DE102010035003B4 - Spatially and temporally high-resolution microscopy - Google Patents

Abstract

Method for spatially and temporally high-resolution detection of the fluorescence of a fluorophore of a sample, comprising the following steps: a) the sample is arranged on the surface of a sample carrier (14); b) adding to the sample a redox buffer comprising at least one reducing agent, and / or at least one oxidizing agent and / or at least one reducing-oxidizing agent to prevent photobleaching of the fluorophore; c) pulsed excitation light (10) with a pulse length less than 1 ns is coupled into the sample carrier (14) in such a way that it either irradiates the sample or leaves the sample carrier (14) only evanescently at the location of the sample; d) the wavelength of the excitation light (10) is selected such that the excitation light is capable of exciting the fluorescence of the fluorophore of the sample; e) fluorescent light emanating from the sample is guided with an optical structure (13, 24, 30) through a pinhole (32) onto a detector (34), wherein the optical assembly (13, 24, 30) by means of the pinhole (32) defines a detection volume (45); f) the detector (34) is selected so that it can detect individual photons of the fluorescent light with a temporal resolution of better than 1 ns; g) the fluorescent light is split and additionally recorded with a spatially resolving detector (22); h) by means of the spatially resolving detector (22) an interesting measurement region within the sample is identified; i) the spatially limited detection volume (45) of the time-resolved detection is positioned on the interesting measurement region in the sample; and j) the interesting measuring region is scanned with the spatially limited detection volume (45) of the time-resolved detection.

Description

Field of the invention

The invention relates to the field of fluorescence microscopy, in particular to cell membranes.

Cell membranes are the contact point of cells to the outside, here is the mass transfer with the environment (release and absorption of substances), but also z. B. virus infections. In this case, internal processes of the membrane are often involved. This field is therefore highly relevant from a biological and medical point of view.

State of the art

Fluorescence-lifetime imaging microscopy

For fluorescence lifetime measurements, confocal methods with pulsed excitation, possibly combined with scanning scanning of the sample or the laser beam, are used. Time-resolved detection is mostly based on the so-called TCSPC technique (time-correlated single photon counting, TCSPC, time-correlated single-photon counting) [see, for example, US Pat. D. O'Connor, D., Phillips, Time Correlated Single Photon Counting, Academic Press 1984]. In this case, the sample by a pulsed excitation light source, i. d. R. stimulated a pulsed laser. Typical pulse lengths are less than 1 ns, often less than 200 ps, sometimes even in the femtosecond range, ie less than 1 ps. The detectors used here are, for example, photomultipliers or avalanche photodiodes, the latter being particularly useful in highly sensitive applications, for example the detection of individual molecules. They can detect single photons and have a temporal resolution of better than 1 ns (typical temporal resolutions in the range of 30 to 3-400 ps (SPADs to 0.5 ns) [see W. Becker, Advanced Time-Correlated Single Photon Counting Techniques , Springer Series in Chemical Physics, 2005].

The fluorescence lifetime extends the available information purely intensity-based measurements and thus the possible analyzes and statements considerably. Two applications are particularly important:

FLIM-FRET

Interactions between different substances, eg. Proteins or nucleic acids can be examined by means of Foerster Resonance Energy Transfer (FRET): Fluorescence Lifetime Imaging Microscopy (FLIM). In this case, the fluorescence lifetime of a donor dye decreases when it interacts with a second biomolecule labeled with an acceptor dye, e.g. As a protein, by spatial proximity in interaction occurs. In this way, z. B. Investigate changes in the internal structure of a membrane [see, eg. J. Lakowicz, "Principles of Fluorescence Spectroscopy", 3rd. Ed., Springer; Cape. 13]

Environmentally sensitive FLIM

The fluorescence lifetime of a dye depends z. B. in many cases also on the polarity of its environment, which is significantly different inside a membrane than at the interface to the aqueous medium. By measuring the fluorescence lifetime conclusions can be drawn on the position of the investigated biomolecule within the membrane structure. A biomolecule may either fluoresce itself (eg a fluorescent protein) or be labeled with a dye (such as a protein, a nucleic acid or a lipid). For this purpose, a spatially resolved image of the lifetime distribution within the sample is often taken with the help of FLIM [s. z. B. Esposito, F.S. Wouters, "Fluorescence Lifetime Imaging Microscopy", Curr. Prot. Cell Biol., Unit 4.14 (2004)].

In far-field microscopy, because of the small depth of focus, given by an axially relatively large examination volume (several microns), always unwanted areas within the cell, which superimpose the actually interesting signal as a disturbing background. This is true even for confocal microscopy in which the axial diameter of the observation volume is still at least 600 nm. This can lead to a superposition of z. B. membrane fluorescence with fluorescence from the cell interior.

Therefore total internal reflection fluorescence (TIRF) fluorescence is commonly used for the selective investigation of cell membranes [s. z. K.N. Fish, Curr. Protoc. Cytom. Chapter 12, Unit 12.18 (2009)]. In TIRF microscopy, a laser beam is coupled into the sample carrier (cover glass) in such a way that total reflection occurs at the interface between sample carrier and sample and the sample is illuminated only by an approximately 100 nm deep evanescent field.

Since this method is a far-field illumination, the emitted fluorescence is usually detected with a CCD camera. With a CCD camera, however, an efficient and accurate, spatially resolved characterization of the fluorescence decay is difficult.

The publication DE 10 2007 030 403 A1 describes the basic approach of the so-called ROXS technology, which will be explained in detail below.

The document Tinnefeld et al. [Tinnefeld, P .; Bauschmann, V .; Weston, K. D .; Biebricher, A .; Herten, D. -P .; Piestert, O .; Heinlein, T .; Heilemann, M .; Sauer, M .: How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interaactions. In: Recent Research Development in Physical Chemistry, Vol. 7, 2004, pp. 95-125] describes a method for spatially and temporally high-resolution detection of the fluorescence of a fluorophore of a sample.

The publication EP 1 291 627 B1 describes a device for the spatially and temporally resolved detection of fluorophores.

task

The object of the invention is to provide a way by which the fluorescence of a sample with high spatial and temporal resolution can be detected.

solution

This object is achieved by the inventions having the features of the independent claims. Advantageous developments of the inventions are characterized in the subclaims. The wording of all claims is hereby incorporated by reference into the content of this specification.

In the following, individual process steps are described in more detail. The steps do not necessarily have to be performed in the order given, and the method to be described may also have other steps not mentioned.

• a) die Probe wird an der Oberfläche eines Probenträgers angeordnet;
• b) der Probe wird ein Redox-Puffer umfassend wenigstens ein Reduktionsmittel, und/oder wenigstens ein Oxidationsmittel und/oder wenigstens ein Reduktions-Oxidations-Mittel zugesetzt, um ein Photobleichen des Fluorophors zu verhindern;
• c) gepulstes Anregungslicht mit einer Pulslänge kleiner als 1 ns, bevorzugt zwischen 10 und 200 ps, wird derart in den Probenträger eingekoppelt, dass es wahlweise die Probe analog zur Standard Epifluoreszenzbeleuchtung durchleuchtet (on axis Einkopplung) oder am Ort der Probe den Probenträger nur evaneszent verlässt (sog. off axis Einkopplung, die zur Totalreflexion führt, total internal reflection, TIR);
• d) die Wellenlänge des Anregungslichts wird derart gewählt, dass das Anregungslicht geeignet ist, die Fluoreszenz des Fluorophors der Probe anzuregen;
• e) von der Probe ausgehendes Fluoreszenzlicht wird mit einem optischen Aufbau durch eine Lochblende auf einen Detektor geleitet, wobei der optischen Aufbau mittels der Lochblende ein Detektionsvolumen definiert;
• f) der Detektor wird derart gewählt, dass er einzelne Photonen des Fluoreszenzlichts mit einer zeitlichen Auflösung von besser als 1 ns, bevorzugt mit einer Auflösung von 10 bis 100 ps, detektieren kann;
• g) das Fluoreszenzlicht wird aufgeteilt und zusätzlich mit einem ortsauflösenden Detektor (22) aufgenommen;
• h) mittels des ortsauflösenden Detektors (22) wird eine interessante Messregion innerhalb der Probe identifiziert;
• i) das räumlich begrenzte Detektionsvolumen (45) der zeitaufgelösten Detektion wird auf die interessante Messregion in der Probe positioniert; und
• j) die interessante Messregion wird mit dem räumlich begrenzten Detektionsvolumen (45) der zeitaufgelösten Detektion abgerastert.

To achieve the object, a method for spatially and temporally high-resolution detection of the fluorescence of a fluorophore of a sample is proposed with the following steps:

• a) the sample is placed on the surface of a sample carrier;
• b) adding to the sample a redox buffer comprising at least one reducing agent, and / or at least one oxidizing agent and / or at least one reducing-oxidizing agent to prevent photobleaching of the fluorophore;
• c) pulsed excitation light with a pulse length of less than 1 ns, preferably between 10 and 200 ps, is coupled into the sample holder in such a way that it selectively illuminates the sample analogously to standard epifluorescence illumination (on-axis coupling) or only evanescently at the site of the sample leaves (so-called off-axis coupling, which leads to total reflection, total internal reflection, TIR);
• d) the wavelength of the excitation light is chosen such that the excitation light is suitable for exciting the fluorescence of the fluorophore of the sample;
• e) fluorescent light emanating from the sample is guided with an optical structure through a pinhole to a detector, wherein the optical structure by means of the pinhole defines a detection volume;
• f) the detector is chosen so that it can detect individual photons of the fluorescent light with a time resolution of better than 1 ns, preferably with a resolution of 10 to 100 ps;
• g) the fluorescent light is split and additionally with a spatially resolving detector ( 22 ) recorded;
• h) by means of the spatially resolving detector ( 22 ) identifies an interesting measurement region within the sample;
• i) the spatially limited detection volume ( 45 ) of the time-resolved detection is positioned on the interesting measurement region in the sample; and
• j) the interesting measuring region is compared to the spatially limited detection volume ( 45 ) of the time-resolved detection.

A sample can z. B. a cell o. Ä. Be. The word sample denotes the object to be examined.

The fluorescence of a fluorophore of the sample is detected. These are z. For example, one or more dye molecules or quantum dots or similar to the fluorescent excitable substances with which the sample was labeled. However, the sample itself may also have a fluorescent part (referred to as a fluorophore). Or the sample is a fluorophore.

The arrangement or fixation of the sample on the surface of the sample carrier takes place for. B. by immobilization, for. B. by a chemical compound with the surface of the sample carrier. But it is also possible to use non-chemical adhesion for immobilization, z. By biochemical bonding, van der Waals forces, and the like.

The punctiform detection in conjunction with a suitable detector achieves the desired high temporal resolution in the determination of the fluorescence lifetime. At the same time, in particular, the TIR illumination allows a high local resolution of about 100 nm along the optical axis (axial) by the evanescent excitation. This allows time-resolved measurements in volumes that are smaller than in the previous confocal Microscopy, in particular with a stronger axial restriction for selective measurements in cell membranes.

The proposed method thus offers a possibility by means of which samples can be evanescently excited in the far field, the detection taking place in such a way that the fluorescence lifetime of selected sample areas can be measured at high temporal resolution without the sample being photobleached.

Since the method of time-correlated single photon counting (TCSPC) used becomes extremely sensitive by using avalanche photodiodes, these measurements are also possible with single-molecule sensitivity.

Wide-field measurement

In order to obtain a spatially resolved image of a sample area with lifetime information, the sample is usually scanned with a confocal setup. In each case, only a small point within the sample is illuminated for a short time, whereby disturbing light-induced processes are minimized.

Without photostability enhancers, such as the ROXS technology described above, permanent TIRF excitation in the far field can not be efficiently combined with a scanned, punctiform fluorescence lifetime measurement.

In order to accelerate the imaging measurement process and thus also further reduce photobleaching, the fluorescent light can additionally be recorded with a spatially resolving detector. The punctiform detection can then be positioned to a location previously identified by means of the spatially resolving detector.

More specifically, the fluorescent light emanating from the sample is split and additionally recorded with a spatially resolved detector. The spatially resolved detector identifies an interesting measurement region within the sample. The spatially limited detection volume of the time-resolved detection is positioned on the interesting measuring region.

Typically, the spatially resolved detector is a camera, e.g. As a high-sensitivity CCD camera that can perform a corresponding wide-field measurement. The camera can identify and locate individual molecules on the sample carrier. Their fluorescence lifetime can then be determined successively in each case a selective measurement in order to draw from this the conclusions reached.

As a result, a significant reduction in the measurement time for determining the fluorescence lifetimes in the sample is achieved in such a way that only selected regions within the sample, known as region-of-interest (ROI), have to be measured in a time-resolved manner. For this purpose, the coordinates of sample areas of interest are determined from the camera images registered in a few milliseconds by means of suitable efficient algorithms, on which time-resolved fluorescence measurements are then carried out in a targeted manner. Since typically selected image sizes of biological preparations are often occupied only in a range up to 10% of the area with a high information content, a measurement time reduction by a factor of 2-20 is possible.

Time-resolved imaging through raster scanning

Confocal optical structures are well known and z. As described in the paper by Tony Wilson [Tony Wilson, "Confocal Microscopy: Basic Principles and Architectures", in: A. Diaspro, "Confocal and Two-Photon Microscopy: Foundations, Applications and Advances", Wiley-Liss, 2002] , There are also described systems in which the scanning is done by moving the sample or the lens.

The detection volume given by the pinhole diaphragm can also be displaced three-dimensionally in the sample with the methods mentioned in the present case. In particular, it is possible to scan the sample two-dimensionally with the detection volume, the latter z. B. is moved with the mirror scanner.

The proposed method replaces the scanned, focused illumination of the confocal microscope by a stationary, wide-area illumination. If the coupling of the light is offset parallel relative to the optical axis of the objective, an evanescent excitation with a low penetration depth into the sample is obtained. Thus, only the sample areas are reached directly at the slide interface. The coupling can also be done directly on the optical axis, whereby a much deeper portion of the sample is irradiated. The principle of a punctiform, scanned detection volume remains analogous to a confocal microscope.

Description of the ROXS technology

The sequential scanning of an image takes a relatively long time. In particular, this applies to the lifetime measurements to be carried out here, in which for each pixel a sufficient Number of photons must be collected for a meaningful lifetime analysis. With wide-area illumination or excitation and scanned detection, the ratio between the "illumination time" of a point in the sample and the "time in which a signal is detected from there" is orders of magnitude higher than in methods in which the excitation light is also scanned synchronously (For example, in the above-mentioned confocal laser scanning microscopy).

This can lead to great problems with photobleaching of the fluorophores in the examined sample area. As a rule, the sample bleaches faster than it can be measured with the scanner.

Therefore, TIRF excitation can not be effectively combined with fluorescence lifetime measurement so far.

In order to prevent, or at least significantly reduce, the fading, reducing and / or oxidizing substances are therefore added to the aqueous sample environment in the method proposed here. This approach is based on the ROXS (Reducing Oxidizing System) technology to improve the photostability of a dye that the WO 2009/003948 A2 as well as In the publication by J. Vogelsang [J. Vogelsang et al., Ang. Chem. Int. Ed. 47 (29), 5465-5469 (2008)] and the contents of which are incorporated herein by reference.

• a) der Licht-induzierte Übergang aus dem Grundzustand SO in den angeregten Zustand S1,
• b) die direkte Rückkehr aus dem S1 in den SO Zustand, bei dem ein Fluoreszenzphoton abgestrahlt wird,
• c) Der spontane Übergang aus dem S1 in den Triplettzustand T1,
• d) Der durch das Oxidations- oder Reduktionsmittel initiierte Übergang aus T1 in einen geladenen Zustand (z. B. ein Radikalkation oder Radikalanion), und
• e) final die durch ein Oxidations- oder Reduktionsmittel ausgelöste Rückkehr aus dem geladenen Zustand in den ungeladenen Grundzustand S0.

The aim of the ROXS technology is, among other things, the maximum shortening of the residence time of a dye in the triplet state, since this is often the primary starting point for a photooxidation or a direct irreversible chemical reaction and thus photobleaching. The reducing agent generates an additional charged state in the photocycle of a dye molecule, which is intended to quickly depopulate the triplet state. Important state transitions are:

• a) the light-induced transition from the ground state SO to the excited state S1,
• b) the direct return from the S1 to the SO state where a fluorescence photon is emitted,
• c) the spontaneous transition from the S1 to the triplet state T1,
• d) the transition initiated by the oxidizing or reducing agent from T1 to a charged state (eg a radical cation or radical anion), and
• e) final return from the charged state to the uncharged ground state S0 triggered by an oxidizing or reducing agent.

Important here are a fast transition to the stable, charged state and then a rapid return to the ground state S0 so that the dye molecule can be excited again to fluorescence. In this way, the photostability can be dramatically increased, so that a permanent illumination of the sample can be tolerated even at positions where it is not permanently detected.

The mode of action of ROXS chemistry in improving photostability is based on the fact that ROXS chemistry transfers the sample as quickly as possible from the excited to the ground state. Upon absorption of a photon from the ground state, the molecule is transferred to the S1 state. The fastest way to depopulate the S1 state is the spontaneous emission associated with the emission of a photon. With a certain probability, however, the dye molecule can also be transferred to other dark states, for example the triplet state. The triplet state normally has a much longer lifetime in water than the S1 state (eg, 100 ns-10 μs vs. 0.2-10 ns), so the probability of an irreversible chemical process from the triplet state is usually much greater, for example , B. can also be promoted by absorption of another photon from the triplet state out.

The agents contained in the ROXS buffer now depopulate this potentially reactive dark state by oxidation or reduction to form a charged particle.

The suitability of the oxidizing or reducing agent depends on its redox potential. For a suitable oxidizing agent, this is preferably in the range of ≥ -1 V to ≦ -0.2 V, preferably in the range of ≥ -600 mV to ≦ 100 mV, preferably in the range of ≥ -250 mV to ≦ -200 mV.

The charged state resulting from the oxidation or reduction process is also a dark state and can now be repopulated by the opposite process, with the dye molecule returning to the ground state. If the charged state is caused by an oxidizing agent, the return to the ground state can be caused by a reducing agent and vice versa.

Important here is the redox potential of the reducing agent. The redox potential of a suitable reducing agent at pH 7 measured against normal hydrogen electrode (NHE) in acetonitrile is preferably in the range of ≥ 0.1 V to ≤ 2 V, preferably in the range of ≥ 500 mV to ≤ 800 mV, preferably in the range of ≥ 540 mV up to ≤ 750 mV.

The most important task of the buffer solution, in which the oxidizing and reducing agents are dissolved, is the setting of a suitable pH, since the redox potentials of the oxidizing and reducing agents as well as of the different states of the dye are dependent on the pH.

Since the cell walls in fixed cells are permeabilized by the fixation, the buffer can also penetrate into the cell interior and allow photostabilization there in the sense of ROXS chemistry.

In principle, this principle can also be applied to living cells since there is an oxidizing agent with dissolved atmospheric oxygen as well as a suitable reducing agent with the amino acid cysteine containing a thiol group; here the choice of the fluorescent dye is decisive.

As a reducing agent is typically a thiol (about 100 mM mercaptoethylamine) and as an oxidizing agent z. B. atmospheric oxygen used. However, the process can be implemented with many other different oxidation and / or reducing agents and / or reduction-oxidation agents.

As the oxidizing agent z. B. - and not conclusively - serve: bipyridinium salts, their derivatives, preferably viologens, in particular methyl viologens, and / or nitroaromatics; substituted nitroaromatics, in particular carboxylic acid-substituted nitroaromatics, preferably nitrobenzenes, or sulfonic acid-substituted nitroaromatics, benzoquinones, substituted benzoquinones, in particular chloro-substituted and / or cyano-substituted benzoquinones, in particular dichlorobenzoquinone, tetrachlorobenzoquinone, tetracyanobenzoquinone, and / or mixtures thereof, and phenols, indophenols , Hydroquinones, catechols, chromans, dihydrobenzofurans, dihydroxinaphthalenes and / or naphthols, and their sulfonic, carboxylic, nitro, chloro and / or cyano-substituted derivatives, such as the oxidizing / reducing agent ubiquinone or cytochromes.

Suitable reducing agents are, for example, aliphatic and aromatic primary, secondary and tertiary amines, mono- and di-naphthylamines, in particular phenylamine, diphenylamine, p-phenylenediamine, hydroxylamines, hydroxylamine derivatives, dihydroquinoline derivatives, piperidine derivatives and / or pyrrolidine derivatives, and thiophenols, thionaphthols, phenol sulfides, uric acid (urate), urea (urea), bilirubin, ascorbic acid and / or flavins, furthermore cyclo-octatetraene (5,6-bis-acetoxymethyl-cycloocta, COT) and 1,4-diaza-bicyclo - (2,2,2) octane (DABCO). Preferred reducing agents are selected from the group comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ascorbic acid, β-mercaptoethanol, β-mercaptoethylamine, β-naphthylamine, dithiothreitol, NaBH3CN, n-propyl gallate and / or mixtures thereof. A particularly preferred reducing agent is 6-hydro-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox ^®).

Suitable reducing oxidants include ubiquinone or cytochromes. In general, these are substances that can function both as an oxidizing agent and as a reducing agent.

A comprehensive description of possible substances is the WO 2009/003948 A2 including, the content of which is hereby incorporated by reference into the content of this specification.

As the oxidizing agent of the redox buffer and oxygen can be selected, since this is the easiest to implement. To reduce the oxygen content of the redox buffer, z. B. be purged with nitrogen. Since the sample is regularly in aqueous solution, it can be flowed through with nitrogen to reduce the oxygen content.

The oxygen content may alternatively be adjusted by adding an enzymatic system comprising glucose oxidase. This enzymatic system consists of an enzyme and substrate. A suitable enzyme is glucose oxidase. A suitable associated substrate is glucose. The glucose is oxidized by the glucose oxidase, which consumes oxygen.

The proposed method also allows specific labeled positions within defined regions of the sample to be identified depending on a change in state of the fluorophores. For example, a dynamic development of the sample can be evaluated in the wide field image. Derived from this selectively the one sample area can be controlled or focused with the punctiform detection, which has the desired dynamics. If, for example, a cell contracts in place x - y, then exactly at this point TCSPC z. As the Ca2 + concentration can be determined, because this affects the fluorescence lifetime of a suitable probe molecule.

high-resolution microscopy

An important trend in light microscopy is the increasingly accurate spatial resolution of cellular structures. Classical light microscopy achieves a resolution that is limited by the wavelength of the light and the optical structure. Given by the Abbé formula, the achievable resolution is in the best case at about half of used wavelength, in the visible range thus between 200 and 300 nm.

• – STED (stimulated emission depletion microscopy, stimulierte Emissions-Entvölkerungs-Mikroskopie), entwickelt von Stefan Hell (s. z. B. WO 1995/021393 A2 ; WO 2009/024529 ], eine konfokale Methode, bei der das Bild über ein Rasterverfahren gewonnen wird.
• – PALM (Photoactivated Localization Microscopy, photoaktivierte Lokalisations-Mikroskopie), entwickelt von Betzig und Hess [ WO 2005/127592 ], ein Verfahren, bei dem die Probe flächig zyklisch zunächst mit einer ersten Wellenlänge beleuchtet wird, die einen Teil der Moleküle von einem Aus- in einen An-Zustand transferriert, und anschließend mit einer zweiten Wellenlänge im An-Zustand bestrahlt wird, wobei die Moleküle nach einigen Zyklen entweder bleichen oder in den Aus-Zustand zurückkehren. Danach wird dieser Zyklus wiederholt. Gleichzeitig wird die Fluoreszenz mit einer ortsauflösenden Kamera aufgenommen. In einer von Stefan Hell entwickelten Variante (PALMIRA: „PALM with Independly Running Acquisition”, A. Egner, Biophys. J. 93, 3285–90 (2007)] erfolgt die Bestrahlung mit beiden Wellenlängen gleichzeitig.
• – STORM (stochastical image reconstruction microscopy, stochastische Bildrekonstruktions-Mikroskopie), entwickelt von Rust und Zhuang [ WO 2008/091296 ], ebenfalls ein Verfahren mit flächiger Beleuchtung mit einer ersten Wellenlänge zum Einschalten eines Farbstoffes über einen Aktivatorfarbstoff, sowie einer zweiten Wellenlänge zum Anregen des detektierten Fluoreszenzfarbstoffs im An-Zustand. Die Detektion erfolgt mit einer ortsauflösenden Kamera.
• – dSTORM (direct STORM, direkte STORM bzw. direkte stochastische Bildrekonstruktions-Mikroskopie), entwickelt von Sauer [Heilemann et al., Ang. Ch. Int. Ed. 48 (37) 6903–8 (2009)], ebenfalls ein Verfahren mit flächiger Beleuchtung, bei der im Gegensatz zu STORM kein Aktivatorfarbstoff benutzt wird. Das Schalten von Farbstoffen kann entweder optisch mit einer zweiten Wellenlänge (bei Carbocyanin-Farbstoffen), oder mit einer einzigen Wellenlänge ausschließlich über die Pufferbedingungen gesteuert werden (z. B. bei Oxazin oder Rhodamin-Farbstoffen). Hier wird ebenfalls mit einer ortsauflösenden Kamera detektiert.
• – Das gezielte Schalten von Dunkelzuständen wird außerdem auch in der GSD-Methode verwendet, allerdings zielt diese Methode auf das Schalten von Farbstoffen durch Verlängerung der Lebensdauer des Triplett-Zustandes ab, z. B. durch Messungen in Polyvinylalkohol, PVA („Ground State Depletion”) [siehe z. B. DE 10 2006 021 317 B3 ; J. Fölling et. al., Nature Methods, 5, 943–5, 2008].
• – Die Unterschiede zwischen PALM, STORM, dSTORM und GSDIM liegen im Wesentlichen darin, welche Prozesse zum Schalten des Farbstoffes genutzt werden, sowie in der Probenpräparation und der Verwendung des Anregungslichtes.

In recent years, various new methods have been developed that circumvent this classic resolution limit. All methods have in common that they are based on the switching on and off of a fluorophore. With these methods, resolutions of up to 20 nm can be achieved. The most important methods are:

• STED (stimulated emission depletion microscopy), developed by Stefan Hell (cf. WO 1995/021393 A2 ; WO 2009/024529 ], a confocal method in which the image is obtained by a raster method.
• PALM (Photoactivated Localization Microscopy, Photoactivated Localization Microscopy) developed by Betzig and Hess WO 2005/127592 ], a method in which the sample is illuminated areal cyclically first with a first wavelength, which transfers a part of the molecules from an off to an on state, and then irradiated with a second wavelength in the on state, wherein the After a few cycles, molecules either bleach or return to the off state. Thereafter, this cycle is repeated. At the same time, the fluorescence is recorded with a spatially resolving camera. In a variant developed by Stefan Hell (PALMIRA: "PALM with Independently Running Acquisition", A. Egner, Biophys J. 93, 3285-90 (2007)], the irradiation with both wavelengths takes place simultaneously.
• - STORM (stochastic image reconstruction microscopy, stochastic image reconstruction microscopy), developed by Rust and Zhuang [ WO 2008/091296 ], also a method with planar illumination with a first wavelength for switching on a dye via an activator dye, and a second wavelength for exciting the detected fluorescent dye in the on state. The detection takes place with a spatially resolving camera.
• DSTORM (direct STORM, direct STORM or direct stochastic image reconstruction microscopy) developed by Sauer [Heilemann et al., Ang. Ch. Int. Ed. 48 (37) 6903-8 (2009)], also a method with surface illumination, in which unlike STORM no activator dye is used. The switching of dyes can be controlled either optically at a second wavelength (for carbocyanine dyes) or at a single wavelength only via the buffer conditions (eg, for oxazine or rhodamine dyes). Here is also detected with a spatially resolving camera.
• - The targeted switching of dark states is also used in the GSD method, but this method aims at the switching of dyes by extending the life of the triplet state, z. B. by measurements in polyvinyl alcohol, PVA ("Ground State Depletion") [see, for. B. DE 10 2006 021 317 B3 ; J. Fölling et. al., Nature Methods, 5, 943-5, 2008].
• - The differences between PALM, STORM, dSTORM and GSDIM lie mainly in which processes are used to switch the dye, as well as in the sample preparation and the use of the excitation light.

An important motivation of such high-resolution measurements are applications in which different structures are marked with spectrally different dyes, so that the spatial arrangement of different structures and their relationship to each other can be determined.

dSTORM

The device described herein uses the dSTORM technology described in Heilemann et al., Ang. Ch. Int. Ed. 48 (37) 6903-8 (2009)]. The disclosure of this document is hereby incorporated by reference into this specification.

dSTORM enables the switching of a single-wavelength dye, which makes labeling with multiple dyes much easier. In this case, a cellular structure is labeled with an antibody which is provided with a fluorescent dye. The sample is overlaid with a buffer solution containing a special buffer additive based on the ROXS technology, so that the dye molecules in the case of planar irradiation in a microscope with their excitation wavelength between on and off states change.

The composition of the ROXS buffer is in this case chosen differently than input described in the context of photostability, without significantly impairing the photostability of the fluorophores. Here, the concentration of the oxidizing and reducing agent is adjusted so that the molecules are for a long time in the non-luminous radical anion state (off state) and thus most molecules are not visible in a microscope image in a single image.

By taking a temporal image stack of approximately 1,000-10,000 individual images with exposure times for a single image of usually 50-100 ms, one obtains a series of images, wherein in each image a different subset of the molecules is switched on stochastically.

This amount must be so small in each image that the molecules in each image are far enough apart that every single molecule can be considered as a individual, spatially separated from the other molecular signals, diffraction-limited spot can be seen.

Now, in each individual image of the image stack, the center - the center of gravity - of the fluorescence is determined in each case for all single-molecule signals found. This can be done with very accurate precision, the theoretically possible accuracy is taking into account the recording time and emitted photon number up to about 20 nm [see R. E. Thompson et al., Biophys. J. 82, 2775-2783, 2002].

From all found molecular positions of the image stack, an image is then generated with a resolution that is given only by the accuracy of the position of the individual molecules in the images, not by the diffraction-limited optical resolution of a standard microscope, which thus improves approximately 10-fold becomes.

The method is based essentially on the switching of the molecules between on and off states of the dye. The ROXS technology was originally developed to suppress bleaching processes by photo-destruction by greatly reducing the residence time of the dye in the triplet state, which is highly susceptible to reactions with atmospheric oxygen. Initially, no value was placed on a subsequent longer residence time (and thus an off state) in the radical anion state.

For dSTORM microscopy, however, longer off-states are a prerequisite for being able to study only a small subset of currently fluorescent fluorophores. The same ROXS buffer additives are used for this, but the concentrations of the buffer additives are specially adapted to longer off times.

In both applications, the fact that the residence time of the dye in different on and off states can be adjusted by the addition of oxidizing and / or reducing substances is essential for the functioning of the method.

A particularly high spatial resolution for the life tests is obtained when using the high-resolution position determination using the dSTORM method or related technologies for the wide-field imaging. The fluorophores of interest can be identified via the camera image and subsequently measured time-resolved, in particular by means of TCSPC.

If the imaging is done via dSTORM, it is sensible to measure precisely those fluorophores that are currently in a fluorescent on state in a time-resolved manner.

The use of the other high-resolution techniques described above for this application is also possible. If other sub-diffraction-limited techniques than dSTORM are used, it may be necessary to use further illumination or de-excitation light, which, depending on the method used, is likewise coupled evanescently to the illumination of the sample.

In this way, spatially resolved fluorescence lifetime information is obtained, with the spatial resolution being below the diffraction limit (see also 7 below with associated description).

Foerster resonance energy transfer

However, the methods described above do not allow sufficiently well the spatial resolution of direct interactions between two labeled molecules, which take place in the range of less than 20 nm.

To measure direct interactions, cell biology often uses the excitation energy transfer from a donor dye excited with a spectrally suitable laser beam to an acceptor dye which emits fluorescence photons in another spectral range. This process is called Förster Resonance Energy Transfer (FRET). In this process, which has a maximum range of 10 nm, the fluorescence lifetime of the donor molecule is shortened [see, for example, US Pat. B. Stryer, Annu. Rev. Biochem. 47, 819-847 (1978)].

FRET measurements cover a distance range up to max. 10 nm down. The measurable distance range is thus in the range of molecular interactions and closes at the previously achieved resolution limit of about 20 nm of the above-mentioned. Procedures STED, GSDIM, STORM, dSTORM and PALM. With a combination of precise FRET measurements based on time-resolved methods with the spatially high-resolution methods, a measurement system is thus possible which can measure from 1 nm to 1000 nm in a spatially and temporally resolved manner.

The proposed method allows u. a. also selective FRET measurements for binding studies within cell membranes, whereby binding stoichiometries can also be determined. Furthermore, investigations of the positioning of individual biomolecules within cell membranes and thus investigations of the membrane structure are possible.

Furthermore, a device is proposed to solve the problem, which is suitable to carry out the proposed method.

• a) eine gepulste Anregungslichtquelle mit einer Pulslänge kleiner als 1 ns;
• b) Mittel zur Auflicht-Beleuchtung eines Probenträgers oder zum evaneszenten Einkoppeln des Lichts der Anregungslichtquelle in einen Probenträger; und
• c) einen optischen Aufbau, um von dem Fluorophor ausgehendes Fluoreszenzlicht durch eine Lochblende auf einen Detektor zu leiten,
• d) wobei der optischen Aufbau mittels der Lochblende ein Detektionsvolumen definiert, und
• e) wobei der Detektor derart ausgebildet ist, dass er einzelne Photonen des Fluoreszenzlichts mit einer zeitlichen Auflösung von besser als 1 ns detektieren kann. Ferner weist die Vorrichtung folgendes auf:
• f) einen ortsaufgelösten Detektor zur Aufnahme von Fluoreszenzlicht;
• g) Mittel zum Identifizieren einer interessanten Messregion innerhalb der Probe ausgehend von mindestens einer Aufnahme des ortsaufgelösten Detektors,
• g1) wobei hierfür ein Verfahren eingesetzt wird, welches eine Ortsauflösung unterhalb der Beugungsbegrenzung erreichen kann, insbesondere direkte stochastische Bildrekonstruktions-Mikroskopie;
• h) Mittel zum Positionieren des Detektionsvolumens auf die interessante Messregion; und
• i) Mittel zum Abrastern der interessanten Messregion mit dem räumlich begrenzten Detektionsvolumen (45) der zeitaufgelösten Detektion.

In particular, the device has:

• a) a pulsed excitation light source with a pulse length less than 1 ns;
• b) means for incident light illumination of a sample carrier or for evanescent coupling of the light of the excitation light source into a sample carrier; and
• c) an optical structure for directing fluorescence emitted by the fluorophore through a pinhole onto a detector,
• d) wherein the optical structure defined by means of the pinhole a detection volume, and
• e) wherein the detector is designed such that it can detect individual photons of the fluorescent light with a temporal resolution of better than 1 ns. Furthermore, the device has the following:
• f) a spatially resolved detector for receiving fluorescent light;
• g) means for identifying an interesting measurement region within the sample from at least one recording of the spatially resolved detector,
• g1) using a method which can achieve a spatial resolution below the diffraction limit, in particular direct stochastic image reconstruction microscopy;
• h) means for positioning the detection volume on the interesting measurement region; and
• i) means for scanning the interesting measuring region with the spatially limited detection volume ( 45 ) of the time-resolved detection.

Further details and features will become apparent from the following description of preferred embodiments in conjunction with the subclaims. In this case, the respective features can be implemented on their own or in combination with one another. The possibilities to solve the problem are not limited to the embodiments. For example, area information always includes all - not mentioned - intermediate values and all imaginable subintervals.

The embodiments are shown schematically in the figures. The same reference numerals in the individual figures designate the same or functionally identical or with respect to their functions corresponding elements. In detail shows:

1 a schematic representation of the optical structure;

2 a schematic representation of the evanescent illumination by total reflection (TIR) and incident light illumination;

3 a schematic representation of the superposition of TIR illumination and spatially limited detection in the sample on a slide; and

4 the schematic sequence of a data acquisition for a sub-diffraction-limited high-resolution fluorescence image and the simultaneous, selective uptake of fluorescence lifetime information.

contraption

1 shows a schematic representation of the optical structure.

Light of the beam source 10 is about a lens 11 , a dichroic beam splitter 12 and a microscope or its lens 13 in the sample carrier 14 coupled. The dichroic beam splitter 12 reflects the excitation light and transmits the fluorescent light.

The unit 37 allows the excitation light both on the optical axis of the lens to be coupled into this or parallel offset to the axis thereof. If the light is so decentralized with respect to the optical axis in the lens 13 coupled to it at a sufficiently large angle relative to the optical axis in the sample carrier 14 is coupled, it is at least at one, namely the upper surface of the sample carrier 14 totally reflected.

For this purpose, the sample preferably sits on that of the lens 13 facing away from the sample carrier 14 , In most cases, the microscope is operated as an inverted microscope, as this makes the sample more easily accessible for replacing the buffer. But also a construction with an upright microscope construction is possible when using suitable sample chambers.

Emitted fluorescent light is emitted from the lens 13 caught and over the beam splitter 12 , a beam splitter 16 and a lens or another lens 20 on a CCD camera 22 passed for the far-field observation of the sample.

To determine the fluorescence lifetime, the fluorescent light in the second beam path after the beam splitter 16 over different lenses 24 , a tiltable mirror unit 26 and a focusing lens 30 on a pinhole 32 behind which a time-resolved detector 34 , z. As a SPAD, (single photon counting avalanche photodiode) or a PMT (photo multiplier tube) behind another lens 36 the light is detected.

The tiltable mirror unit 26 allows the positioning of the detection volume in two mutually perpendicular directions. For measuring a sample area, either the sample carrier 14 be scanned, whereby an image can be generated with fluorescence lifetimes. Or - as explained above - it can with the CCD camera 22 directly a complete, spatially resolved image are taken. Thereafter, the fluorescent positions of the probe molecules can be located, whereupon the detection volume by means of the tilting mirror 26 can be directed to the selected areas.

The tiltable mirror unit 26 can be realized by a singular, two-axis tilting mirror (eg with piezo drive and / or miniaturized on MOEMS basis) or two uniaxial tilting mirrors (eg with galvo drives) with mutually orthogonal tilt axes. For quick start-up and regular scanning (and skipping uninteresting sample areas), a single-step operation (bridge & settle mode) is required, which allows positioning within less than 1 ms.

The following will be on 2 Referenced. 2 shows a schematic representation of the evanescent illumination by total reflection (TIR) and incident light illumination.

The beam path 41 becomes laterally in the microscope objective 13 coupled in that he - after passing through the immersion oil 40 - at the top of the sample holder 14 is totally reflected. As a result, only an excitation volume narrowly defined along the optical axis occurs 43 , This has an exponentially decreasing intensity profile along the optical axis with an extension of approximately 100 nm. This allows a selective examination of this limited area of the sample 50 ,

The light beam 42 schematically indicates incident light illumination.

The following will be on 3 Referenced. 3 shows a schematic representation of the superposition of TIR illumination 41 and spatially limited detection 45 in the sample 50 on a microscope slide 14 , The evanescent (TIR) illumination 41 leads to a spatially limited excitation volume along the optical axis 43 on the surface 46 of the sample carrier 14 , On the other hand, the detection using a pinhole leads 32 to a senkecht limited to the optical axis detection volume 45 , The overlap of these two volumes is smaller than the diffraction limit. The spatial extent of the overlapping area is about 100 nm × 500 nm × 500 nm.

measuring procedure

The typical procedure for a measurement is as follows:
The sample is placed on a sample carrier 14 arranged. Typically, the sample is a cell in whose membrane to be examined a fluorescently-labeled biomolecule is incorporated or attached.

A pulsed laser 10 is used as an excitation light either on the axis or off axis (laterally offset from the optical axis of the microscope objective 13 ) in this coupled.

The coupling of the excitation light into the sample carrier 14 can thus take place such that total reflection of the excitation light at the interface between sample carrier 14 and sample is achieved. As a result, the sample is selectively excited in an axially (with respect to the optical axis of the microscope) only about 100 nm deep area (total internal reflection fluorescence, TIRF). The excitation region in this case is significantly thinner than in conventional wide-field or confocal microscopy.

The limiting photobleaching of the sample is also greatly reduced by the addition of certain buffer substances (see the above description of the ROXS technology).

Examples of measurement conditions are z. In Heilemann et al., Ang. Chem. Int. Ed. 48 (37), 6903-8 (2009), which we hereby incorporated by reference into the content of this specification. For many dyes (eg, ATTO520 or Alexa Fluor 532), a pH 7.4 phospate buffered saline buffer (pH 7.4) is used with the addition of a thiol as a reducing agent, such as mercaptoethylamine (MEA) Concentration of the thiol is often 10-100 mM.

The fluorescent light is transmitted through the lens 13 collected again.

That on the pinhole 32 Focused light comes with a fast detector 34 detected via single photon counting. In this way, both measurements of the intensity and the fluorescence lifetime in one point are possible.

In order to obtain a time-resolved image, the imaged detection volume now appears above the pinhole 32 attached tiltable detection mirrors 26 scanned. In this way, a raster image of a sample area is created.

Among other things, the proposed method differs from existing confocal microscopy methods in that it does not scan excitation and detection volumes simultaneously. In the present case, only the detection volume is scanned. This preserves the possibility of simultaneously recording a stationary wide-field image with the same illumination.

Alternative to the tiltable mirrors 26 can also do the sample with the sample carrier 14 be moved.

Simultaneously with the scanning of the punctiform detection volume is by means of a beam splitter 16 a part of the fluorescent light on a surface detector 22 directed. The beam splitting in the beam splitter 16 can be due to a variety of discrimination characteristics (intensity ratios, polarization states, spectral splitting, etc.).

That with the CCD camera 22 The recorded wide field image of larger parts of the surface of the sample carrier is analyzed to identify interesting areas. This can be z. B. by means of a simple intensity threshold or more complex algorithms.

The information obtained in this way is used so that subsequently only the areas of interest are scanned or scanned. The other areas are skipped, resulting in a considerable time savings.

• – Der einfachste Fall ist derjenige, bei dem ein Nutzer auf dem Weitfeldbild einen ihn interessierenden Bereich markiert. Dieser wird anschließend abgescannt.
• – Für die Detektion einzelner Moleküle bzw. Fluorophore bestimmt ein Algorithmus automatisch aus dem Weitfeldbild Einzelmolekülsignale. Anschließend fährt der Scanner diese sequentiell an, wobei die Aufnahmezeit der konfokalen Aufnahme unabhängig einstellbar ist.
• – Zur Bestimmung zellulärer Strukturen werden über eine Schwelle (Threshold) oder andere Auswahlkriterien die interessanten Bereiche ausgewählt. Der Scanner 26 fährt die entsprechenden Bereiche ab. Dabei kann auch ein Bild generiert werden.
• – Es ist auch ein Feedback-Loop-denkbar, in dem z. B. nach Anfahren eines konfokalen Punktes das Molekül selektiv gebleicht oder geschaltet wird.
• – Durch die gleichzeitige Weitfeldaufnahme ohne Umschaltung von optischen Elementen im Strahlengang können auf dem Flächendetektor 22 permanent intensitätsbasierte Bilder der gesamten Probe aufgenommen werden. Dies ermöglicht z. B. die Untersuchung von Dynamiken im ms-Bereich.
• – Es können weiterhin auf Basis des Kamerabildes spezielle Bereiche in der Probe (Englisch: Region of Interest (ROI)) durch geeignete Algorithmen sehr schnell ermittelt werden, in welchen selektiv die zeitaufgelöste Detektion stattfindet

The interactive feedback between capturing the wide field image with the CCD camera 22 and the control of the confocal scanner and the tilting mirror 26 can be done in different ways:

• The simplest case is the one where a user marks an area of interest on the wide field image. This is then scanned.
• - For the detection of single molecules or fluorophores, an algorithm automatically determines single-molecule signals from the wide-field image. Subsequently, the scanner scans them sequentially, whereby the recording time of the confocal recording is independently adjustable.
• - To determine cellular structures, the areas of interest are selected via a threshold or other selection criteria. The scanner 26 moves off the corresponding areas. An image can also be generated.
• - It is also a feedback loop conceivable in the z. B. after approaching a confocal point, the molecule is selectively bleached or switched.
• - Due to the simultaneous far field recording without switching of optical elements in the beam path can on the area detector 22 permanently intensity-based images of the entire sample are taken. This allows z. For example, the study of dynamics in the ms range.
• - It can also be based on the camera image specific areas in the sample (English: Region of Interest (ROI)) determined very quickly by suitable algorithms in which selectively takes place the time-resolved detection

Description of the ROXS preparation

The limiting photobleaching of the sample is also greatly reduced by the addition of certain buffer substances [see WO 2009/003948 A2 ]. This also allows long far-field illumination times, which are necessary for the comparatively slow-scanning, sequential detection and for the repeated acquisition of CCD images for superresolution microscopy. This is particularly important for those pixels that are queried only at the end of the image capture, but have been illuminated since the beginning of recording.

The ROXS technology can be used in two ways: If only photobleaching is to be minimized, the buffer additives used in the ROXS technology are selected so that the molecule is returned to the S0 ground state as quickly as possible.

This method is described in the publication by J. Vogelsang et al., Ang. Chem. Int. Ed. 47 (29), 5465-5469 (2009) and in the patent WO 2009/003948 A2 described. Here it is shown that z. B. for individual molecules of the dye ATTO647N the time to photobleaching by addition of 1.8 mM of the oxidizing agent methylviologen and 0.5 mM of the reducing agent Trolox in the sample buffer of ~ 20 s (in an exemplary construction) can be increased to over 20 min, ie to 60 times. The principle is usually based on that z. B. the reducing agent, the dye, when it is currently in the triplet state, reduced to a radical anion, which is oxidized by the oxidant back to the ground state. Generally, with the ROXS technology, a dye can be converted to a charged state by the oxidizing or reducing agent. This shortens the temporal duration in the bleach-sensitive triplet state and makes the dye less susceptible to photobleaching.

Since the charged state itself represents a normally long-lived dark state, also by lowering the concentration of the oxidizing agent, the residence time of the dye in the dark state, as is the case with the dSTORM method.

In this second use of the ROXS method described, for example, in the publication M. Heilemann et al., Angew. Chem. Int. Ed. Engl. 48 (37) 6903-8 (2009), the concentrations of the oxidizing and / or reducing agent in the sample buffer are adjusted so that the dye stays in the charged reversible dark state for a statistically longer time. Furthermore, by adjusting the ratio between the reducing agent and the oxidizing agent, the duration of the non-fluorescent "off" state of a fluorescent dye can be adjusted in the range of nanoseconds down to the millisecond range. Thus, the duration of the non-fluorescent "off" state of a fluorescent dye in the range of ≥ 10 ns to ≤ 200 ms, preferably in the range of ≥ 100 ns to ≤ 100 ms, preferably in the range of ≥ 1 ms to ≤ 100 ms, especially preferably in the range of ≥ 5 ms to ≤ 20 ms.

In addition, the duration of the fluorescence "on" state of the fluorescent dye may be controlled by the laser power, for example, and may be about 100 ms / cm 2 for about 5 ms. For example, an increase in the laser power can cause a corresponding antiproportionale shortening of the duration of the fluorescent "on" state of the fluorescent dye. In this case, the number of emitted photons during an "on" state preferably does not change approximately. On the other hand, the duration of the "on" state can additionally be influenced by an increased addition of oxidizing agent or reducing agent by adding at concentrations ≥ 10 μmol / l to ≤ 1 mol / l, preferably ≥ 100 μmol / l to ≤ 50 mmol / l, also the singlet states are depopulated by photoinduced electron transfer. In this type of "on" state control, the number of emitted photons may decrease accordingly during an "on" state.

Preferred buffers and agents for this method correspond to the agents of the photostability enhancement already described. Preferred solvents are, for. B. Buffer solutions, preferably selected from the group comprising phosphate buffered saline (PBS) and / or BSS (balanced salt solution), but also solvents other than water, such as alcohols, dimethyl sulfoxide (DMSO), glycerol, organic solvents and / or mixtures thereof ,

Preferred oxidizing agents are, as already described above, bipyridinium salts, their derivatives, preferably viologens, in particular methyl viologens, and / or nitroaromatics, in particular carboxylic acid-substituted nitroaromatics or sulfonic acid-substituted nitroaromatics, preferably nitrobenzenes, benzoquinones, substituted benzoquinones, in particular chlorine-substituted and / or cyano -substituted benzoquinones, especially dichlorobenzoquinone, tetrachlorobenzoquinone and / or mixtures thereof, and z. As phenols or quinones.

Preferred reducing agents come from the group of aliphatic or aromatic amines, thiophenols, thionaphthols, phenol sulfides, uric acid (urate), urea (urea), bilirubin, ascorbic acid and / or flavins, and 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid, ascorbic acid, β-mercaptoethanol, β-mercaptoethylamine, β-naphthylamine, dithiothreitol, NaBH 3 CN, n-propyl gallates and / or mixtures thereof.

In further embodiments, the concentration of a redox buffer comprising at least one reducing agent and / or at least one oxidizing agent or a reduction-oxidizing agent, for example in an aqueous solution, preferably in the range of ≥ 0 mM to ≤ 100 mM, preferably in the range of ≥ 0 , 01 mM to ≤ 50 mM, more preferably in the range of ≥ 0.1 mM to ≤ 10 mM, also preferably in the range of ≥ 0.2 mM to ≤ 5 mM, particularly preferably in the range of ≥ 0.5 mM to ≤ 2 mM.

To adjust the fluorescence state of a fluorescent dye, the ratio of reducing agent to oxidizing agent may be shifted significantly to one side or the proportion of reducing agent or oxidizing agent may be zero. For example, the ratio of reducing agent to oxidizing agent in the range of ≥ 1000: 1 to ≤ 1: 1000, preferably in the range of ≥ 1000: 1 to ≤ 1: 100 or in the range of ≥ 100: 1 to ≤ 1: 1000, more preferred in the range of ≥ 100: 1 to ≤ 1: 100, more preferably in the range of ≥ 10: 1 to ≤ 1:10.

Single-molecule measurement

The following will be on 4 Referenced.

When using dSTORM usually only a few fluorophores light up on the detection surface and are correspondingly far apart.

To find these, then to measure and finally to generate a high-resolution wide field image or to gain point statistics, is typically with the CCD camera 22 started recording a picture batch. The single pictures 65 . 66 . 67 are analyzed in real time to the position of a switched on fluorophore 54 to identify. This should shine as bright as possible, none Neighboring molecules and can be reached quickly with the scanner.

After identifying such a fluorophore 54 in the first frame 65 the punctual detection volume is moved to its position within the on-time of the identified fluorophore. Subsequently, the fluorescence decay curve 56 this fluorophore was added. Time-shifted with the CCD-detector 22 already another single picture 66 in which a new fluorophore can be identified, targeted and characterized. Thus one obtains additional lifetime information for (at least) one fluorophore per image.

The result is in 4 shown schematically. The frame 53 is a schematic representation of a sample labeled with individual fluorophores. The frame 61 shows the sub-diffraction-limited fluorescence image, built from all the midpoints of the in the individual images 65 . 66 . 67 recorded diffraction-limited spots. In 62 the fluorophores are additionally coded life-coded. In this example, the fluorophores in different structures show different lifetimes (1 ns and 3 ns). For a part of the fluorophores 63 there is no life information.

In this way one obtains a sub-diffraction-limited, spatially resolved image with fluorescence lifetime information.

Description of the FRET measurements

In order to still be able to use FRET for determining molecular interactions in addition to the high-resolution position determination, a second structure, for. As a protein or a lipid o. Ä., Which is incorporated in certain membrane structures, directly excited (fluorescent protein) or labeled with a second dye (acceptor). This absorbs in a long-wavelength shifted region of the optical spectrum, wherein for the energy transfer, the emission of the first, excited dye (donor) with the absorption of the second dye (acceptor) has to overlap spectrally.

The first dye (donor) is not only prevented from bleaching by the addition of certain buffering agents, but is also caused to switch between on and off states at the picking conditions (see WO 2009/003948 A2 ).

Examples of measurement conditions are z. In Heilemann et al., Angew. Chem. Int. Ed. 48 (37), 6903-8 (2009). For many dyes (eg, ATTO520 or Alexa Fluor 532), pH 7.4 phospate buffered saline (PBS) is used with the addition of a thiol as a reducing agent, such as mercaptoethylamine (MEA) Of the thiol is often 10-100 mM.

The fluorescent light is transmitted through the lens 13 collected again and by means of the beam splitter 16 split for simultaneous wide-field and time-resolved, point-like detection.

With the CCD camera 22 a picture stack is taken. When switching the molecules in the dSTORM process, only a single molecule of the donor may be in its on state at all times within the optical resolution. This is ensured by the setting of the buffer as well as by the intensity of the excitation light and / or by the applications of a second wavelength with which the first dye can possibly be photo-switched. Examples of this can be found in the publication by M. Heilemann et al., Angew. Chem. Int. Ed. 48 (37): 6903-8 (2009) for dyes switched at one wavelength only, or in the publication by M. Heilemann et al, Angew. Chem. Int. Ed. 47 (33): 6172-6 (2008) for dyes in which two wavelengths are necessary for switching. For the former case, for example, for the dye ATT0655 the use of a PBS buffer with pH 7.4 with an addition of 100 mM mercaptoethylamine (MEA) is common, the illumination intensity z. B. 1.5 kW / cm ² at 647 nm. Within a single image (recording time, eg 50 ms) of the image stack, only signals of individual molecules are present.

With subpixel resolution, the center of gravity of the diffraction-limited fluorescence signal and thus its position is determined for each molecule found (localization microscopy) [s. z. R. E. Thompson et al., Biophys. J., 82, 2775-2783 (2002) or S. Wolter et al., J. Microsc., 237, 12-22 (2010)].

From all found positions of a picture stack, a high-resolution picture of all found positions is generated. It is important that for each position found still the information about the temporal occurrence within the recording is available.

The second part of the detected fluorescence light is on the pinhole 32 and behind it on the fast and sensitive detector 34 focused. This detector registers single photons and their time arrival in relation to the exciting laser pulse via single photon counting. In this way measurements of the fluorescence lifetime in one point are possible.

As a shortened fluorescence lifetime in a fluorescence signal on a By suggesting resonance energy transfer with the second dye (acceptor), the localization of these interactions is unambiguously assigned to the signals on the calculated positioning image, ie the interaction sites between donor and acceptor molecule can be unambiguously assigned on the basis of their lifetime. Not only structures can be displayed with high resolution, but also interaction centers.

Possibilities of use

First possibility: spatially high-resolution lifetime imaging

The sample is scanned continuously with the confocal, temporally high-resolution detection channel and in parallel with the camera 22 added.

After the measurement becomes the fast detector 34 measured fluorescence decay time at different times of recording with the information of the camera 22 correlated to the different times of the measurement.

Thus, within the range of the camera image which corresponds to the imaging range of the fast point detector, the fluorescence signals of the camera image can be assigned to specific fluorescence lifetimes. From this can be z. B. determine distances using FRET, which are an order of magnitude more accurate than the distances can be determined with a camera based on the high-resolution approaches such as STORM, PALM etc ..

second possibility: Single Molecule Feedback

Since the point-shaped detection area, given by the pinhole 32 , can be scanned during the camera recording a feedback done in such a way that by online image analysis of the camera data coordinates of molecules are identified, which are currently in the on state.

These coordinates are passed on within a few milliseconds to the control unit of the scanner, so that the point-shaped detection volume shifts within a desired image region.

The blinking times of the molecules (one molecule is typically about 100 ms) are here by means of the specially selected buffer on the recording rate of the camera (about 20 Hz corresponds to 2 frames per 100 ms) tuned by exploiting the ROXS technology concentration of the oxidizing or reducing agent is adjusted so that the on times of the dye reach the desired length of on average 100 ms.

• – Erstes Bild (Frame) der Kamera 22 (50 ms): es werden Daten mit der Kamera gesammelt.
• – Zweites Bild (50 ms):
• – In den ersten 10 ms erfolgt die Datenverarbeitung (Kamera auslesen, Position der gerade leuchtenden Moleküle innerhalb der Probe identifizieren).
• – In den nächsten 10 ms wird das konfokale Detektionsvolumen auf das zuvor identifizierte Molekül positioniert.
• – In den folgenden 40 ms werden im konfokalen Detektionsvolumen zeitaufgelöste Daten zur Bestimmung der Fluoreszenzlebensdauer aufgenommen.
• – Während der gesamten 50 ms wird im Weitfeld-Detektionsvolumen ein neues Kamerabild aufgenommen, um anschließend ein neues Molekül auszuwählen und den Scanner zu repositionieren.

This allows the following scenario (see also 4 ). All mentioned times are only exemplary and may vary in magnitude as well as relation to each other.

• - First picture (frame) of the camera 22 (50 ms): data is collected with the camera.
• - Second image (50 ms):
• - Data processing takes place in the first 10 ms (read out the camera, identify the position of the currently illuminated molecules within the sample).
• In the next 10 ms, the confocal detection volume is positioned on the previously identified molecule.
• - In the following 40 ms time-resolved data are recorded in the confocal detection volume to determine the fluorescence lifetime.
• - During the entire 50 ms, a new camera image is recorded in the far-field detection volume, in order to then select a new molecule and reposition the scanner.

Using this algorithm, the lifespan image acquisition time can be significantly reduced under the high resolution buffer conditions, since the ratio of on to off times of the molecules must be about 1: 100, depending on the desired high resolution. This means that in the worst case, without this method, one would have to spend more than 100 times the actually required measurement time for the determination of a fluorescence lifetime.

In this way, alternatively, the lifetime of a significantly larger number of molecules can be determined per unit of time.

LIST OF REFERENCE NUMBERS

10

laser

11

lens

12

dichroic beam splitter

13

lens

14

sample carrier

16

Beam combination / division z. 50/50

20

tube lens

22

CCD camera

24

Telescope (telecentric system)

26

Scan mirror

30

tube lens

32

pinhole

34

temporally high-resolution single-photon detector

36

lens

37

Beam Shifter (module for parallel beam offset)

40

Immersion oil

41

Excitation light for evanescent lighting

42

Excitation light for incident illumination

43

TIR excitation volume

45

Spatially limited detection volume

46

Top of the slide 14

50

sample

53

Fluorophore labeled probe

54

Spot of a luminous fluorophore

56

Decay curve of a single, luminous fluorophore

61

sub-diffraction-limited fluorescence image

62

Fluorescence image with additional life information

63

Fluorophore without life information

65

CCD single frame

66

CCD single frame

67

CCD single frame

quoted literature

cited patent literature

• DE 10 2006 021317 B3
• DE 10 2007 030 403 A1
• EP 1 291 627 B1
• WO 1995/021393 A2
• WO 2006/127692
• WO 2009/024529
• WO 2009/003948 A2

quoted non-patent literature

• W. Becker, Advanced Time-Correlated Single Photon Counting Techniques, Springer Series in Chemical Physics, 2005
• A. Egner: "PALM with Independently Running Acquisition", Biophys. J. 93, 3285-90 (2007)
• A. Esposito, F.S. Wouters, "Fluorescence Lifetime Imaging Microscopy", Curr. Prot. Cell Biol., Unit 4.14 (2004)
• K.N. Fish, Curr. Protoc. Cytom., Chapter 12, Unit 12.18 (2009)
• J. Folling et al., Nature Methods, 5, 943-5 (2008)
• M. Heilemann et al., Angew. Chem. Int. Ed. 47 (33) 6172-6 (2008)
• M. Heilemann et al., Angew. Chem. Int. Ed. 48 (37) 6903-8 (2009)
• J. Lakowicz, "Principles of Fluorescence Spectroscopy", 3rd. Ed., Springer; Cape. 13 (2006)
• D. O'Connor, D, Phillips, Time Correlated Single Photon Counting, Academic Press (1984)
• L. Stryer, Annu. Rev. Biochem. 47, 819-847 (1978)
• R. E. Thompson et al., Biophys. J. 82, 2775-2783 (2002)
• J. Vogelsang et al., Ang. Chem. Int. Ed. 47 (29), 5465-5469 (2008)
• Tinnefeld, P .; Bauschmann, V .; Weston, K. D .; Biebricher, A .; Herten, D. -P .; Piestert, O .; Heinlein, T .; Heilemann, M .; Sauer, M .: How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interaactions. In: Recent Research Development in Physical Chemistry, Vol. 7, 2004, pp. 95-125.
• Tony Wilson, "Confocal Microscopy: Basic Principles and Architectures," in: A. Diaspro, "Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances," Wiley-Liss (2002)
• Wolter, S., et al., J. Microsc., 237, 12-22 (2010).

Claims (8)

Method for spatially and temporally high-resolution detection of the fluorescence of a fluorophore of a sample, comprising the following steps: a) the sample is deposited on the surface of a sample carrier ( 14 ) arranged; b) adding to the sample a redox buffer comprising at least one reducing agent, and / or at least one oxidizing agent and / or at least one reducing-oxidizing agent to prevent photobleaching of the fluorophore; c) pulsed excitation light ( 10 ) with a pulse length less than 1 ns is in such a way in the sample carrier ( 14 ) that it either radiates the sample or at the location of the sample the sample carrier ( 14 ) leaves only evanescently; d) the wavelength of the excitation light ( 10 ) is chosen so that the excitation light is capable of exciting the fluorescence of the fluorophore of the sample; e) fluorescent light emanating from the sample is coated with an optical structure ( 13 . 24 . 30 ) through a pinhole ( 32 ) to a detector ( 34 ), the optical structure ( 13 . 24 . 30 ) by means of the pinhole ( 32 ) a detection volume ( 45 ) Are defined; f) the detector ( 34 ) is selected so that it can detect individual photons of the fluorescent light with a temporal resolution of better than 1 ns; g) the fluorescent light is split and additionally with a spatially resolving detector ( 22 ) recorded; h) by means of the spatially resolving detector ( 22 ) identifies an interesting measurement region within the sample; i) the spatially limited detection volume ( 45 ) of the time-resolved detection is positioned on the interesting measurement region in the sample; and j) the interesting measuring region is compared with the spatially limited detection volume ( 45 ) of the time-resolved detection. Process according to the preceding claim, characterized in that the reducing agent of the redox buffer is selected from the group comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ascorbic acid, β-mercaptoethanol, β-mercaptoethylamine, β-nathylamine, dithiothreitol, NaBH 3 CN, n-propyl gallate and / or mixtures thereof; and / or that the oxidizing agent of the redox buffer is selected from the group comprising Bipyridinium salts, preferably viologens, especially methyl viologens, nitroaromatics, in particular carboxylic acid-substituted nitroaromatics or sulfonic acid-substituted nitroaromatics, preferably nitrobenzenes, benzoquinones, substituted benzoquinones, in particular chlorine-substituted and / or cyano-substituted benzoquinones, in particular dichlorobenzoquinone, tetrachlorobenzoquinone and / or mixtures thereof; and / or that the reduction-oxidation agent of the redox buffer is selected from the group comprising ubiquinone or cytochrome. Method according to one of the preceding claims, characterized in that oxygen is selected as the oxidizing agent of the redox buffer; that the sample is in aqueous solution; and that the oxygen content of the redox buffer is adjusted by adding an enzymatic system comprising glucose oxidase or by flowing the aqueous solution with nitrogen. A method according to claim 1, characterized in that the position of a fluorophore to be examined is identified as a function of a change in state of the fluorophore. A method according to claim 1, characterized in that for identifying the location of the sample, a method is used, which can achieve a spatial resolution below the diffraction limit, in particular direct stochastic image reconstruction microscopy. Method according to one of the preceding claims, characterized in that for the measurement of the sample additionally Förster resonance energy transfer is utilized. Apparatus for carrying out the method according to one of the preceding method claims. Device for spatially and temporally high-resolution detection of the fluorescence of a fluorophore of a sample comprising: a) a pulsed excitation light source ( 10 ) with a pulse length less than 1 ns; b) means for incident illumination of a sample carrier ( 14 ) or for the evanescent coupling of the light of the excitation light source ( 10 ) in a sample carrier ( 14 ); c) an optical structure ( 13 . 24 . 30 ) fluorescent light emanating from the sample through a pinhole ( 32 ) to a detector ( 34 ), the optical design ( 13 . 24 . 30 ) by means of the pinhole ( 32 ) a detection volume ( 45 ) and e) where the detector ( 34 ) is designed so that it can detect individual photons of the fluorescent light with a temporal resolution of better than 1 ns; f) a spatially resolved detector ( 22 ) for receiving the fluorescent light emitted by the fluorophore; g) means for identifying an interesting measurement region within the sample from at least one recording of the spatially resolved detector, g1) using a method which can achieve a spatial resolution below the diffraction limit; h) means ( 26 ) for positioning the detection volume ( 45 ) on the interesting measuring region; and i) means for scanning the interesting measuring region with the spatially limited detection volume ( 45 ) of the time-resolved detection.

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