DE102016119262B4 - A method for spatially high-resolution determination of the location of a singled, excitable light for the emission of luminescent light molecule in a sample - Google Patents

Abstract

In a method for spatially high resolution determination of the location (x) of a singulated molecule in one or more spatial directions in a sample, the molecule being excitable with excitation light for emission of luminescent light (12), the excitation light is directed to the sample with an intensity distribution In each of the spatial directions, at least one intensity increase region (22) with a known strictly monotonous profile of the intensity of the excitation light has a distance to a reference point of the intensity distribution. An initial location area (23) in the sample in which the molecule is located is determined. (i) in each of the spatial directions, at least one position (x) of the point of intensity distribution is set such that the at least one intensity increasing region (22) extends in the respective spatial direction over the initial spatial region (23); the point of the intensity distribution is located at the positions (x) in the sample, and at each position (x) of the point of intensity distribution in the sample, the luminescent light (12) emitted from the molecule is registered. (ii) from intensity values (I; I) of the luminescent light (12) which comprise two intensity values (I; I) for each of the spatial directions, of which one the intensity (I) of the at least one position (x) of the point of view Indicates intensity distribution of registered luminescent light (12), another location area (24) is determined in the sample in which the molecule is located and which is smaller than the initial location area (23). Steps (i) and (ii) are repeated at least once using the further location area (24) as the new initial location area (23).

Description

TECHNICAL FIELD OF THE INVENTION

The invention relates to a method for spatially high-resolution determination of the location of an isolated molecule in one or more spatial directions in a sample, wherein the molecule is excitable with excitation light for emission of luminescent light. In particular, the present invention relates to a method having the features of the preamble of independent claim 1.

An isolated molecule excitable with excitation light for emission of luminescent light is understood here to be a molecule which has a minimum distance from other similar molecules which can be excited by the same excitation light for emission of luminescent light in the same wavelength range. This minimum distance is certainly maintained if the distance of the separated molecule to such similar molecules is at least equal to the diffraction limit at the wavelength of the luminescent light, because then the luminescent light can be detected separately from the different excitable excitation light for the emission of luminescent light molecules. However, the invention also includes embodiments in which this minimum distance is smaller than the diffraction limit at the wavelength of the luminescent light.

The luminescent light, for the emission of which the molecule can be excited by the excitation light, can be in particular fluorescent light. However, the emission of the luminescent light by the molecule can be based on any photophysical process that is excited by the excitation light. These include all processes of photoluminescence and, for example, an emission of luminescent light excited by the excitation light by quantum dots.

STATE OF THE ART

verbesserten Präzision bestimmt werden. Dabei ist „n“ die Anzahl der registrierten Photonen. Eine deutliche Unterschreitung der Beugungsgrenze bei diesem als Lokalisierung bezeichnet Verfahren setzt daher eine Vielzahl von Photonen des Lumineszenzlichts voraus, die von dem vereinzelten Molekül emittiert und registriert werden. Damit besteht die Gefahr, dass das vereinzelte Molekül beim und evtl. schon vordem Bestimmen seines Orts in der Probe mit der gewünschten Präzision weggeblichen wird. Insbesondere ein wiederholtes Bestimmen des Orts desselben vereinzelten Moleküls, wie es zum Tracking eines sich in einer Probe bewegenden Moleküls erforderlich ist, ist deshalb oft nicht möglich.The simplest method of determining the location of a singulated molecule excitable with excitation light for emission of luminescent light is to excite the molecule with the excitation light to emit luminescent light and to image the luminescence light onto a camera or more generally onto a spatially resolving detector. In principle, the diffraction limit at the wavelength of the luminescent light applies to the spatial resolution achievable in this figure. However, when a plurality of photons of the luminescent light are detected with the spatially resolving detector derived from a single molecule at a fixed location in the sample, the spatial distribution of these photons across the detector may change the location of the molecule by one factor $1\text{/}\sqrt{n}$

improved precision can be determined. Where "n" is the number of registered photons. A clear drop below the diffraction limit in this process called localization therefore requires a large number of photons of the luminescent light, which are emitted and registered by the isolated molecule. There is thus the danger that the isolated molecule will be lost in the sample with the desired precision during and possibly even before its location in the sample. In particular, a repeated determination of the location of the same isolated molecule, as required for tracking a moving in a sample molecule is therefore often not possible.

When an isolated molecule emits luminescent light with a directional spatial distribution, this results in the determination of its location from the distribution of the photons of the luminescent light via a spatially resolving detector, ie by localization, in the form of a spatial error. This location error depends on the orientation of the molecule in the respective sample. A directional distribution of the emitted luminescence light is evident, for example, in molecules whose rotational diffusion times are longer than their residence time in their excited state, from which they emit the luminescent light (see Engelhardt, et al. J. et al. "Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy", NanoLett 2011 Jan. 12; 11 (1): 209-13) ,

From the WO 2006/127692 A2 It is known to label a structure of interest in a sample of activatable molecules that are in a non-fluorescent starting state but can be converted to a fluorescent state with activating light in which they are excitable with excitation light to emit luminescent light. Thus, with the activation light, a small proportion of the total existing molecules can be brought into the fluorescent state, in which the next adjacent molecules in the fluorescent state have a greater distance than the diffraction limit, ie are isolated. Upon subsequent exposure of the sample to excitation light, fluorescent light is emitted only by the molecules in the fluorescent state. Thus, the fluorescent light can be separately registered by the individual isolated molecules in the fluorescent state; and the locations of the individual molecules can be determined despite high absolute density of the molecules in the sample by localization with a precision beyond the diffraction limit. An illustration of the distribution of the molecules in the sample and thus of the structure of interest marked with them achieved step-by-step by repeating the steps of activating a small proportion of the molecules, exciting these activated molecules to emit fluorescent light and registering the fluorescence light with a spatially resolving detector until the respective activated molecules are repeating, and thus more and more of the molecules which mark the structure of interest.

The WO 2006/127692 A2 also describes that activation of only a subset of the total molecules present can be transferred to other imaging methods. In this case, the scattered molecules can be excited to emit fluorescent light, especially in certain planes or other spatial subunits of the sample, by means of an intensity distribution of the excitation light with maxima limited by minima.

That from the WO 2006/127692 A2 known method is also referred to as PALM, that is, as photoactivated localization microscopy. A very similar process, referred to as STORM (Stochastic Optical Reconstruction Microscopy), has basically the same advantages and disadvantages.

From the US 8,174,692 B2 It is known that molecules of a normal dye which are not activatable but have a fluorescent starting state, and also can not be switched between two conformational states, only one of which is fluorescent, can be used to localize the location of individual molecules to determine. For this purpose, the sample with such a high intensity of excitation light, which at the same time transfers the molecules into a relatively long-lived electronic dark state with a certain transition probability, is subjected to distances above the diffraction limit between the molecules currently in the fluorescent state.

The respective molecules present in the fluorescent state are excited by the excitation light for the emission of fluorescent light, which is registered with a camera as a spatially resolving detector. In this way, successively different molecules of the dye are localized, since the molecules, from which photons have already been registered, reach the dark state, from which other molecules with a certain transition probability return to the fluorescent state. This known method can be carried out continuously, d. H. frames can be continuously read out of the camera while the sample is exposed to the high intensity of the excitation light which keeps the dye substantially in the dark state and at the same time excites the scattered fluorescent molecules to emit luminescent light.

That from the US 8,174,692 B2 known method is also referred to as GSDIM (Ground State Depletion of Individual Molecule Return Microscopy).

A fundamentally different method for spatially high-resolution determination of locations of excitable excitation light for the emission of luminescent light molecules in a sample is used in spatially high-resolution variants of the scanning fluorescence microscopy. In scanning fluorescent microscopy, the precision in determining the locations of fluorescent molecules in a sample is based on only local excitation of the sample at any time, so that fluorescent light registered at that particular time can be assigned to the spatial region of the excitation. When the molecules in the sample are excited with focused excitation light, the diffraction limit at the wavelength of the excitation light sets the lower limit for the spatial dimensions of the region of excitation and, thus, for the spatial resolution involved in imaging a structure labeled with the fluorescent molecules Probe is reachable.

In STED (Stimulated Emission Depletion) fluorescence microscopy, the excitation of molecules with which a structure of interest in a sample is marked is eliminated by directed emission in the vicinity of each measuring point. The directed emission is stimulated with STED light and prevents the emission of fluorescent light by the molecules. After that registered fluorescent light can only come from the area in which the excitation with the STED light was not eliminated. This range, in which the excitation is not eliminated, can be kept very small by being defined by a zero of the intensity distribution of the STED light and the maximum intensity of the STED light in the zero point adjacent intensity maxima is set high, so it completely eliminates the excitation of the molecules already very close to its zero.

Instead of de-excitation of previously induced excitation of the molecules in parts of the sample, fluorescence-preventing light having a zero-intensity intensity distribution can also be used to switch fluorescent molecules outside the null to a non-fluorescent dark state. This is done, for example, in RESOLFT (Reversible Saturable Optical (Fluorescence) Transitions) fluorescence microscopy or GSD (Ground State Depletion) fluorescence light microscopy.

In the method known as STED, RESOLFT or GSD scanning fluorescence microscopy, each molecule, even before it is detected by fluorescent light, is applied in the regions adjacent to the zero of the fluorescence-preventing light next to the excitation light with the high intensities of the fluorescence-inhibiting light therewith significant risk of bleaching of the molecules are connected. The danger of bleaching also exists when, in these known methods, a singular molecule excitable with excitation light for emission of luminescent light is traced in a sample having the zero of the fluorescence preventing light, in order to continuously maximize the rate of photons emitted from the molecule of the luminizing light.

From the WO 2012/171999 A1 For example, a method is known in which a sample with an excitation light beam superimposed with an intensity distribution of STED light with a minimum at the focus of the excitation light beam is scanned so rapidly that in each of many consecutive scans fluorescent light is only in the form of single photons is registered, each derived from a single molecule. The locations of the various individual molecules from which the photons originate are assigned to the associated positions of the minimum of the STED light in the sample.

In this method, although only a few photons, in extreme cases only a single, registered by each molecule to which a location in the sample is assigned. However, the molecules are typically subjected to several cycles of stimulation and stimulated emission so that their bleaching is not excluded. In addition, targeted tracking of a particular isolated molecule is not meaningfully possible with this known method.

From the DE 10 2011 055 367 A1 For example, a method of following a single fluorescent molecule is known. The molecule is excited with excitation light to emit fluorescent light and the fluorescent light is registered. In this case, the excitation light is directed onto the sample with a local minimum intensity distribution, and the minimum is tracked to the molecule moving in the sample. For this purpose, the intensity distribution of the excitation light is continuously shifted relative to the sample such that a rate of photons of the fluorescence light emitted by the molecule is minimized. This means holding the molecule in the minimum of the intensity distribution of the excitation light, which may be a zero of the intensity distribution of the excitation light.

From the WO 2015/097000 A1 For example, a method is known for determining the locations of isolated molecules in a sample, wherein the singulated molecules are in a fluorescent state in which they are excitable with excitation light for emission of fluorescent light, and wherein distances of the singulated molecules of the substance keep a minimum value. The isolated molecules are excited with the excitation light for emitting the fluorescent light, wherein an intensity distribution of the excitation light has a zero point. The fluorescent light from the excited singulated molecules of the substance is registered for different positions of the zero of the intensity distribution of the excitation light in the sample. In this case, distances between next adjacent positions of the zero point are not greater than half the minimum value of the distance of the separated molecules. Specifically, the sample is scanned at the zero, with a pitch when sampling the sample is not greater than half the minimum value. The locations of the singulated molecules in the sample are derived from a progression of the intensity of the fluorescence light from the respective isolated molecule over the positions of the zero point of the intensity distribution of the excitation light. In this case, a function with zero point can be fitted to the course of the intensity of the luminescence light of the particular isolated molecule; and the location of each molecule can be equated to the position of the zero of the fitted function. The function can be a quadratic function. However, the fitted function can also be adapted individually to the intensity profile of the luminescence light from an isolated molecule of the respective substance in response to the intensity profile of the excitation light in the vicinity of its zero position.

In the from the WO 2015/097000 A1 In known methods, the particular isolated molecule is exposed to the higher intensities of the adjacent intensity maxima of the excitation light before it reaches the vicinity of the zero point of the intensity distribution of the excitation light during scanning of the sample. These intensities exceed a saturation intensity of the excitation light, above which there is no longer an increase in the intensity of the luminescence light from the respective separated molecule, so that its profile is constant at a saturation value and thus without information content. Accordingly, each individual molecule of the substance emits a large number of photons before emitting the photons from which the location of the molecule is determined. As a result, there is a considerable risk of bleaching of the molecule, even if only a limited number of photons are registered at each position of the zero point of the excitation light, before the zero point is shifted further.

From the DE 10 2010 028 138 A1 For example, a method for determining the distribution of a substance in a measurement area by sensing with a measurement front is known. Over a depth of the measurement front, which is smaller than the diffraction limit at the wavelength of an optical signal, the intensity of the optical signal increases such that a proportion of the substance in a measurement state initially increases from not present and then drops back to none. The measurement front is shifted in the opposite direction to the increase in the intensity of the optical signal over the measurement area. The measurement signal, which may be fluorescent light, is detected and assigned to the respective position of the measurement front in the measurement area.

From the DE 10 2014 111 167 A1 For example, there is known a method of high resolution scanning microscopy in which a sample of illumination radiation is excited to emit fluorescence radiation such that the illumination radiation is collimated to a point in or on the sample to form a diffraction limited illumination spot. The point is diffraction-limited imaged in a diffraction image on a spatially resolving surface detector, which has a spatial resolution that dissolves a diffractive structure of the diffraction image. The sample is scanned by means of various scan positions with a pitch smaller than half the diameter of the illumination spot. From the data of the area detector and from the scan positions assigned to these data, an image of the sample is generated which has a resolution that is increased above a resolution limit of the image. Such a method and corresponding microscopes are also from the WO 2013/135487 A1 , of the DE 10 2013 019 348 A1 , of the DE 10 2012 204 128 A1 , of the DE 10 2012 023 024 A1 and the EP 2 317 362 A1 known.

The US 7,772,569 B2 discloses a microscope system for generating three-dimensional images from individually located sample molecules. The microscope system comprises a sample holder, an activation light source, a readout light source, a beam splitter, at least one camera and a controller. The activation light source activates photosensitive luminescent probes, and the readout light source causes luminescent light from the activated probes. The beam splitter splits the luminescent light into at least two paths to produce at least two detection planes corresponding to different object planes of the sample. The at least one camera simultaneously detects the at least two detection levels. The controller is programmed to combine a signal from different object levels into three-dimensional data.

The DE 10 2015 004 104 A1 discloses a method for locating an emitter of electromagnetic emission radiation by means of a localization microscope. The method comprises directing the electromagnetic emission radiation through at least two objectives onto at least one optical detector, wherein the optical axes of the at least two objectives extend in mutually linearly independent directions. The electromagnetic emission radiation is detected by the at least one optical detector, wherein the optical detector receives measured values from which the position of the at least one emitter is determined.

OBJECT OF THE INVENTION

The invention has for its object to provide a method for spatially high-resolution determination of the location of a singular, excitable light for the emission of luminescent light molecule in a sample, which is the molecule in which the number of photons, the emission of which scattered molecule must be based on the achieved precision compared to the known methods for spatially high-resolution determination of the location of a scattered molecule is significantly reduced.

SOLUTION

The object of the invention is achieved by a method having the features of independent claim 1. The dependent claims 2 to 20 relate to preferred embodiments of the method according to the invention. The claims 21 and 22 relate to repeated implementations of the method according to the invention for determining the location of a plurality of sequentially separated molecules or for tracking the separated molecule; and claim 23 relates to a use of a STED scanning fluorescent light microscope for carrying out the method according to the invention.

DESCRIPTION OF THE INVENTION

The invention is based on one of WO 2015/097000 A1 known methods for spatially high-resolution determination of the location of a separated molecule in one or more spatial directions in a sample, wherein the molecule is excitable with excitation light for emission of luminescent light. As in the case of the starting method, the excitation light is directed onto the sample with an intensity distribution which has at least one intensity increase region in each of the spatial directions with a known strictly monotonous progression of the intensity of the excitation light over a distance to an observation point of the intensity distribution. This point of the intensity distribution is arranged in each of the spatial directions at different positions in the sample. At each position of the point of intensity distribution in the sample, the luminescent light emitted from the molecule is registered; and the location of the molecule in the sample is derived from intensities of the registered luminescent light.

In addition to the known method, an initial local area in the sample in which the molecule is arranged is first determined according to the invention. Then, (i) in each of the spatial directions, at least one position of the point of intensity distribution is set so that the at least one intensity increasing region in the respective spatial direction extends over the entire initial spatial region, and (ii) from intensity values of the luminescent light corresponding to each of the first If spatial directions include two intensity values, one of which indicates the intensity of the luminescent light registered to the at least one position of the intensity distribution point, another location region in the sample in which the molecule is located and which is smaller than the initial local area is determined. Steps (i) and (ii), including arranging the point of the intensity distribution of the excitation light at the positions determined in step (i) and registering the luminescent light emitted by the molecule to each of these positions, are performed at least once using the further location area repeated as a new initial location area.

In this way, the location of the singulated molecule in the sample in each spatial direction becomes very fast, i. H. based on a small number of emitted and registered by the scattered molecule photons, limited to a small local area. The dimensions of this small area correspond to the precision with which the location of the isolated molecule is determined. You can easily below 10 nm and thus the diffraction limit at the wavelength of the luminescent light significantly.

In the method according to the present invention, the sample is not spatially scanned even in the initial locus of the molecule, but the positions of the intensity distribution point become intelligent, i.e., due to the intensities of the luminescent light registered relative to the locus at the previous positions of the intensity distribution point. H. with maximum utilization of all information available so far on the registered intensities of the luminescent light about the actual location of the isolated molecule. The fixed position of the point of view can completely eliminate the local area itself.

The fact that the inventive method is used for spatially high-resolution determination of the location of the isolated molecule means, as can be read from the achievable precision of 10 nm and much better that in particular a spatial resolution well below the diffraction limit at the wavelength of the excitation light and the luminescence is reached. In this context, it should be noted that the term "spatial resolution" is used here as a synonym for the precision with which the location of each individual molecule in the sample is determined.

The fact that the method according to the invention serves to determine the location of a singulated molecule means, as already mentioned, that the location of the singulated molecule maintains a minimum distance to nearest adjacent homogeneous molecules which can be excited by the same excitation light for emission of luminescent light in the same wavelength range that the luminescent light from the next adjacent like molecules can not be separated due to different wavelengths. In the method according to the invention, this minimum distance is of the order of the diffraction limit at the wavelength of the luminescent light or smaller. In fact, the minimum distance may be the same as in the case of the original method according to WO 2015/097000 A1 also be considerably smaller than the diffraction limit at the wavelength of the luminescent light, as long as it is not smaller than an extension of the intensity increase range of the intensity distribution of the excitation light in the respective spatial direction, above the intensity of the excitation light below a saturation intensity and thus the intensity of the luminescent light from the molecule remains below a saturation value. If the distance of the singulated molecule to next adjacent excitable light excitable molecules is greater than the diffraction limit at the wavelength of the luminescent light, the luminescent light emitted from the singulated molecule can be spatially separated from the luminescent light adjacent to it by a spatial resolution detector Molecules is emitted. When the distance of the scattered molecules is at least not smaller than the range of the intensity distribution of the excitation light affected by the molecules from the intensity increasing region with respect to the intensity of the luminescent light, the difference of the intensity of the luminescent light from the saturation value at the saturation intensity of the excitation light can be close to that of the scattered one Molecule be assigned, because it is the molecule then alone in this area influenced by the intensity increase range.

That the isolated molecule with the excitation light for the emission of luminescent light Excitable, means that the isolated molecule is photoluminescent. Specifically, the isolated molecule can be fluorescent, ie be excited with the excitation light for emission of fluorescent light. It should be noted at this point that the term "luminescent" or "fluorescent" here only indicates that the isolated molecule can be excited with the excitation light for emission of luminescent light or fluorescent light, but not that it already luminesces or fluoresces, ie is excited.

The fact that the excitation light is directed onto the sample with an intensity distribution having in each of the spatial directions at least one intensity increase region with a known, strictly monotonous progression of the intensity of the excitation light over a distance to an observation point of the intensity distribution may mean that the intensity increase region is at a different distance is formed to the Aufpunkt different destructive interference. Furthermore, the intensity of the excitation light in the at least one intensity increase range of zero, d. H. for example, from complete destructive interference of the excitation light. In particular, the intensity increase range then includes small intensities of the excitation light, which only slightly stress the singulated molecule by exciting it to emit luminescent light. In this case, the intensity increase range can adjoin a zero point of the intensity distribution of the excitation light to which a further intensity increase region adjoins on the opposite side in the respective spatial direction, wherein the two intensity increase regions can be formed symmetrically with respect to the zero point. The zero can be used at the same time as the point of the intensity distribution of the excitation light, which is then favorably close to the smallest intensities of the excitation light, which only make little use of the isolated molecule. In general, the point of origin of the intensity distribution of the excitation light is a reference point defined within the intensity distribution, whose respective position is defined in the sample and which can be positioned correspondingly at defined positions in the sample. When the point of the intensity distribution of the excitation light is a zero point adjoining the intensity increasing regions, the intensity of the excitation light above each of these intensity increasing regions increases strictly monotonically, that is, continuously, with increasing distance to the receptor point. Depending on the nature of the point of view of the intensity distribution, the intensity of the excitation light can decrease with increasing distance to the receptor point but also in a strictly monotonic manner over the intensity increase regions.

Regardless of whether two intensity increase regions adjoining a zero point or another point of the intensity distribution of the excitation light are symmetrical to one another, in the method according to the invention use can be made of both intensity increase regions, for example by positioning the point on different sides of the imaginary one in step (i) Local area.

The zero can be an ideal zero formed by interference, in which the intensity of the excitation light actually goes back to zero. However, a low residual intensity of the excitation light at the zero point is harmless, since it is not the core of the method according to the invention to position the zero, if present in the intensity distribution of the excitation light so, in the sample such that its position coincides with the location of the molecule in FIG the sample coincides. For the same reason, the zero point bounded by the intensity increase ranges can in principle also be formed by Gaussian intensity distributions spaced apart in the respective spatial direction, specifically by the minimum intensity remaining between them. Furthermore, the at least one intensity increase range can also be provided by an edge of only a Gaussian intensity distribution. In this case, however, the center of gravity of the excitation light focussed to the Gaussian intensity distribution is a point of the intensity distribution which is relatively far from the particularly interesting small intensities of the excitation light in the intensity increase region.

Even with an intensity increase range extending in only one spatial direction, the location of the molecule in the sample can be determined to be highly resolved in two or all three spatial directions. Likewise, with only two spatial directions extending intensity increase areas of the location of the isolated molecule in the sample not only in these two but also in all three spatial directions are determined high resolution. For this purpose, the method according to the invention is to be carried out by aligning the intensity increase range or regions in different spatial directions. This can be done successively or quasi simultaneously for the different orientations of the intensity increase range or areas, for example by repeating steps (i) and (ii) only if they have been performed at least once for all spatial directions of the orientation of the intensity increase range or regions.

In the method according to the invention, the initial location area in which the molecule is arranged can be determined in different ways. Examples of this are given below.

That in the method according to the invention in step (i) the at least one position of the point of intensity distribution is determined in each of the spatial directions such that the at least one intensity increase range in the respective spatial direction extends beyond the initial local area, means that the molecule is safely embedded in is the region of the intensity distribution of the excitation light affected by the intensity increase region with respect to the intensity of the luminescent light from the molecule.

That in step (ii) intensity values of the luminescent light, which comprise two intensity values for each of the spatial directions, one of which is the intensity of the luminescent light registered to the at least one position of the intensity distribution point, does not preclude the respective second intensity value is the same value for the respective spatial direction for all spatial directions, so that in the step (ii) a total of only n + 1 intensity values are evaluated, where "n" is the number of spatial directions in which the location of the singulated molecule is determined. In principle, however, an additional intensity value can also be taken into account for each spatial direction, so that a total of 2n intensity values are then evaluated.

In order to determine the further location area in the sample in the step (ii) in which the molecule is located and which is smaller than the initial location area, the dependence of the intensity of the luminescence light emitted from the separated molecule on the distance of the singulated molecule to the point of the intensity distribution of the excitation light. This dependence depends on the known course of the intensity of the excitation light, which increases or decreases monotonically in the direction of this distance, in the intensity increase range and also on the photophysical process on which the excitation of the molecule for emission of the luminescence light is based. Thus, with an intensity increase range adjacent to a zero formed by destructive interference and photoluminescence based on a one-photon process, there is approximately a quadratic dependence of the intensity of the luminescent light emitted by the molecule on its distance from the zero. In a photoluminescence based on a two-photon process, the dependence is even more pronounced and follows a function x ⁴ . In fact, in the method according to the invention, the strictly monotonous course of the intensity of the excitation light in the intensity increase region only has to be known to the extent that it affects the intensity of the luminescent light from the molecule. That is, the dependence of the intensity of the luminescent light from the distance of the molecule to the point of the intensity distribution of the excitation light must be known so that it can be taken into account in the determination of the further local area. However, this dependency can be easily determined, for example empirically by scanning the environment of the molecule with the intensity increase range in small steps.

The fact that in step (ii) the smaller further local area of the molecule in the sample is determined from intensity values of the luminescent light which comprise two intensity values of the luminescence light for each of the spatial directions does not only include the possibility of considering different rates of photons of the luminescent light, but also the possibility to look at temporal distances of the registered photons. It is understood that the mean value of the time intervals of the registered photons is equal to the reciprocal of the rate of the photons of the luminescent light registered to a position of the point of the intensity distribution of the excitation light.

In the method according to the invention, steps (i) and (ii) are repeated at least once. That is, the location area in which the molecule is located in the sample is reduced at least two times from the initial location area. This means that with the aid of the method according to the invention at least twice the local area of the molecule in the sample is narrowed compared to its first estimate. There may also be other restrictions of the location area in the same or in another way. However, the smaller local area obtained after the at least two narrowings of the initial location area can also be used directly to indicate the location of the separated molecule, for example as the center of the location area with the radius of the location area as a possible error.

The second intensity value for the respective spatial direction, which is evaluated in step (ii) of the method according to the invention, can be an intensity of the luminescent light registered to a second position of the point of the intensity distribution in the sample. This second position may lie in the respective spatial direction elsewhere on the same side as the at least one position of the point of intensity distribution or on the side of the initial local region opposite the at least one position of the point of the intensity distribution. In any case, the effect of the different intensities of the excitation light in the intensity increasing region in this embodiment of the method according to the invention is detected at two positions of the point of the intensity distribution of the excitation light, of which it is known where respective spatial direction with respect to the initial location of the molecule.

Taking into account the known profile of the intensity of the excitation light in the intensity increase region, the associated intensities of the luminescent light on the actual location of the molecule in the respective spatial direction can be inferred in conjunction with the distance of the positions of the point of view in the respective spatial direction. Also in this embodiment of the method according to the invention, two separate positions of the point of view with respect to the intensities of the luminescent light registered therefor need not be taken into account for each spatial direction. Rather, z. B. for determining the location of the molecule in two spatial directions, the positions of the point in a plane spanned by these two spatial directions plane in the corners of a triangle, so a total of only three positions. Correspondingly, the positions of the point for determining the location can lie in three spatial directions in the corners of a tetrahedron. In principle, of course, two separate positions of the Aufpunkts per spatial direction are possible in which the location of the molecule is determined. However, from the viewpoint of determining the location of the molecule in the sample with as few photons as possible of the luminescent light, the greater number of positions of the spot in the sample to which the luminescent light is registered is not necessarily preferred.

In another embodiment, the second intensity value to the respective spatial direction in step (ii) is a measure of the relative luminous intensity of the separated molecule. Concretely, this measure may be a maximum intensity of the luminescent light from the molecule upon excitation with the excitation light or an intensity value directly correlated therewith. The maximum intensity or the intensity value correlated therewith can be measured concretely for the separated molecule or can also be estimated for all potential isolated molecules with a specific value.

In the method according to the invention, the luminescent light can be registered to the various positions of the point of the intensity distribution in the sample quasi simultaneously by the reference point is repeatedly shifted between the positions in the sample. For this purpose, the same intensity distribution of the excitation light can be shifted by means of a scanner. However, it is also possible to re-form the intensity distribution for each of the different positions of its point of view in the sample, for example with the aid of a spatial light modulator (SLM) arranged in the beam path of the excitation light. Then, at least insofar can be dispensed with a scanner. Furthermore, it is possible to shift the point of the intensity distribution of the excitation light in the sample by providing the excitation light successively by wholly or partially different light sources. In all these possible embodiments of the method according to the invention, the point of view of the intensity distribution of the excitation light between its positions in the sample can be repeatedly shifted back and forth. It goes without saying that the luminescence light belonging to the individual positions of the point of view is registered separately from the molecule. The quasi-simultaneous registration of the luminescence light to the different positions of the point of the intensity distribution of the excitation light has the advantage that the application of the sample to the excitation light and the registration of the luminescence light from the sample can be stopped immediately, if it is to the individual positions of the Aufpunkts the intensity distribution of the excitation light registered photons of the luminescent light already allow the initial location area in the sample in which the molecule is arranged to narrow by a predetermined amount.

Although a corresponding termination criterion can be applied to a continuous recording of the luminescence light to any position of the point of the intensity distribution of the excitation light in the sample. However, there are cases where observing the intensities of the luminescent light registered to all positions of the point of the intensity distribution of the excitation light after only a few total photons from the molecule indicates that the initial local area can be limited by a desired amount.

In the method of the present invention, when the luminescent light is registered to the positions of the intensity distribution of the excitation light only until the intensities of the registered luminescent light to the positions are detected with sufficient accuracy that the further local area is smaller than the initial one by a predetermined value Value can be determined, this predetermined value can be in a range of 5% to 75%. In this case, this specification refers to the dimension of the location area in the respective spatial direction. It is understood that the intensities of the luminescent light registered at the different positions of the point of view of the intensity distribution of the excitation light must be detected more accurately, the more the further location area is to be delimited from the initial location area. A smaller confinement requires less accuracy in detecting the intensities of the luminescent light to the different positions of the point of view, but also more repetitions of the Steps (i) and (ii) of the method according to the invention to achieve the same precision. Here, optimization can be made with respect to the number of photons of the luminescent light needed to achieve a certain precision in determining the location of the molecule in the sample.

As already mentioned, the entire initial location area at each position of the point of view should be in a range of the intensity distribution of the excitation light in which the intensities in the intensity increase range remain below the saturation intensity of the excitation light, above which a further increase in the intensity of the excitation light will not be higher Intensity of the luminescent light of the isolated molecule more result. It is preferred in the method according to the invention if a maximum intensity, i. H. an absolute intensity level of the excitation light is respectively set so that the initial location area is in a range of not more than 90% of the saturation intensity of the excitation light with respect to each position of the intensity distribution of the excitation light set in the step (i).

With decreasing local area, the maximum intensity of the excitation light can be increased. In this way, the decreasing local area can be divided back to the full bandwidth of the different intensities of the excitation light in the intensity increase range and thus to the full bandwidth of the different intensities of the luminescent light from the separated molecule.

Specifically, the maximum intensity of the excitation light can be increased by at least 50% over all repetitions of steps (i) and (ii). It can also be increased by at least 100%, or 3 times, 4 times, or more times its initial value.

The steps (i) and (ii) of the method according to the invention can be repeated until the further location area in the last performed step (ii) is no longer greater than a predetermined precision. This predetermined precision may be in the range of 20 nm or smaller. It can also be less than 10 nm. Also, a predetermined precision on the order of 1 nm and thus down to 0.5 nm is possible. Basically, the method according to the invention has no inherent limit on the precision achievable in determining the location of the singulated molecule in the sample. However, circumstances occurring in each case, such as a decreasing signal-to-noise ratio, may limit the precision that can be achieved in practice. Thus, as a termination criterion for repeating steps (i) and (ii) in the method according to the invention, the undershooting of a predetermined signal-to-noise ratio or a lower limit for an achieved further reduction of the location area can be determined, in which the isolated Molecule is located in the sample.

In the method according to the invention, at the beginning of determining the location of the singulated molecule, an area of the sample comprising the singulated molecule can be scanned in each of the spatial directions with the at least one intensity increase range, wherein the first initial location area is determined from the course of the intensity of the luminescence light during the scanning becomes. This spatial scanning of at least one region of the sample can be carried out with comparatively low intensity of the excitation light and in comparatively large spatial and small temporal steps, because the initial local area has to be determined only roughly from this. Instead of having the at least one intensity increase region, the portion of the sample comprising the singulated molecule can be detected at the beginning of determining the location of the singulated molecule with a Gaussian intensity distribution of the excitation light, i. H. with a simple focused beam of excitation light, scanned in each of the spatial directions. This corresponds to a confocal microscopic image of the isolated molecule in the sample.

In another embodiment of the method according to the invention, at the beginning of determining the location of the singulated molecule, the excitation light having a Gaussian intensity distribution is directed only pointwise or on circular or spiral paths onto an area of the sample comprising the singulated molecule, the first initial location area being derived from the course the intensity of the luminescent light is determined via the points or tracks. In order to limit the number of photons emitted by the isolated molecule of the luminescent light, the Gaussian intensity distribution is very fast to move and / or small with respect to the maximum intensity of the excitation light. When moving the Gaussian intensity distribution on circular or spiral paths, the intensity increase region in the periphery of the Gaussian intensity distribution of the excitation light can be used to apply the isolated molecule only with low intensity of the excitation light, but sufficient to localize the initial local area by localization of the excitation light Molecule to determine.

In still another embodiment of the method of the invention, at the beginning of determining the location of the singulated molecule, an area of the singulated molecule is obtained Probe spatially, ie in total, subjected to the excitation light and imaged on a the luminescence with spatial resolution registering detector, such as a camera. Then, the first initial location area can be determined from the spatial distribution of the luminescence light registered with the detector. Since the local area does not have to be particularly narrowed down in this determination, only a few photons from the isolated molecule are sufficient for this purpose.

In all of the steps described here, which can be carried out at the beginning of determining the location of the singulated molecule in order to determine the first initial location area, a maximum intensity of the luminescence light from the molecule upon excitation with the excitation light or a correlated intensity value can be detected at the same time is a measure of the relative luminosity of the isolated molecule.

In the method according to the invention, the luminescent light can also be registered, in principle, spatially, for example with a spatially resolving detector, such as a camera, but also a smaller array of, for example, only 2x2 or 3x3 point sensors. From the sum of the photons of the luminescent light registered by the different positions of the point of view of the excitation light from the separated molecule, its position can additionally be determined by localization. In this case, a location of the molecule in the sample resulting from the localization may indicate a specific orientation of the molecule in the sample, because the localization for determining the location of the molecule in the sample is influenced by its orientation, but not the determination according to the invention the location of the molecule in the sample. In the method according to the invention, it does not matter where the particular photon emitted by the molecule is registered. Accordingly, the luminescent light can also be registered with a point detector in the method according to the invention.

In the method according to the invention, prior to determining the location of the singulated molecule, the sample may be subjected to a switching signal which singles the molecule from adjacent like molecules by switching the neighboring like molecules to a dark state, as opposed to the singulated molecule which they can not be excited by the excitation light to emit luminescent light. This dark state may be another conformational state of the molecules that is not luminescent. It can also be an electronic dark state. Alternatively, the switching signal can switch only the molecule to be separated into a luminescent state. The switching signal may be switching light of a different wavelength and intensity than the excitation light. However, it can also have the same wavelength as and only a different intensity than the excitation light. In principle, the switching signal can also be the excitation light itself.

A repeated implementation of the method according to the invention can be used to determine the location of a multiplicity of molecules that are sequentially separated and excitable with excitation light for emission of the luminescent light and that mark a structure of interest in the sample. In another embodiment, a repeated implementation of the method according to the invention serves to track the separated molecule moving in the sample. In this case, the first initial location area for each repetition of the method according to the invention may be the smallest previously determined further location area of the molecule plus an error range surrounding it. The width of this error range is to be matched to the maximum movement speed of the molecule in the sample.

For carrying out the method according to the invention, an STED scanning fluorescent microscope can be used, the STED light provided by the STED scanning fluorescent microscope being used with a zero point adjacent to intensity maxima as the excitation light.

Advantageous developments of the invention will become apparent from the claims, the description and the drawings. The advantages of features and of combinations of several features mentioned in the description are merely exemplary and can take effect alternatively or cumulatively, without the advantages having to be achieved by embodiments according to the invention. Without thereby altering the subject matter of the appended claims, as regards the disclosure of the original application documents and the patent, further features can be found in the drawings, in particular the illustrated geometries and the relative dimensions of several components and their relative arrangement and operative connection. The combination of features of different embodiments of the invention or of features of different claims is also possible deviating from the chosen relationships of the claims and is hereby stimulated. This also applies to those features which are shown in separate drawings or are mentioned in their description. These features can also be combined with features of different claims. Likewise, in the Patent claims listed features omitted for further embodiments of the invention.

The features mentioned in the patent claims and the description are to be understood in terms of their number that exactly this number or a greater number than the said number is present, without requiring an explicit use of the adverb "at least". For example, if we are talking about an isolated molecule, it is to be understood that there is exactly one isolated molecule, two isolated molecules or more isolated molecules whose locations are determined. The features cited in the claims may be supplemented by other features or be the only features exhibited by the claimed subject matter.

The reference numerals contained in the claims do not limit the scope of the objects protected by the claims. They are for the sole purpose of making the claims easier to understand.

Further information on possible embodiments of the present invention can be made of WO 2015/097000 A1 be removed. Everything disclosed there and not inconsistent with the present invention may also be realized in the practice of the present invention.

list of figures

• 1 zeigt schematisch ein STED-Mikroskop, das zur Durchführung des erfindungsgemäßen Verfahrens zum Bestimmen des Orts eines vereinzelten, mit Anregungslicht zur Emission von Lumineszenzlicht anregbaren Moleküls in einer Probe verwendbar ist.
• 2 zeigt einen Schnitt durch eine Intensitätsverteilung von Anregungslicht mit einer von Intensitätsmaxima benachbarten Nullstelle, das bei der Durchführung des erfindungsgemäßen Verfahrens mit dem STED-Mikroskop gemäß 1 auf eine Probe gerichtet wird, und resultierende Intensitäten von Lumineszenzlicht, das von dem an den jeweiligen Ort in der Probe angeordneten lumineszenten Molekül emittiert wird.
• 3 illustriert eine erste Durchführung von Schritten des erfindungsgemäßen Verfahrens in Bezug auf einen ersten anfänglichen Ortsbereich des vereinzelten Moleküls in der Probe.
• 4 illustriert eine zweite Durchführung der Schritte des erfindungsgemäßen Verfahrens in Bezug auf einen verkleinerten anfänglichen Ortsbereich des vereinzelten Moleküls in der Probe.
• 5 illustriert eine alternative Ausführungsform einer Durchführung der Schritte des erfindungsgemäßen Verfahrens mit einer gaußförmigen Intensitätsverteilung des Anregungslichts.
• 6 illustriert zwei aufeinanderfolgende Durchführungen der Schritte des erfindungsgemäßen Verfahrens mit einer ersten Relativanordnung von Positionen eines Aufpunkts der Intensitätsverteilung des Anregungslichts in Bezug auf die anfänglichen Ortsbereiche.
• 7 illustriert zwei aufeinanderfolgende Durchführungen der Schritte des erfindungsgemäßen Verfahrens mit einer anderen Relativanordnung der Positionen des Aufpunkts der Intensitätsverteilung des Anregungslichts in Bezug auf die anfänglichen Ortbereiche.
• 8 ist ein Blockdiagramm einer Ausführungsform des erfindungsgemäßen Verfahrens zur Bestimmung des Orts eines vereinzelten Moleküls in einer Probe.
• 9 ist ein Blockdiagramm einer wiederholten Durchführung des erfindungsgemäßen Verfahrens zur Abbildung einer mit vereinzelbaren Molekülen markierten Struktur in einer Probe; und
• 10 ist ein Blockdiagramm einer wiederholten Durchführung des erfindungsgemäßen Verfahrens zum Tracken einer Bewegung eines vereinzelten Moleküls in einer Probe.

In the following the invention will be further explained and described with reference to preferred embodiments shown in the figures.

• 1 1 schematically shows an STED microscope which can be used to carry out the method according to the invention for determining the location of an isolated molecule excitable with excitation light for emitting luminescent light in a sample.
• 2 shows a section through an intensity distribution of excitation light with a zero intensity adjacent to the zero point, which in the implementation of the method according to the invention with the STED microscope according to 1 is directed to a sample, and resulting intensities of luminescent light emitted from the luminescent molecule located at the respective location in the sample.
• 3 illustrates a first implementation of steps of the inventive method with respect to a first initial location area of the singulated molecule in the sample.
• 4 illustrates a second implementation of the steps of the method according to the invention with respect to a reduced initial local area of the isolated molecule in the sample.
• 5 illustrates an alternative embodiment of carrying out the steps of the method according to the invention with a Gaussian intensity distribution of the excitation light.
• 6 illustrates two successive implementations of the steps of the method according to the invention with a first relative arrangement of positions of a point of the intensity distribution of the excitation light with respect to the initial location areas.
• 7 illustrates two successive implementations of the steps of the method according to the invention with another relative arrangement of the positions of the point of the intensity distribution of the excitation light with respect to the initial location areas.
• 8th Figure 3 is a block diagram of one embodiment of the method of the invention for determining the location of a singulated molecule in a sample.
• 9 Figure 10 is a block diagram of a repeated procedure of the method of the invention for imaging a singly-labeled structure in a sample; and
• 10 Figure 10 is a block diagram of a repeated practice of the method of the invention for tracking a movement of a singulated molecule in a sample.

DESCRIPTION OF THE FIGURES

1 schematically shows a STED fluorescent light microscope 1 with which a method according to the invention can be carried out. In carrying out the method according to the invention, not all components of the STED fluorescent light microscope are necessarily 1 used. The STED fluorescent light microscope 1 but includes all components necessary for carrying out the method according to the invention. A light source 2 in the usual use of the STED fluorescent light microscope 1 STED light provides provides when using the STED fluorescent light microscope 1 for carrying out the method according to the invention excitation light 3 ready. With beam shaping agents 4 becomes the excitation light 3 shaped so that it is in the focus of a lens 5 has an intensity distribution with at least one intensity increase range. In one embodiment of the method according to the invention, the excitation light 3 in the focus of the lens 5 an intensity distribution with a zero on, in all Spatial directions in which the location of an isolated molecule in a sample 6 is to be determined, intensity maxima are adjacent on both sides. The flanks of these intensity maxima then form the intensity increase ranges used in the method according to the invention. The beam shaping agent 4 may include only passive components or also an active optic, such as a Spatial Light Modulator (SLM). Another light source 7 of the STED fluorescent light microscope 1 , which provides excitation light in its usual use, switching light can in the implementation of the method according to the invention 8th provide to the molecule in the sample 6 to be singulated, whose location in the sample is subsequently determined. The singulation of the molecule can be based on the fact that other similar molecules with the switching light 8th be switched to a dark state in which they are not with the excitation light 3 can be excited to emit luminescent light. The switching light 8th is coupled into the beam path of the excitation light 3, including here a dichroic beam splitter 9 is provided. With a scanner 10 becomes the rise area or the zero point of the excitation light 3 in the sample 6 postponed. About a dichroic beam splitter 11 that from the sample 6 out in front of the scanner 10 is arranged, becomes luminescent light 12 from the sample 6 from the beam path of the excitation light 3 decoupled and with an optic 13 on a camera 14 displayed. The camera 14 is an example of a spatial resolution detector for the luminescent light 12 , Alternatively, using a dichroic beam splitter 15 the luminescent light 12 from the sample 6 a point detector 16 with an upstream pinhole 17 fed. The luminescent light 12 from the sample 6 comes from the isolated molecule that is excited by the excitation light 3 is excited to emit the luminescent light. The underlying of this emission of luminescent light process is photoluminescence, in particular fluorescence. The sample 6 is on a sample holder 18 arranged. With the sample holder 18 For example, the sample may additionally be in the z-direction, ie in the direction of the optical axis of the objective 5 be shifted to the intensity increase range or the zero point of the intensity distribution of the excitation light 3 also in this direction in the sample 6 to shift, especially if the zero is also adjacent in the z direction of intensity maxima. On the camera 14 will be the sample 6 as shown, that a localization of the isolated molecule from the spatial distribution of the photons of the luminescent light 12 from the molecule over the camera 14 is possible. In the method according to the invention, however, at least in addition the location of the molecule in the sample 6 due to the intensities of the luminescent light 12 with different arrangement of the intensity increase range or the zero point of the intensity distribution of the excitation light 3 in the sample 6 certainly.

In 2 is the intensity distribution 19 of the excitation light 3 along a section in the x-direction transverse to the optical axis of the lens 5 according to 1 shown. The intensity distribution comprises a central zero 20 in which the intensity I _A of the excitation light 3 goes down to zero or at least close to zero. This zero point 20 are on both sides of intensity maxima 21 adjacent. Between the zero point 20 and the intensity maxima 21 are intensity increase ranges 22 educated. In these intensity increase ranges 22 the intensity I _{A of} the excitation light increases 3 from zero to a saturation intensity I _S and beyond. At the saturation intensity I _S reaches the intensity L with the excitation light 3 excited luminescent light 12 a saturation value I _SW beyond which the intensity L does not increase any further. The intensity distribution 19 of the excitation light 3 is symmetric to the zero 20 formed, ie the intensity increase ranges are symmetrical to each other.

3 gives for a location of the zero 20 at x _N the resulting intensity L of the luminescent light 12 depending on the location x of the isolated molecule in the sample. The intensity L rises above the intensity increase ranges 22 from zero to the saturation value I _SW at. This is used in the method according to the invention as follows. For example, from the distribution of the luminescent light 12 from the isolated molecule over the camera 14 with spatial illumination of the sample 6 with the excitation light 3 according to 1 becomes an initial location area 23 determines the location of the molecule in the sample 6 located. By positioning the zero point 20 with the help of the scanner 10 according to 1 at a position x _N1 becomes the left-hand intensity area 22 so in the sample 6 arranged it to be the initial location area 23 spans. Then, while the sample is being charged 6 with the excitation light 3 that of the actual place x ₀ Luminescent light emitted molecule located 12 registered and its intensity I ₁ determined. The accuracy with which the intensity I _{1 is} determined depends on the number of photons of the luminescent light 12 off to that position x _N1 the zero is registered. If the intensity I _1, as well as the saturation value Isw, were known exactly, the known progression of the intensity could L over the distance to the zero point 20 exactly to the place x ₀ of the isolated molecule. However, with a limited number of photons registered to determine intensity I ₁ , there remains an error here. Likewise, an error remains in the determination of the saturation value Isw, for example, from the initial one with the camera 14 registered photons of the luminescent light 12 from the molecule. On the other hand is the Course of intensity L of the luminescent light 12 quite accurately determinable. Near the zero point 20 it is at excitation of the molecule for the emission of luminescent light 12 by a one-photon process usually square of the distance to the zero 20 dependent. Despite the inaccuracies in the determination of I ₁ and I _SW can therefore be due to the measurement of I ₁ another location area 24 determine in which the place x ₀ the molecule is located and the significantly smaller than the location area 23 is. This further location area 24 is in 3 for certain faulty measured values of I ₁ and I _SW drawn.

4 shows how the knowledge of the wider local area 24 in the method according to the invention for the renewed positioning of the zero point 20 now at the position x _N2 is used to the actual place x ₀ of the molecule with even higher precision, ie spatial resolution, in the sample 6 to determine. This is the position x _N2 the zero point 20 again arranged so that the left intensity increase range 22 over the local area 24 extends. In addition, the absolute intensity of the excitation light 3 increased so that the saturation value Isw of the luminescent light 12 over a smaller distance to the zero point 20 is reached. Then get to this position x _N2 the zero point 20 the intensity I _{2 of} the luminescent light 12 determined by the molecule. From this intensity I ₂ and the saturation value Isw can then another location area 25 be determined in which the molecule is in the sample 6 is located and again smaller than the Ortsbereich 24 is. This further location area 25 can then be used to position the zero 20 at yet another position in the sample 6 be used to the place x ₀ of the molecule in the sample with even higher precision. The local area 25 but gives the place x ₀ of the molecule in the sample already with much higher accuracy than the initial location area 23 at. In fact, the place x ₀ of the molecule in the sample 6 be determined with the inventive method practically with an error of not more than the order of 1 nm.

Instead of the other local area 24 . 25 from the intensity I ₁ or I ₂ and the saturation value Isw of the intensity L of the luminescent light 12 can determine the root 20 also be arranged at two different positions x _N1a and x _N1b in the sample to the associated intensities of the luminescent light 12 to measure and then can from these two intensities and the course of the intensity L over the distance to the zero point 20 the additional location area 24, 25 can be determined without having to resort to the saturation value Isw.

5 illustrates a corresponding procedure with a Gaussian intensity distribution of the excitation light 3 , which, in the absence of reaching a saturation limit value in a likewise Gaussian intensity distribution of the luminescent light 12 results. The intensity increase ranges 22 can be found on the flanks of the Gaussian intensity distributions. Around the initial location area 23 of the place x ₀ of the molecule in the sample 6 Narrow down, here is the focus 26 the Gaussian intensity distribution of the excitation light 3 in two different positions x _G1a and x _G1b positioned so that each of the in 5 left intensity increase range 22 the initial location area 23 spans. To each of these positions x _G1a and x _G1b becomes the intensity I _1a and I _1b , respectively, of the luminescent _light 12 registered by the molecule. From the intensities I _1a and I _1b , along with the course of the intensity L of the luminescent light 12 above the distance to the center of gravity 26 a smaller further local area 24 determined in which the place x ₀ of the molecule in the sample 6 located. It should be noted that 5 not shown on the same scale as the 3 and 4 and that then, if advantageously, the lowest possible intensities of the excitation light 3 in the rise areas 22 be used to claim the isolated molecule as little as possible, the possitions x _G1a , x _{G1b of} the point of intensity distribution I _A of the excitation light 3 much further away from the place of interest x ₀ of the molecule in the sample must be arranged as the positions x _N1 respectively. x _N2 the zero point 20 , This is because the Gaussian intensity distribution I _A of the excitation light 3 a half width of the diffraction limit at the wavelength of the excitation light 3 and the areas of low intensity I _A of the excitation light 3 about this half-width of the center of gravity 26 are removed while the small intensities of the excitation light 3 in the intensity increasing regions 22 in the intensity distribution of the excitation light 3 with the zero point 20 directly adjacent to the zero point 20.

6 shows the initial location area 23 of the molecule in the sample 6 in an xy plane perpendicular to the optical axis of the objective 5 according to 1 , Around this location area are four equally spaced positions of the zero 20 as a point of reference of the intensity distribution of the excitation light 3 according to 2 located. If at each of these positions the zero point 20 the intensity of the luminescent light from the molecule 27 is registered, it can become the more local area 24 be determined. It is one of the in 6 shown position of the zero point 20 only optionally, which is indicated by the drawing in the position shown above right here. Accordingly, in a next step of the method according to the invention, the further location area 24 by positioning the zero 20 at adjacent positions continue on the other local area 25 be restricted to the place of the molecule 27 in the sample 6 with even smaller error indicates. Again, one of the in 6 illustrated position of the zero point 20 optional, which is again indicated in the drawing above.

7 shows a different arrangement of the zero 20 opposite the initial location area 23 and the further location area 24 in each case three equally spaced positions. These three positions also include x and y in each spatial direction in which the location of the molecule 27 is determined, at least two different positions.

That in the 6 and 7 The procedure illustrated for two dimensions can also be extended to three dimensions. These are to each local area 23 - 25 in each case at least four positions of the zero point 20 set.

8th is a block diagram of one embodiment of the method according to the invention. After the start 28 a determination is made 29 of the initial location area 23 , Then a setting is made for the specific location area 30 from positions of the point of view, ie the zero point 20 or the center of gravity 26 , In the following step 31 becomes the intensity distribution of the excitation light 3 placing the point at the positions on the sample 6 directed and that of the isolated molecule in the sample 6 emitted luminescent light is registered. Based on in the step 31 detected intensities of the luminescent light is then determined 32 of a further location area 24 optionally with additional use of the saturation value Isw of the luminescent light from the molecule. Subsequently, a review takes place 33 instead of whether by the dimension of the location area 24 given precision already meets a specification for this precision. If the review 33 shows that the precision is not high enough, the steps become 30 - 32 repeated, while setting 30 the positions of the point of view relative to the other local area 24 be determined. If the review 33 has a positive result is the end 34 the method for determining the location of the molecule 27 in the sample 6 reached.

9 is a block diagram of a method in which the method according to 8th concerning his steps 29 - 33 is carried out repeatedly. Here takes place after the start 28 first a singling 35 of the molecules. Then be in a routine 36 using the steps 29 -33 for each of the isolated molecules determines their locations. In a review 37 it is determined whether a structure marked with the molecules is already displayed with sufficient degree of detail. If not, some of the molecules that mark the structure are again singled out. If the marked structure in the review 37 is considered sufficient, is the end 34 reached.

10 is a block diagram for repeating the steps 29 - 33 according to 8th for tracking an isolated molecule 27 in the sample 6 , This will be after the start 28 the momentary place of the molecule 27 determined with the desired accuracy by performing steps 29-33. Subsequently, it is optionally maintained 38 or it will immediately re-the current location of the molecule in the sample 6 certainly. At each of the repetitions of determining the location of the molecule 27 with the steps 29 - 33 may be the last certain smallest area of the molecule 27 in the sample 6 be widened centrically to a new initial location area, then as the initial location area 23 for re-performing steps 29-33. The expansion of the last determined smallest location area is to make it so that the molecule 27 in the meantime, have not yet moved out of the expanded area.

LIST OF REFERENCE NUMBERS

1

STED fluorescent light microscope

2

light source

3

excitation light

4

Beam shaping means

5

lens

6

sample

7

light source

8th

switching light

9

dichroic beam splitter

10

scanner

11

dichroic beam splitter

12

luminescence

13

optics

14

camera

15

dichroic beam splitter

16

point detector

17

pinhole

18

sample holder

19

intensity distribution

20

Zero (Aufpunkt) of the intensity distribution 19 of the excitation light 3

21

maximum

22

Intensity increase area

23

initial location area

24

further local area

25

further local area

26

Center of gravity (point of view) of the intensity distribution 19 of the excitation light 3

27

molecule

28

begin

29

Determine

30

Establish

31

step

32

Determine

33

Verification

34

The End

35

seperate

36

routine

37

Verification

38

Waiting

I _A

Intensity of the excitation light 3

L

Intensity of the luminescent light 12

I _S

Saturation intensity of the excitation light 3

I _SW

Saturation value of the intensity L of the luminescent light 12

x ₀

Place of the molecule 27 in the sample 6

x _N1

Position of the zero point 20 the intensity distribution 19 of the excitation light 3

x _N2

Position of the zero point 20 the intensity distribution 19 of the excitation light 3

x _G1a

Position of the center of gravity 26 the intensity distribution 19 of the excitation light 3

_XG1b

Position of the center of gravity 26 the intensity distribution 19 of the excitation light 3

Claims (22)

Method for spatially high-resolution determination of the location of an isolated molecule (27) in one or more spatial directions in a sample (6), the molecule (27) being excitable with excitation light (3) for emitting luminescent light (12), the excitation light (3) with an intensity distribution (19) is directed to the sample (6), in each of the spatial directions at least one intensity increase region (22) with known strictly monotonic course of the intensity (I _A ) of the excitation light (3) over a distance to a Having the point of origin (20, 26) of the intensity distribution (19), - wherein the point of view (20, 26) of the intensity distribution (19) in each of the spatial directions is arranged at different positions in the sample (6), - to each position of the point of view (20, 26) of the intensity distribution (19) in the sample (6), the luminescent light (12) emitted by the molecule (27) is registered, and - wherein the location of the molecule in the sample (x ₀ ) of Inte derived from the registered luminescent light (12), characterized in that - an initial location region (23) is determined in the sample (6) in which the molecule (27) is arranged, - (i) in each of the spatial directions at least a position of the point of view (20, 26) of the intensity distribution (19) is set such that the at least one intensity increase region (22) extends beyond the initial spatial region (23) in the respective spatial direction, - (ii) from intensity values (I ₁ , I _SW , I _1a , I _1b ) of the luminescent light (12), which for each of the spatial directions comprise two intensity values (I ₁ , I _SW or I _1a , I _1b ), one of which (I ₁ or I _1a ) indicates the intensity of the luminescent light (12) registered to the at least one position of the point of view of the intensity distribution (19), a further location area (24, 25) in the sample (6) in which the molecule (27) is arranged, and the smaller than the initial one e local area (23), and - that steps (i) and (ii) are repeated at least once using the further location area as the new initial location area (23). Method according to Claim 1 , characterized in that the intensity (I _A ) of the excitation light (3) in the at least one intensity increase region (22) increases from zero. Method according to Claim 1 or 2 , characterized in that the Aufpunkt a zero point (20) of the intensity distribution (19) is followed by in each of the spatial directions on one side of the at least one intensity increase region (22) and on the opposite side of a further intensity increase region (22). Method according to Claim 3 , characterized in that the at least one intensity increase region (22) and the further intensity increase region (22) are formed symmetrically to the zero point (20). Method according to one of the preceding claims, characterized in that the second intensity value to the respective spatial direction is an intensity (I1 _b ) of the luminescent light (12) registered to a second position of the point of view of the intensity distribution (19) Spatial direction elsewhere on the same side as the at least one position of the point of the intensity distribution (19) or on the at least one position of the point of the intensity distribution (19) opposite side of the initial location area (23). Method according to one of the preceding claims, characterized in that the number of positions determined in each execution of step (i) is between n + 1 and 2n, where n is the number of spatial directions in which the molecule (27 ) is located. Method according to one of Claims 1 to 4 , characterized in that the second intensity value to the respective spatial direction is a saturation value (I _SW ) of the intensity (I _L ) of the luminescent light (12) of the molecule (27) when excited with the excitation light (3). Method according to one of the preceding claims, characterized in that the luminescent light (12) is registered to the positions of the point of view of the intensity distribution (19) quasi simultaneously by the Aufpunkt (20, 26) is repeatedly shifted between the positions. Method according to one of the preceding claims, characterized in that the luminescent light (12) is registered to the positions of the point of view (20, 26) of the intensity distribution (19) only until the intensities of the registered luminescent light (12) with the positions a sufficient accuracy are detected, so that the further location area (24, 25) by a predetermined value smaller than the initial location area (23) can be determined. Method according to Claim 9 , characterized in that the predetermined value by which the further location area (24, 25) in the respective spatial direction is smaller than the initial location area (23) can be determined, in a range of 5% to 75%. Method according to one of the preceding claims, characterized in that a maximum intensity of the excitation light (3) is adjusted so that the initial location area (23) is in a range of. With respect to each position of the point (20, 26) of the intensity distribution (19) is not more than 90% of a saturation intensity (Is) of the excitation light (3). Method according to Claim 11 , characterized in that the maximum intensity of the excitation light (3) is increased in at least one repetition of steps (i) and (ii). Method according to Claim 12 , characterized in that the maximum intensity of the excitation light (3) over all repetitions of steps (i) and (ii) is increased by at least 50%. Method according to one of the preceding claims, characterized in that the steps (i) and (ii) are repeated until the further location area (24, 25) is no greater than a predetermined precision. Method according to Claim 14 , characterized in that the predetermined precision is in the range between 0.5 and 20 nm. Method according to one of the preceding claims, characterized in that a region of the sample (6) comprising the singulated molecule (27) at the beginning of determining the location of the singulated molecule (27) with the at least one intensity increase region (22) or a Gaussian intensity distribution ( 19) of the excitation light (3) is scanned in each of the spatial directions, wherein from the course of the intensity (I _L ) of the luminescence light (12) during the scanning, the first initial location area (23) is determined. Method according to one of the preceding claims, characterized in that the excitation light (3) at the beginning of determining the location of the isolated molecule (27) with a Gaussian intensity distribution (19) pointwise or on circular or spiral paths on a isolated molecule (27) 6), wherein the first initial location area (23) is determined from the course of the intensity (I _L ) of the luminescent light (12) over the points or tracks. Method according to one of the preceding claims, characterized in that a region of the sample (6) comprising the singulated molecule (27) is spatially applied to the excitation light (3) at the beginning of determining the location of the singulated molecule (27) and to a spatially resolving one Detector is shown, wherein from a spatial distribution of the registered with the detector luminescent light (12) of the first initial location area (23) is determined. Method according to one of the preceding claims, characterized in that the luminescent light (12) is registered with spatial resolution, wherein additionally a spatial distribution of the total registered luminescent light (12) with respect to the location of the molecule (27) in the sample (6) is evaluated , Method according to one of the preceding claims, characterized in that the sample (6) prior to determining the location of the singulated molecule (27), is applied with a switching signal which singulates the molecule (27) of adjacent like molecules by the adjacent like molecules (27) after singulation - as opposed to the singulated molecule (27) - can not be excited by the excitation light (3) to emit luminescent light (12). Repeated performance of the method according to one of the preceding claims for determining the location of a multiplicity of molecules (27) which can be excited one after the other and excite light (3) for emission of the luminescent light (12), marking a structure of interest in the sample (6). Repeated performance of the procedure according to one of Claims 1 to 20 for tracing the isolated molecule (27) moving in the sample (6). 23. Use of a STED scanning fluorescence microscope for performing the method according to one of Claims 1 to 22 wherein STED light provided by the STED scanning fluorescent microscope is used as the excitation light (3).

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