DE102008024568A1 - 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 - Google Patents

Abstract

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 limit of the imaging of the specimen onto the sensor array; registering the fluorescent light which is spontaneously emitted from the region of molecules of the substance, in several images recorded by the sensor array during continued exposure of the specimen to the light of the one wavelength; and determining the position in the specimen of the molecules of the substance that are respectively in the first state, which lie at a distance from their closest neighboring molecules in the first state which is greater than the spatial resolution limit of the imaging of the specimen onto the sensor array, from the images recorded by the sensor array, where the first state and the second state are different electronic states of the substance. The first state is a singlet state and the second state is a triplet state of the substance.

Description

TECHNICAL FIELD OF THE INVENTION

The The invention relates to a method for spatially high resolution Imaging an interesting structure of a sample with the features the preamble of independent claim 1 and a fluorescent light microscope with the features of the preamble of independent claim 17.

STATE OF THE ART

From the WO 2006/127692 A2 For example, there is known a method for spatially high resolution imaging of a sample structure of interest, wherein the structure of interest is labeled with phototransformable optical labels. Of the markers, a subgroup is in each case activated in a state in which they can be excited to emit fluorescent light. In this case, the respective subgroup comprises so few of the markers that they have a distance from each other that is greater than the spatial resolution limit when imaging the sample onto a sensor array. This makes it possible to localize, after excitation of the fluorescence subgroup markers, the origins of the fluorescence light at a resolution better than the diffraction limit that applies to the spatial resolution in imaging the sample on the sensor array, thereby also indicating a point of the labeled structure of interest is detected with this increased resolution. The phototransformable optical markers are in the WO 2006/127692 defined so that they can be turned on by an activating signal in the state in which they are excitable for the emission of fluorescent light. In this case, this activating signal can be identical to the excitation light that the markers then excited to fluorescence.

More concrete embodiments of phototransformable optical markers disclosed in U.S. Pat WO 2006/127692 include only photoactivatable fluorescent proteins, ie molecules that only after they have absorbed at least one quantum of light to become a fluorophore or, in other words, have to be switched on before they are fuoreszent. This activation or switching process is accompanied by a change in the molecular structure of the molecules (rearrangement of atomic groups or even breakage or formation of a bond). That from the WO 2006/127692 known method is also referred to as PALM (Photoactivated Localization Microscopy).

In a similar, STORM (Stochastic Optical Reconstruction Microscopy) called and of Rust et al. in Nature Methods, 3, 793-796 (2006) described methods are also used in a fluorescent state switchable molecules, which are not proteins but photoswitchable organic fluorophores, specifically the fluorescent dyes Cy3 and Cy5, is. It is known of these cyanine dyes that they can be switched between different conformational, concretely isomeric states.

Out C. Geisler et al .: Resolution of λ / 10 in fluorescence microscopy using fast single molecule photo-switching, Appl. Phys. A 88, 223-226 (2007) is known as PALMIRA method with the features of the preamble of independent claim 1 known. Again, an interesting structure of a sample is labeled with a switchable fluorescent protein. Specifically, this is a protein called rsFastLime, which is excited to fluoresce not only in its initial state by light of wavelength 488 nm, but also proportionately turned off in a non-fluorescent state and is partially switched back into its fluorescent state. The underlying mechanism is a conformational change of the fluorophore. These properties of the switchable fluorescent protein make it possible to tune with light of only a single wavelength changing subgroups of fluoresceable molecules of the protein, in which the fluorescence-capable molecules have a distance greater than the diffraction limit, and to excite the fluorescence-capable molecules to fluorescence. In this context, images can be taken continuously, ie at high frequency, which record the changing subgroups of the fluorescent molecules and in which the position of the respectively detected molecules can be determined with an accuracy beyond the diffraction limit.

With the sum of the images becomes the structure in the sample with a Diffraction limit below spatial resolution detected.

The switchable fluorescent dyes used in the method described above are the first time in a designated as RESOLFT (Reversible Saturable OticaL Fluorescence Transition) and from DE 103 25 460 A Known methods have been used for spatially high resolution imaging of a substance-labeled structure of a sample. Here, the switchable fluorescent dyes are explained as being selected from a group of substances which can be repeatedly transferred from a first state having first optical properties to a second state having second optical properties by means of an optical switching signal and which can return to the first state from the second state, wherein the two states differ in at least one of the following criteria: conformational state of a molecule; Structural formula of a molecule; spatial arrangement of atoms within a molecule; spatial arrangement of bonds within a molecule; Addition of further atoms or molecules to a molecule; Grouping of atoms and / or molecules; spatial orientation of a molecule; Orientation of adjacent molecules to each other and from a variety of molecules and / or atoms formed order.

The Selection of Switchable Proteins and Fluorophores Used for the above-described methods PALM, STORM, PALMIRA and RESOLFT are usable compared to the total of the principle known and available fluorescent dyes very small. Dyes which are both switchable and (in one of the switching states) are capable of fluorescence, are very rare. They will therefore synthesized and optimized in complex procedures. Come in addition, that the switching and the fluorescence behavior are very different from the depend on the chemical environment of the molecule. The applies to both switchable fluorescent proteins as well for switchable organic fluorophores. This defect is as fundamental and depends inter alia together with that fluorescence and switching of the molecule are competing molecular processes that often out of one and the same excited state in competition with each other stand. Also the brightness of the switchable fluorescent dyes in their fluorescent state, ie. the relative yield of fluorescent light from a molecule during repeated stimulation against a variety of non-switchable organic Fluorophores and non-switchable fluorescent proteins frequently just small. The strong restrictions through switchable However, proteins or fluorophores have been accepted so far the achievable by the above-mentioned methods high spatial resolutions in the picture to realize structures of interest.

In the so-called GSD (Ground State Depletion) microscopy ( S. Bretschneider et al .: Breaking the diffraction barrier in fluorescence microscopy by optical shelving, Phys. Rev. Lett. 98, 218103 (2007) ) the diffraction limit is exceeded when imaging a structure labeled with a fluorescent dye in a sample in that the respective fluorescent dye is transferred outside the respective measurement point from its electronic ground state, from which it can be excited with excitation light to fluorescence, into an electronic dark state he is not fluorescent. This is done prior to excitation of the remaining molecules at the measurement point for fluorescence with de-population light of the same wavelength as the excitation light. Typically, the electronic dark state is a triplet state while the ground state of the fluorescent dye is a singlet state. Typically, the molecules return thermally, ie not switched (optically), from this dark state to the electronic ground state, so that only light of a single wavelength, namely the excitation light, is necessary for carrying out the experiment.

OBJECT OF THE INVENTION

Of the Invention is based on the object, a method for spatial high-resolution imaging of a structure of interest a sample having the features of the preamble of the independent claim 1 and a fluorescence light microscope suitable for implementing this method, with which the resolution advantages of as PALM, STORM or PALMIRA known methods are usable without their disadvantages due to the Limited to switchable fluorescent dyes in purchase to have to take.

SOLUTION

The The object of the invention is achieved by a method for spatial high-resolution imaging of a structure of interest a sample having the features of the independent claim 1 and by a fluorescent light microscope with the features of solved in addition to ordered claim 17. Preferred embodiments of the new method and the new fluorescent light microscope defined in the dependent claims.

DESCRIPTION OF THE INVENTION

In the new method, the first and second states are different electronic states of the substance, ie, states of the substance that differ only from an electronic point of view. At the beginning of loading the sample with the light of one wavelength, its intensity is adjusted so that the substance is converted from its fluorescent first state to its second non-fluorescent or at least distinguishable fluorescent state, up to at least 10%, preferably at least 50% more Preferably, at least 90% and most preferably at least 95% of the molecules remaining in the first state of the substance have the critical distance to their closest adjacent molecules in the first state that is greater than the spatial resolution limit of imaging the sample on the sensor array. This is usually synonymous with more than 90%, preferably More than 95%, more preferably more than 98% and most preferably more than 99% of the molecules of the substance are converted into the second electronic state. Subsequently, images in which the fluorescent light spontaneously emitted from the substance is registered are recorded with the sensor array while the sample is further supplied with the light of the one wavelength. In this case, the intensity of the light, which is reduced as required relative to the initial intensity, causes the molecules, which are still each in the first state, to be excited for the spontaneous emission of fluorescent light which is registered in the images, and to a lesser extent into the or otherwise fluorescent second state can be transferred. This smaller part of the molecules of the substance is ideally just as large as a proportion of the molecules of the substance which return from the second state spontaneously or under the action of the same light of the one wavelength simultaneously to the first state. The images taken continuously with the exposure of the sample to the light of the one wavelength thus always record fluorescent light from the individual molecules of the substance so far that their position in the sample can be determined with an accuracy beyond the diffraction limit. From the sum of the individual positions registered in this way, the structure of interest in the sample, which is marked with the substance, can also be detected with a spatial resolution beyond the diffraction limit.

It is not always necessary, the intensity of light one wavelength for taking pictures but only if the initial concentration the fluorophore was very large. At middle and low Concentrations can be the intensity of set in front so that the singulating and registering of molecules with the same value of intensity is to perform. This is related to that the number of photons acting as fluorescent light from the fluorescent dye in the first state in succession until the transfer emitted in the second state (in the same fluorescence burst), largely independent of the intensity of the light used is.

It is quite surprising that the process according to the invention, although having essentially the same procedure as the process known as PALMIRA, manages without switchable proteins or fluorophores. Rather, in the new method, a substance is used as the fluorescent dye, which does not fall under the definitions of DE 103 25 460 A and which may be any conventional, non-switchable fluorescent dye. Almost all conventional fluorescent dyes, in addition to their electronic ground state from which they are excitable for fluorescence, an electronic dark state in which they can be converted at a relevant rate due to excitation with light of the same wavelength, as it is used to excite the fluorescence , As a rule, this is a singlet state as a fluorescence-capable ground state and a triplet state as a dark state. In normal fluorescent light microscopy, the partial transfer of a fluorescent dye instead of its singlet excited state into its non-fluorescent triplet state - especially at higher intensities of the excitation light - is disadvantageous because it reduces the yield of fluorescent light from a sample. In the present invention, this effect is exploited precisely because the position of each individual molecule of the substance with which the structure of interest in the sample is labeled can be detected with a resolution better than the diffraction limit only when the fluorescent light is isolated from the molecule that is, can be registered separately from the fluorescent light of adjacent molecules. In each case only a few of the molecules may be in the first state.

at In many cases, it is beneficial for the new process to take measures to seize the life of the non-fluorescent second Change the state of the fluorescent dye in the sample. In contrast to conventional fluorescence microscopy is However, it is often not an extension but a shortening of the life of the non-fluorescent second state. Among the measures that such a reduction effect the life, include a cooling the sample is at low temperatures at which thermal stimuli reduced to collision-induced transitions are a lowering of the concentration to the triplet state of the Fluorescent dye quenching oxygen in the sample, for example, with oxygen-binding glucose oxidase or by Measuring in vacuum, or fixing or embedding the sample in polymers, such as PVA. The increased life of not fluorescent second state also allows it with lower intensities the light of the one wavelength, large proportions of the fluorescent dye in the non-fluorescent second state to keep.

The known risk of photobleaching of a conventional fluorescent dye from its triplet state is also not a problem in the new process. Strictly speaking, it is sufficient if a substantial proportion of the fluorescent dye molecules are pumped into their second non-fluorescent or otherwise fluorescent second state once to return to their fluorescent first state. After this return, the molecules are registered individually riert. their subsequent fate is irrelevant. So they can get back into the triplet state and be bleached out of it.

One partial photobleaching of the fluorescent dye can be carried out in the new procedures can even be carried out specifically before the remaining unbleached molecules are registered. The new method can be carried out in a particularly advantageous manner, if the molecules of the fluorescent dye have a certain do not exceed the spatial density in the sample, because then a certain percentage remaining in the first state the molecules also a certain spatial Density does not exceed what is essential for the separate registrability of the individual molecules is. When the actual concentration of the fluorescent dye in the sample exceeds the certain spatial density, conversely, the molecules may become difficult individually to register. To eliminate this difficulty, the surplus to fluorescent dye by means of a high intensity the light of one or another wavelength by photobleaching switched off, that is converted into a persistent dark state which differs from the first state and the second state. Typically, the persistent dark state differs in which the fluorescent dye is no longer at the steps to Take the pictures for registering each one Molecules involved, not only electronically, but for example also chemically from the first state and the second state, the for this registration of the individual molecules used according to the invention.

The new process was using commercially available and each Professional as non-switchable known fluorescent dyes such Rhodamine 6G successfully performed. His execution required compared to conventional fluorescence light microscopy using this fluorescent dye - except of different preferred details in sample preparation - only one Tuning the intensity of the light of a wavelength with the frequency at which the images were taken with the sensor array become. The necessary apparatus requirements are present in many fluorescent light microscopes. Here must just controlling the intensity of the light of one wavelength according to the method of the invention be modified. Alternatively, change the frequency the image recording of the sensor array or a sensor array having Camera. Accordingly, the new fluorescent light microscope is only through a special training of control for the intensity the light of the one wavelength. Preferably is an online image processing for the individual provided with the sensor array images.

These Evaluation is useful to the intensity of the light set a wavelength to such a value actually the registration of fluorescent light individual, spatially separated molecules allowed in the individual pictures. The set value of the intensity The light of one wavelength can be a constant value be. This also includes a very fast pulse sequence a much higher frequency than the frame rate of the recorded Images. The intensity of the light of a wavelength but also one with the sequence of recording the pictures in time have modulated intensity curve, for example between the individual pictures the subgroup of the first State-targeted molecules of the substance targeted and during the recording of each picture primarily the molecules of the selected subgroup fluoresce to stimulate. Here, too, the light of a wavelength both continuously (with the time-modulated intensity curve) or in the unreleased pulses when taking pictures (also with the temporally modulated intensity curve) be directed to the area of interest.

The Online evaluation of each captured image can serve the non-spatially separable fluorescent molecules to determine the substance, according to which the intensity of the light can be varied until a density limit for such not separable fluorescent molecules is below. In this way, there is an upper limit to the intensity the light of the one wavelength defined. A lower limit can be defined by the fact that the individual recorded Images evaluated online for maximum density in which they are separable fluorescent molecules of the Show substance, and by the intensity of the light of the one Wavelength is varied until a density limit for reached such separable fluorescent molecules from below is. While it is important on the one hand that the molecules in the fluorescent state are not so high in concentration that they should no longer be registered separately their concentration below this limit as high as possible Be sure to get as much information about each picture as possible to obtain the structure of interest.

The initially applying the substance to the light a wavelength that primarily serves them essentially to convert to their second state, can also be used with the sensor array an intensity distribution of Fluorescence light of the entire substance in the sample record. This intensity distribution corresponds to a concentration distribution the substance in the sample with the spatial resolution the image of the sample on the sensor array.

These Concentration distribution of the substance in the sample provides an overview the location of the structure of interest labeled with the substance ready in the sample. This simplifies the further steps of the new process, as it applies to such areas the sample can be concentrated in which ever Parts of the marked structure are located. This is usually with switchable and especially with activatable fluorophores not possible, since this is the largest at first Part are not in the fluorescent state, due to the missing signal does not allow an overview.

Dependent from the concentration distribution of the substance in the sample can In addition, the intensity of the light of a wavelength set for the respective closer examined area or at least to an approximately appropriate value for the Fine adjustment to be preset. Furthermore, due to the Concentration distribution of the substance in the sample a local termination criterion for taking more pictures of the same area Sample can be defined. The information content of additional images a portion of the sample has a decreasing information content on, where the drop in the information content of the concentration the substance in the respective area depends. When in an area only very few molecules of the substance are, A relatively small number of images are enough for the position of a high percentage to capture the molecules. More pictures are only available here redundant information ready. It is different at a very high concentration of the substance in one area. Here also with many pictures only smaller portions of the substance become captured in the sample, and each additional image provides new information to disposal.

Concrete With the new procedure, each one in the consecutive pictures registered position of a molecule not only in a high resolved overall picture of the structure of interest in the sample, but folded with the PSF (Point Spread Function) mapping the sample onto the sensor array also into a reconstruction the initially recorded intensity distribution be registered. If this reconstruction reaches a certain level Measure of the initially recorded intensity distribution approximates the positions of other molecules from further pictures no noteworthy further information about the structure to be expected. When folding with the PSF can the brightness of each molecule as a weighting factor be taken into account. As a measure of approach the reconstruction of the initially recorded intensity distribution different values, such as a cross-correlation, a simple difference or quadratic deviation of the normalized Intensity distributions or deviations between the Spatial frequencies (Fourier transforms) of the intensity distributions, be taken.

The novel method is particularly well suited to label the structure of interest of the sample with the non-switchable fluorophores by genetically engineering a biological sample to contain even the non-switchable fluorescent dyes or specific binding sites for the non-switchable fluorescent dyes or linkers coupled thereto expressed. In this way, the structure of interest in the sample with non-switchable organic dyes is particularly advantageously marked via so-called small labels or self-labeling protein tags such as FlAsh, snap-tags or halo-tags. These and similar concepts are generally known to the person skilled in the art, see, for example http://www.promega.com/cnotes/cn011/cn011_02.htm or http://www.covalys.com/ or BioForum Europe 6, 51-59 (2005) , This makes it possible to image proteins in a biological sample with widespread, conventional fluorescent dyes with high resolution.

at the fluorescent dye used in the new method it does not matter that it's second electronic Condition not fluorescent, d. H. not fluorescent and completely dark. He can also fluoresce differently to be the first electronic state. When the fluorescent dye while in the second state by the same light for fluorescence it is stimulated as in its first electronic state important to get fluorescent light from the fluorescent dye is emitted in the first electronic state, of the fluorescent light, that of the fluorescent dye in the second electronic state is emitted, can be distinguished.

It is understood that the new method can be combined with various measures that are familiar to the person skilled in the art, in particular in the field of methods known as PALM, STORM or PALMIRA. These include, in particular, measures for the three-dimensional resolution of the registered positions of the molecules in the sample, ie for the spatial resolution of these positions also in the z-direction. These measures include multiphoton excitation of the fluorescent dye from its first state both for fluorescence and for conversion to its non-fluorescent second state, focusing the stimulating light of one wavelength onto the plane of interest and using two opposing high numerical aperture objectives in 4Pi configuration for exposing the sample to the light of one wavelength and / or registering the fluorescence zenzlichts from the sample. As far as the light is focused in each case only in one or more individual points of the plane, the plane with these points in all steps of the process, for. B. during the recording of each image to scan. The focusing of the light of the one wavelength into individual points of the sample can be advantageously combined with a confocal registration of the fluorescent light from the sample. Alternatively, it is possible to apply the probe orthogonal to the direction of imaging of the sample onto the sensor array with the light of the one wavelength from which a light cut is formed by means of a cylindrical lens. This procedure is known to the person skilled in the art as SPIM (Selective Plane Illumination).

advantageous Further developments of the invention will become apparent from the claims, the description and the drawings. The in the introduction to the description advantages of features and combinations of several Features are merely exemplary and may be alternative or cumulatively without compelling advantages of embodiments of the invention must be achieved. Other features are the drawings - in particular the illustrated geometries and the relative dimensions of several Components to each other and their relative arrangement and operative connection - to remove. The combination of features of different embodiments the invention or features of different claims is also different from the chosen relationships the claims possible and is hereby stimulated. This also applies to such features, in separate drawings are shown or mentioned in their description. These Features may also be consistent with features of different claims be combined. Likewise, in the claims listed features for further embodiments the invention omitted.

BRIEF DESCRIPTION OF THE FIGURES

in the The invention will now be described with reference to the appended drawings Figures on the basis of preferred embodiments on explained and described.

1 shows the structure of a fluorescent light microscope, which is suitable for carrying out the new method.

2 (A) shows a total image of microtubules of a PtK2 cell taken as a structure of interest with the new method. The structure is stained with the dye rhodamine 6G. The medium in which the cell is located is an aqueous buffer solution with glucose oxidase and catalase (50 mM Tris, pH 7.5, 10 mM NaCl, glucose oxidase (Sigma, G2133), 40 μg / mL catalase (Roche Applied Science, 106810), 10% (w / v) glucose). The number of frames taken for the entire image is 61440 with exposure times of 5 ms. The light intensity was constant at 50 kW / cm ² .

2 B) is a reconstruction that is a resolution-bound image of the same object as in 1 (A) equivalent. The reconstruction was generated by the sum of 61440 frames. The small figure at the bottom right in (B) shows a profile at the marked points of 1 (A) and (B), which clearly shows the increase in resolution of the new process.

EMBODIMENTS

At the in 1 sketched fluorescent light microscope 1 When carrying out the method according to the invention, a high-resolution imaging of a structure of interest of a sample is performed 2 light 3 a wavelength (black line) from a suitable light source 4 provided and by means of the lens 5 in a lens 6 focused. That light 3 is used for large-area illumination of the entire region of interest of the sample 2 , fluorescent light 7 (gray line) of fluorescent dye in the sample 2 is also from a lens, in this case the same lens 6 , collected and from the light 3 by means of a dichroic mirror 8th separated and, if necessary, by a suitable fluorescent light filter 9 further purified. A lens 10 ensures interaction with the lens 5 for a suitable mapping of the fluorescent molecules of the fluorescent dye on a sensor array 11 ,

When carrying out the method according to the invention on the fluorescent light microscope 1 take the following steps:
First, a structure of interest in a sample is stained with a non-switchable fluorescent dye.

Thereafter, the sample is embedded in a suitable environment. This can be z. B. PVA, or even an aqueous medium (eg., For living cells) is deprived of oxygen. This measure is usually necessary because with today's technology and conventional fluorescent dyes, the life of the dark triplet state in aqueous solutions without Sauerstoffkonzentrationsreduktion is not long enough to separate single molecule events can. The oxygen reduction can, for. B. done by adding glucose oxidase and catalase. Such aqueous buffers are well-known media for microscopy. A medium that is in principle also suitable for living cell applications is: 88% (v / v) Gibco-DMEM (In Vitrogen Corporation, Carlsbad, California) with 10 mM HEPES, 10% (v / v) glucose oxidase (5 mg / ml, Sigma, G2133), 2% (v / v) catalase (2 mg / ml, Roche Applied Science , 106810)

Before starting the actual measurement u. For example, if the label density, ie, the spatial density of the fluorescent dye, is too high, a sufficient portion of the molecules of the fluorescent dye will be irreversibly bleached by suitably irradiating the sample with the light. In any case, before starting the measurement by irradiating the light, a sufficiently large proportion of the molecules must be pumped from their first, fluorescent to their second, dark state, so that the images of the few molecules remaining in the fluorescent state on the sensor array are wider than the Resolution limit on a sensor array away from each other. Typical intensities are between 1 and 100 kW / cm ² , depending on the environment and fluorescent dye. The intensity distribution of the fluorescent light, which can be recorded at the beginning of the irradiation of the light with the sensor array, shows the resolution-limited image of the structure of interest. This can later serve as a reference for a termination criterion. In practice, the exposure time of a camera having the sensor array or the gain thereof must be adapted or an intensity filter used to record the resolution-limited image of the structure of interest, since the camera will be optimized for the detection of single-molecule signals. Alternatively, a low-intensity light signal may be irradiated in front of the light signal, which serves to transfer the majority of the molecules to the dark state, in order to record a diffraction-limited reference image.

Such as a sufficient proportion of the molecules in the dark state pumped, the actual measurement can be seamless but in any case this must be done in a timeframe which is much smaller than the life of the dark state. The Exposure time of the individual images depends on the average Duration, over which a molecule, which in the first, luminous state is emitted, fluorescent light emitted before it goes back to the second, dark state. at This leads to a typical Exposure time from 2 to 10 ms. In this time, on average, one Order of magnitude of 1000 photons per molecule collected on the detector before returning to the dark state.

While The measurement can be made after irreversible molecules Bleaching are lost, the intensity of light be reduced to an optimal density of the molecules reach in the first state.

The Duration of the entire measurement depends on the number of individual images and their exposure time. The required number of frames depends on the selected termination criterion. For more complex Structures typically record up to 100,000 frames. Thus, the total recording time is on the order of magnitude of minutes.

1

light microscope

2

sample

3

light

4

light source

5

lens

6

microscope objective

7

fluorescent light

8th

dichroic mirror

9

Fluorescent light filter

10

lens

11

sensor array

12

mirror

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• WO 2006/127692 A2 [0002]
• - WO 2006/127692 [0002, 0003, 0003]
• - DE 10325460 A [0007, 0014]

Cited non-patent literature

• Rust et al. in Nature Methods, 3, 793-796 (2006) [0004]
• C. Geisler et al .: Resolution of λ / 10 in fluorescence microscopy using fast single molecule photo-switching, Appl. Phys. A 88, 223-226 (2007) [0005]
• Bretschneider et al .: Breaking the diffraction barrier in fluorescence microscopy by optical shelving, Phys. Rev. Lett. 98, 218103 (2007) [0009]
• - http://www.promega.com/cnotes/cn011/cn011_02.htm [0025]
• - http://www.covalys.com/ [0025]
• - BioForum Europe 6, 51-59 (2005) [0025]

Claims (18)

A method of spatially high resolution imaging of a sample structure of interest comprising the steps of: selecting one of a group of substances having a first state with first fluorescence properties and a second state with second fluorescence properties; - Which are excitable in their first state with light of a wavelength for the spontaneous emission of fluorescent light; - Which can be converted from its first state with the light of a wavelength in its second state and - which can return spontaneously and / or by the action of the light of the one wavelength from its second state to its first state; - marking the structure of interest of the sample with the substance; Imaging the sample onto a sensor array, wherein a spatial resolution limit of the image is greater (ie worse) than an average distance between nearest neighboring molecules of the substance in the sample; Subjecting the sample in a region which has larger dimensions than the spatial resolution limit of the image of the sample on the sensor array, with the light of a wavelength, wherein changing proportions of the substance with the light of the one wavelength for spontaneous emission of fluorescent light is excited and their second state is transferred and wherein at least 10% of the molecules of the substance respectively in the first state have a distance to their closest adjacent molecules in the first state, which is greater than the spatial resolution limit of the image of the sample on the sensor array; and - registering the fluorescent light spontaneously emitted from the region of the substance in a plurality of images taken during the continuous exposure of the sample to the light of the one wavelength with the sensor array; characterized in that the first state and the second state are different electronic states of the substance. Method according to claim 1, characterized in that at the beginning of the loading of the sample ( 2 ) with the light ( 3 ) of one wavelength whose intensity is adjusted so large that the substance is converted to its second state, to more than 90%, preferably more than 95%, even more preferably more than 98% and most preferably more than 99% of the molecules the substance are transferred to the second electronic state. Method according to claim 1 or 2, characterized that the substance is not switchable. Method according to claim 1, 2 or 3, characterized that the first state is a singlet state and the second state is a triplet state. Method according to one of claims 1 to 4, characterized in that at least one measure is taken, the lifetime of the second state of the fluorescent dye in the sample ( 2 ) changed, in particular extended. Method according to one of claims 1 to 5, characterized in that a portion of the substance before taking the images by means of a high intensity of the light ( 3 ) of one or another wavelength is converted by photobleaching into a persistent dark state that differs from the first state and the second state. Method according to one of claims 1 to 6, characterized in that the intensity of the light ( 3 ) of one wavelength is set to a constant value during the recording of the images. Method according to one of claims 1 to 6, characterized in that the intensity of the light ( 3 ) of the one wavelength during the recording of the images is set to a temporally modulated with the sequence of recording the images intensity profile. Method according to one of claims 1 to 8, characterized in that the light ( 3 ) of a wavelength continuously or in fast, unresolved in the recording of the images pulses is directed to the area. Method according to one of claims 1 to 9, characterized in that the individual recorded images are evaluated online, whether they show non-separable fluorescent molecules of the substance, and that the intensity of the light ( 3 ) is varied until a density limit for such non-separable fluorescent molecules is exceeded. Method according to one of claims 1 to 10, characterized in that the individual recorded images are evaluated online with respect to the maximum density, in which they show separable fluorescent molecules of the substance, and that the intensity of the light ( 3 ) is varied until a density limit for such separable fluorescent molecules is reached. Method according to one of claims 1 to 11, characterized in that at the beginning of the loading of the sample ( 2 ) with the light ( 3 ) of one wavelength with the sensor array, an intensity distribution of the fluorescent light of the entire substance in the sample ( 2 ) with the spatial resolution of the image of the sample ( 2 ) is recorded on the sensor array. A method according to claim 12, characterized in that an abort criterion for taking further images of the same area of the sample ( 2 ) based on the intensity distribution of the fluorescent light of the whole substance in the sample ( 2 ) To be defined. Method according to claim 13, characterized in that each position of a molecule of the substance registered in the successive images folded with the PSF (Point Spread Function) of the image of the sample ( 2 ) on the sensor array ( 11 ) is entered into a reconstruction of the initially recorded intensity distribution and that this reconstruction is compared with the initially recorded intensity distribution. Method according to one of claims 1 to 14, characterized in that the structure of interest of the sample ( 2 ) is labeled with the substance by a biological sample ( 2 ) is genetically engineered to express the substance itself. Method according to one of claims 1 to 14, characterized in that the structure of interest of the sample ( 2 ) is labeled with the substance by a biological sample ( 2 ) is genetically engineered to express proteins having specific binding sites for the substance or a linker coupled thereto. Fluorescence light microscope with a sensor array for registering fluorescent light from a sample in successive images, with an imaging optics for imaging the sample on the sensor array, wherein a grid of pixels of the sensor array smaller than the spatial resolution limit of the image of the sample on the sensor array times the imaging factor of 1, with a light source for exposing the sample to light of a wavelength in a region larger in size than the spatial resolution limit of imaging the sample on the sensor array and having intensity control of the light of the one wavelength, characterized that the controller controls the intensity of the light ( 3 ) which controls a wavelength according to the method of any one of claims 1 to 14. Fluorescence light microscope according to claim 17, characterized in that the controller has an online image processing for the individual with the sensor array ( 11 ) has recorded images.