Method, computer program and device for determining the position of molecules in a sample

Abstract

The invention relates to a method for determining the position of two or more mutually spaced molecules in one or more spatial directions in a sample by means of a localization microscope, a computer program having instructions and a device. Furthermore, the invention also relates to a localization microscope which uses a device according to the invention. For determining the position of the molecules, light distributions which arise as a result of the interference of coherent light are used. In the method, a plurality of light distributions is generated using a first light modulator having a plurality of switchable pixels (S1). The first light modulator is arranged in the imaging plane of the localization microscope. Each light distribution has a local intensity minimum and an adjoining region of increasing intensity. Each of the two or more molecules is illuminated with a respective light distribution (S2). For each light distribution, the photons emitted by the molecule are detected (S4) for different positions of the light distribution. Here, the light distributions are positioned independently of one another (S3). Finally, based on the photons detected for the different positions of the light distribution, the position of the molecule is derived (S5).

Description

Technical Field

[0001] This invention relates to a method, a computer program with instructions, and an apparatus for determining the position of molecules in a sample. More particularly, this invention relates to a method, a computer program with instructions, and an apparatus for determining the position of two or more spaced-apart molecules in a sample along one or more spatial directions using a positioning microscope, wherein a light distribution generated by the interference of coherent light is used to determine the position of the molecules. Furthermore, this invention also relates to a positioning microscope using the apparatus according to the invention. Background Technology

[0002] MINFLUX microscopy involves the application of positioning microscopy techniques. The localization of fluorophores in a sample is achieved using a structured laser distribution. The term MINFLUX is based on the scheme of determining the coordinates of molecules using a minimum number of fluorescent photons (minimal emission fluxes). A fundamental characteristic of MINFLUX microscopy is that fluorophore excitation is performed such that the fluorophore to be located is always placed near or within the minimum of the excitation light distribution, ideally at zero, where the excitation light distribution must have an intensity-increasing region adjacent to the minimum. This allows for better utilization of fluorescent photons in obtaining information about the location of the corresponding emitting fluorophore. This also applies to applications where the fluorophore moves over time. The principle of MINFLUX microscopy is known from DE 10 201 1 055 367 B4, US 2014 / 0042340 A1, and DE 10 201 3 114 860 B3.

[0003] DE 10 2011 055 367 B4 describes a method for tracking the motion of particles in a sample. In this method, light is used to induce particles to emit photons, and the photons emitted by the particles are recorded. Here, the light is aligned with the sample with an intensity distribution having a spatially constrained minimum. The minimum is used to track particles moving in the sample in such a way that the intensity distribution is shifted relative to the sample such that the ratio of photons emitted by the particles remains minimal.

[0004] US 2014 / 0042340 A1 further describes how images of the traces of tracked particles can be obtained based on the residence time of the particles in a segment of the sample. Furthermore, this document describes how a combination of excitation and cutoff light distributions can be reasonably used for particle tracking, particularly the distribution of light that excites the excited fluorophores via excitation rays. Here, the local central minima of the two light distributions should coincide with each other. When tracking particles that are always located at the common minima of the two light distributions, other fluorophores reaching the influence region of the light distribution are prevented from emitting fluorescence, thus avoiding interference with the tracking of the corresponding particle.

[0005] Reference DE 10 2013 114 860 B3 describes a method for determining the positions of molecules in a sample. The molecules are in a fluorescent state and are excited to emit fluorescence under excitation light, wherein the intensity distribution of the excitation light has at least one zero point. Fluorescence originating from the excited molecules is recorded at different positions relative to the at least one zero point of the excitation light intensity distribution. The positions of the individual molecules are then derived from the curves showing the change in fluorescence intensity relative to the positions of the at least one zero point of the excitation light intensity distribution.

[0006] Based on this, a series of detailed information collection methods are formed. For a detailed description of MINFLUX microscopy, see F. Balzarotti et al.'s "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes" [1]. In order to locate the fluorophore by means of MINFLUX microscopy, in principle, the minimum and maximum intensity values ​​among a large number of locations must be located relative to the position of the fluorophore. For this purpose, in the preliminary step, the position of the fluorophore is estimated with a low first accuracy. This can be achieved by means of conventional positioning microscopes, such as PALM (photosensitive positioning microscope) or STORM (random optical reconstruction microscope), or by means of other known methods, such as based on the use of a structured illumination device. Subsequently, the intensity distribution of the excitation light is located at a known location, which is selected such that the fluorophore is close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is measured. The same is repeated for one or more other locations of the intensity distribution. The position of the fluorophore can be determined more accurately by means of an evaluation of the intensity ratio, similar to a triangulation method. In principle, the farther the fluorophore is from the excitation minimum, or the closer the fluorophore moves towards the range of increasing intensity, the higher the emissivity. This more accurately determined position can now be used as the starting point for repeating the aforementioned steps, where the position can be closer to the estimated position of the fluorophore. In particular, considering the tracking of fluorophore movement, the change in emissivity as the fluorophore moves towards the range of increasing intensity or towards the minimum can also be used to estimate the fluorophore movement.

[0007] Some aspects of MINFLUX microscopy are disclosed in US 2019 / 0235220 A1, US 2019 / 0234882 A1 and US 2019 / 0234879 A1.

[0008] US 2019 / 0235220 A1 describes a method for determining the location of a fluorophore, wherein only a small or minimal number of locations (where a minimum intensity is placed) are required. The minimum intensity values ​​are adjacent to each other on either side of the region of increasing intensity in each spatial direction in which the location of the fluorophore should be determined.

[0009] US 2019 / 0234882 A1 describes a method in which location information obtained by a first MINFLUX step is used to place the minimum of intensity light distribution closer to the fluorophore in subsequent steps and thereby derive more accurate location information.

[0010] US 2019 / 0234879 A1 describes a method in which a minimum intensity is very rapidly placed at multiple locations around an estimated location of the fluorophore. Then, if a higher emissivity is determined at a certain location, that location is pushed closer to the assumed minimum. This method is particularly suitable for observing the movement of fluorophores.

[0011] MINFLUX microscopy enables the location of fluorophores in two spatial directions with an unreliability of only 1 nm using laboratory methods; that is, the accuracy of the location is similar to the size of the fluorophore itself. Determining the location of a single fluorophore with a given measurement unreliability requires significantly less time compared to determining its location using conventional localization microscopy. However, a drawback of MINFLUX microscopy is that determining the location of a large number of fluorophores has, to date, been extremely time-consuming.

[0012] Among all localization microscopy methods, this technique achieves highly accurate positioning of individual fluorophores. A large ensemble of such localizations allows for the creation of images of the sample with a resolution far exceeding the resolution limit of wide-field imaging defined by the Abbe limit. This finely resolved microscopy method is known as super-resolution microscopy or nanomicroscopy.

[0013] Another microscopy method can achieve a resolution slightly below the classical Abbe limit or specifically allows for the resolution of structures that are half the size of the Abbe limit. This other microscopy method is known by the term SIM (Structured Illumination Microscopy), for example, in reference MGL Stefsson’s “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy” [2]. Here, the sample in a wide field of view is loaded with a periodically varying intensity distribution, the period of which is approximately equal to the diffraction limit of the imaging system. The resulting fluorescence is imaged on a wide field of view detector. Subsequently, the intensity distribution is shifted relative to the sample by less than the period of the pattern in the sample, typically by one-third of the period, and the image is taken again. These steps are repeated again. The captured fluorescence images are different from each other, where the difference arises from the known displacement of the illumination light distribution relative to the unknown, but identical, distribution of the fluorophore. The unknown distribution of the fluorophore can be calculated from the set of images. To obtain resolution in multiple spatial directions, the grating must be rotated. In MGLostafsson’s paper “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution”[3], the method is described under the name SSIM (Saturated Structured Illumination Microscopy).

[0014] Another concept used to achieve super-resolution is called ROSE (Repetitive optical selective exposure), which is known, for example, by the paper “Molecular resolution imaging by repetitive optical selective exposure” [4] by L. Gu et al. In this method of combining localization microscopy with structured illumination, individual fluorescent molecules are excited sequentially with multiple interference-based strip intensity distributions, and the fluorescent molecules are localized by means of ratiometric position determination based on the sequentially detected fluorescence. This can achieve approximately twice the improvement in localization accuracy compared to conventional localization microscopy with the same photon budget. To generate the interference fringe pattern, the laser beam is modulated and split using an amplitude modulator. Rapid detection is achieved by a switching device in the detection optical path, which directs the fluorescence of each intensity distribution of the excitation sequence to different regions of the same CCD detector (CCD: Charge Coupled Device).

[0015] The paper “Simple: Structured Illumination based Point Localization Estimator with Enhanced Precision” [5] by L. Reymond et al. also describes a method that achieves twice the accuracy of single-molecule localization compared to traditional centroid estimation methods. SIMPLE also combines the SIM scheme with ratiometric localization, using a precise phase-shifted sine wave pattern as a nanoscale to simultaneously localize particles based on the change in the number of photons in a 20µm field of view. The sine wave pattern is generated by a micromirror actuator.

[0016] The paper “Localization microscopy at doubled precision with patterned illumination”[6] by J. Cnossen et al. describes a method called SIMFLUX, which combines centroid estimation and photon number variation caused by sinusoidal illumination patterns in a conventional wide field-of-view imaging device to extract positional information about a typical micrometer-range field of view. In addition to positional information on the camera, information about the corresponding position of the excitation intensity pattern at the detection time point is used during localization. The authors point out that the same resolution improvement as MINFLUX can be achieved in principle if the position of the excitation intensity pattern does not move over the entire period, but only within a small range. According to the related supplementary notes, this applies to fluorophores with a specific position relative to the middle phase of the excitation intensity pattern used.

[0017] US 2019 / 0353912 A1 describes an apparatus for beam shaping and scanning. This apparatus includes a digital micromirror device (DMD) with multiple micromirrors. The micromirror device allows adjustment of the axial position of the laser beam's focus along the beam's propagation direction and its lateral position in a plane perpendicular to the direction of motion. This apparatus can be applied, for example, in super-resolution microscopy. Summary of the Invention

[0018] The technical problem to be solved by the present invention is to provide an improved scheme for determining the position of molecules in a sample, thereby reducing the time cost for determining the location of multiple fluorophores.

[0019] According to a first aspect of the invention, the method is used to determine the positions of two or more spaced-apart molecules in a sample along one or more spatial directions by means of a positioning microscope, wherein, to determine the positions of the molecules, a light distribution generated by interference of coherent light is used, the method comprising the steps of:

[0020] - In the case of using a first light modulator with multiple switchable pixels, multiple light distributions are generated, the first light modulator being arranged in the imaging plane of the positioning microscope, wherein each light distribution has a local minimum intensity (or minimum intensity) and an adjacent region of increasing intensity.

[0021] - Illuminate each of two or more molecules individually (or accordingly) using a light distribution;

[0022] - For each light distribution, photons emitted by molecules are detected for different locations within the light distribution, wherein the light distributions are located independently of each other; and

[0023] - The location of molecules is deduced based on photons detected at different locations for light distribution.

[0024] According to another aspect of the invention, a computer program includes instructions that, when executed by a computer, cause the computer to perform the following steps to determine the positions of two or more mutually spaced molecules in a sample along one or more spatial directions using a positioning microscope, wherein, to determine the positions of the molecules, a light distribution generated by interference of coherent light is used:

[0025] - In the case of using a first light modulator with multiple switchable pixels, multiple light distributions are generated, the first light modulator being arranged in the imaging plane of the positioning microscope, wherein each light distribution has a local intensity minimum and an adjacent intensity increase region.

[0026] - Illuminate each of two or more molecules using a single light distribution;

[0027] - For each light distribution, photons emitted by molecules are detected for different locations within the light distribution, wherein the light distributions are located independently of each other; and

[0028] - The location of molecules is deduced based on photons detected at different locations for light distribution.

[0029] The term "computer" should be interpreted broadly here. In particular, it also includes microcontrollers, embedded systems, and other processor-based data processing devices.

[0030] The computer program may be provided, for example, for electronic retrieval, or may be stored on a computer-readable storage medium.

[0031] According to another aspect of the invention, the device is used to determine the positions of two or more spaced-apart molecules in a sample along one or more spatial directions by means of a positioning microscope, wherein, to determine the positions of the molecules, a light distribution generated by interference of coherent light is used, and the device comprises:

[0032] - A control unit designed to control a first light modulator having multiple switchable pixels, the first light modulator being arranged in the imaging plane of a positioning microscope, for generating multiple mutually independent (or mutually unrelated) positioned light distributions, wherein each light distribution has a local intensity minimum and an adjacent intensity increasing region; and

[0033] - An evaluation unit designed to deduce the position of a molecule based on the photons emitted by the molecule for each light distribution, in order to determine the different locations of the corresponding light distributions.

[0034] Multiple light distributions are used in the technical solution according to the invention to determine the positions of multiple molecules or fluorophores in parallel using a positioning microscope. For this purpose, individual light distributions are generated using light modulators arranged in the imaging plane of the positioning microscope. The light distributions can be positioned independently of each other through corresponding control of the light modulators. Parallel detection of molecules significantly reduces the time cost for scanning samples. The detected molecules or fluorophores must be simultaneously activated and have a detection spacing that allows for simultaneous, limited diffraction. Minimal spacing is required in the light distributions. If the active molecules have too small a spacing in the sample region within a time segment, these active molecules will not be observed within that time segment. In the technical solution of the invention, multiple molecules or fluorophores are observed separately, spaced so far apart that the light distributions do not contact each other.

[0035] Minflux microscopy is typically performed using fluorophores, which "flicker," meaning they change between fluorescing and non-fluorescing states. These state transitions can occur either purely randomly under fixed environmental conditions or through a switching process, i.e., by deactivation light, activation light, or a combination of deactivation light (deactivating all molecules) and activation light (activating a small number of molecules). Therefore, it is possible for only one fluorophore to fluoresce in a specific region, but the entire sample can still be imaged using most of the fluorophores. Suitable fluorophores and methods for switching between switchable fluorophores are known to those skilled in the art.

[0036] The advantage of arranging optical modulators in the imaging plane is that the movement of the light distribution in the sample can be achieved particularly simply and rapidly through direct imaging of the structure or pattern onto the optical modulator in the imaging plane. The individualized movement of the light distribution corresponds to the individualized movement of the pattern onto the optical modulator. The required movements can be calculated very quickly, thus achieving high decision-making speed.

[0037] According to one aspect of the invention, a second optical modulator for influencing the light distribution is arranged in the Fourier plane of the positioning microscope. The second optical modulator is specifically designed to block individual diffraction orders. The second optical modulator allows for targeted further influence on the light distribution generated by the first optical modulator. If, for example, multiple diffraction orders are generated by the first optical modulator, those diffraction orders that should not be used in further optical paths can be filtered out by the second optical modulator. Here, the filtering can be adjusted as needed, thereby achieving great flexibility.

[0038] According to one aspect of the invention, the first optical modulator is an amplitude modulator or a phase modulator. Preferably, the first optical modulator is an optical modulator switchable between two fixed states. Binary optical modulators have the advantage of being very fast, while analog phase modulators (as commonly used in holographic methods) are significantly slower. The optical modulator can be implemented, for example, as a liquid crystal modulator or a microelectromechanical system modulator, particularly as a micromirror array. Such optical modulators are commercially available, easy to implement, and capable of achieving frame rates from 30 kHz to 50 kHz. Higher frame rates are expected in the future. Exemplarily, an optical modulator with switchable pixels of 1920 × 1080 or 2560 × 1600 can be used.

[0039] According to one aspect of the invention, to generate a light distribution, a one-dimensional or two-dimensional structure is constructed using switchable pixels of a first light modulator. Preferably, the one-dimensional structure is a grating structure, and the two-dimensional structure is a ring structure. Through each one-dimensional grating structure, a grating-like light distribution consisting of successive maximum and minimum intensity values ​​is achieved in the sample, using which the position of, for example, a fluorophore can be determined in a spatial direction. For position determination, in particular, the central maximum intensity value and one or both of the two brightest maximum intensity values, along with the minimum intensity value located between the selected maximum values, can be used. For position determination in two spatial directions, light distributions with other orientations must be additionally provided. A ring structure can achieve a light distribution in the form of a so-called "partially hollow beam," that is, a light distribution having zeros restricted in three spatial directions, and using this light distribution, position determination in three spatial directions can be achieved. Another feasible approach is to generate a donut-shaped light distribution, i.e., a light distribution having zeros restricted in two spatial directions, and using this light distribution, position determination in two spatial directions can be achieved. For this purpose, dots or disks can be displayed on the first optical modulator. In addition, static or dynamic phase modulators, as known from STED microscopy (Stimulated Emission Depletion), are subsequently used to modulate (or display) the phase ramp.

[0040] According to one aspect of the invention, in order to locate the light distribution, a one-dimensional or two-dimensional structure constructed of switchable pixels using a first light modulator is moved. Here, the displacement of the structure is preferably less than the size of the structural elements of the structure, wherein the size of the structural elements is larger than the size of the pixels in both dimensions. For example, the grid lines formed by the pixels can have a linewidth of ten pixels. If the optical system is designed such that a 250 nm period of light distribution in the sample is obtained, then a movement of the grid lines around a pixel results in a 25 nm movement of the light distribution in the sample.

[0041] According to one aspect of the invention, in order to determine the position of a light distribution sequentially, the intensity curve of the light distribution is altered by changing the structural properties of the associated one-dimensional or two-dimensional structure. For example, by changing the lattice constant and / or aspect ratio of the one-dimensional structure or the aperture diameter of the two-dimensional structure, the edge steepness of the intensity-increasing region adjacent to the local intensity minimum can be changed. By varying the light distribution used for sequential position determination or measurement, the accuracy of position determination can be progressively improved. In this way, the position of a molecule or fluorophore can be determined with increasing accuracy in iterative steps with multiple zoom levels. This iterative position determination can be performed very rapidly, especially when using a binary light modulator.

[0042] According to one aspect of the invention, the light distribution is generated by the temporal superposition of different partial light distributions. For this purpose, for example, different diffraction orders of an interference pattern generated by a first light modulator can be interfered with in successive steps, thereby forming partial light distributions, each having a tilted axis to some extent. The direction of the tilt depends on which diffraction orders interfere. If the formed partial light distributions are superimposed temporally, a three-dimensionally additive light distribution is produced, which has a minimum value in the axial direction at the intersection of the tilted axes, with an intensity-increasing region adjacent to the minimum value. By relative movement in the radial direction between two sub-light distributions, i.e., by movement of the associated structure on the first light modulator, the intersection point, and thus the position of the minimum value, can be moved in the axial direction. This allows for very rapid determination of the position of a molecule or fluorophore in three dimensions.

[0043] According to one aspect of the invention, drift is determined by detecting a marker in the sample using at least one light distribution. Alternatively, drift can be determined from changes in the same direction of molecular positions measured in successive measurements. These methods are particularly advantageous when it is necessary not only to determine the position of molecules but also to determine the motion of molecules from successive measurements. If motion of a fixed-position marker is detected, the measured molecular motion can be corrected. However, for this purpose, a suitable marker needs to be provided in the sample. In MINFLUX microscopy, such a marker can be, for example, a fluorophore, as it is virtually non-fading due to the MINFLUX principle. To provide the marker, a bead can be decolorized, for example, from a stained structure using a locally hollow beam of light, such as in fibers within fixed cells stained with a non-switchable fluorophore. If a location is irradiated for a period of time, the fibers near that point are decolorized. In this way, a continuously emitting light spot that is, in principle, arbitrarily small can be generated. This can, for example, have a size of 40 nm to 100 nm, and then be gently positioned using the MINFLUX principle. Alternatively, the relative displacement between the sample and the measuring device can be inferred from the motion of multiple molecules in the same direction. The measured motion of individual molecules can then be corrected by the portion of the motion in the same direction.

[0044] According to one aspect of the invention, the light distribution in the sample has a lateral dimension in the range of 0.5 µm to 4 µm. It has been shown that reliable position determination can be achieved with these dimensions of the light distribution in the sample, and that multiple light distributions that can be positioned independently of each other can be realized simultaneously.

[0045] In one embodiment of the invention, the location of an active luminescent body or fluorophore can be estimated before applying localization according to the MINFLUX principle by preferably generating a sequence of one-dimensional grid-like illumination distributions using a first light modulator, wherein each illumination distribution in the sequence covers the same number of separate fluorophores. The locations of the multiple fluorophores can preferably be estimated using methods known in the art as SIMPLE, SIMFLUX, or ROSE. Subsequently, more precise localization is performed for multiple fluorophores among the multiple fluorophores or for all fluorophores simultaneously and individually according to the MINFLUX principle.

[0046] In one embodiment of the invention, a suitable wavelength of light distribution, when using a suitable projection pattern, can first be used to selectively place the fluorophore in a fluorescent state only in a defined region, or selectively maintain it in a fluorescent state in a partial region. For this purpose, in addition to the aforementioned microgrids and donuts, other basic patterns can be used. Specifically, the light distribution can be a dot matrix, either globally or locally. Furthermore, for example, by using a DMD in grayscale operations, it is possible to create a continuous planar illumination pattern with continuously varying additive light intensity, which matches the density distribution of the structure to be observed, thereby selectively generating a desired density distribution on the activated molecule. Here, when using a corresponding fluorophore, for example for activation (or so-called activation), it is preferable to use the same light modulator in the imaging plane as for subsequent excitation.

[0047] Sequences or combinations of different light distributions can also be used, such as sequences of activation light patterns with such adapted intensity distributions that inactive fluorophores switch to an active state with appropriate probability in regions of maximum intensity; and sequences of corresponding deactivation light patterns that have zero points in regions of maximum activation light intensity and thus keep the fluorophores in an active state there. It is known in principle to those skilled in the art that the activation light is adjusted to activate only a suitable small number of fluorophores. The MINFLUX principle can then be used in parallel in such prepared regions.

[0048] In other words, the focus is on knowing quite accurately from the outset where fluorophores can be in a fluorescent state, or in which regions the density of fluorescent fluorophores is suitable for single fluorophore detection, and then utilizing the fact that multiple sub-grids (Teilgitter) can move independently of each other.

[0049] For each fluorophore to be localized, a combination of excitation and deactivation light distributions can advantageously be used, where the deactivation light is STED light. Advantageously, at least one local central minimum of the STED light distribution coincides with the minimum of the excitation light distribution, and the minimum of the STED light distribution is used for localization; that is, based on previous position estimates, the fluorophores to be localized are located within the minimum. Here, the multiple light distributions do not need to correspond perfectly. For example, the STED light distribution can be selected as a donut shape, and the excitation light distribution can be selected as a one-dimensional grid. For fluorophore localization, the excitation and STED light distributions are simultaneously relocated, i.e., moved and reoriented if necessary; or the excitation light distribution can be relocated while the STED light distribution remains fixed, so that the excitation minimum is located multiple times, differently, within the region of the STED minimum; or the excitation light distribution can remain fixed while the STED light distribution is relocated. Combinations of these three schemes can also be considered. As an alternative or supplement to relocation, the size and / or intensity of the excitation and / or STED light distributions can also be changed. For example, a minimum STED donut with a larger spatial size can be initially superimposed on the excitation light distribution, and in subsequent steps, the intensity of the STED donut can be narrowed and / or increased, for example, based on the achieved positioning accuracy. In all cases described herein, to generate light distributions for two light types, it is preferable to use one light modulator for each light type, respectively arranged in the imaging plane within the corresponding excitation light path or STED light path. The light paths can be merged along the optical path direction, for example, by means of a beam combiner, which can be a dichroic element.

[0050] In one extended approach, a combination of excitation and deactivation light distributions is used, where the deactivation light is STED light, the STED light distribution having a local minimum, while the excitation light distribution does not have a local minimum. In this variant, the Minflux principle is not implemented, but the location of the molecule to be localized is still determined based on photons detected at different locations of the light distribution, preferably by means of a ratio method. For example, a luminescent or phosphor capable of switching to a protected state can be used. Such luminescent materials and their use in specific STED methods are known, for example, from WO 2014 108455 A1. In one embodiment of the invention, the corresponding luminescent material (part of which is in an excitable state in the initial state of the sample) is switched from the excitable state to the protected state at least in the region outside the local minimum of the switching light distribution. Subsequently, the sample may be loaded with corresponding light distributions, each consisting of an excitation light distribution and an STED light distribution, wherein the STED light distribution has a minimum value that overlaps positionally with the minimum value of the corresponding switching light distribution. The switching light and the STED light can be identical in wavelength. This is achieved by ensuring that only a single luminescent element can be excited with a high probability within the STED minimum, and that the luminescent element can then be localized using a ratio method. Outside the minimum, at least multiple luminescent elements are in a protected state, in which they are neither excited nor decolorized by STED light. By using STED light, the random excitable state of fluorophores adjacent to a single group of fluorophores during localization is prevented, thus avoiding interference with or prevention of localization of the fluorophores involved.

[0051] Correspondingly, fluorophores in a protected state in their initial state can also be used. These can then be locally activated by means of an activation light distribution, preferably having a diffraction-limited central maximum, and subsequently localized by means of a combination of excitation light and STED light as described. Alternatively, localization can also be performed using only the excitation light distribution.

[0052] According to one aspect of the invention, photons emitted by molecules are detected by a camera or a photon counter array. The advantage of using a camera is its low cost and ability to achieve rapid readout. The advantage of using a photon counter is its high efficiency in detecting photons and its very high temporal resolution. The photon counter can be, for example, a single-photon avalanche diode (SPAD).

[0053] According to another aspect of the invention, the microscope according to the invention comprises:

[0054] - A light source used to generate coherent light;

[0055] - A first light modulator having multiple switchable pixels, the first light modulator being arranged in the imaging plane of the microscope for generating multiple light distributions formed by the interference of coherent light, wherein each light distribution has a local intensity minimum and an adjacent intensity increasing region.

[0056] - Optical devices used to illuminate two or more spaced molecules in a sample using a single light distribution;

[0057] - A detection unit for detecting photons emitted by molecules at different locations for different light distributions, wherein the light distributions are located independently of each other; and

[0058] - An apparatus according to the invention for determining the position of two or more molecules in one or more spatial directions. Attached Figure Description

[0059] Other features of the invention will be derived from the following description taken in conjunction with the accompanying drawings.

[0060] Figure 1 The illustration schematically shows a method for determining the positions of two or more spaced-apart molecules in a sample in one or more spatial directions;

[0061] Figure 2 A first embodiment of an apparatus for determining the position of two or more spaced-apart molecules in a sample in one or more spatial directions is shown;

[0062] Figure 3 A second embodiment of an apparatus for determining the position of two or more spaced-apart molecules in a sample in one or more spatial directions is shown;

[0063] Figure 4 A sample containing multiple molecules is shown;

[0064] Figure 5 The intensity variation curve is shown on the cross section passing through the light distribution;

[0065] Figure 6 The principle structure of a microscope is shown, wherein the technical solution according to the present invention is implemented;

[0066] Figure 7 The structure is explained using a light modulator with multiple pixels;

[0067] Figure 8 This illustrates a first example of generating multiple light distributions that can be positioned independently of each other;

[0068] Figure 9 A second example of generating multiple light distributions that can be positioned independently of each other is shown;

[0069] Figure 10 An example of generating a light distribution in the form of a locally hollow beam is shown, wherein the locally hollow beam has zeros restricted in three spatial directions;

[0070] Figure 11 An example is shown of generating multiple independently locating light distributions in the form of locally hollow beams;

[0071] Figure 12 This illustrates an example of light distribution generated by the temporal superposition of different local light distributions;

[0072] Figure 13 The axial section passing through the interference pattern used for superposition is shown; and

[0073] Figure 14 Showing through according to Figure 12 Axial cross section of light distribution generated by different lateral movements of local light distribution. Detailed Implementation

[0074] To better understand the principles of the present invention, embodiments of the invention are described in more detail below with the aid of the accompanying drawings. Of course, the present invention is not limited to these embodiments, and the described features can be combined or modified without departing from the scope of protection defined in the appended claims.

[0075] Figure 1This diagram schematically illustrates a method for determining the positions of two or more molecules spaced apart from each other in one or more spatial directions in a sample. To determine the molecular positions, a light distribution generated due to interference of coherent light is used. In this method, multiple light distributions S1 are generated using a first light modulator with multiple switchable pixels. The first light modulator, such as an amplitude modulator or a phase modulator, is arranged in the imaging plane of a positioning microscope and is preferably switchable between two fixed states. Preferably, a second light modulator for influencing the light distribution is arranged in the Fourier plane, for example, to block individual diffraction orders. Each light distribution has a local intensity minimum and an adjacent region of intensity increase. Each of the two or more molecules is illuminated using one light distribution, S2. For each light distribution, photons emitted by the molecules are detected for different locations within the light distribution, S4. Here, the light distributions are positioned independently of each other, S3. Finally, the molecular positions S5 are deduced based on the photons detected for different locations within the light distributions. For the generation of the light distributions S1, a one-dimensional structure, such as a lattice structure, or a two-dimensional structure, such as a ring structure, can be constructed using the switchable pixels of the first light modulator. To position the light distribution S3, the constructed structure is then moved. Here, the displacement of the structure is preferably less than the size of the structural elements (or structural components) of the structure, wherein the size of the structural elements is larger than the pixel size in both dimensions. The structural elements are grid lines in the case of a grating structure and rings in the case of a ring structure. Here, the light distribution can also be generated by the temporal superposition of different interference patterns of coherent beams. For this purpose, for example, in successive steps, different diffraction orders of the interference pattern generated by the first light modulator can interfere, and the resulting interference patterns can move relative to each other in the axial direction. Furthermore, it is possible to change the intensity curve of the light distribution by altering the structural characteristics of the associated one-dimensional or two-dimensional structure in order to determine the successive positions of the light distribution. For example, by changing the grating constant of the one-dimensional structure or the aperture diameter of the two-dimensional structure, the edge steepness of the intensity-increasing region adjacent to the local intensity minimum can be changed. The availability of multiple light distributions can be used to detect and compensate for sample drift. For example, a marker in a sample can be detected by means of at least one light distribution. Alternatively, drift can be determined by changes in the same direction of the measured position of molecules in successive measurements.

[0076] Figure 2A simplified schematic diagram of a first embodiment of device 20 is shown, used to determine the positions of two or more spaced-apart molecules in a sample in one or more spatial directions. Device 20 has an interface 21 through which a control unit 22 controls a first light modulator 7 having multiple switchable pixels to generate multiple independently locating light distributions. The first light modulator 7 (e.g., an amplitude modulator or a phase modulator) is arranged in the imaging plane of a positioning microscope and is preferably switchable between two fixed states. Each light distribution has a local intensity minimum and an adjacent region of intensity increase. Furthermore, the control unit 22 can control a second light modulator 12 arranged in a Fourier plane via the interface 21 to influence the light distributions, for example, by blocking individual diffraction orders, and the control unit can control a light source 15 via the interface. An evaluation unit 23 receives data from a detection unit 16 via the interface 21 and uses the detection unit to detect photons emitted by the molecules. Based on the photons emitted by the molecules for each light distribution to accommodate different positioning of the corresponding light distributions, the evaluation unit 23 derives the positions of the molecules. To generate a light distribution, a one-dimensional structure, such as a grating structure, or a two-dimensional structure, such as a ring structure, can be constructed using switchable pixels of a first light modulator. The constructed structure is then moved to position the light distribution. Here, the displacement of the structure is preferably less than the size of the structural elements, where the size of the structural elements is larger than the pixel size in both dimensions. The structural elements are grating lines in the case of a grating structure and rings in the case of a ring structure. The light distribution can also be generated by the temporal superposition of different interference patterns of coherent beams. For this purpose, for example, in successive steps, different diffraction orders of the interference patterns generated by the first light modulator can interfere, and the resulting interference patterns can move relative to each other in the axial direction. Furthermore, it is possible to change the intensity curve of the light distribution by altering the structural characteristics of the associated one-dimensional or two-dimensional structure in order to determine the successive positions of the light distributions. For example, by changing the grating constant of the one-dimensional structure or the aperture diameter of the two-dimensional structure, the edge steepness of the intensity-increasing region adjacent to the local intensity minimum can be changed. The availability of multiple light distributions can be used to detect and compensate for sample drift. For example, markers in a sample can be detected using at least one light distribution. Alternatively, drift can be determined by changes in the same direction of the measured position of the molecule in consecutive measurements.

[0077] Control unit 22 and evaluation unit 23 can be controlled by monitoring unit 24. The settings of control unit 22, evaluation unit 23, or monitoring unit 24 can be changed as needed via user interface 26. Data generated in device 20 can be stored in the memory 25 of device 20 when needed, for example, for subsequent evaluation or use by components of device 20. Control unit 22, evaluation unit 23, and monitoring unit 24 can be implemented as fixed hardware, such as integrated circuits. However, they can of course be partially or completely combined or implemented as software, which runs on a suitable processor, such as a CPU or GPU. Interface 21 can also be implemented as separate input and output terminals.

[0078] Figure 3 A simplified schematic diagram of a second embodiment of device 30 is shown, used to determine the positions of two or more spaced-apart molecules in a sample in one or more spatial directions. Device 30 has a processor 32 and a memory 31. Device 30 is, for example, a microcontroller, a computer, or an embedded system. Instructions are stored in memory 31 that, when executed by processor 32, cause device 30 to perform steps according to one of the methods. Thus, the instructions stored in memory 31 embody a program executable by processor 32 that implements the method according to the invention. Device 30 has an input terminal 33 for receiving information. Data generated by processor 32 is provided through output terminal 34. Furthermore, this data can be stored in memory 31. Input terminal 33 and output terminal 34 can be combined to form a bidirectional interface.

[0079] Processor 32 may include one or more processor units, such as a microprocessor, a digital signal processor, or a combination thereof.

[0080] The memories 25 and 31 of the embodiments may have volatile or non-volatile storage areas and include different storage devices and storage media, such as hard disks, optical storage media or semiconductor memories.

[0081] Figure 4 A sample 1 containing multiple molecules 2 is shown. Five molecules 2 are exemplarily shown, and the positions of these molecules, P1 to P5, should be determined. For this purpose, molecules 2 are labeled with fluorophores, or molecules 2 are fluorophores directly. Fluoresceins can be excited by light of a suitable wavelength to emit photons. In MINFLUX microscopy, the excitation of fluorophores is thus performed such that the fluorophore to be located is always placed near or within the minimum of the light distribution used for excitation, wherein the light distribution must have an intensity-increasing region adjacent to the minimum. This achieves better utilization of fluorescent photons in obtaining information about the location of the corresponding emitting fluorophore. Ideally, the minimum of the excitation light distribution is zero.

[0082] Figure 5 An exemplary intensity variation curve is shown on a cross-section of a light distribution 4 suitable for use according to the invention. The intensity I relative to a position within the light distribution 4 (here, along the x-axis) is plotted. The intensity variation curve of the light distribution 4 has a central intensity minimum 5, with intensity increasing regions 6 adjacent to both sides of the minimum. This light distribution 4 can be implemented, for example, in the form of an intensity distribution in the shape of a donut. In this case, the central intensity minimum 5 is surrounded by intensity increasing regions 6 on all sides, thus allowing direct position determination in two dimensions. Alternatively, this light distribution 4 can also be implemented as an interference pattern of a striped grating. In this case, the light distribution 4 has more than one intensity minimum, but only one is used. Furthermore, the intensity increasing regions 6 are adjacent to the intensity minimum 5 only on two opposite sides, thus requiring a correspondingly rotated striped grating for position determination in two dimensions. As further explained below, position determination in three dimensions can also be achieved as long as the intensity distribution is appropriately varied along the beam propagation direction.

[0083] Figure 6 An exemplary principle structure of microscope 40 is shown, in which the technical solution according to the invention is implemented. A light source 15, such as a laser, emits coherent light 3 with a wavelength of, for example, 640 nm. The coherent light 3 is incident on a first light modulator 7, which is arranged in an imaging plane and has multiple switchable pixels, such as 1920 × 1080 or 2560 × 1600 pixels. Multiple light distributions that can be independently positioned can be generated by means of the first light modulator 7, in that the switchable pixels form a one-dimensional or two-dimensional structure. The light emitted from the first light modulator 7 is imaged in a Fourier plane 13 by lens 14, in which a second light modulator 12 is located. The second light modulator is used to influence the light distribution, for example, by blocking individual diffraction orders. The light emitted from the second light modulator 12 is imaged onto the sample by another lens 14 and can be used there for position determination. Photons emitted by molecules in sample 1 are deflected toward detector unit 16 via beamsplitter 17, such as a wavelength-selective beamsplitter, and detected by the detector unit. Detector unit 16 can be a camera, such as a camera with 2048×2048 pixels. Considering the Nyquist criterion used for scanning, a field of view of 64µm×40µm only requires 640×400 pixels, thus allowing for faster readout, and the use of multiple wavelengths can also be considered.

[0084] The first light modulator 7, the second light modulator 12, and the light source 15 are controlled by a device 20 according to the invention for determining molecular positions, which also evaluates data from the detection unit 16. For simplicity, other components of the positioning microscope 40, such as those affecting the polarization or movement of the lens 14 or the sample 1, are not described. Figure 6As shown in the diagram. These components are known to those skilled in the art and can also be controlled by device 20.

[0085] Ideally Figure 6 The structure shown has multiple elements precisely positioned in either the imaging plane 11 or the Fourier plane 13. A feasible structure further exists where these two conditions are only approximately satisfied. Here, the tolerable deviation along the axial direction scales with the Rayleigh length of the optics surrounding the element, which, when viewed along the beam direction, is related not only to the beam quality of the laser itself but also to the focal length of the upstream optics and, when viewed from the object plane, to the downstream optics along the beam direction.

[0086] In practice, the simplest implementation is to place the second light modulator 12 as a spatial filter behind the first light modulator 7 in the focal plane of the lens 14, and further place the first light modulator 7 in the imaging plane relative to the object plane. Deviations from the ideal state at other locations can be easily tolerated, however it is certainly extremely simple to ideally construct the structure from the first light modulator 7 through the lens 14 to the spatial filter 12. Typically, an STED microscope consists of a microscope stand with built-in objectives and associated tubular lenses. It is known here where the (virtual) back focal plane is located. This is commonly referred to in practice as the pupil plane or simply the pupil. This plane is the plane that other optical elements must reference; that is, if collimated illumination is to be achieved in the object plane, the incident beam must be focused into this plane (which ultimately lies, or in any case, within the geometry of the objective), i.e., the incident beam must be set such that, without the objective, the incident beam would form a focal point there. This plane is also the plane in which the field distribution should lie, and its Fourier transform field distribution should lie within the object. In practice, when the objective lens is removed, the point source can be placed in this plane and imaged through the tube lens. Then, a plane conjugate to the Fourier plane is obtained precisely at the location where the point source is imaged, which is also the Fourier plane. Other Fourier planes 13 or imaging planes 11 can then be easily determined.

[0087] Figure 7 The structure 9 is illustrated by utilizing a light modulator 7 with multiple pixels 8. Figure 7 a) shows a structure 9 having multiple structural elements 10, in this case a striped grid. The structural element 10 is therefore a single grid line. Figure 7(b) shows a magnified portion of each grid line. The grid line is composed of multiple pixels 8 of the first light modulator 7, meaning the size of the structural element 10 is larger than the size of the pixel 8 in two dimensions. To position the light distribution, the structure 9 implemented by the light modulator 7 is moved, meaning each structural element 10 is moved in integer multiples of the pixel size. In the binary light modulator 7, the minimum movement of the structure is defined by the pixel size and is therefore smaller than the size of the structural element 10. Subpixel movement can be achieved, for example, by a tilted pattern (aliasing effect), a perforated pattern, or a multi-stage or analog modulator, combined with an additional Fourier filter if necessary.

[0088] Figure 8 This illustrates a first example of generating multiple light distributions that can be positioned independently of each other. Here, Figure 8 a) shows the structure implemented by the first optical modulator, which in this case consists of two striped grids, each having four grid lines. Figure 8 b) shows the interference pattern in the Fourier plane. Here, in this example, the 0th order of the interference pattern is blocked by the second optical modulator, which in Figure 8 As shown in c), in subsequent processes, the +1 and -1 orders of the interference pattern interfere, which results in the sample plane, i.e., in the xy plane. Figure 8 The interference pattern shown in b) can be used to determine the position, in particular, the minimum intensity of ±1 order and the adjacent maximum intensity, that is, the maximum intensity of 0 order and ±1 order, which have approximately the same maximum intensity.

[0089] Figure 9 A second example of generating multiple light distributions that can be positioned independently of each other is shown. Here, Figure 9 a) shows a structure implemented by a first optical modulator, in this case, consisting of two striped gratings, each with only two grating lines. The lateral dimension of the striped grating is smaller than... Figure 8 The situation is shown in the example. Figure 9 b) also shows the interference pattern in the Fourier plane. Here, as previously stated, in this example, the 0th order of the interference pattern is blocked by the second optical modulator, which in Figure 9 As shown in the diagram. In subsequent processes, the +1 and -1 orders of the interference pattern interfere, which results in [something] in the plane of the sample. Figure 9 The interference pattern shown in d) can also be used, in particular, for position determination, with the minimum intensity of the ±1 order and the maximum intensity of the adjacent 0th and ±1 orders. Although these maximum intensity values ​​have different maximum intensities, these deviations do not impair the position determination.

[0090] Figure 10 An example of generating a light distribution in the form of locally hollow beams is shown. Here, Figure 10a) shows the structure implemented by the first optical modulator, which in this case is a ring structure. Figure 10 b) Shows the interference pattern in the Fourier plane. Formed in the plane of the sample. Figure 10 Image c) shows the image. The minimum intensity at the center is surrounded by the maximum intensity from all sides. This is formed in the axial direction, i.e., in the beam propagation direction. Figure 10 c) shows the intensity variation curve as a cross-section in the xz plane. As clearly shown, this light distribution also has a minimum intensity along the axial direction, with the intensity-increasing region adjacent to this minimum. This light distribution thus allows for position determination in three dimensions. For this purpose, for example, the sample can be moved along the axial direction. Alternatively, additional dynamically focusing elements can be arranged in the optical path, using which the light distribution can be moved along the axial direction, such as deformable mirrors.

[0091] Figure 11 An example is shown of generating multiple independently positionable light distributions in the form of locally hollow beams. Here, Figure 11 a) shows the structure implemented by the first optical modulator, which in this case consists of nine ring structures that can be positioned independently of each other. Figure 11 b) Shows the interference pattern in the Fourier plane. Formed in the plane of the sample. Figure 11 The image shown in c) shows that in all nine light distributions, the minimum intensity at the center is surrounded by the maximum intensity on all sides. This is formed in the axial direction... Figure 11 c) shows the intensity variation curve as a cross-section in the xz plane. As clearly shown in the figure, all light distributions also have a minimum intensity value along the axial direction, and the intensity increase region is adjacent to this minimum intensity value, thus enabling position determination in three dimensions.

[0092] Figure 12 An example is shown where a light distribution is generated by the temporal superposition of different local light distributions. Here, Figure 12 a) shows the structure implemented by the first optical modulator, in this case a striped grid with two grid lines. Figure 12 b) shows the interference pattern in the Fourier plane. Here, in this example, the +1 order of the interference pattern is blocked by the second optical modulator, which in Figure 12 As shown in c), in subsequent processes, the 0th and -1st orders of the interference pattern interfere, which results in the sample plane, i.e., in the xy plane. Figure 12b) shows a partial light distribution. As clearly shown, a light distribution with a minimum intensity and maximum intensity adjacent to both sides is formed. Similarly, the -1st order of the interference pattern is subsequently blocked in the Fourier plane by a second light modulator, thereby causing interference between the 0th and +1st orders of the interference pattern in subsequent processes. In this case, attention should be paid to the intensity variation curve along the axial direction, which... Figure 13 The section in the xz plane is shown here. Figure 13 a) Shows the intensity variation curves of the interference pattern between the 0th and -1st orders. Figure 13 b) Shows the intensity variation curves of the interference pattern between the 0th and +1st orders. As clearly shown in the figure, some light distributions exhibit, to a certain extent, the intensity variation between the 0th and +1st orders. Figure 13 a) and Figure 13 The inclined axis is shown as a dotted line in b). The two partial light distributions are superimposed in time, meaning that the light distribution radiates to the same locations on the sample at short time intervals. Figure 14 The intensity variation curve shown in b) also serves as a cross-section in the xz plane. As clearly shown, this superimposed light distribution also has a minimum intensity along the axial direction, and the region of increased intensity is adjacent to this minimum intensity. Figure 14 b) and Figure 14 As shown in c), the minimum intensity can move in the axial direction by the two partial light distributions moving radially relative to each other.

[0093] References

[0094] [1]F. Balzarotti et al.: “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes”, Science, Vol. 355 (2017), pp. 606-612.

[0095] [2]MGL Gustafsson: "Surpassing the lateral resolution limit by afactor of two using structured illumination microscopy", Journal of Microscopy, Volume 198 (2000), Pages 82-87.

[0096] [3]MGL Gustafsson: "Nonlinear structured-illuminationmicroscopy: Wide-field fluorescence imaging with theoretically unlimited resolution", Proceedings of the National Academy of Sciences, Volume 102 (2005), Pages 13081-13086.

[0097] [4] L. Gu et al.: “Molecular resolution imaging by repetitive optical selective exposure”, Nature Methods, Vol. 16 (2019), pp. 1114-1118.

[0098] [5] L. Reymond et al.: "SIMPLE: Structured illumination based pointlocalization estimator with enhanced precision", Optics Express, Volume 27 (2019), Pages 24578-24590.

[0099] [6] J. Cnossen et al.: “Localization microscopy at doubled precision with patterned illumination”, Nature Methods, Volume 17 (2020), Pages 59-63.

[0100] List of reference numerals

[0101] 1 sample

[0102] 2 molecules

[0103] 3-coherent light

[0104] 4. Light distribution

[0105] 5. Minimum strength

[0106] 6 areas of increased intensity

[0107] 7 First optical modulator

[0108] 8 pixels

[0109] 9 structure

[0110] 10 structural components

[0111] 11 Imaging Planes

[0112] 12 Second optical modulator

[0113] 13 Fourier plane

[0114] 14 lenses

[0115] 15 light sources

[0116] 16 detection units

[0117] 17-beam splitter

[0118] 20 devices

[0119] 21 interface

[0120] 22 control units

[0121] 23 assessment units

[0122] 24 monitoring units

[0123] 25 memory

[0124] 26 User Interface

[0125] 30 devices

[0126] 31 memory

[0127] 32 processor

[0128] 33 input terminals

[0129] 34 output terminals

[0130] 40 microscopes

[0131] Pi location

[0132] S1 generates multiple light distributions

[0133] S2 irradiates molecules using light distribution.

[0134] S3 mutually independent positioning light distribution

[0135] S4 detects photons emitted from different locations within the light distribution.

[0136] S5 infers the position of molecules based on emitted photons.

Claims

1. A method for determining the position (Pi) of two or more mutually spaced molecules (2) in a sample (1) in one or more spatial directions by means of a localization microscope (40), wherein, To determine the position (Pi) of the molecule (2), a light distribution (4) generated by the interference of coherent light is used, the method comprising the following steps: - Multiple light distributions (4) are generated using a first light modulator (7) with a large number of switchable pixels (8), the first light modulator being arranged in the imaging plane (11) of the positioning microscope (40), wherein each light distribution (4) has a local intensity minimum (5) and an intensity increase region (6) adjacent to it. - Irradiate (S) each of two or more molecules (2) using a light distribution (4); - For each light distribution (4), photons emitted by the molecule (2) are detected for different locations of the light distribution (4), wherein the light distributions (4) are located independently of each other; and - The position (Pi) of molecule (2) is deduced based on the photons detected at different locations for light distribution (4).

2. The method according to claim 1, wherein, A second light modulator (12) for influencing the light distribution (4) is arranged in the Fourier plane (13) of the positioning microscope (40).

3. The method according to claim 2, wherein, The second optical modulator (12) is designed to block individual diffraction levels.

4. The method according to claim 1, wherein, The first optical modulator (7) is an amplitude modulator or a phase modulator.

5. The method according to claim 4, wherein, The first optical modulator (7) is an optical modulator that can switch between two fixed states.

6. The method according to any one of claims 1 to 5, wherein, In order to generate light distribution (4), a one-dimensional structure or a two-dimensional structure (9) is constructed by means of switchable pixels (8) of the first light modulator (7).

7. The method according to claim 6, wherein, The one-dimensional structure (9) is a grid structure, while the two-dimensional structure (9) is a ring structure.

8. The method according to claim 6, wherein, In order to locate the light distribution (4), the structure (9) constructed by the switchable pixels (8) of the first light modulator (7) moves.

9. The method according to claim 8, wherein, The movement of structure (9) is less than the size of the structural element (10) of structure (9).

10. The method according to claim 9, wherein, The size of the structural element (10) of the structure (9) is larger than the size of the pixel (8) in two dimensions.

11. The method according to claim 6, wherein, In order to use the light distribution (4) to determine the successive positions, the intensity change curve of the light distribution (4) is changed by changing the structural characteristics of the one-dimensional or two-dimensional structure (9).

12. The method according to claim 11, wherein, By changing the lattice constant and / or aspect ratio of a one-dimensional structure or the aperture diameter of a two-dimensional structure, the edge steepness of the intensity-increasing region (6) adjacent to the local intensity minimum (5) is changed.

13. The method according to claim 1, wherein, The light distribution (4) is formed by the time superposition of different partial light distributions.

14. The method according to claim 13, wherein, The partial light distribution is formed by the interference of different diffraction orders of the interference pattern generated by the first light modulator (7).

15. The method according to claim 1, wherein, The markers in the sample (1) are detected by means of at least one light distribution (4) to determine the drift.

16. The method according to claim 1, wherein, The drift is determined by the change in the same direction of the position (Pi) of the molecule (2) measured in successive measurements.

17. The method according to claim 1, wherein, The light distribution (4) in sample (1) has a lateral dimension in the range of 0.5µm to 4µm.

18. The method according to claim 1, wherein, The photons emitted by molecule (2) are detected by a camera or a photon counter array.

19. A computer program product having instructions which, when executed by a computer, cause the computer to perform the steps of the method according to any one of claims 1 to 18, for determining, by means of a positioning microscope (40), the positions (Pi) of two or more mutually spaced molecules (2) in a sample (1) along one or more spatial directions.

20. An apparatus (20) for determining the positions (Pi) of two or more spaced molecules (2) in a sample (1) along one or more spatial directions by means of a positioning microscope (40), wherein, To determine the position (Pi) of the molecule (2), a light distribution (4) generated by the interference of coherent light is used, and the device has: - A control unit (22) designed to control a first light modulator (7) having a large number of switchable pixels (8), the first light modulator being arranged in the imaging plane (11) of a positioning microscope (40) for generating multiple mutually independent light distributions (4), wherein each light distribution (4) has a local intensity minimum (5) and an adjacent intensity increasing region (6); and - Evaluation unit (23), which is designed to deduce the position (Pi) of molecule (2) based on the photons emitted by molecule (2) for each light distribution (4) for different locations of the corresponding light distribution (4).

21. A microscope (40) having: - Light source (15) for generating coherent light (3); - A first light modulator (7) having a large number of switchable pixels (8), the first light modulator being arranged in the imaging plane (11) of the microscope (40) for generating multiple light distributions (4) formed by the interference of coherent light, wherein, Each light distribution (4) has a local minimum intensity (5) and an adjacent region of increasing intensity (6); - Optical device (14) for irradiating two or more spaced molecules (2) in sample (1) with a light distribution (4); - Detection unit (16) for detecting photons emitted by molecules (2) at different locations for light distributions (4), wherein the light distributions (4) are mutually independent of each other; and - The apparatus (20) for determining the position (Pi) of two or more molecules (2) in one or more spatial directions according to claim 20.

Images