetauschereinrichtung (12) für das aus der Kammer (5) über den Gasauslass (17) ausgeleitete Biogas angeordnet ist.

Three dimensional memory arrangement uses fluorescent protein crystals with optically controlled state changes

The three dimensional memory [2] is formed by a solid state body that has a pattern of green fluorescent protein, GFP, [4] crystalline elements that represent data bits. A '1' state is provided by a fluorescent condition and a '0' by a non fluorescent state. The states of the protein bits is controlled by an optical system [1] and output by a reader [7].

Verfahren zur Herstellung räumlicher Feinstrukturen

Verfahren zur Herstellung räumlicher Feinstrukturen mit den Schritten: – Zugeben eines Luminophors zu einem strahlungsempfindlichen Material; – Auftragen einer Schicht aus dem Material auf ein Substrat; – Bestrahlen der aufgetragenen Schicht in definierten räumlichen Bereichen; – Entwickeln der bestrahlten Schicht, wobei Teile der Schicht entfernt werden und die gewünschte Feinstruktur zurückbleibt, und – mikroskopisches Überprüfen, ob die gewünschte Feinstruktur vorliegt, wobei von dem Luminophor emittiertes Lumineszenzlicht gemessen wird, dadurch gekennzeichnet, dass der Luminophor (2) zwei Zustände aufweist, die sich in Bezug auf ihre Lumineszenzeigenschaften unterscheiden, wobei der Luminophor (2) reversibel, aber vollständig durch ein optisches Signal von dem einen in den anderen Zustand überführbar ist, dass der Luminophor (2) beim mikroskopischen Überprüfen, ob die gewünschte Feinstruktur vorliegt, jeweils außerhalb räumlich begrenzter Bereiche durch einen bis zur Sättigung getriebenen Übergang in einen inaktiven Zustand überführt wird, in dem er kein Lumineszenzlicht (13) emittiert, und dass... Method for producing spatial fine structures with the steps: Adding a luminophore to a radiation-sensitive material; - applying a layer of the material to a substrate; - irradiation of the applied layer in defined spatial areas; Developing the irradiated layer, wherein parts of the layer are removed and the desired fine structure remains, and Microscopically checking whether the desired fine structure is present, whereby luminescent light emitted by the luminophore is measured, characterized in that the luminophore (2) has two states which differ with regard to their luminescence properties, wherein the luminophore (2) is reversibly but completely convertible from one to the other state by an optical signal such that the luminophore ( 2) when microscopically checking whether the desired fine structure is present, is transferred outside of spatially limited areas by a ...

Advanced methods for automated high-performance identification of carbohydrates and carbohydrate mixture composition patterns and systems therefore as well as methods for calibration of multi wavelength fluorescence detection systems therefore, based on new fluorescent dyes

The present invention relates to improved (simplified/easier, more robust and more reproducible) methods for identification of carbohydrates compositions, e.g. out of complex carbohydrate mixtures, as well as the determination of carbohydrate mixture composition patterns (e.g.: of glycosylation patterns) based on advanced internal standards to determine precise and highly reproducible migration and retention time indices using novel fluorescent dyes in combination with high performance separation technologies, like capillary (gel) electrophoresis (C(G)E) or (ultra)high performance liquid chromatography (U)HPLC with a highly sensitive detection like (laser induced) fluorescence detection. In a first aspect, the present invention relates to methods for an automated determination and/or identification of carbohydrates and/ or carbohydrate mixture composition pattern profiling as well as a method for an automated carbohydrate mixture composition pattern profiling based on the use of at least ...

Scanning microscope in which a sample is simultaneously and optically excited at various points

An optical apparatus, especially a scanning microscope (1), wherein an expanded laser beam (2) is divided into several partial beams (4) by micro lenses (5) arranged next to one another. Each partial beam (4) is focused onto a focal point (11) by a common objective lens (7) to optically excite a sample (8). Fluorescent light emanating from the individual focal points (11) of the sample (8) is registered by a photo sensor (13) arranged behind the objective lens (7) as seen from the sample (8). Each photon of the fluorescent light coming from the sample (8) and being registered by the photo sensor (13) is excited by at least two photons of the laser beam (2).

Fluoreszenzmikroskop

Bei einem Fluoreszenzmikroskop (12) mit einer Anregungslichtquelle (14) für Anregungslicht (5), um einen Fluoreszenzfarbstoff in einer Probe (19) während eines begrenzten Zeitraums in einem räumlichen Bereich zur spontanen Emission von Fluoreszenzlicht (6) mit Wellenlängen in einem Wellenlängenbereich anzuregen, und mit einer gepulsten Abregungslichtquelle (15) für Abregungslicht (7), um den Fluoreszenzfarbstoff bis auf einen gegenüber dem räumlichen Bereich verkleinerten Restbereich wieder abzuregen, wobei Licht von der Probe (19) mit anderen Wellenlängen als denjenigen des Anregungslichts (5) und des Abregungslichts (7) der spontanen Emission von Fluoreszenzlicht (6) aus dem Restbereich des räumlichen Bereichs zuordbar ist, ist die Abregungslichtquelle (15) ein modengekoppelter Gaslaser (13) mit einer Linienbreite des Abregungslichts (7) von weniger als 2 nm.

Method and apparatus for storing a three-dimensional arrangement of data bits in a solid-state body

A method which serves for writing a three-dimensional arrangement of data bits to a solid-state body comprises the steps of selecting a protein having fluorescence properties that can be altered by means of an optical write signal; providing the solid-state body made from the protein, the protein being present in the solid-state body in crystalline form; setting a spatial distribution which corresponds to the three-dimensional arrangement of data bits of the fluorescence properties of the protein of the solid-state body by means of the optical write signal.

RASTERMIKROSKOP, BEI DEM EINE PROBE IN MEHREREN PROBENPUNKTEN GLEICHZEITIG OPTISCH ANGEREGT WIRD

VERFAHREN UND VORRICHTUNG ZUM RÄUMLICH ENG BEGRENZTEN ANREGEN EINES OPTISCHEN ÜBERGANGS

Verfahren zur Herstellung räumlicher Feinstrukturen

Bei der Herstellung räumlicher Feinstrukturen mit den Schritten Ausbilden einer räumlichen Feinstruktur aus einem Material (3) und mikroskopisch Überprüfen, ob die gewünschte Feinstruktur vorliegt, wird dem Material (3) vor dem Ausbilden der Feinstruktur ein Luminophor (2) zugegeben, und zum mikroskopischen Überprüfen, ob die gewünschte Feinstruktur vorliegt, wird von dem Luminophor emittiertes Lumineszenzlicht gemessen (10).

VERFAHREN ZUR MIKROSKOPISCHEN UNTERSUCHUNG EINER RÄUMLICHEN FEINSTRUKTUR

Method for irradiating sample with light in stimulated emission depletion-fluorescent light microscope, involves synchronizing polarizations of input light beams to beam splitter such that partial beams interferes in common focus point

The method involves using a polarization beam splitter (24) as a beam splitter, and adjusting polarizations of two pairs of partial beams between the polarizing beam splitter and lenses (28, 29) such that the partial beams interfere in a region of a common focal plane (30). Polarizations of input light beams (13, 14) that are different from each other are synchronized to the polarization beam splitter such that one pair of partial beams interferes in a common focus point (22) with a phase that is offset relative to another phase by pi. An independent claim is also included for a fluorescent light microscope comprising a light unit for irradiating a sample with light.

Verfahren zur mikroskopischen Untersuchung einer räumlichen Feinstruktur

Zur mikroskopischen Untersuchung einer räumlichen Feinstruktur unter Belegen einer Oberfläche der räumlichen Feinstruktur mit einer Hilfssubstanz wird als Hilfssubstanz ein Luminophor ausgewählt, der zwei Zustände aufweist, die sich in Bezug auf ihre Lumineszenzeigenschaften unterscheiden, wobei der Luminophor reversibel, aber im Wesentlichen vollständig durch ein optisches Signal von dem einen in den anderen Zustand überführbar ist, und zum Bestimmen des Verlaufs der mit dem Luminophor belegten Oberfläche der räumlichen Feinstruktur wird von dem Luminophor emittiertes Lumineszenzlich gemessen, wobei der Luminophof in der Umgebung jedes aktuelle betrachteten Messpunkts in den anderen seiner beiden Zustände überführt wird.

Method and Device for Optically Measuring a Sample

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 mus which is optimized with respect to photon yield from the substance.

Rastermikroskop, bei dem eine Probe in mehreren Probenpunkten gleichzeitig optisch angeregt wird

Verfahren und Vorrichtung zum räumlich hochaufgelösten Abbilden einer mit einer Substanz markierten Struktur

Zum räumlich hochaufgelösten Abbilden einer Struktur in einer Probe (2) wird eine Substanz aus einer Gruppe von Substanzen ausgewählt, die mit einem ersten elektromagnetischen Signal (5) aus einem ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt für ein zweites elektromagnetisches Signal (3) aufweisen, in einen zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal (3) aufweisen, überführbar sind oder die mit einem ersten elektromagnetischen Signal (5) in einen ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt weisen, aus einem zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal (3) aufweisen, überführbar sind. Mit dieser Substanz wird die Struktur in der Probe (2) markiert. Mittels des ersten elektromagnetischen Signals (5) wird dann eine räumliche Verteilung eines Anteils der Substanz in dem ersten Zustand eingestellt, bei der mindestens ein Bereich, in dem die Substanz in dem ersten Zustand vorliegt, räumlich begrenzt ist; und nach der Einstellung der räumlichen Verteilung des Anteils der Substanz in dem ersten Zustand mittels des ersten elektromagnetischen Signals (5) wird die Probe (2) mit dem zweiten elektromagnetischen Signal (3) beaufschlagt. Anschließend wird mindestens eine aus dem größeren Absorptionsquerschnitt der Substanz in dem ersten Zustand beim Beaufschlagen mit dem ... For spatially high-resolution imaging of a structure in a sample (2), a substance is selected from a group of substances which are connected to a first electromagnetic signal (5) from a first state in which they have a larger absorption cross-section for a second electromagnetic signal (3). have in a second state in which they have a smaller absorption cross section for the second electromagnetic signal (3) can be converted or with a first electromagnetic signal (5) in a first state in which they have a larger absorption cross-section of a ...

Method of microscopically examining a spatial finestructure

A method of microscopically examining a spatial fine structure comprises the steps of selecting a luminophore from the group of luminophores which have two physical states, the two states differing from each other with regard to the luminescence properties displayed by the luminophore, and which are reversibly, but essentially completely transferable out of one into the other state of their two states by means of an optical signal; overlaying a surface of the spatial fine structure with the luminophore; and determining the profile of the surface overlaid with the luminophore. The step of determining the profile of the surface comprises the sub-steps of transferring the luminophore by means of the optical signal out of the one into the other of its two states outside a presently observed measurement point, measuring luminescence light emitted by the luminophore, and repeating the sub-steps of transferring and measuring for further measurement points distributed over the surface.

Creating a high-resolution structure with a substance that can be altered with an optical signal comprises applying the signal so that an intensity minimum excludes a restricted area, e.g. for writing to data storage media

Creation of a permanent structure with high spatial resolution comprises providing a substance that can be altered with an optical signal in a writing zone and applying the optical signal so that a local intensity minimum excludes a spatially restricted area of the writing zone from being altered while alteration of the rest of the writing zone reaches saturation. An independent claim is also included for a device with a permanently structurable writing zone comprising a substance that can be altered with an optical signal, where the substance can be repeatedly converted from a state A to a state B, each with different optical properties, and converted back from state B to state A, but only in state A can the substance be permanently changed to another state C with a writing signal.

Verfahren und Vorrichtungen zum Speichern einer dreidimensionalen Anordnung von Datenbits in einem Festkörper

Confocal microscope comprising two microlens arrays and a pinhole diaphragm array

A confocal microscope comprises a microlens array having a plurality of microlenses for splitting a ray bundle of illumination light into a plurality of convergent partial ray bundles which illuminate a sample simultaneously at several measuring points; a beam splitter for separating a beam path of the illuminating light and a beam path of sample light originating from the illumination of the sample and captured in an inverse direction with regard to the illumination light; a pinhole diaphragm array having a plurality of pinhole diaphragms arranged in the beam path of the sample light and corresponding to said microlenses of said microlens array splitting the illumination light; and a further microlens array having a plurality of microlenses corresponding to said microlenses of said microlens array splitting the illumination light. Said microlenses of said microlens array splitting the illumination light and said microlenses of said further microlens array are arranged in the beam path ...

Confocal microscope comprising two microlens arrays and a pinhole diaphragm array

A confocal microscope comprises a microlens array having a plurality of microlenses for splitting a ray bundle of illumination light into a plurality of convergent partial ray bundles which illuminate a sample simultaneously at several measuring points; a beam splitter for separating a beam path of the illuminating light and a beam path of sample light originating from the illumination of the sample and captured in an inverse direction with regard to the illumination light; a pinhole diaphragm array having a plurality of pinhole diaphragms arranged in the beam path of the sample light and corresponding to said microlenses of said microlens array splitting the illumination light; and a further microlens array having a plurality of microlenses corresponding to said microlenses of said microlens array splitting the illumination light. Said microlenses of said microlens array splitting the illumination light and said microlenses of said further microlens array are arranged in the beam path ...

Light sensitive compound, particularly photoresist, has photo initiation system and matrix, and photo initiation system has initiator and is transversed reversably into inhibition condition by application with optical in inhibition signal

The light sensitive compound has a photo initiation system and a matrix. The photo initiation system has an initiator and is transversed reversably into an inhibition condition (PIS) by application with an optical in inhibition signal (hv1). The inhibition signal is differentiated from the reading and writing signal (hv3) and the application of photo initiation system does not guided to an activation of initiation condition with the reading and writing signal.

Verfahren und Vorrichtung zum räumlich eng begrenzten Anregen eines optischen Übergangs

Verfahren zum räumlich eng begrenzten Anregen eines optischen Übergangs, wobei ein sich über einen Fokuspunkt hinweg erstreckender Fokusbereich eines Anregungslichtstrahls, dessen Wellenlänge auf den anzuregenden optischen Übergang abgestimmt ist, mit einem räumlichen, ein Intensitätsminimum an dem Fokuspunkt und mehrere Intensitätsmaxima auf verschiedenen Seiten des Fokuspunkts aufweisenden Interferenzmuster eines den optischen Übergang auf irgendeine Weise beeinflussenden, in Teilstrahlen aufgespaltenen und in dem Fokusbereich in Form seiner fokussierten Teilstrahlen aus unterschiedlichen Richtungen mit sich selbst zur Interferenz gebrachten Abregungslichtstrahls überlagert wird, dadurch gekennzeichnet, dass die Wellenfronten (28) der aus unterschiedlichen Richtungen auf den Fokuspunkt (11) fokussierten Teilstrahlen (22, 23) vor dem Fokussieren auf den Fokuspunkt (11) aberriert werden, so dass auf jeder der Seiten des Fokuspunkts (11) die Intensitätsmaxima (31, 32) des Interferenzmusters (30) räumlich aufgeweitet werden, ohne das Intensitätsminimum am Fokuspunkt (11) zu beseitigen.

Fluoreszenzmikroskop

Fluoreszenzmikroskop mit einer Anregungslichtquelle für Anregungslicht, um einen Fluoreszenzfarbstoff in einer Probe während eines begrenzten Zeitraums in einem räumlichen Bereich zur spontanen Emission von Fluoreszenzlicht mit Wellenlängen in einem Wellenlängenbereich anzuregen, und mit einer gepulsten Abregungslichtquelle für Abregungslicht, um den Fluoreszenzfarbstoff bis auf einen gegenüber dem räumlichen Bereich verkleinerten Restbereich wieder abzuregen, wobei Licht von der Probe mit anderen Wellenlängen als denjenigen des Anregungslichts und des Abregungslichts der spontanen Emission von Fluoreszenzlicht aus dem Restbereich des räumlichen Bereichs zuordbar ist, dadurch gekennzeichnet, dass die Abregungslichtquelle (15) ein modengekoppelter Gaslaser (13) mit einer Linienbreite des Abregungslichts (7) von weniger als 2 nm ist. fluorescence microscope with an excitation light source for Excitation light to a fluorescent dye in a sample during a limited period in a spatial Sphere for spontaneous emission of fluorescent light with wavelengths in a wavelength range and with a pulsed depletion light source for de-excitation light, to the fluorescent dye down to one compared to the spatial area reduced Restrain remaining area, taking light from the sample with others wavelength as those of the excitation light and the depletion light of the spontaneous Emission of fluorescent light from the residual area of the spatial Area is assignable, characterized in that the de-excitation light source (15) a mode-locked gas laser (13) having a line width of the depletion light (7) is less than 2 nm.

Method and device for multi photon excitation of a sample

In a method for multi photon excitation of a sample a laser beam is split into at least two coherent partial beams each having a beam axis and a same intensity distribution about its beam axis. The partial beams are directed from different directions towards a common measuring plane running transversely to the beam axes at an inclination angle <1 between the beam axes of the partial beams; and the partial beams are projected onto the measuring plane by means of a common lens system. Thus, an interference pattern formed by the coherent partial beams within the measuring plane provides areas of maximum light intensity adjacent to areas of minimum light intensity.

Räumlich hochauflösendes Abbilden

Bei einem Verfahren zum räumlich hochauflösenden Abbilden einer mit einer Substanz markierten Struktur einer Probe, mit den Schritten: Auswählen der Substanz aus einer Gruppe von Substanzen, die mit einem optischen Umschaltsignal (3) wiederholt aus einem ersten Zustand (1) mit ersten optischen Eigenschaften in einen zweiten Zustand (2) mit zweiten optischen Eigenschaften überführbar sind und die aus dem zweiten Zustand (2) in den ersten Zustand (1) zurückkehren können, Überführen der Substanz in Bereichen der Probe (7) mit dem Umschaltsignal (3) in den zweiten Zustand (2), wobei ein definierter Bereich gezielt ausgelassen wird, und Registrieren eines optischen Messsignals (5), das der Substanz in dem ersten Zustand (1) zuzuordnen ist, für einen Registrierbereich, der neben Bereichen, in denen die Substanz in den zweiten Zustand überführt ist, den gezielt ausgelassenen Bereich umfasst, wird die Substanz aus einer Untergruppe von Substanzen ausgewählt, bei denen sich die beiden Zustände (1, 2) mindestens hinsichtlich eines der folgenden Kriterien unterscheiden: Konformationszustand eines Moleküls, Strukturformel eines Moleküls, räumliche Anordnung von Atomen innerhalb eines Moleküls, räumliche Anordnung von Bindungen innerhalb eines Moleküls, Anlagerung weiterer Atome oder Moleküle an ein Molekül, Gruppierung von Atomen und/oder Molekülen, räumliche Orientierung eines Moleküls, Orientierung benachbarter Moleküle zueinander und von einer Vielzahl von Molekülen und/oder ... In a method for spatially high-resolution imaging of a structure of a sample marked with a substance, with the steps: selecting the substance from a group of substances which is repeated with an optical switching signal (3) from a first state (1) with first optical properties a second state (2) with second optical properties can be transferred and which can return from the second state (2) to the first state (1), transferring the substance in areas of the sample (7) with the switching signal (3) into ...

METHOD OF FLUORESCENCE-MICROSCOPICALLY IMAGING A STRUCTURE IN A SAMPLE WITH HIGH THREE-DIMENSIONAL SPATIAL RESOLUTION

For imaging a structure in a sample with three-dimensional spatial resolution, a fluorophore is selected which is transferable by means of an optical transfer signal out of a first into a second photochromic state having specific fluorescence properties, and which displays a return rate back into the first photochromic state. The structure is labeled with the fluorophore. Via a common objective, the sample with the labeled structure is subjected both to the optical transfer signal in a spatially limited transfer-volume, and to an optical excitation signal exciting a portion of the fluorophore being in its second photochromic state for fluorescence in a spatially limited excitation-volume, the transfer-volume and the excitation-volume having a common centre of maximum intensity of the transfer signal and of the excitation signal, and a decrease of intensity of the transfer signal with the distance to the common centre of maximum intensity being substantially stronger than any decrease of ...

Multiple photon excitation of a sample for optical microscopy, has excitation laser beam that is split into two partial beams of equal intensity distribution and directed towards sample from different directions so that they interfere

Method in which partial beams are directed onto a measurement plane that is perpendicular to the beam axes from different directions so that the two beams interfere with each other. The two beams (11, 12) are incident on each other with an inclination angle of less than 1 degree and then are directed via a lens system (21) on to the sample (24) where an interference pattern is generated with adjacent areas of high and low intensity. The invention also relates to a corresponding device for implementation of the inventive method.

Konfokales Mikroskop mit zwei Mikrolinsenarrays und einem Lochblendenarray

Method of producing spatial fine structures

A method of producing spatial fine structures comprises the steps of: selecting a luminophore from the group of luminophores displaying two different states, one of the two states being an active state in which luminescence light is obtainable from the luminophore, the other of the two states being an inactive state in which no luminescence light is obtainable from the luminophore, and the luminophore being reversibly, but essentially completely, transferable out the one state into the other state by means of an optical signal; adding the luminophore to a material; forming a spatial fine structure of the material; and fluorescence-microscopically examining whether the desired fine structure is present. The step of fluorescence-microscopically examining comprises the sub-steps of: outside measuring points of interest, transferring the luminophore into the other state with the optical signal, the luminophore being essentially completely transferred into the inactive state outside the measuring ...

Method and apparatus for spatially limited excitation of an optical transition

A method of exciting an optical transition in a narrowly limited area of a material comprising the steps of focusing an excitation light beam whose wavelength is tuned to the optical transition to be excited into a focal area extending beyond a focal point; splitting up a de-excitation light beam which is at least somehow influencing the optical transition into at least two partial beams; focusing the at least two partial beams of the de-excitation light beam out of different directions onto the focal point to form a spatially extending interference pattern in the focal area; adjusting a relative phase of the at least two partial beams of the de-excitation light beam so that the interference pattern has an intensity minimum at the focal point and a plurality of intensity maxima on different sides of the focal point; and aberrating the wave fronts of the at least two partial beams of the de-excitation light beam so that the intensity maxima of the interference pattern on different sides ...

High spatial resolution imaging

In a method of high spatial resolution imaging a structure of a sample, the structure is marked with a substance. The substance is selected from a group of substances which are capable of being repeatedly transferred out of a first state having first optical properties into a second state having second optical properties by means of an optical switch over signal, and which are capable of returning out of the second state into the first state, the two states differing with regard to at least one criteria. Within areas of the sample the sample is transferred into the second state by means of the optical switch over signal with which at least one spatially limited area of the sample is purposefully omitted. An optical measurement signal is detected, which is associated with the substance in the first state and which comes out of a detection area including both the area purposefully omitted with the switch over signal and areas in which the substance has been transferred into to second state ...

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.

METHOD OF PRODUCING SPATIAL FINE STRUCTURES

A method of producing spatial fine structures comprises the steps of: selecting a luminophore from the group of luminophores displaying two different states, one of the two states being an active state in which luminescence light is obtainable from the luminophore, the other of the two states being an inactive state in which no luminescence light is obtainable from the luminophore, and the luminophore being reversibly, but essentially completely, transferable out the one state into the other state by means of an optical signal; adding the luminophore to a material; forming a spatial fine structure of the material; and fluorescence-microscopically examining whether the desired fine structure is present. The step of fluorescence-microscopically examining comprises the sub-steps of: outside measuring points of interest, transferring the luminophore into the other state with the optical signal, the luminophore being essentially completely transferred into the inactive state outside the measuring ...

Hydrophilic and lipophilic rhodamines for labelling and imaging

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 R1=an unsubstituted or substituted alkyl group, including a cycloalkyl group, or heterocycloalkyl group; R2=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=CH2, C=O, C=NORa, C=NNRaNRb, CH(ORa), O, S, SO, SO2, 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 ...

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.

Method and device for optically measuring a sample

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.

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

For high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have two different electronic states: 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 ...

High spatial resolution imaging

In high spatial resolution imaging, a structure of a sample is marked with a substance selected from a group of substances capable of being repeatedly transferred out of a first state having first optical properties into a second state having second optical properties by means of an optical switch over signal, and capable of returning out of the second state into the first state, the two states differing in at least one criteria. Within areas the sample is transferred into the second state by means of the optical switch over signal with which at least one spatially limited area is purposefully omitted. An optical measurement signal is detected, which is associated with the substance in the first state and which comes out of a detection area including both the area omitted with the switch over signal and areas in which the substance has been transferred into to second state.

Method of fluorescence-microscopically imaging a structure in a sample with high three-dimensional spatial resolution

For imaging a structure in a sample with spatial resolution, the structure is labeled with a fluorophore which is transferable by an optical transfer signal out of a first into a second photochromic state. Via a common objective, the sample is subjected to both the focussed optical transfer signal and a focussed optical excitation signal only exciting a portion of the fluorophore being in its second photochromic state for fluorescence. The transfer and the excitation signal have a common centre of maximum intensity; and a decrease of intensity of the transfer signal with the distance to this common centre is substantially stronger than any decrease of the effective return rate of the fluorophore back into the first photochromic state. Fluorescence light emitted by the excited fluorophore is detected. Then, the common centre is shifted with regard to the sample; and the steps of subjecting and detecting are repeated.

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

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.

METHOD OF MICROSCOPICALLY EXAMINING A SPATIAL FINESTRUCTURE

A method of microscopically examining a spatial fine structure comprises the steps of selecting a luminophore from the group of luminophores which have two physical states, the two states differing from each other with regard to the luminescence properties displayed by the luminophore, and which are reversibly, but essentially completely transferable out of one into the other state of their two states by means of an optical signal; overlaying a surface of the spatial fine structure with the luminophore; and determining the profile of the surface overlaid with the luminophore. The step of determining the profile of the surface comprises the sub-steps of transferring the luminophore by means of the optical signal out of the one into the other of its two states outside a presently observed measurement point, measuring luminescence light emitted by the luminophore, and repeating the sub-steps of transferring and measuring for further measurement points distributed over the surface.

ADVANCED METHODS FOR AUTOMATED HIGH-PERFORMANCE IDENTIFICATION OF CARBOHYDRATES AND CARBOHYDRATE MIXTURE COMPOSITION PATTERNS AND SYSTEMS THEREFORE AS WELL AS METHODS FOR CALIBRATION OF MULTI WAVELENGTH FLUORESCENCE DETECTION SYSTEMS THEREFORE, BASED ON NEW FLUORESCENT DYES

High spatial resoulution imaging and modification of structures

In a method of high spatial resolution imaging or modifying a structure, the structure is marked with a substance which is selected from the group of substances which can be transferred from a first state having first optical properties to a second state having second optical properties by means of an optical switch over signal. Then, the second state of the substance is adjusted with the switch over signal except for a spatially limited area. If the substance and the switch over signal are adapted to each other in such a way, that everywhere where the switch over signal exceeds a threshold value essentially the second state of the substance is adjusted, and if the spatial area purposefully omitted by the switch over signal is an intensity minimum of an interference pattern, the spatial area of the structure in which the substance is within the first state becomes smaller than the diffraction limit for the switch over signal.

Creating a permanent structure with high spatial resolution

In a method of creating a permanent structure with high spatial resolution, a substance which may be modified by an optical signal is provided in a writing area. The optical signal is applied to the writing area in such a way that a spatially limited partial area of the writing area is purposefully omitted, the spatially limited partial area being a local intensity minimum of the optical signal, and the optical signal, outside of the spatially limited partial area, being applied to the writing area in such a way that saturation is achieved in modifying the substance with the optical signal. Then, different states of the substance in the spatially limited partial area and of the substance in the partial areas of the writing area covered by the optical signal are permanently adjusted.

Method and apparatus for the high spatial resolution imaging of a structure marked with a substance

For the high spatial resolution imaging of a structure in a sample ( 2 ) the structure is marked with a substance which can be changed over by means of a first electromagnetic signal ( 5 ) from a first state having a larger absorption cross section for a second electromagnetic signal ( 3 ) into a second state having a smaller absorption cross section for the second signal ( 3 ) or which can be changed over by means of a first electromagnetic signal ( 5 ) into a first state having a larger absorption cross section for a second electromagnetic signal ( 3 ) from a second state having a smaller absorption cross section for the second signal ( 3 ). A spatially delimited distribution of a portion of the substance in the first state is then set by means of the first signal ( 5 ). Afterward, the second electromagnetic signal ( 3 ) is applied to the sample ( 2 ), and a local temperature increase in the sample ( 2 ) which results from the larger absorption cross section of the substance in the first state is detected.

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

Compounds of formula I are disclosed: wherein X1, X2, X3, X4are independently H, F, Cl, Br, I, CN, NO2, OR1, SR1, NR1R2, COR1, COOR1, CONR1R2, PO3R1R2, SO2R1, SO3R1or R3; R1and R2are, e.g., H, alkyl or aryl or optionally a ring; R3is, e.g., alkyl, alkenyl, alkynyl, aryl or cycloalkyl; Y is OR1, NR1R2, or NR1R3; Q is O, S, SO2, NR, C(R3)2, Si(R3)2, Ge(R3)2, P(═O)R3or P(═O)OR3; Q and X1can optionally form part of a ring; L and M are independently OR1, SR1, NR1R2and R3; L and M can optionally form part of a ring; Z is O, S, NR1, CR1R3or aryl; and Z and X4can optionally form part of a ring.

Method and apparatus for the high spatial resolution imaging of a structure marked with a substance

For the high spatial resolution imaging of a structure in a sample (2) the structure is marked with a substance which can be changed over by means of a first electromagnetic signal (5) from a first state having a larger absorption cross section for a second electromagnetic signal (3) into a second state having a smaller absorption cross section for the second signal (3) or which can be changed over by means of a first electromagnetic signal (5) into a first state having a larger absorption cross section for a second electromagnetic signal (3) from a second state having a smaller absorption cross section for the second signal (3). A spatially delimited distribution of a portion of the substance in the first state is then set by means of the first signal (5). Afterward, the second electromagnetic signal (3) is applied to the sample (2), and a local temperature increase in the sample (2) which results from the larger absorption cross section of the substance in the first state is detected.

Creating a permanet structure with high spatial resolution

In a method of creating a permanent structure with high spatial resolution, a substance which may be modified by an optical signal is provided in a writing area. The optical signal is applied to the writing area in such a way that a spatially limited partial area of the writing area is purposefully omitted, the spatially limited partial area being a local intensity minimum of the optical signal, and the optical signal, outside of the spatially limited partial area, being applied to the writing area in such a way that saturation is achieved in modifying the substance with the optical signal. Then, different states of the substance in the spatially limited partial area and of the substance in the partial areas of the writing area covered by the optical signal are permanently adjusted.

NOVEL FLUORINATED RHODAMINES AS PHOTOSTABLE FLUORESCENT DYES FOR LABELLING AND IMAGING TECHNIQUES

The present invention relates to novel fluorinated -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. 4. A method for preparing 4 ,5-disulfono-3 ,6-bis[N ,N′-(2 ,2 ,2-trifluoroethyl)amino]xanthenes Ib ,e or 4-sulfono-6-amino-3-[N′-(2 ,2 ,2-trifluoroethyl)amino]xanthene derivatives Ig ,j by direct sulfonation of a corresponding unsulfonated N[ ,N′-bis](2 ,2 ,2-trifluoroethyl)-substituted 3 ,6-diaminoxanthene derivative at positions 4 and 5 in the course of a one-step procedure by reacting with a sulfonating agent.6. The method according to claim 5 , wherein the reaction is conducted at a temperature in a range from −5 to +20° C.7. A method of using compounds according to or any of their stable conjugates with biomolecules or any other chemical substances as fluorescent dyes.8. The method according to claim 7 , wherein the compound is used in spectroscopy or microscopy.9. A method of using hydrophilic compounds of in a free form or attached to antibodies or other biomolecules for microinjections into cells and for immunostainings.10. A method of using compounds according to or any of their conjugates with biomolecules or with any other chemical substances as molecular probes.11. The method according to wherein absorption and/or emission spectra and/or fluorescence quantum yields or lifetimes of the molecular probes are changed in response to a change in a polarity of a medium or microenvironment.12. The method according to claim 11 , wherein the change of the absorption and/or emission spectra of the molecular probes involves a ...

Three-Dimensionally Localizing Light Emitting Molecules of Unknown Orientation and Unknown Z-Position

To the end of three-dimensionally localizing light emitting marker entities of unknown orientation and unknown position in a sample, the light emitted by each single marker entity is imaged in at least two different ways onto at least one detection plane which corresponds to a focal plane () in the sample resulting in at least two images of the marker entity. Virtual x- and y-positions of the marker entity in parallel to the focal plane () are separately determined from the emitted light intensity distribution over each image of the marker entity. Further, the z-position of the marker entity normal to the focal plane is determined from the emitted light intensity distributions over the images of the marker entity. The real x- and y-positions of the marker entity in parallel to the focal plane () are determined based on its virtual x- and y-positions and on its z-position. 1. A method of three-dimensionally localizing light emitting marker entities of unknown orientation and unknown position in a sample , the method comprising:imaging light emitted by each single marker entity in at least two different ways onto at least one detection plane which corresponds to a focal plane in the sample resulting in at least two images of the marker entity;separately determining virtual x- and y-positions in parallel to the focal plane of each marker entity from the emitted light intensity distribution over each image of the marker entity;determining a z-position of each marker entity normal to the focal plane from the emitted light intensity distributions over the images of the marker entity; anddetermining real x- and y-positions of each marker entity in parallel to the focal plane based on its virtual x- and y-positions and on its z-position.2. The method of claim 1 , wherein the real x- and y-positions of the marker entity in parallel to the focal plane are extrapolated for its z-position normal to the focal plane from its virtual x- and y-positions.3. The method of claim 2 , ...

Determining the Distribution of a Substance by Scanning with a Measuring Front

For determining the distribution of a substance, a measuring front is formed of a first and a second optical signal. Intensities of the first and second optical signals, over a depth of the measuring front which is smaller than the diffraction limit at the wavelengths of the first and second optical signals, increase so steeply that a portion of the substance in a measurement state in which a measurement signal is available from the substance increases from essentially zero due to transferring the substance by means of the first optical signal into the measurement state, and decreases to essentially zero again due to transferring the substance by means of the second optical signal back out of the measurement state. The measuring front is moved over a measurement region. The measurement signal is recorded for different positions of the measuring front in the measurement region and assigned to these positions.

STED Microscopy With Pulsed Excitation, Continuous Stimulation, And Gated Registration Of Spontaneously Emitted Fluorescence 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 (). 2. The method of claim 1 , wherein claim 1 , in the step of registering claim 1 , the fluorescence light emitted by the excited fluorescent entities is registered at a temporal resolution which is higher than a life time of the excited state of the fluorescent entities decaying by spontaneous emission of fluorescence light only.3. The method of claim 2 , wherein claim 2 , in the step of registering claim 2 , the fluorescence light emitted by the excited fluorescent entities is registered at a temporal resolution which is at least as high as a life time of the excited state of the fluorescent entities decaying both by spontaneous emission of fluorescence light and by de-excitation due to the de-excitation light at its maximum intensity applied.4. The method of claim 2 , wherein claim 2 , in the step of registering claim 2 , the fluorescence light emitted by the excited fluorescent entities is registered at a temporal resolution which is at least 200 ps.5. The method of claim 4 , wherein claim 4 , in the step of registering claim 4 , the fluorescence light emitted by the excited fluorescent entities is registered at a temporal resolution which is at least 100 ps.6. The method of claim 1 , wherein claim 1 , in the step of registering claim 1 , at least one time gate is set for ...

Method and Apparatus for Tracking a Particle, Particularly a Single Molecule, in a Sample

For the purpose of tracking a movement of a particle in a sample, the particle is driven by light to emit photons, and the photons emitted by the particle are detected. The light applied to the sample features a light intensity distribution with a spatially limited minimum. The particle is tracked with the minimum of the light intensity distribution by moving the light intensity distribution with respect to the sample such that a rate of photons emitted by the particle remains minimal, and by taking an actual position of the minimum of the light intensity distribution in the sample as an actual position of the particle in the sample.

Method and Apparatus for Imaging a Structure Marked with a Fluorescent Dye

In a method for imaging a structure () marked with a fluorescent dye in a sample, the sample is repeatedly scanned in a scanning range () with a light intensity distribution localised around a focal point () of a focused fluorescence excitation light beam. The light intensity distribution further comprises a focused fluorescence inhibiting light beam () whose wave fronts are modulated so that a fluorescence inhibiting light intensity distribution comprises a minimum at the focal point () of the fluorescence excitation light beam (). The scanning conditions are coordinated in such a way that the fluorescence light is emitted out of the scanning range () as individually detectable photons. When these photons are detected, the location () of the focal point () at the respective point in time is allocated to them. An image of the structure () is composed of the locations () to which the detected photons have been allocated during several repetitions of scanning the scanning range (). 1. A method of imaging a structure marked with a fluorescent dye in a sample , comprisingrepeatedly scanning the sample in a scanning range with a light intensity distribution localised around a focal point of a focused fluorescence excitation light beam of fluorescence excitation light, wherein the light intensity distribution further comprises a focused fluorescence inhibiting light beam of fluorescence inhibiting light whose wave fronts are modulated such that a fluorescence inhibiting light intensity distribution comprises a minimum at the focal point of the fluorescence excitation light beam;detecting fluorescence light emitted out of the scanning range; andallocating the detected fluorescence light to the actual location of the focal point in the sample;wherein scanning conditions which, besides a scanning speed at which the focal point is shifted with regard to the sample and light intensities of the light intensity distribution, include properties and a concentration of the ...

Advanced methods for automated high-performance identification of carbohydrates and carbohydrate mixture composition patterns and systems therefore as well as methods for calibration of multi wavelength fluorescence detection systems therefore, based on new fluorescent dyes

The present invention relates to improved (simplified/easier, more robust and more reproducible) methods for identification of carbohydrates compositions, e.g. out of complex carbohydrate mixtures, as well as the determination of carbohydrate mixture composition patterns (e.g.: of glycosylation patterns) based on advanced internal standards to determine precise and highly reproducible migration and retention time indices using novel fluorescent dyes in combination with high performance separation technologies, like capillary (gel) electrophoresis (C(G)E) or (ultra)high performance liquid chromatography (U)HPLC with a highly sensitive detection like (laser induced) fluorescence detection. In a first aspect, the present invention relates to methods for an automated determination and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling as well as a method for an automated carbohydrate mixture composition pattern profiling based on the use of at least a first and second fluorescent label for labelling the migration/retention time alignment standard and sample or different samples, respectively, whereby the at least one of that fluorescent dye is a compound as defined herein. Moreover, the present invention relates to a method for calibration of multi wavelength fluorescence detection systems as well as calibration systems or calibration standards and new compounds suitable for calibration are described. The present invention relates further to a kit or system for determining or identifying carbohydrate mixture composition patterns as well as a kit or system for determining and/or identifying carbohydrate mixture composition pattern. Further, a carbohydrate dye conjugate comprising the dye as defined herein for use in a method according to the present invention is provided. The dyes employed for forming the carbohydrate dye conjugate have formula A or B below:

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 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. 1. A method of high resolution imaging a structure in a sample , the structure being marked with luminescence markers , the method comprisingdirecting light that has an effect on the emission of luminescence light by the luminescence markers onto the sample with an intensity distribution which has a zero point and intensity maxima neighboring the zero point in at least one direction and having a distance in the at least one direction;scanning scan areas with the zero point, the scan areas being parts of the sample;while scanning the scan areas, registering luminescence light emitted out of a local area including the zero point in the sample;assigning the registered luminescence light to a respective location of the zero point in the sample; andlimiting dimensions of the scan areas, in the at least one direction in which the intensity maxima are neighboring the zero point in the sample, to not more than 75% of the distance of the intensity maxima in the at least one direction,wherein each of a plurality of copies of an object of interest is arranged such that it overlaps with one of ...

FLUORESCENT DYES WITH PHOSPHORYLATED HYDROXYMETHYL GROUPS AND THEIR USE IN LIGHT MICROSCOPY AND IMAGING TECHNIQUES

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: 3. A method for preparing a fluorescent dye according to claim 1 , comprising the following steps:{'sup': a', 'a', 'a', 'a, 'sub': '2', 'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'a) providing a precursor compound wherein W in formulae IIa-IIb is replaced by a reactive group W′ selected from the group consisting of halogen (Cl, Br, I), hydroxy (OH), ether (OR), acyloxy (OCOR), or sulfonyloxy (OSOR) residues; with Rdefined as in above;'}b) reacting said group W′ of the precursor compound with a phosphorylating agent to obtain the fluorescent dye; andc) optionally performing post-synthetic modifications of the (protected) phosphate group.4. The method of claim 3 , wherein step c) comprises deprotection of a tertiary phosphate ester with two equal groups to obtain a target compound with a primary phosphate group or claim 3 , alternatively claim 3 , saponification of the tertiary phosphate group to obtain a secondary phosphate group; and claim 3 , optionally claim 3 , amidation of the primary or secondary phosphate group in a presence of an amine and a coupling reagent to obtain a functionally substituted amide (or amido ester) of the target phosphoric acid.7. A method of using a fluorescent dye according to claim 1 , or a stable conjugate thereof with an organic substance.8. The method according to claim 7 , wherein the fluorescent dye is used in spectroscopy claim 7 , far-field optical microscopy fluorescence correlation spectroscopy claim 7 , ground state depletion with individual molecular return (GSDIM) imaging methods claim 7 , fluorescence lifetime imaging (FLIM) claim 7 , as a donor or an acceptor in fluorescent resonance energy transfer studies claim 7 , or confocal microscopy ...

Photoactivatable fluorescent dyes with hydrophilic caging groups and their use

Disclosed are photoactivable fluorescent dye compounds of formula I: wherein: n=0, 1, 2, 3; X is selected from O, CRR′, SiRR′ and GeRR′, where R and R′ represent independently alkyl, cycloalkyl, alkenyl, alkynyl or aryl; Y is H, SO 3 H or SO 3 M, with M being a positively charged counterion, in particular selected from NH 4 + and cations of organic ammonium compounds; R 1 is H, CO 2 H, C(O)NH-linker-CO 2 H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; R 2 may represent H, unsubstituted or substituted alkyl (including cycloalkyl); R 3 and R 4 may represent independently H or F; R 5 is H, Me, CO 2 H, C(O)NH-linker-CO 2 H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; wherein the ligand moiety at each occurrence represents a reactive group or tag, capable to form a covalent or non-covalent bond or molecular complex with a target chemical entity or substance. Methods of using the compounds in imaging of fixed and living cells are also disclosed.

Locally Imaging a Structure in a Sample at High Spatial Resolution

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. 1. A method of high resolution imaging a structure in a sample , the structure being marked with luminescence markers , the method comprisingdirecting light that has an effect on the emission of luminescence light by the luminescence markers onto the sample with an intensity distribution which has a zero point and intensity maxima neighboring the zero point in at least one direction;scanning a scan area with the zero point, the scan area being a part of the sample;while scanning the scan area registering luminescence light emitted out of a local area including the zero point in the sample; andassigning the registered luminescence light to a respective location of the zero point in the sample;wherein dimensions of the scan area, in the at least one direction in which the intensity maxima are neighboring the zero point in the sample, are not larger than 75% of a distance of the intensity maxima in the at least one direction.2. The method of claim 1 , wherein the dimensions of the scan area in the at least one direction in which the intensity maxima are neighboring the zero point in the sample are not larger than 25% of the distance of the intensity ...

METHOD AND SYSTEM FOR IMAGING A MOLECULAR STRAND

The present disclosure concerns a method and system for imaging a molecular strand (MS). The method comprises providing a sample volume (SV) comprising the strand (MS); providing an excitation beam (EB) having an excitation focus (EF) in the sample volume (SV); scanning the excitation focus (EF) in the sample volume (SV) along a one dimensional scanning line (SL); trapping an end of the strand (MS) in the sample volume (SV) and extending the strand (MS) along a one-dimensional trapping line (LL) parallel to the scanning line (SL); aligning the trapping line (LL) to coincide with the scanning line (SL) to have the scanning excitation focus (EF) coincide with the strand (MS); and recording the fluorescence response (FR) as a function of a plurality of distinct scanning positions (X) of the excitation focus (EF) along the scanning line (SL). 1. Method for imaging a molecular strand , the method comprisingproviding a sample volume comprising the strand;providing an excitation beam having an excitation focus in the sample volume wherein an excitation of a fluorophore on the strand by the excitation focus results in a fluorescence response when the excitation focus coincides with the fluorophore;scanning the excitation focus in the sample volume along a one dimensional scanning line;trapping an end of the strand in the sample volume and extending the strand along a one-dimensional trapping line parallel to the scanning line;aligning the trapping line to coincide with the scanning line to have the scanning excitation focus coincide with the strand; andrecording the fluorescence response as a function of a plurality of distinct scanning positions of the excitation focus along the scanning line.2. Method according to claim 1 , comprising providing a depletion beam having a depletion focus with a depletion profile coinciding with an excitation profile of the excitation focus and causing stimulated emission depletion of the excitation of the fluorophore according to the ...

FLUORESCENT DYES WITH PHOSPHORYLATED HYDROXYMETHYL GROUPS AND THEIR USE IN LIGHT MICROSCOPY AND IMAGING TECHNIQUES

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: 4. A method for preparing a fluorescent dye according to claim 1 , comprising the following steps:{'sup': a', 'a', 'a', 'a, 'sub': '2', 'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'a) providing a precursor compound wherein W in formulae Ia-Ib and IIa-IIb is replaced by a reactive group W′ selected from the group consisting of halogen (Cl, Br, I), hydroxy (OH), ether (OR), acyloxy (OCOR), or sulfonyloxy (OSOR) residues; with Rdefined as in above;'}b) reacting said group W′ of the precursor compound with a phosphorylating agent to obtain the fluorescent dye; andc) optionally performing post-synthetic modifications of the (protected) phosphate group.5. The method of claim 4 , wherein step c) comprises deprotection of a tertiary phosphate ester with two equal groups to obtain a target compound with a primary phosphate group or claim 4 , alternatively claim 4 , saponification of the tertiary phosphate group to obtain a secondary phosphate group; and claim 4 , optionally claim 4 , amidation of the primary or secondary phosphate group in a presence of an amine and a coupling reagent to obtain a functionally substituted amide (or amido ester) of the target phosphoric acid.8. A method of using a fluorescent dye according to claim 1 , or a stable conjugate thereof with an organic substance.9. The method according to claim 8 , wherein the fluorescent dye is used in spectroscopy claim 8 , far-field optical microscopy fluorescence correlation spectroscopy claim 8 , ground state depletion with individual molecular return (GSDIM) imaging methods claim 8 , fluorescence lifetime imaging (FLIM) claim 8 , as a donor or an acceptor in fluorescent resonance energy transfer studies claim 8 , or confocal ...

Method of and Apparatus for Forming and Shifting a Light Intensity Distribution in a Focal Area of an Objective Lens

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. 1. A method of forming and shifting a light intensity distribution in a focal area of an objective lens , the method comprising wherein each of the plurality of portions of coherent input light is collimated in the pupil of the objective lens, and', 'wherein the plurality of non-identical two-dimensional pupil areas include at least one pair of two two-dimensional pupil areas which are axially symmetrically arranged on opposite sides of an optical axis of the objective lens, and, 'directing a plurality of portions of coherent input light one by one into a plurality of non-identical two-dimensional pupil areas of a pupil of the objective lens,'}separately modulating at least one of the two discrete portions of coherent input light that are directed into the two two-dimensional pupil areas of the at least one pair with regard to its phase by means of an electro optical modulator such as to form the light intensity distribution in the focal area of the objective lens with a local intensity minimum delimited by intensity maxima and to shift the local intensity minimum in the focal area laterally with regard to the ...

NOVEL DYES WITH PHOSPHINIC ACID, PHOSPHINATE, PHOSPHONATE AND PHOSPHONAMIDATE SUBSTITUENTS AS AUXOCHROMIC GROUPS AND METHODS FOR PREPARING THE SAME

Compounds of formula I are disclosed: 2. The compound according to claim 1 , wherein the amine is a member selected from the group consisting of NH claim 1 , NH(alkyl) claim 1 , NH(aryl) claim 1 , N(alkyl)(aryl) and N(alkyl).3. The compound according to claim 1 , where Z and X claim 1 , taken with the atoms to which they are bonded claim 1 , form a substituted or unsubstituted 5-7 membered ring claim 1 , substituted with at least one additional heteroatom selected from the group consisting of N claim 1 , O and S and/or at least one substituent selected from the group consisting of F claim 1 , Cl claim 1 , Br claim 1 , I claim 1 , CN claim 1 , N claim 1 , B(OR)(OR) claim 1 , OR claim 1 , SR claim 1 , NRR claim 1 , COR claim 1 , COOR claim 1 , CONRR claim 1 , PORR claim 1 , SOR claim 1 , SORand R claim 1 , where R claim 1 , R claim 1 , Rare defined as in .5. The compound according to claim 1 , wherein Rof COORis N-succinimidyl claim 1 , N-phthalimidyl claim 1 , N-tetrachlorphthalimidyl claim 1 , pentachlorophenyl claim 1 , pentafluorophenyl claim 1 , 2 claim 1 ,3 claim 1 ,5 claim 1 ,6-tetrafluorophenyl claim 1 , 4-(hydroxysulfonyl)-2 claim 1 ,3 claim 1 ,5 claim 1 ,6-tetrafluorophenyl [p-(HOSO)CF] claim 1 , 1-benzotriazolyl or cyanomethyl.6. The compound according to claim 1 , wherein ORis OCOOR claim 1 , where Ris N-succinimidyl claim 1 , N-phthalimidyl claim 1 , N-tetrachlorophthalimidyl claim 1 , pentachlorophenyl claim 1 , pentafluorophenyl claim 1 , 2 claim 1 ,3 claim 1 ,5 claim 1 ,6-tetrafluorophenyl claim 1 , 4-(hydroxysulfonyl)-2 claim 1 ,3 claim 1 ,5 claim 1 ,6-tetrafluorophenyl [p-(HOSO)CF] claim 1 , 1-benzotriazolyl or cyanomethyl.9. The compound according to in a form of a salt with organic or inorganic counterion(s) claim 1 , its cocrystal with another organic or inorganic compound(s) claim 1 , or a composition containing any of the dyes of .10. A conjugate or bioconjugate comprising a compound according to coupled via at least one covalent chemical bond ...

Method of High Spatial Resolution Determining a Position of a Singularized Molecule Which is Excitable for Emission of Luminescence Light

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. 1. A method of spatial high resolution determining , in n spatial dimensions , a position of a singularized molecule in a sample , the singularized molecule being excitable with excitation light for emission of luminescence light , and n being 1 , 2 or 3 , the method comprisingproviding the excitation light with an intensity distribution which comprises a zero point and intensity increasing regions delimiting the zero point on both sides in each of the n spatial dimensions,determining a preliminary local area in the sample which includes the singularized molecule,defining at least one preliminary position of the zero point per each of the n spatial dimensions which is on a known side of the preliminary local area in the respective one of the n spatial dimensions,directing the excitation light with the intensity distribution comprising the zero point onto the sample, andseparately registering the luminescence light emitted by the singularized molecule for each of the positions of the zero point in the sample,wherein, ...

Method of High Spatial Resolution Determining a Position of a Singularized Molecule Which is Excitable for Emission of Luminescence Light

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. 1. A method of spatial high resolution determining , in n spatial dimensions , a position of a singularized molecule in a sample , the singularized molecule being excitable with excitation light for emission of luminescence light , and n being 1 , 2 or 3 , the method comprisingproviding the excitation light with an intensity distribution which, in each of the n spatial dimensions, comprises at least one intensity increasing region with a known strictly monotonic course of an intensity of the luminescence light from the singularized molecule over a distance of the singularized molecule to a model point of the intensity distribution,determining a preliminary local area in the sample which includes the singularized molecule,directing the excitation light with the intensity distribution onto the sample, andarranging the model point of the intensity distribution, in each of the n spatial directions, at different positions in the sample,separately registering the luminescence light ...

Method of High Spatial Resolution Determining a Position of a Singularized Molecule Which is Excitable for Emission of Luminescence Light

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×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×3 different positions of the zero point. 1. A method of spatial high resolution determining , in n spatial dimensions , a position of a singularized molecule in a sample , the singularized molecule being excitable with excitation light for emission of luminescence light , and n being 1 , 2 or 3 , the method comprisingdirecting the excitation light with an intensity distribution onto the sample, the intensity distribution having a zero point and intensity increasing regions which are adjoining the zero point on both sides in each of the spatial dimensions,separately registering the luminescence light emitted by the singularized molecule for each of the different positions of the zero point in the sample, anddeducing the position of the singularized molecule in the sample from intensities of the luminescence light separately registered for the different positions of the zero point,wherein the zero point is arranged at not more than n×3 different positions in the sample to deduce the position of the singularized molecule in the n spatial dimensions from the intensities of the luminescence light separately registered for the different positions of the zero point.2. The method of claim ...

Method of and Apparatus for Spatially Measuring Nano-Scale Structures

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.

HIGH-RESOLUTION FLUORESCENCE MICROSCOPY USING A STRUCTURED BEAM OF EXCITATION 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. 1. A method of determining the locations of individual molecules of a substance in a sample , wherein the individual molecules of the substance are in a fluorescent state in which they are excitable for emission of fluorescence light by means of excitation light , the method comprising:forming an intensity distribution of the excitation light comprising at least one local minimum of the intensity of the excitation light;exciting the individual molecules of the substance in the fluorescent state for the emission of the fluorescence light by means of the excitation light;registering an intensity of the fluorescence light emitted by the excited individual molecules of the substance in the fluorescent state for different positions of the at least one minimum in an area of interest of the sample; anddeducing the locations of the individual molecules of the substance in the fluorescent state from a course of the registered intensity of the fluorescence light over the positions of the at least one minimum in the area of interest of the sample;{'sub': 's', 'claim-text': λ is a wavelength of the excitation light,', 'n is the refraction index of an optical material in which the intensity distribution of the excitation light with the at least one minimum is formed,', 'α is half an aperture angle of an optical arrangement by which the ...

Method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore

For spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, the sample, in a measurement area, is subjected to an intensity distribution of luminescence inhibiting light comprising a local minimum. Then, the sample, in the measurement area, is subjected to luminescence excitation light which excites the luminophore out of an electronic ground state into a luminescent state, and luminescence light emitted out of the measurement area is registered. This registered luminescence light is assigned to the position of the local minimum within the sample. The luminescence inhibiting light disturbs the electronic ground state of the luminophore such that the luminophore, in the disturbed electronic ground state, has an absorption cross-section for the luminescence excitation light which is reduced by at least 50% as compared to the undisturbed electronic ground state.

METHOD OF SPATIAL HIGH RESOLUTION IMAGING OF A STRUCTURE OF A SAMPLE, THE STRUCTURE COMPRISING A LUMINOPHORE

For spatial high resolution imaging of a structure of a sample comprising a luminophore, the sample is subjected to excitation inhibiting light transferring the luminophore out of an excitable electronic ground state into a protection state in which the luminophore is protected against electronic excitation by luminescence excitation light and luminescence de-excitation light. The excitation inhibiting light comprises a first local minimum. Next, the sample is subjected to the luminescence excitation light exciting the luminophore within the first local minimum into an excited luminescent state. Then, the sample is subjected to the luminescence de-excitation light returning the luminophore out of the excited luminescent state into the excitable electronic ground state. The luminescence de-excitation light comprises a second local minimum overlapping with the first local minimum. Luminescence light emitted out of the measurement area is measured and assigned to the position of the second local minimum within the sample. 1. A method of high spatial resolution imaging of a structure of a sample , the structure comprising a luminophore , the method comprisingin a measurement area, subjecting the sample to an intensity distribution of excitation inhibiting light transferring the luminophore out of an excitable electronic ground state into a protection state in which the luminophore is protected against electronic excitation by luminescence excitation light and by luminescence de-excitation light, the intensity distribution of the excitation inhibiting light comprising a first local minimum;in the measurement area, subjecting the sample to the luminescence excitation light exciting the luminophore which, within the first local minimum of the intensity distribution of the excitation inhibiting light, is still in its excitable electronic ground state into an excited luminescent state;in the measurement area, subjecting the sample to an intensity distribution of the ...

Scanning Luminescence Light Microscope with Gratings of Luminescence Inhibition Light and Further Light

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. 1. A scanning luminescence light microscope for spatial high resolution imaging a structure in a sample , the structure comprising a luminescent marker , the microscope comprisinga light source configured to provide luminescence inhibition light and further light differing from the luminescence inhibition light; wherein at least one beam of the further light enters the light shaping and aligning device together with one of the two beams of the luminescence inhibition light so that the light shaping and aligning device, from the at least one beam of the further light, by means of one of the optical gratings, forms two coherent partial of the further light and focuses and superimposes the partial beams of the further light by means of the objective so that the partial beams of the further light form a line grating of the further light in the area of the sample which comprises a plurality of intensity maxima delimited in one direction and intensity minima extending in parallel to the local ...

CELL-PENETRATING FLUORESCENT DYES WITH SECONDARY ALCOHOL FUNCTIONALITIES

The invention relates to novel cell-penetrating fluorescent dyes with secondary alcohol functionalities having one of the following general formulae I-III and 4: The invention also relates to the use of these compounds for optical microscopy and imaging techniques. 120-. (canceled)23. A fluorescent dye according to claim 22 , wherein the term “a functional group as defined below” on each occurrence refers to a hydroxy group.24. A compound of the structures IIIa and IIIb according to which contains two hydroxyl groups and wherein either R=R═OH and R=R═H claim 21 , or R=R═H and R=R═OH.25. A compound of the structures IIIa and IIIb according to which contains one hydroxyl group and wherein either R=R═OH and R=R═H claim 22 , or R=R═H and R=R═OH.26. A compound of the structures IIIa and IIIb according to which contains one hydroxyl group and wherein either R═OH and R=R=R═H claim 21 , or R═OH and R=R=R═H.27. A compound of the structures IIIa and IIIb according to which contains one hydroxyl group and wherein either R═OH and R=R=R═H claim 22 , or R═OH and R=R=R═H.32. A fluorescent dye of structures 4a and 4b according to where alkyl is a straight or branched C-Calkyl chain; R is COH or a reactive ester claim 21 , or a functional group capable of participating in a ligation reaction selected from the group consisting of an azide claim 21 , alkyne claim 21 , strained alkene and tetrazine reactive group claim 21 , which functional group is connected with the phenyl group through a linker claim 21 , or R is any ligand forming a covalent bond with a biological target of interest claim 21 , or a noncovalent ligand binding to a biological target of interest selected from the group consisting of a docetaxel and jasplakinolide derivative.33. A fluorescent dye of structures 4a and 4b according to where alkyl is a straight or branched C-Calkyl chain; R is COH or a reactive ester claim 22 , or a functional group capable of participating in a ligation reaction selected from the group ...

Method of and Apparatus for Spatially Measuring Nano-Scale Structures

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.

Verfahren zum optischen Messen eines Probenpunkts einer Probe und Vorrichtung zur Durchführung des Verfahrens

Verfahren und fluoreszenzlichtmikroskop zum räumlich hochauflösenden abbilden einer struktur einer probe

Zum räumlich hochauflösenden Abbilden einer interessierenden Struktur einer Probe werden die Schritte durchgeführt: Auswählen einer Substanz aus einer Gruppe von Substanzen, die mit einem Umschaltsignal wiederholt aus einem ersten Zustand mit ersten optischen Eigenschaften in einen zweiten Zustand mit zweiten optischen Eigenschaften überführbar sind und die aus dem zweiten Zustand in den ersten Zustand zurückkehren können; Markieren der interessierenden Struktur der Probe mit der Substanz; Überführen wechselnder Anteile der Substanz mit dem Umschaltsignal in den zweiten Zustand; Abbilden der Probe auf ein Sensorarray, wobei eine räumliche Auflösungsgrenze der Abbildung größer (also schlechter) ist als ein mittlerer Abstand zwischen nächst benachbarten Molekülen der Substanz in der Probe, und räumlich aufgelöstes Registrieren eines optischen Messsignals, das von dem jeweiligen Anteil der Substanz in dem zweiten Zustand ausgeht, mit dem Sensorarray. Beim Überführen des Anteils der Substanz in den zweiten Zustand wird eine Intensität des Umschaltsignals so eingestellt, dass mindestens 10 % der jeweils in den zweiten Zustand überführten Moleküle einen Abstand zu den ihnen nächst benachbarten Molekülen in dem zweiten Zustand aufweisen, der größer als die räumliche Auflösungsgrenze der Abbildung der Probe auf das Sensorarray ist.

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

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.

Process and device for optically measuring a point on a sample with high local resolution

The invention relates to a device for the optical measurement of a point (7) on a sample (8) with high local resolution, with a light source (1) to emit a beam (16) suitable for exciting an energy state in the sample (8), and object lens (6) to focus the exciting beam (16) on the point (7) on the sample (8) arranged in the focal field of the object lens (6), a separating device (5) to separate the emitted light spontaneously radiated owing to the excitation of the energy state and a detector (9) to detect the emitted light, and a corresponding process. The lateral resolution of the device and the process is improved in that there is a stimulation light beam (17) from the exciting light source (1) to generate stimulated emission at the point (7) on the sample (8) excited by the light beam (16), in which the exciting light beam (16) and the stimulation light beam (17) are arranged in such a way that their intensity distributions partly overlap in the focal region.

Process and device for optically measuring a point on a sample with high local resolution

The invention relates to a device for the optical measurement of a point (7) on a sample (8) with high-local resolution, with a light source (1) to emit a beam (16) suitable for exciting an energy state in the sample (8), and a detector (9) to detect the emitted light. The lateral resolution of the device is improved in that there is a stimulation light beam (17) from the exciting light source (1) to generate stimulated emission at the point (7) on the sample (8) excited by the light beam (16), in which the exciting light beam (16) and the stimulation light beam (17) are arranged in such a way that their intensity distributions partly overlap in the focal region.

Process and device for optically measuring a point on a sample with high local resolution

Method for the optical excitation of an energy state of a sample in a sample point and device for carrying out the method

STED fluorescence microscopy with two-photon excitation

Verfahren zum räumlich hochaufgelösten Abbilden einer mit einem Fluoreszenzfarbstoff markierten Struktur in einer Probe, wobei gepulstes Anregungslicht in die Probe fokussiert wird, um den in einem Fokusbereich befindlichen Fluoreszenzfarbstoff zur spontanen Emission von Fluoreszenzlicht anzuregen, wobei die Wellenlänge des Anregungslichts (5) so ausgewählt wird, dass das Anregungslicht (5) den Fluoreszenzfarbstoff über einen Mehrphotonenprozess anregt, wobei Abregungslicht, das eine andere Wellenlänge als das Anregungslicht aufweist, auf die Probe gerichtet wird, um angeregten Fluoreszenzfarbstoff außerhalb eines gegenüber dem Fokusbereich verkleinerten Messbereichs vor seiner spontanen Emission abzuregen, und wobei von dem Fluoreszenzfarbstoff spontan emittiertes Fluoreszenzlicht registriert wird, dadurch gekennzeichnet, dass das Abregungslicht (10), das eine kürzere Wellenlänge als das Anregungslicht (5) aufweist, über eine Vielzahl von Pulsen (19) des Anregungslichts (10) hinweg kontinuierlich auf die Probe (2) gerichtet wird. Method for spatially high-resolution imaging of a structure marked with a fluorescent dye in a sample, wherein pulsed excitation light is focused into the sample in order to excite the fluorescent dye located in a focal area to spontaneously emit fluorescent light, the wavelength of the excitation light (5) being selected so that the excitation light (5) excites the fluorescent dye via a multiphoton process, with the de-excitation light, which has a different wavelength than the excitation light, being directed onto the sample in order to excite excited fluorescent dye outside a measurement area smaller than the focal area before its spontaneous emission, and from the fluorescent dye spontaneously emitted fluorescent light is registered, characterized in that the de-excitation light (10), which has a shorter wavelength than the excitation light (5), over a plurality of pulses (19) of the excitation light (10) ko n is continuously directed ...

Process for the optical excitation of a sample

Method for imaging fluorescent dye with labeled structure in sample, involves emitting fluorescent light in form of detectable photons, and assigning image of structure from location to photons over repetitive scanning of scanning area

The method involves scanning sample (21) in scanning area repeatedly. The fluorescent light (22) emitted from scanning region is detected and the respective location is assigned to focal point. The scanning conditions are shifted relative to sample. The light intensities of light intensity distribution characteristics and concentration of fluorescent dye in scanning area are matched, so that fluorescent light in the form of detectable photons is emitted from scanning area, and image of structure assembled from location is assigned to photons over repetitive scanning of scanning area. An independent claim is included for device for imaging fluorescent dye with labeled structure in sample.

High resolution spatial imaging

High three-dimensional resolution representation

Disclosed is a method for the high three-dimensional resolution representation of a sample structure marked with a substance. Said method comprises the following steps: the substance is selected among a group of substances which can be repeatedly transformed from a first state (1) having first optical characteristics into a second state (2) having second optical characteristics by means of an optical switching signal (3) and can return from the second state (2) into the first state (1); the substance is transformed into the second state (2) in certain areas of the sample (7) by means of the switching signal (3), a defined area being specifically omitted; and an optical test signal (5) that is to be associated with the substance in the first state (1) is registered for a registration range comprising the specifically omitted area in addition to areas in which the substance has been transformed into the second state. The substance used in the inventive method is selected among a subgroup of substances in which the two states (1, 2) differ at least regarding one of the following criteria: conformation state of a molecule; structural formula of a molecule; spatial arrangement of atoms within a molecule; spatial arrangement of bonds within a molecule; attachment of additional atoms or molecules to a molecule; grouping of atoms and/or molecules; spatial orientation of a molecule; orientation of adjacent molecules relative to each other; and order embodied by a plurality of molecules and/or atoms.

Photosensitive composition, in particular photoresist

Lichtempfindliche Zusammensetzung mit einem Photoinitiationssystem und einer Matrix, wobei das Photoinitiationssystem einen Initiator aufweist, der durch Beaufschlagung des Photoinitiationssystems mit einem Schreibsignal lokal aktivierbar ist und der dort, wo er aktiviert ist, die Matrix chemisch verändert, wobei das Photoinitiationssystem durch Beaufschlagung mit einem optischen Inhibierungssignal, das sich in seinen physikalischen Eigenschaften von dem Schreibsignal unterscheidet, reversibel in einen inhibierten Zustand überführbar ist, in dem eine Beaufschlagung des Photoinitiationssystems mit dem Schreibsignal nicht zu einer Aktivierung des Initiators führt, wobei ein Grad der Inhibierung des Photoinitiationssystems von der lokalen Intensität des Inhibierungssignals abhängt und wobei das Photoinitiationssystem mit dem Inhibierungssignaktivierbaren Zustands in seinen inhibierten Zustand überführbar ist. Photosensitive composition with a photoinitiation system and a matrix, wherein the photoinitiation system has an initiator which can be activated locally by applying a write signal to the photoinitiation system and which, where it is activated, chemically changes the matrix, the photoinitiation system being activated by an optical inhibition signal , which differs in its physical properties from the write signal, can be reversibly converted into an inhibited state in which exposure of the photoinitiation system with the write signal does not lead to activation of the initiator, a degree of inhibition of the photoinitiation system depending on the local intensity of the inhibition signal depends and wherein the photoinitiation system with the inhibition sign activatable state can be converted into its inhibited state.

Determining the distribution of a substance by scanning with a measuring front

Zum Bestimmen der Verteilung einer Substanz in einem Messgebiet, wobei die Substanz mittels eines optischen Signals (1) (a) aus einem Zustand, in dem kein Messsignal (3) von ihr erhältlich ist, in einen Messzustand überführbar ist, in dem ein Messsignal (3) von ihr erhältlich ist, und mittels desselben optischen Signals (1) oder eines anderen optischen Signals (5) (b) aus dem Messzustand, in dem das Messsignal (3) von ihr erhältlich ist, in den einen oder einen weiteren Zustand überführbar ist, in dem kein Messsignal von ihr erhältlich ist, wird in dem Messgebiet eine Messfront (4) aus dem optischen Signal (1) und/oder aus dem weiteren optischen Signal (5) ausgebildet, wobei die Intensität des optischen Signals (1) und/oder des weiteren optischen Signals (5) über eine Tiefe der Messfront (4), die kleiner als die Beugungsgrenze bei der Wellenlänge des optischen Signals (1) und/oder des weiteren optischen Signals (5) ist, derart ansteigt, das der Anteil der Substanz (10) in dem Messzustand durch Überführen der Substanz (a) aus dem einen Zustand in den Messzustand von nicht vorhanden anwächst und durch Überführen der Substanz (b) aus dem Messzustand in den einen oder den weiteren Zustand wieder auf nicht vorhanden anwächst. Die Messfront (4) wird. in Gegenrichtung zu dem Anstieg der Intensität des optischen Signals (1) und/oder des weiteren optischen Signals (5) über das Messgebiet verschoben. Das Messsignals (3) wird zumindest aus dem Bereich der Messfront (4) erfasst und zu zugehörigen Positionen der Messfront (4) in dem Messgebiet zugeordnet. To determine the distribution of a substance in a measurement area, whereby the substance can be converted from a state in which no measurement signal (3) can be obtained from it into a measurement state in which a measurement signal ( 3) can be obtained from it, and by means of the same optical signal (1) or another optical signal (5) (b) from the measurement state in which the measurement signal (3) is available from it, into ...

Method and device for determining the locations of individual molecules of a substance in a sample

Zur Bestimmung des Ortes (xM) einzelner Moleküle einer Substanz in einer Probe, wobei sich die einzelnen Moleküle der Substanz in einem fluoreszenten Zustand befinden, in dem sie mit Anregungslicht zur Emission von Fluoreszenzlicht anregbar sind und wobei Abstände der einzelnen Moleküle der Substanz in einem interessierenden Bereich der Probe einen Mindestwert d = λ/(2nsinα √(1 + I/IS)) einhalten, werden die einzelnen Moleküle der Substanz mit dem Anregungslicht zur Emission von Fluoreszenzlicht angeregt, wobei eine Intensitätsverteilung des Anregungslichts mindestens eine Nullstelle aufweist. Das Fluoreszenzlicht von den angeregten einzelnen Molekülen der Substanz wird für verschiedene Positionen (xN) der mindestens einen Nullstelle der Intensitätsverteilung des Anregungslichts in dem interessierenden Bereich der Probe registriert. Dabei sind Abstände zwischen nächstbenachbarten Positionen (xN) der mindestens einen Nullstelle der Intensitätsverteilung des Anregungslichts, in denen das Fluoreszenzlicht von den angeregten einzelnen Molekülen der Substanz registriert wird, nicht größer als der halbe Mindestwert d. Dann werden die Orte (xM) der einzelnen Moleküle der Substanz aus dem Verlauf der Intensität (I) des Fluoreszenzlichts von dem jeweiligen Molekül über den Positionen (xN) der mindestens einen Nullstelle der Intensitätsverteilung des Anregungslichts in dem interessierenden Bereich der Probe abgeleitet. For determining the location (xM) of individual molecules of a substance in a sample, wherein the individual molecules of the substance are in a fluorescent state in which they are excitable with excitation light for emission of fluorescent light and wherein distances of the individual molecules of the substance in an interesting Area of the sample a minimum value d = λ / (2nsinα √ (1 + I / IS)), the individual molecules of the substance are excited with the excitation light for emission of fluorescent light, wherein an intensity distribution of the excitation ...

Method and device for spatially high-resolution imaging of a structure marked with a substance

1. Verfahren zum räumlich hochaufgelösten Abbilden einer Struktur in einer Probe, – wobei eine Substanz aus einer Gruppe von Substanzen ausgewählt wird, – die mit einem ersten elektromagnetischen Signal aus einem ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt für ein zweites elektromagnetisches Signal aufweisen, in einen zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal aufweisen, überführbar sind oder – die mit einem ersten elektromagnetischen Signal in einen ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt für ein zweites elektromagnetisches Signal aufweisen, aus einem zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal aufweisen, überführbar sind, – wobei die Struktur mit der Substanz markiert wird, – wobei mittels des ersten elektromagnetischen Signals eine räumliche Verteilung eines Anteils der Substanz in dem ersten Zustand eingestellt wird, bei der mindestens ein Bereich, in dem die Substanz in dem ersten Zustand vorliegt, räumlich begrenzt ist, und – wobei die Probe nach der Einstellung der räm ersten Zustand mittels des ersten elektromagnetischen Signals mit dem zweiten elektromagnetischen Signal beaufschlagt wird, dadurch gekennzeichnet, dass mindestens eine aus dem größeren Absorptionsquerschnitt der Substanz in dem ersten Zustand beim Beaufschlagen mit dem zweiten elektromagnetischen Signal (3) resultierende lokale Temperaturerhöhung der Probe (2) detektiert wird. 1. A method for spatially high-resolution imaging of a structure in a sample, - whereby a substance is selected from a group of substances, - which have a first electromagnetic signal from a first state in which they have a larger absorption cross-section for a second electromagnetic signal, into a second state in which they have a smaller absorption cross section for the second electromagnetic signal, or - those with a first electromagnetic signal ...

Method and device for spatially high-resolution imaging of a structure marked with a substance

Verfahren zum räumlich hochaufgelösten Abbilden einer Struktur in einer Probe, – wobei eine Substanz aus einer Gruppe von Substanzen ausgewählt wird, – die mit einem ersten elektromagnetischen Signal aus einem ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt für ein zweites elektromagnetisches Signal aufweisen, in einen zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal aufweisen, überführbar sind oder – die mit einem ersten elektromagnetischen Signal in einen ersten Zustand, in dem sie einen größeren Absorptionsquerschnitt für ein zweites elektromagnetisches Signal aufweisen, aus einem zweiten Zustand, in dem sie einen kleineren Absorptionsquerschnitt für das zweite elektromagnetische Signal aufweisen, überführbar sind, – wobei die Struktur mit der Substanz markiert wird, – wobei mittels des ersten elektromagnetischen Signals eine räumliche Verteilung eines Anteils der Substanz in dem ersten Zustand eingestellt wird, bei der mindestens ein Bereich, in dem die Substanz in dem ersten Zustand vorliegt, räumlich begrenzt ist, und – wobei die Probe nach der Einstellung der räumlichen Verteilung des Anteils der Substanz in dem ersten Zustand mittels des ersten elektromagnetischen Signals mit dem zweiten elektromagnetischen Signal beaufschlagt wird ...

High three-dimensional resolution representation

Disclosed is a method for the high three-dimensional resolution representation of a sample structure marked with a substance. Said method comprises the following steps: the substance is selected among a group of substances which can be repeatedly transformed from a first state (1) having first optical characteristics into a second state (2) having second optical characteristics by means of an optical switching signal (3) and can return from the second state (2) into the first state (1); the substance is transformed into the second state (2) in certain areas of the sample (7) by means of the switching signal (3), a defined area being specifically omitted; and an optical test signal (5) that is to be associated with the substance in the first state (1) is registered for a registration range comprising the specifically omitted area in addition to areas in which the substance has been transformed into the second state. The substance used in the inventive method is selected among a subgroup of substances in which the two states (1, 2) differ at least regarding one of the following criteria: conformation state of a molecule; structural formula of a molecule; spatial arrangement of atoms within a molecule; spatial arrangement of bonds within a molecule; attachment of additional atoms or molecules to a molecule; grouping of atoms and/or molecules; spatial orientation of a molecule; orientation of adjacent molecules relative to each other; and order embodied by a plurality of molecules and/or atoms.

Method for high-resolution local imaging of a structure in a sample

Verfahren zum hochaufgelösten Abbilden einer mit Lumineszenzmarkern markierten Struktur in einer Probe (8),- wobei Licht, das sich auf die Emission von Lumineszenzlicht durch die Lumineszenzmarker auswirkt, mit einer Intensitätsverteilung auf die Probe (8) gerichtet wird, die eine von Intensitätsmaxima (3) benachbarte Nullstelle (4) aufweist,- wobei ein abzutastender Teilbereich (7) der Probe (8) mit der Nullstelle (4) abgetastet wird und- wobei aus dem Bereich der Nullstelle (4) emittiertes Lumineszenzlicht registriert und dem Ort der Nullstelle (4) in der Probe (8) zugeordnet wird, dadurch gekennzeichnet,- dass Abmessungen des abzutastenden Teilbereichs (7) der Probe (8) in mindestens einer Richtung, in der die Intensitätsmaxima (3) der Nullstelle (4) in der Probe benachbart sind, nicht größer sind als 75 % eines Abstands (Do) der Intensitätsmaxima (3) in dieser Richtung. Method for high-resolution imaging of a structure marked with luminescence markers in a sample (8), - whereby light which has an effect on the emission of luminescence light by the luminescence markers is directed onto the sample (8) with an intensity distribution which is one of intensity maxima (3 ) has an adjacent zero point (4), - a partial area (7) of the sample (8) to be scanned being scanned with the zero point (4) and - wherein luminescent light emitted from the area of the zero point (4) is registered and the location of the zero point (4 ) in the sample (8), characterized in that - dimensions of the portion (7) of the sample (8) to be scanned in at least one direction in which the intensity maxima (3) are adjacent to the zero point (4) in the sample, are not greater than 75% of a distance (Do) of the intensity maxima (3) in this direction.

Creation of a permanent structure with high three-dimensional resolution

Method for a high-resolution local imaging of a structure in a sample in order to detect reactions of an object of interest to altered environmental conditions

The aim of the invention is a high-resolution imaging of a structure marked with luminescent markers in a sample. This is achieved in that light (2) which affects the emission of luminescent light by means of the luminescent markers is oriented towards the sample with an intensity distribution which has a zero point (4) adjoined by intensity maxima (3). Sub-regions of the sample to be scanned are scanned using the zero point (4), and luminescent light emitted from the region of the zero point (4) is registered and assigned to the location of the zero point (4) in the sample. Each of a plurality of copies of an object of interest is arranged so as to overlap with one of the sub-regions of the sample (8) to be scanned, and the plurality of copies of the object of interest are subjected to altered environmental conditions in order to detect reactions of the object of interest to the altered environmental conditions. The individual sub-regions of the sample (8) are scanned using the respective zero point (4) during and/or prior to and after the environmental conditions are altered. Dimensions of the sub-regions of the sample to be scanned are delimited in at least one direction in which the intensity maxima (3) adjoin the zero point (4) in the sample such that the sub-regions are not larger than 75% of the spacing (D o ) of the intensity maxima (3) in said direction.

Determination of the distribution of a substance by scanning with a measuring front

Method for producing three-dimensional fine structures

The invention relates to a method for producing three-dimensional fine structures involving the following steps: adding a luminophore to a material (3); forming a three-dimensional fine structure from the material (3), and; conducting fluorescence microscopic examination in order to determine whether the desired fine-structure exists during which luminescent light (10) emitted by the luminophore is measured. The luminophore has two states, which differ with regard to their luminescence properties, and it is reversible but can be, in essence, entirely converted from one state and into the other state by an optical signal (12). The luminophore is, during microscopic examination for determining whether the desired fine structure exists, converted, at measuring points of respective interest, by a transition, which is driven till saturation, between its two states into an inactive state in which it does not emit luminescent light.

Method for the high spatial resolution localization of an individualized molecule which can be excited with excitation light in order to emit luminescent light in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten Moleküls (24) in einer oder mehreren Raumrichtungen (x, y) in einer Probe, wobei das Molekül (24) mit Anregungslicht zur Emission von Lumineszenzlicht anregbar ist, wird das Anregungslichts mit einer Intensitätsverteilung auf die Probe gerichtet wird, die eine Nullstelle (20) und Intensitätsanstiegsbereiche aufweist, welche in jeder der Raumrichtungen (x, y) beidseitig an die Nullstelle (20) angrenzen. Zu jeder von verschiedenen Positionen (A-D) der Nullstelle (20) in der Probe wird das von dem Molekül (24) emittierte Lumineszenzlicht registriert; und der Ort des Moleküls (24) in der Probe wird von den Intensitäten des zu den verschiedenen Positionen (A-D) der Nullstelle (20) registrierten Lumineszenzlichts abgeleitet. Dabei wird die Nullstelle (20) an nicht mehr als n x 3 verschiedenen Positionen (A-D) in der Probe angeordnet wird, um den Ort des Moleküls (24) in den n Raumrichtungen (x, y) von den zu den verschiedenen Positionen (A-D) der Nullstelle (20) registrierten Intensitäten des Lumineszenzlichts abzuleiten.

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten Moleküls (24) in einer oder mehreren Raumrichtungen (x., y) in einer Probe, wobei das Molekül (24) mit Anregungslicht zur Emission von Lumineszenzlicht anregbar ist, wird das Anregungslichts mit einer Intensitätsverteilung auf die Probe gerichtet wird, die eine Nullstelle (20) und Intensitätsanstiegsbereiche aufweist, welche in jeder der Raumrichtungen (x, y) beidseitig an die Nullstelle (20) angrenzen. Zu jeder von verschiedenen Positionen (A-D) der Nullstelle (20) in der Probe wird das on dem Molekül (24) emittierten Lumineszenzlicht registriert; und der Ort des Moleküls (24) in der Probe wird von den Intensitäten des zu den verschiedenen Positionen (A-D) der Nullstelle (20) registrierten Lumineszenzlichts abgeleitet. Dabei wird die Nullstelle (20) an nicht mehr als n x 3 verschiedenen Positionen (A-D) in der Probe angeordnet wird, um den Ort des Moleküls (24) in den n Raumrichtungen (x, y) von den zu den verschiedenen Positionen (a-D) der Nullstelle (20) registrierten Intensitäten des Lumineszenzlichts abzuleiten. In a method for spatially high-resolution determination of the location of a singulated molecule (24) in one or more spatial directions (x, y) in a sample, wherein the molecule (24) is excitable with excitation light for emission of luminescent light, the excitation light with a Intensity distribution is directed to the sample having a zero point (20) and intensity increase ranges, which in both the spatial directions (x, y) on both sides of the zero point (20). At each of different positions (A-D) of the zero point (20) in the sample, the luminescent light emitted on the molecule (24) is registered; and the location of the molecule (24) in the sample is derived from the intensities of the luminescent light registered to the various positions (A-D) of the null (20). In this case, the zero point (20) is arranged at not more than nx 3 different positions (AD) in the ...

Method for exciting molecules in a first state into a second state using an optical signal

The invention relates to a method for converting a sample from a first state into a second state by means of an optical signal, said signal having a spatial intensity distribution comprising at least one zero point and areas that lie adjacent to said zero point, in which the intensity of the signal attains such a level that a saturation point is reached during the conversion of the sample into the second state. According to said method, the sample is cooled to a temperature of below 5 °C before being exposed to the optical signal.

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten Moleküls (24) in einer oder mehreren Raumrichtungen (x, y) in einer Probe, wobei das Molekül (24) mit Anregungslicht zur Emission von Lumineszenzlicht anregbar ist, wird das Anregungslichtmit einer Intensitätsverteilung auf die Probe gerichtet, die eine Nullstelle (20) und Intensitätsanstiegsbereiche aufweist, welche in jeder der Raumrichtungen beidseitig an die Nullstelle (20) angrenzen. Die Nullstelle wird in jeder der Raumrichtungen in der Probe verschoben, wobei zu jeder Position (A-C) der Nullstelle in der Probe das von dem Molekül (24) emittierte Lumineszenzlichts registriert wird. Dabei wird zunächst ein anfänglicher Ortsbereich (23) in der Probe bestimmt, in dem das Molekül (24) angeordnet ist. Dann wird in jeder der Raumrichtungen mindestens eine anfängliche Position (A-C) der Nullstelle (20) so festgelegt, dass sie in der jeweiligen Raumrichtung auf einer Seite des anfänglichen Ortsbereichs (23) liegt. Das Lumineszenzlicht wird zu den allen Raumrichtungen (x, y) zugeordneten Positionen der Nullstelle quasi gleichzeitig registriert, indem die Nullstelle (20) wiederholt zwischen den Positionen (A-C) verschoben wird. Die Positionen (A-C) der Nullstelle werden abhängig von den zu jeder der Positionen (A-C) registrierten Photonen des Lumineszenzlichts sukzessive in den anfänglichen Ortsbereich (23) hinein verschoben. In a method for spatially high resolution determination of the location of a singulated molecule (24) in one or more spatial directions (x, y) in a sample, the molecule (24) being excitable with excitation light for emission of luminescent light, the excitation light having an intensity distribution becomes directed the sample, which has a zero point (20) and intensity increase ranges, which adjoin the zero point (20) on both sides in each of the spatial directions. The zero is shifted in each of the spatial directions in the sample, and at each position (A-C) of ...

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts (x0) eines vereinzelten Moleküls in einer oder mehreren Raumrichtungen in einer Probe, wobei das Molekül mit Anregungslicht zur Emission von Lumineszenzlicht (12) anregbar ist, wird das Anregungslichtmit einer Intensitätsverteilung auf die Probe gerichtet, die in jeder der Raumrichtungen mindestens einen Intensitätsanstiegsbereich (22) mit bekanntem streng monotonem Verlauf der Intensität des Anregungslichts über einem Abstand zu einem Aufpunkt der Intensitätsverteilung aufweist. Ein anfänglicher Ortsbereich (23) in der Probe, in dem das Molekül angeordnet ist, wird bestimmt. (i) in jeder der Raumrichtungen wird mindestens eine Position (xN1) des Aufpunkts der Intensitätsverteilung so festgelegt wird, dass sich der mindestens eine Intensitätsanstiegsbereich (22) in der jeweiligen Raumrichtung über den anfänglichen Ortsbereich (23) erstreckt; der Aufpunkt der Intensitätsverteilung wird an den Positionen (xN1) in der Probe angeordnet und zu jeder Position (xN1) des Aufpunkts der Intensitätsverteilung in der Probe wird das von dem Molekül emittierte Lumineszenzlicht (12) registriert. (ii) aus Intensitätswerten (I1; ISW) des Lumineszenzlichts (12), die zu jeder der Raumrichtungen zwei Intensitätswerte (I1; ISW) umfassen, von welchen einer die Intensität (I1) des zu der mindestens einen Position (xN1) des Aufpunkts der Intensitätsverteilung registrierten Lumineszenzlichts (12) angibt, wird ein weiterer Ortsbereich (24) in der Probe bestimmt wird, in dem das Molekül angeordnet ist und der kleiner als der anfängliche Ortsbereich (23) ist. Die Schritte (i) und (ii) werden mindestens einmal unter Verwendung des weiteren Ortsbereichs (24) als neuer anfänglicher Ortsbereich (23) wiederholt. In a method for spatially high resolution determination of the location (x0) of a singulated molecule in one or more spatial directions in a sample, the molecule being excitable with excitation light for emission of ...

Raster luminescence light microscope with luminescence-preventing light and other light lattices

Ein Rasterlumineszenzlichtmikroskop (43) zum räumlich hochaufgelösten Abbilden einer einen Lumineszenzmarker aufweisenden Struktur einer Probe (15) weist eine Lumineszenzverhinderungslicht (38) und sich von dem Lumineszenzlicht unterscheidendes weiteres Licht bereitstellende Lichtquelle (46), eine Lichtformungs- und -ausrichteinrichtung und einen Detektor (56) auf. Die Lichtformungs- und -ausrichteinrichtung formt aus zwei nicht kohärenten Strahlen (19, 20) des Lumineszenzverhinderungslichts (38) mittels optischer Gitter (21, 22) vier paarweise kohärente Teilstrahlen und fokussiert diese mit einem gemeinsamen Objektiv (25), so dass sie im Bereich der Probe (15) zwei sich kreuzende Liniengitter ausbilden. Die sich kreuzenden Liniengitter weisen jeweils eine Mehrzahl von eindimensional begrenzten lokalen Intensitätsminima auf, so dass eine Intensitätsverteilung des Lumineszenzverhinderungslichts (38) in der Probe ein zweidimensionales Feld aus gleichartigen, mindestens zweidimensional begrenzten lokalen Intensitätsminima aufweist. Mindestens ein Strahl (39, 40) des weiteren Lichts (17) tritt zusammen mit einem der beiden Strahlen (19, 20) des Lumineszenzverhinderungslichts (38) in die Lichtformungs- und -ausrichteinrichtung ein, so dass die Lichtformungs- und -ausrichteinrichtung aus dem Strahl (39, 40) des weiteren Lichts (17) mittels eines der optischen Gitter (21, 22) zwei kohärente Teilstrahlen des weiteren Lichts (17) formt und die Teilstrahlen des weiteren Lichts (17) mit dem Objektiv (25) gemeinsam fokussiert, so dass sie im Bereich der Probe (15) ein Liniengitter ausbilden, das eine Mehrzahl von eindimensional begrenzten und sich parallel zu den lokalen Intensitätsminima eines der Liniengitter des Lumineszenzverhinderungslichts (38) erstreckenden lokalen Intensitätsmaxima und Intensitätsminima aufweist. Weiterhin richtet die Lichtformungs- und -ausrichteinrichtung das Liniengitter des weiteren Lichts (17) so gegenüber dem einen der Liniengitter des ...

Method and device for tracking a movement of a particle, in particular a single molecule, in a sample

Verfahren zum Verfolgen einer Bewegung eines Partikels (2) in einer Probe (3), wobei der Partikel (2) mit Licht (4) zur Emission von Photonen veranlasst wird und wobei von dem Partikel (2) emittierte Photonen registriert werden, dadurch gekennzeichnet, dass das Licht (4) mit einer ein räumlich begrenztes Minimum (19) aufweisenden Intensitätsverteilung (18) auf die Probe (3) gerichtet wird und dass das Minimum (18) dem sich in der Probe (3) bewegenden Partikel (2) nachgeführt wird, indem die Intensitätsverteilung (18) so gegenüber der Probe (3) verschoben wird, dass eine Rate der von dem Partikel (2) emittierten Photonen minimal bleibt. Method for following a movement of a particle (2) in a sample (3), wherein the particle (2) is caused to emit photons with light (4) and wherein photons emitted by the particle (2) are registered, characterized in that the light (4) is directed onto the sample (3) with an intensity distribution (18) having a spatially limited minimum (19) and that the minimum (18) is tracked in the particle (2) moving in the sample (3) in that the intensity distribution (18) is shifted relative to the sample (3) such that a rate of the photons emitted by the particle (2) remains minimal.

A method for determining a measurement based on single molecule events

Zum Bestimmen mindestens eines Messwerts auf der Basis von Einzelmolekülereignissen von gleichartigen Markermolekülen (13) in einer Probe, wobei der Messwert einen anderen Parameter als die Position und Orientierung einzelner Markermoleküle (13) betrifft, wobei die Markermoleküle (13) einen messbaren Zustand, in dem ein zum Bestimmen des mindestens einen Messwerts benötigtes Messsignal von ihnen erhältlich ist, und einen nicht messbaren Zustand, in dem sie das zum Bestimmen des mindestens einen Messwerts benötigte Messsignal nicht abgeben, aufweisen und wobei die Markermoleküle (13) in einer so hohen absoluten Konzentration in der Probe vorliegen, dass der mindestens eine Messwert auf der Basis von Einzelmolekülereignissen der Markermoleküle (13) nicht bestimmbar ist, wenn sich alle Markermoleküle (13) in dem messbaren Zustand befinden, wird mit einem auf die Probe aufgebrachten Einstgellsignal (4) eine solche Messkonzentration der Markermoleküle (13) in dem messbaren Zustand eingestellt, dass der Messwert innerhalb eines definierten Messbereichs (14) auf der Basis von Einzelmolekülereignissen der Markermoleküle (13) bestimmbar wird, indem die Markermoleküle (13) mit dem auf die Probe aufgebrachten Einstellsignal (4) entweder (a) aus ihrem nicht messbaren Zustand in ihren messbaren Zustand überführt werden oder (b) aus ihrem messbaren Zustand in ihren nicht messbaren Zustand überführt werden, wobei die Markermoleküle (13) aus einer Gruppe von Markermolekülen (13) ... For determining at least one measured value on the basis of single-molecule events of similar marker molecules (13) in a sample, wherein the measured value relates to a parameter other than the position and orientation of individual marker molecules (13), wherein the marker molecules (13) have a measurable state in which a measurement signal required for determining the at least one measured value is obtainable from them, and a non-measurable state in which they do not emit the measurement signal ...

Method of fluorescence-microscopically imaging a structure in a sample with high three-dimensional spatial resolution

A method of imaging a structure in a sample with three-dimensional spatial resolution comprises selecting a fluorophore (19) from a group of photochromic fluorophores which are repeatedly temporarily transferable by means of an optical transfer signal (7) out of a first (20) Into a second (21) photochromic state having specific fluorescence properties, and which display a spontaneous and/or inducible return rate out of the second photochromic state (21) into the first photochromic state (20); labelling the structure with the fluorophore (19); via a common objective, subjecting the sample both to the optical transfer signal (7) in a spatially limited transfer-volume, and to an optical excitation signal (5) exciting a portion of the fluorophore (19) being In its second photochromic state (21) for fluorescence In a spatially limited excitation-volume, the transfer-volume and the excitation-volume having a common centre of maximum intensity of the transfer signal (7) and of the excitation signal (5), wherein, at least in the direction of the optical axis of the common objective, a decrease of intensity of the transfer signal with the distance to the common centre of maximum intensity is substantially stronger than any decrease of the effective return rate of the fluorophore (19); detecting fluorescence light (18) emitted by the excited fluorophore (19); shifting the common centre of maximum intensity with regard to the sample; and repeating the preceding steps of subjecting and detecting.

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

For high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have two different electronic states: 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.

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten Moleküls (24) in einer oder mehreren Raumrichtungen (x., y) in einer Probe, wobei das Molekül (24) mit Anregungslicht zur Emission von Lumineszenzlicht anregbar ist, wird das Anregungslichts mit einer Intensitätsverteilung auf die Probe gerichtet wird, die eine Nullstelle (20) und Intensitätsanstiegsbereiche aufweist, welche in jeder der Raumrichtungen (x, y) beidseitig an die Nullstelle (20) angrenzen. Zu jeder von verschiedenen Positionen (A–D) der Nullstelle (20) in der Probe wird das on dem Molekül (24) emittierten Lumineszenzlicht registriert; und der Ort des Moleküls (24) in der Probe wird von den Intensitäten des zu den verschiedenen Positionen (A–D) der Nullstelle (20) registrierten Lumineszenzlichts abgeleitet. Dabei wird die Nullstelle (20) an nicht mehr als n × 3 verschiedenen Positionen (A–D) in der Probe angeordnet wird, um den Ort des Moleküls (24) in den n Raumrichtungen (x, y) von den zu den verschiedenen Positionen (a–D) der Nullstelle (20) registrierten Intensitäten des Lumineszenzlichts abzuleiten. In a method for spatially high-resolution determination of the location of a singulated molecule (24) in one or more spatial directions (x, y) in a sample, wherein the molecule (24) is excitable with excitation light for emission of luminescent light, the excitation light with a Intensity distribution is directed to the sample having a zero point (20) and intensity increase ranges, which in both the spatial directions (x, y) on both sides of the zero point (20). At each of different positions (A-D) of the zero point (20) in the sample, the luminescent light emitted on the molecule (24) is registered; and the location of the molecule (24) in the sample is derived from the intensities of the luminescent light registered to the various positions (A-D) of the null (20). In this case, the zero point (20) is placed at not more than n × 3 different positions (A-D) in the ...

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts eines vereinzelten Moleküls (24) in einer oder mehreren Raumrichtungen (x, y) in einer Probe, wobei das Molekül (24) mit Anregungslicht zur Emission von Lumineszenzlicht anregbar ist, wird das Anregungslichtmit einer Intensitätsverteilung auf die Probe gerichtet, die eine Nullstelle (20) und Intensitätsanstiegsbereiche aufweist, welche in jeder der Raumrichtungen beidseitig an die Nullstelle (20) angrenzen. Die Nullstelle wird in jeder der Raumrichtungen in der Probe verschoben, wobei zu jeder Position (A–C) der Nullstelle in der Probe das von dem Molekül (24) emittierte Lumineszenzlichts registriert wird. Dabei wird zunächst ein anfänglicher Ortsbereich (23) in der Probe bestimmt, in dem das Molekül (24) angeordnet ist. Dann wird in jeder der Raumrichtungen mindestens eine anfängliche Position (A–C) der Nullstelle (20) so festgelegt, dass sie in der jeweiligen Raumrichtung auf einer Seite des anfänglichen Ortsbereichs (23) liegt. Das Lumineszenzlicht wird zu den allen Raumrichtungen (x, y) zugeordneten Positionen der Nullstelle quasi gleichzeitig registriert, indem die Nullstelle (20) wiederholt zwischen den Positionen (A–C) verschoben wird. Die Positionen (A–C) der Nullstelle werden abhängig von den zu jeder der Positionen (A–C) registrierten Photonen des Lumineszenzlichts sukzessive in den anfänglichen Ortsbereich (23) hinein verschoben. In a method for spatially high resolution determination of the location of a singulated molecule (24) in one or more spatial directions (x, y) in a sample, the molecule (24) being excitable with excitation light for emission of luminescent light, the excitation light having an intensity distribution becomes directed the sample, which has a zero point (20) and intensity increase ranges, which adjoin the zero point (20) on both sides in each of the spatial directions. The zero is shifted in each of the spatial directions in the sample, and at each position (A-C) of ...

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

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.

Method and fluorescent light microscope for the high-resolution three-dimensional representation of the structure of a specimen

Zum räumlich hochauflösenden Abbilden einer interessierenden Struktur einer Probe werden die Schritte durchgeführt: Auswählen einer Substanz aus einer Gruppe von Substanzen, die mit einem Umschaltsignal wiederholt aus einem ersten Zustand, in dem sie nicht fluoreszent sind, in einen zweiten Zustand, in dem sie mit einem optischen Anregungssignal zur spontanen Emisson von Fluoreszenzlicht anregbar sind, überführbar sind und die aus dem zweiten Zustand in den ersten Zustand zurückkehren können; Markieren der interessierenden Struktur der Probe mit der Substanz; Überführen wechselnder Anteile der Substanz mit dem Umschaltsignal in den zweiten Zustand; Anregen der Substanz in dem zweiten Zustand durch das optische Anregungssignal zur spontanen Emission von Fluoreszenzlicht; Abbilden der Probe auf ein Sensorarray, wobei eine räumliche Auflösungsgrenze der Abbildung größer (also schlechter) ist als ein mittlerer Abstand zwischen nächst benachbarten Molekülen der Substanz in der Probe, und räumlich aufgelöstes Registrieren des spontan emittierten Fluoreszenzlichts als ein optisches Messsignal, das von dem jeweiligen Anteil der Substanz in dem zweiten Zustand ausgeht, mit dem Sensorarray. Beim Überführen des Anteils der Substanz in den zweiten Zustand wird eine Intensität des Umschaltsignals so eingestellt, dass mindestens 10 % der jeweils in den zweiten Zustand überführten Moleküle einen Abstand zu den ihnen nächst benachbarten Molekülen in dem zweiten Zustand aufweisen, der größer als die räumliche Auflösungsgrenze der Abbildung der Probe auf das Sensorarray ist. Dabei wird das von den Molekülen in dem zweiten Zustand, die einen kleineren Abstand voneinander aufweisen, ausgehende Messsignal von dem Messsignal getrennt, das von den Molekülen in ausreichendem Abstand zueinander ausgeht, und nur für den Fall dass eine Entsprechung zu einem einzelnen Molekül vorliegt, erfolgt eine Bestimmung der Ortslage dieses Moleküls aus der Intensitätsverteilung des Messsignals über das ...

Method for the high-resolution three-dimensional representation of the structure of a specimen

Zum räumlich hochauflösenden Abbilden einer interessierenden Struktur einer Probe werden die Schritte durchgeführt: Auswählen einer Substanz aus einer Gruppe von Substanzen, die mit einem Umschaltsignal wiederholt aus einem ersten Zustand mit ersten optischen Eigenschaften in einen zweiten Zustand mit zweiten optischen Eigenschaften überführbar sind und die aus dem zweiten Zustand in den ersten Zustand zurückkehren können; Markieren der interessierenden Struktur der Probe mit der Substanz; Überführen wechselnder Anteile der Substanz mit dem Umschaltsignal in den zweiten Zustand; Abbilden der Probe auf ein Sensorarray, wobei eine räumliche Auflösungsgrenze der Abbildung größer (also schlechter) ist als ein mittlerer Abstand zwischen nächst benachbarten Molekülen der Substanz in der Probe, und räumlich aufgelöstes Registrieren eines optischen Messsignals, das von dem jeweiligen Anteil der Substanz in dem ersten Zustand ausgeht, mit dem Sensorarray. Beim Überführen des Anteils der Substanz in den zweiten Zustand wird eine Intensität des Umschaltsignals so eingestellt, dass mindestens 10 % der jeweils in dem ersten Zustand verbleibenden Moleküle einen Abstand zu den ihnen nächst benachbarten Molekülen in dem ersten Zustand aufweisen, der größer als die räumliche Auflösungsgrenze der Abbildung der Probe auf das Sensorarray ist. For spatially high-resolution imaging of a structure of interest of a sample, the steps are carried out: selecting a substance from a group of substances which can be repeatedly transferred with a switching signal from a first state with first optical properties to a second state with second optical properties and which consists of second state can return to the first state; Marking the structure of interest of the sample with the substance; Transferring changing portions of the substance with the switching signal in the second state; Imaging the sample onto a sensor array, wherein a spatial resolution limit of the image is greater (ie worse) than ...

Method and fluorescent light microscope for the high-resolution three-dimensional representation of the structure of a specimen

Zum räumlich hochauflösenden Abbilden einer interessierenden Struktur einer Probe werden die Schritte durchgeführt: Auswählen einer Substanz aus einer Gruppe von Substanzen, die mit einem Umschaltsignal wiederholt aus einem ersten Zustand, in dem sie nicht fluoreszent sind, in einen zweiten Zustand, in dem sie mit einem optischen Anregungssignal zur spontanen Emisson von Fluoreszenzlicht anregbar sind, überführbar sind und die aus dem zweiten Zustand in den ersten Zustand zurückkehren können; Markieren der interessierenden Struktur der Probe mit der Substanz; Überführen wechselnder Anteile der Substanz mit dem Umschaltsignal in den zweiten Zustand; Anregen der Substanz in dem zweiten Zustand durch das optische Anregungssignal zur spontanen Emission von Fluoreszenzlicht; Abbilden der Probe auf ein Sensorarray, wobei eine räumliche Auflösungsgrenze der Abbildung größer (also schlechter) ist als ein mittlerer Abstand zwischen nächst benachbarten Molekülen der Substanz in der Probe, und räumlich aufgelöstes Registrieren des spontan emittierten Fluoreszenzlichts als ein optisches Messsignal, das von dem jeweiligen Anteil der Substanz in dem zweiten Zustand ausgeht, mit dem Sensorarray. Beim Überführen des Anteils der Substanz in den zweiten Zustand wird eine Intensität des Umschaltsignals so eingestellt, dass mindestens 10 % der jeweils in den zweiten Zustand überführten Moleküle einen Abstand zu den ihnen nächst benachbarten Molekülen in dem zweiten Zustand aufweisen, der größer als die räumliche Auflösungsgrenze der Abbildung der Probe auf das Sensorarray ist. Dabei wird das von den Molekülen in dem zweiten Zustand, die einen kleineren Abstand voneinander aufweisen, ausgehende Messsignal von dem Messsignal getrennt, das von den Molekülen in ausreichendem Abstand zueinander ausgeht, und nur für den Fall dass eine Entsprechung zu einem einzelnen Molekül vorliegt, erfolgt eine Bestimmung der Ortslage dieses Moleküls aus der Intensitätsverteilung des Messsignals über das ...

Method for the high-resolution three-dimensional representation of the structure of a specimen

The method involves marking an interesting structure of a sample (2) with a substance. Portions of the substance with a switching signal (7) are transferred into a specific condition. An intensity of the switching signal is adjusted in such a manner that 10 percentage of molecules of the substance have a specific distance to the adjacent molecules during transferring the portion of the substance, where the distance is larger than spatial resolution limit of imaging of the sample. An independent claim is also included for a fluorescence light-optical microscope.

Method for determining a measurement value based on single molecule events

In order to determine at least one measurement value based on single molecule events of marker molecules (13) of the same kind in a sample, wherein the measurement value relates to a different parameter than the position or orientation of individual marker molecules (13), wherein the marker molecules (13) have a measurable state in which a measurement signal necessary for determining the at least one measurement value can be obtained thereof, and a state that cannot be measured in which the measurement signal necessary for determining the at least one measurement value cannot obtained, and wherein the marker molecules (13) are present in such a high concentration in the sample, that the at least one measurement value cannot be determined based on single molecule events of the marker molecules (13) when all marker molecules (13) are in the state that can be measured, a measurement concentration of the marker molecules (13) is set in the measurable state via a setting signal (4) that is applied to the sample such that the measurement value can be determined within a defined measurement region (14) based on single molecule events of the marker molecules (13), in that the marker molecules (13) with the setting signal (4) applied to the sample either (a) are brought from a state that cannot be measured into a state that can be measured, or (b) from a state that can be measured into the state that cannot be measured, wherein the marker molecules (13) are selected from a group of molecule markers (13) that transition at a transfer rate from the state that cannot be measured into the state that can be measured.

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

High-resolution fluorescence microscopy with a structured excitation beam

In order to determine the location (X M ) of individual molecules of a substance in a sample, wherein the individual molecules of the substance are in a fluorescent state in which they are excitable with excitation light for emission of fluorescent light, and wherein distances between the individual molecules of the substance in a region of interest in the sample comply with a minimum value d = λ/(2nsinα √(1 + l/ls)), the individual molecules of the substance are excited with the excitation light for emission of fluorescent light, wherein an intensity distribution of the excitation light has at least one zero. The fluorescent light from the excited individual molecules of the substance is registered for different positions (X N ) of the at least one zero of the intensity distribution of the excitation light in the region of interest in the sample. In this case, distances between closest adjacent positions (X N ) of the at least one zero of the intensity distribution of the excitation light in which the fluorescent light from the excited individual molecules of the substance is registered are not greater than half the minimum value d. The locations (X M ) of the individual molecules of the substance are then derived from the profile of the intensity (I) of the fluorescent light from the respective molecule against the positions (X N ) of the at least one zero of the intensity distribution of the excitation light in the region of interest in the sample.

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

Raster luminescence light microscope with luminescence-preventing light and other light lattices

Rasterlumineszenzlichtmikroskop (43) zum räumlich hochaufgelösten Abbilden einer einen Lumineszenzmarker (13) aufweisenden Struktur (14) einer Probe (15) mit – einer Lumineszenzverhinderungslicht (38) und sich von dem Lumineszenzverhinderungslicht (38) unterscheidendes weiteres Licht bereitstellenden Lichtquelle (46), – einer Lichtformungs- und -ausrichteinrichtung, die aus zwei nicht kohärenten Strahlen (19, 20) des Lumineszenzverhinderungslichts (38) mittels optischer Gitter (21, 22) vier paarweise kohärente Teilstrahlen des Lumineszenzverhinderungslichts (38) formt, die die Teilstrahlen des Lumineszenzverhinderungslichts (38) mit einem Objektiv (25) so gemeinsam in die Probe (15) fokussiert, dass sie im Bereich der Probe (15) zwei sich kreuzende Liniengitter (1, 2) ausbilden, die jeweils eine Mehrzahl von eindimensional begrenzten lokalen Intensitätsminima (3, 4) aufweisen, so dass eine Intensitätsverteilung (7) des Lumineszenzverhinderungslichts (38) in der Probe (15) ein zweidimensionales Feld aus gleichartigen, mindestens zweidimensional begrenzten lokalen Intensitätsminima (9) aufweist, ... A scanning luminescence light microscope (43) for spatially high-resolution imaging of a structure (14) of a sample (15) having a luminescence marker light (38) and a further light source (46) which differs from the luminescent prevention light (38) A light-shaping and aligning device which forms from two non-coherent beams (19, 20) of the luminescence-preventing light (38) by means of optical gratings (21, 22) four pairwise coherent partial beams of the luminescence-preventing light (38) which transmits the partial beams of the luminescence-preventing light (38) a lens (25) together in the sample (15) focused so that they form in the region of the sample (15) has two intersecting line grids (1, 2), each having a plurality of one-dimensional limited local intensity minima (3, 4) such that an intensity distribution (7) of the luminescence-preventing light (38) in the sample ...

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

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.

A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample

Bei einem Verfahren zum räumlich hochauflösenden Bestimmen des Orts (x) eines vereinzelten Moleküls in einer oder mehreren Raumrichtungen in einer Probe, wobei das Molekül mit Anregungslicht zur Emission von Lumineszenzlicht (12) anregbar ist, wird das Anregungslichtmit einer Intensitätsverteilung auf die Probe gerichtet, die in jeder der Raumrichtungen mindestens einen Intensitätsanstiegsbereich (22) mit bekanntem streng monotonem Verlauf der Intensität des Anregungslichts über einem Abstand zu einem Aufpunkt der Intensitätsverteilung aufweist. Ein anfänglicher Ortsbereich (23) in der Probe, in dem das Molekül angeordnet ist, wird bestimmt. (i) in jeder der Raumrichtungen wird mindestens eine Position (x) des Aufpunkts der Intensitätsverteilung so festgelegt wird, dass sich der mindestens eine Intensitätsanstiegsbereich (22) in der jeweiligen Raumrichtung über den anfänglichen Ortsbereich (23) erstreckt; der Aufpunkt der Intensitätsverteilung wird an den Positionen (x) in der Probe angeordnet und zu jeder Position (x) des Aufpunkts der Intensitätsverteilung in der Probe wird das von dem Molekül emittierte Lumineszenzlicht (12) registriert. (ii) aus Intensitätswerten (I; I) des Lumineszenzlichts (12), die zu jeder der Raumrichtungen zwei Intensitätswerte (I; I) umfassen, von welchen einer die Intensität (I) des zu der mindestens einen Position (x) des Aufpunkts der Intensitätsverteilung registrierten Lumineszenzlichts (12) angibt, wird ein weiterer Ortsbereich (24) in der Probe bestimmt wird, in dem das Molekül angeordnet ist und der kleiner als der anfängliche Ortsbereich (23) ist. Die Schritte (i) und (ii) werden mindestens einmal unter Verwendung des weiteren Ortsbereichs (24) als neuer anfänglicher Ortsbereich (23) wiederholt. In a method for spatially high resolution determination of the location (x) of a singulated molecule in one or more spatial directions in a sample, the molecule being excitable with excitation light for emission of luminescent light (12), ...

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

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×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×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 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.

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

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.

Method and device for physically defined excitation of an optical transition

Method for a high-resolution local imaging of a structure in a sample in order to detect reactions of an object of interest to altered environmental conditions

The aim of the invention is a high-resolution imaging of a structure marked with luminescent markers in a sample. This is achieved in that light (2) which affects the emission of luminescent light by means of the luminescent markers is oriented towards the sample with an intensity distribution which has a zero point (4) adjoined by intensity maxima (3). Sub-regions of the sample to be scanned are scanned using the zero point (4), and luminescent light emitted from the region of the zero point (4) is registered and assigned to the location of the zero point (4) in the sample. Each of a plurality of copies of an object of interest is arranged so as to overlap with one of the sub-regions of the sample (8) to be scanned, and the plurality of copies of the object of interest are subjected to altered environmental conditions in order to detect reactions of the object of interest to the altered environmental conditions. The individual sub-regions of the sample (8) are scanned using the respective zero point (4) during and/or prior to and after the environmental conditions are altered. Dimensions of the sub-regions of the sample to be scanned are delimited in at least one direction in which the intensity maxima (3) adjoin the zero point (4) in the sample such that the sub-regions are not larger than 75% of the spacing (D o ) of the intensity maxima (3) in said direction.

Method for spatially high-resolved imaging of a structure of a sample that has a luminophore

In a method for the spatially high-resolved imaging of a structure (2) of a sample (3) that has a luminophore (1), the sample (3) is subjected to luminescence excitation light (7) in a measurement range (5), which luminescence excitation light(7) excites the luminophore (1) from an excitable electronic basic state into an excited luminescent state. The sample (3) is subjected to a intensity distribution of luminescence damping light (8) in the measurement range (5), the intensity distribution having a local minimum (9), and the luminescence damping light returning the luminophore (1) from the excited luminescent state into the excitable electronic basic state. Luminescence light (10) emitted from the measurement range (5) is recorded and is assigned to the position of the local minimum (9) in the sample (3). Prior to being subjected to the luminescent excitation light (7), the sample (3) is subjected to an intensity distribution of excitation prevention light (4) in the measurement range (5), which excitation prevention light transfers the luminophore (1) from the excitable electronic basic state into a protected state, in which the luminophore (1) is protected against electronic excitations from the luminescence excitation light (7) and the luminescence damping light (8). The intensity distribution of the excitation prevention light (4) has a local minimum (6), which overlaps with the local minimum (9) of the intensity distribution of the luminescence damping light (8).

Scanning microscope in which a sample is simultaneously and optically excited at various points

The invention relates to an optical device, especially a scanning microscope (1), wherein an expanded laser beam (2) is divided into several partial beams (4) by micro lenses (5) that are arranged next to each other. Each partial beam (4) is focused onto a focal point (11) by means of a common lens (7) in order to optically excite a sample (8). The fluorescent light emanating from the individual focal points (11) of the sample (8) is registered by a photosensor (13) placed behind the lens (7) from the sample (8) outwards. Each photon of fluorescent light coming from the sample (8) and registered by the photosensor is excited by at least two photons from the laser beam (2).

A method for spatially high resolution imaging of a luminophor structure of a sample

Bei einem Verfahren zum räumlich hochaufgelösten Abbilden einer einen Luminophor (1) aufweisenden Struktur (2) einer Probe (3) wird die Probe (3) in einem Messbereich (5) mit Lumineszenzanregungslicht (7) beaufschlagt, das den Luminophor (1) aus einem anregbaren elektronischen Grundzustand in einen angeregten lumineszierenden Zustand anregt. Die Probe (3) wird in dem Messbereich (5) zudem mit einer ein lokales Minimum (9) aufweisenden Intensitätsverteilung von Lumineszenzabregungslicht (8) beaufschlagt, das den Luminophor (1) aus dem angeregten lumineszierenden Zustand in den anregbaren elektronischen Grundzustand zurückbringt. Aus dem Messbereich (5) emittiertes Lumineszenzlicht (10) wird registriert und der Position des lokalen Minimums (9) in der Probe (3) zugeordnet. Vor dem Beaufschlagen mit dem Lumineszenzanregungslicht (7) wird die Probe (3) in dem Messbereich (5) mit einer Intensitätsverteilung von Anregungsverhinderungslicht (4) beaufschlagt, das den Luminophor (1) aus dem anregbaren elektronischen Grundzustand in einen Schutzzustand überführt, in welchem der Luminophor (1) vor elektronischen Anregungen durch das Lumineszenzanregungslicht (7) und das Lumineszenzabregungslicht (8) geschützt ist. Die Intensitätsverteilung des Anregungsverhinderungslichts (4) weist ein lokales Minimum (6) auf, das mit dem lokalen Minimum (9) der Intensitätsverteilung des Lumineszenzabregungslichts (8) überlappt. In a method for spatially high-resolution imaging of a structure (2) of a sample (3) having a luminophore (1), the sample (3) is exposed to luminescence excitation light (7) in a measuring area (5), which illuminates the luminophore (1) from a excitable electronic ground state into an excited luminescent state. In the measuring area (5), the sample (3) is also exposed to an intensity distribution of luminescence de-excitation light (8) which has a local minimum (9) and which brings the luminophore (1) back from the excited luminescent state to the excitable electronic ...

Cell-penetrating fluorescent dyes with secondary alcohol functionalities

The invention relates to novel cell-penetrating fluorescent dyes with with secondary alcohol functionalities having one of the following general formulae I-III and 4: The invention also relates to the use of these compounds for optical microscopy and imaging techniques.

High spatial resoulution imaging and modification of structures

In a method of high spatial resolution imaging or modifying a structure, the structure is marked with a substance which is selected from the group of substances which can be transferred from a first state having first optical properties to a second state having second optical properties by means of an optical switch over signal. Then, the second state of the substance is adjusted with the switch over signal except for a spatially limited area. If the substance and the switch over signal are adapted to each other in such a way, that everywhere where the switch over signal exceeds a threshold value essentially the second state of the substance is adjusted, and if the spatial area purposefully omitted by the switch over signal is an intensity minimum of an interference pattern, the spatial area of the structure in which the substance is within the first state becomes smaller than the diffraction limit for the switch over signal.

Method and device for optically measuring a sample

Zum optischen Messen einer Probe wird ein elektromagnetisches Signal (2) wiederholt vorübergehend auf die Probe gerichtet, um eine Substanz in der Probe von einem ersten elektronischen Zustand (1 ) in einen zweiten elektronischen Zustand (3) zu überführen, wobei zumindest ein Teil der Substanz aus dem zweiten Zustand (3) heraus Photonen aussendet, die zum optischen Messen der Probe verwendet werden, wobei das Signal (2) mit einem zeitlichen Wiederholungsabstand auf dieselben Bereiche der Probe gerichtet wird, und wobei der zeitliche Wiederholungsabstand des Signals (2) bei einer Substanz, bei der eine Lebensdauer des zweiten Zustands eine Größenordnung von 1 ns aufweist, auf einen Wert von mindestens 0,1 µs eingestellt wird, der in Bezug auf eine Ausbeute an Photonen von der Substanz optimiert ist.

Method for spatial measuring of a nanoscale structure