Method and device for simultaneously tracking two emitters

Abstract

The purpose of the present invention is to provide a solution that enables simultaneous tracking of two closely adjacent light-emitting particles or light-emitting units, which are optically distinguishable, with high spatial and temporal resolution. The task is solved by a MINFLUX method or a STED-MINFLUX method or a combination of the two methods. The position of the first emitter is determined, or the positions of the first emitter and the second emitter, or a common average position of both emitters is determined, based on different paths of an intensity increase region of the excitation light or the emission suppression light and associated measurements of the emissions. The above steps are repeated in chronological order as the path of the intensity increasing region is adjusted according to the current position of the emitter.

Description

[0001] Technical field of the invention

[0002] The present invention relates to the field of high-resolution optical microscopy. In particular, the present invention relates to methods and apparatus for simultaneously tracking the movement of multiple emitters (e.g., fluorescently labeled biological structures) with high temporal resolution, the emitters being at a distance from one another less than the diffraction limit during tracking. Existing technology

[0003] Interferometric scattering (iSCAT) microscopy is a microscopy method for tracking fast-moving particles. In the publication "Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane" (Taylor, RW et al., 2019, Nature Photonics 13, 480–487, https: / / doi.org / 10.1038 / s41566-019-0414-6 ), describes the application of iSCAT microscopy to tracking proteins in living cell membranes. The method achieves a spatial resolution better than 10 nm at a temporal resolution better than 20 μs. In this method, interference is observed between a reference light and light scattered at a marker (e.g., gold nanoparticles), which is associated with the particle to be tracked (e.g., a biomolecule). Although this method can achieve very good spatiotemporal resolution, it has the disadvantage that the markers to be used are relatively large and therefore have a significant impact on the properties of the sample. Due to the size of the markers, this method is not suitable for individually observing two objects that are only a few nanometers apart to track their movement.

[0004] Camera-based localization microscopy methods for fluorescence microscopy are known in the prior art. These methods typically enable high spatial resolution fluorescence imaging in multiple colors over a range of tens of nanometers, using fluorophores with very different excitation spectra, emission spectra, or both. These methods are limited in temporal and spatial resolution by the camera and the brightness of the emitter, making them suitable only for observing slow movements of the emitter. Suitable emitters are, in particular, functionalized core-shell quantum dots, which, despite being highly photostable and extremely bright, have been rarely used in fluorescence microscopy to date. For example, this method is described in the publication "Differential Labeling of Myosin V Heads with QuantumDots Allows Direct Visualization of Hand-Over-Hand Processivity" (Warshaw DM et al., Biophysical Journal, 88(5): L30-2, doi:10.1529 / biophysj.105.061903, May 2005, e-published March 11, 2005, PMID: 15764654; PMCID: PMC1305523). A spatial resolution of 6 nm was achieved with a temporal resolution of 83 ms. In order to be able to observe the movement of the dual-stained motor protein alone and thus the movement of the dual-stained motor protein end alone, the ATP concentration in the sample was set very low. Similar studies using more common fluorophores in fluorescence microscopy (i.e., Cy3 and Cy5) using two excitation wavelengths with TIRF illumination to minimize background fluorescence are described in the publication "Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time" (L. Stirling Churchman et al., Proceedings of the National Academy of Sciences of the United States of America, 102(5):1419-23, doi:10.1073 / pnas.0409487102, epub 24 Jan 2005, PMID: 15668396; PMCID: PMC545495, February 1, 2005). A lateral resolution of approximately 6 nm was achieved with an integration time of 0.5 s.

[0005] Multicolor scanning microscopy or a corresponding method for achieving high spatial resolution fluorescence imaging in the range of a few nanometers is known from the publication "Ultrahigh-resolution multicolor colocalization of single fluorescent probes" (Thilo D. Lacoste et al., Proceedings of the National Academy of Sciences of the United States of America, 97(17):9461-6, Doi: 10.1073 / pnas.170286097, PMID: 10931959; PMCID: PMC16886, August 15, 2000). Different fluorophores are used here, all excited with the same wavelength, because when using multiple excitation wavelengths, the required accuracy of overlapping the excitation beams is not possible with the resolution to be achieved. The sample is scanned confocally, the fluorescence is spectrally separated into two or more channels, and a multicolor image is generated from the measured values. This method is used to determine the distances between fluorophores of different colors within a range of approximately 10 nm. Like the camera-based localization microscopy approach presented here, this method is not suitable for rapid tracking of multiple emitters simultaneously and individually.

[0006] In the publication “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes” (Balzarotti, F. et al., Science, 355(6325):606-612, February 10, 2017, doi:10.1126 / science.aak9913, epub December 22, 2016, PMID: 28008086), a method for localizing and tracking individual emitters is proposed, which is called MINFLUX. According to this method, the emitter is illuminated with focused excitation light having an intensity distribution having a central local intensity minimum (in particular, a zero point) and adjacent regions of increased intensity, wherein the center of the intensity distribution is placed in a temporally sequential manner at different positions in a vicinity around the presumed position of the emitter, so that the emitter is in each case located within the region of increased intensity surrounding the minimum, wherein the fluorescence emission is measured and associated with the corresponding position of the center, and wherein the position of the emitter is determined from these positions and the associated number of photons or fluorescence intensity. A method for real-time estimation of the emitter's position is proposed for real-time tracking of the emitter within a certain area. This requires tracking the particle using an intensity distribution such that the center of the intensity distribution is located at different positions in the vicinity around the emitter's estimated position in a temporal sequence, so that the emitter, whose movement is tracked, lies within a region of increasing intensity surrounding the minimum. The essence of the MINFLUX method described in this publication is to position the local minimum of the excitation light distribution close to the emitter, so that the emitter lies within the region of increasing intensity adjacent to the minimum. The potential implications are that, on the one hand, the illumination position can be illuminated with different intensity distributions, and, on the other hand, the method could be further developed for multicolor imaging in the future. In this context, a distinction is made between emission spectra, polarization, and fluorescence lifetime. However, no specific corresponding solutions are provided.

[0007] In Jasmin Pape's doctoral thesis "Multicolor 3D MINFLUX nanoscopy for biological imaging" (Göttingen, 2020, http: / / dx.doi.org / 10.53846 / goediss-8248 ) describes the theoretical background for applying the MINFLUX method to simultaneously track emitters of different colors. While a first experimental demonstration of feasibility is mentioned, this demonstration only involves immobilized emitters and therefore does not involve the actual simultaneous tracking of two emitters. The theoretical considerations concern the case where emitters with mutually different spectral emission distributions are co-located in a nearby scanned area, and the fluorescence emission is detected in multiple spectral channels. The spectral separation can be complete or incomplete, meaning that crosstalk effects are taken into account. The theoretical interpretation refers to the information content inherent in the corresponding recorded data. The paper notes that position estimation using the maximum likelihood estimator (MLE) exhibits bias (i.e., systematic errors), which is particularly relevant with low photon counts. Consequently, the paper argues that information about the emitter positions cannot be extracted as simply as by determining the position of only one emitter, because, at least in the presence of non-zero crosstalk, the emitter positions cannot be determined independently of each other. In particular, it is determined that achieving high temporal resolution when tracking multiple emitters requires the development of a bias-free position estimator. The aforementioned doctoral dissertation does not mention such a position estimator that is actually suitable for the simultaneous tracking of two fluorophores with different spectral emissions. Therefore, the feasibility of simultaneous tracking of two emitters was verified by experimentally determining the position of each of the two immobilized emitters as part of a subsequent detailed analysis of the dataset recorded in the vicinity of the fixed positions.

[0008] In the aforementioned doctoral thesis and the publication "Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins" (Pape et al., 2020, Proceedings of the National Academy of Sciences of the USA, 117(34), 20607-20614, doi: 10.1073 / pnas.2009364117), the imaging of samples stained with two different dyes using MINFLUX is described. Here, individual emitters are localized once or more times, each associated with one of the dyes, and the different localization results for the same emitter are correlated with one another as part of a cluster analysis.

[0009] In the publication “MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells” (Gwosch et al., 2020, Nature Methods 17, 217–224, https: / / doi.org / 10.1038 / s41592-019-0688-0 ), also describes the use of MINFLUX to image samples stained with two different dyes. Fluorophores that can be excited with a common excitation wavelength and have measurably different emission spectra are used for imaging. Each fluorophore is individually activated with activation light before being localized. The MINFLUX measurement is now performed as follows: the fluorescence emission is spectrally separated with the aid of a dichroic beam splitter and detected separately in each of two spectral channels. The emitters are localized based on the summed signal, using a modified least mean square estimator (mLMSE) as an estimator (see the publication cited above, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes”). This estimator is used to estimate the positions of the individual fluorophores. As part of the subsequent data analysis, which includes a first assignment based on the number of photons detected in each channel and a subsequent principal component analysis for refining the assignment, the localization results are assigned to the individual dyes. This type of image recording is referred to in the introductory part of the publication as “simultaneous two-color registration.” Looking ahead, it was suggested, among other things, that measurement times could be reduced by localizing multiple molecules as part of the localization procedure, but no specific details on this were provided.

[0010] A method for improving positioning according to the MINFLUX method by detecting and taking into account interferences that occur during the measurement is disclosed in the international patent publication WO 2021 / 122407 A1. A corresponding microscope is described. The method is used, on the one hand, to determine the trajectory of fluorescent dye molecules, i.e. to track the molecules, and on the other hand to determine the position of a plurality of different dye molecules. The microscope has two excitation lasers of different wavelengths. The light paths of the two lasers pass together through a deflection device with two electro-optical deflectors (EODs), after which the light paths are separated, with a liquid crystal modulator (spatial light modulator - spatial light modulator, SLM) in each light path, in order to form a light distribution with a local intensity minimum at the excitation focus in the sample after focusing in a common beam path by an objective lens. Among other components, the microscope also contains a beam splitter for splitting the fluorescence into two spectrally separated detection channels, which correspond to the emission ranges of the two different dyes. Regarding multi-color imaging, reference is made to techniques known from PALM and STORM microscopy. Furthermore, it is mentioned that simultaneous or quasi-simultaneous localization of multiple fluorescent dye molecules can be achieved with appropriate illumination and detection devices when the fluorescent dye molecules are spaced apart by distances greater than the diffraction limit. Further details are not provided. Simultaneous localization of the positions of multiple fluorophores spaced apart by distances less than the diffraction limit is not disclosed.

[0011] International Patent Publication WO 2022 / 136361 A1 describes a method in which particle tracking is limited to areas of a sample where relevant information about the sample is obtained by tracking the particles. To achieve this, a second measured variable, which can also be fluorescence intensity, is detected in the sample. Tracking of the relevant particles is then interrupted or terminated when a control value determined based on the second measured variable meets a termination criterion.

[0012] International Patent Publication WO 2021 / 239679 A1 describes a method for simultaneously localizing and tracking the movement of multiple fluorophores based on the MINFLUX method, as well as an apparatus designed to perform this method. The fluorophores to be localized are located at a distance significantly greater than the diffraction limit. Information regarding the camera used to detect fluorescence indicates that multiple wavelengths can be used. Further details regarding this aspect are not provided.

[0013] In the international patent publication WO 2022 / 079265 A1, methods for localization microscopy of samples stained with multiple dyes are described, in particular the MINFLUX method and MINFLUX-like methods, in which a shift of the intensity distribution of fluorescence suppression light with a central minimum replaces the shift of the corresponding excitation light, as well as a microscope configured to perform one of the methods. All methods aim to place nanoimaging in the spatial context of microscopic imaging or to perform nanotracking of single molecules in the spatial context of microscopic imaging. To this end, methods are proposed that use multiple detection channels (possibly used sequentially) and, in many cases, also multiple excitation wavelengths. Some methods aim to achieve an optimized ratio separation of the dyes, such as the method described in the above-mentioned publication "MINFLUX nanoscopy Delivers 3D multicolor nanometerresolution in cells". Simultaneous localization or tracking of multiple fluorophores is not described.

[0014] In the publication “Two-photon MINFLUX with doubled localization precision” (Zhao, K. et al., 2022, published in eLight 2,5, https: / / doi.org / 10.1186 / s43593-021- 00011-x ) describes the theory of localization using two-photon excitation according to the MINFLUX method. The paper shows that multiple fluorophores can be excited using the same excitation wavelength via the two-photon process and have emission spectra that are easily and completely separable. This enables, among other things, the simultaneous tracking of multiple fluorophores, which is crucial for studying molecular interactions such as protein-protein interactions, protein-nucleic acid interactions, or virus-cell interactions.

[0015] In the publication US2022 / 163440 A1, a method and apparatus for nano-inspection based on the observation of single molecules (particularly in biological samples) are described. During the inspection, the movement of the target in the sample is compensated by moving the sample relative to the focused intensity distribution, keeping the target in the region of the local zero point of the intensity distribution. A time resolution of about 10 ms is sufficient for compensation. At the same time, the movement of the target in the sample is tracked in this way. In several embodiments, the intensity distribution at which the target is kept at its zero point is the intensity distribution of fluorescence suppression light. In other embodiments, the intensity distribution is the intensity distribution of a tracking beam with a wavelength of 488 nm, which is used to locate the target with the aid of MINFLUX. This positioning can adopt a higher time resolution than mentioned above, so that the movement of the target can also be tracked with this higher time resolution. Even in the case where the target is kept in the region of the local zero point of the focused intensity distribution of fluorescence suppression light, positioning can be performed with the aid of MINFLUX, and compensation for movement is performed based on this positioning. When localization is performed using MINFLUX, the target is maintained in the region of the local zero of the focused intensity distribution of the fluorescence suppression light, or in the region of both the local zero of the tracking beam and the central maximum of the intensity distribution of the excitation light. While the target is tracked, further single-molecule events are detected in the vicinity around the target using excitation light of multiple wavelengths. This allows, for example, the temporal sequence of attachment of different fluorescent samples (fluorescently labeled biomolecules) to the target to be identified. While the tracking beam itself can be moved rapidly using an electro-optical deflector, the remaining intensity distribution is moved much more slowly relative to the sample using a nanopositioning stage. This method does not observe the relative movement of the two fluorophores.

[0016] In the publication "MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope" (Schmidt, R et al., 2021, Nature Communications 12, 1478, https: / / doi.org / 10.1038 / s41467-021-21652-z ) describes, on the one hand, a MINFLUX method for tracking fast-moving particles with a temporal resolution of approximately 100 μs and a spatial resolution better than 20 nm, and, on the other hand, 3D-MINFLUX image recording with a resolution in the range of a few nanometers, both in a single color. Both particle tracking and 3D-MINFLUX image recording were performed using commercially available microscopes. The high resolution is achieved, on the one hand, by active sample stabilization. On the other hand, background correction is performed during data recording, thereby optimizing the real-time estimation of the respective positions of the fluorophores and, therefore, the overall data recording. In each case, the MINFLUX localization of the fluorophores was performed by exciting the fluorophores with a CW laser at a wavelength of 642 nm.

[0017] In the publication "MINFLUX dissects the unimpeded walking of kinesin-1" (Wolff, Jan O. et al. published in bioRxiv 2022.07.25.501426, doi: https: / / doi.org / 10.1101 / 2022.07.25.501426 ) describes tracking motor proteins with high spatiotemporal resolution using MINFLUX. In one variant, the motor protein is labeled with a fluorophore at each of its two binding sites, where the two fluorophores are identical. The two fluorophores are tracked simultaneously using a pair of excitation light profiles, one of which has a minimum extending in the y-direction at the illumination focus, while the other has a minimum extending in the x-direction. The profiles are used iteratively to determine the fluorophore positions, with the new position of the fluorophore estimated in each case based on the fluorescence measurement results and the path of the associated region of increased intensity. At least two intensity profiles are used to determine each position in a spatial direction. Each of these intensity profiles has a minimum and regions of increased intensity adjacent to the minimum on either side, and the shapes of the intensity profiles differ from one another. At the zero position, the intensity distribution is symmetrical about the central intensity minimum. At positions deviating from the zero position, the central minimum is now shifted, and the symmetry of the intensity distribution is disrupted, so that the intensity profiles used differ not only in the position of the local intensity minimum but also in its shape. When estimating the position, the position of the midpoint between the two fluorophores is inherently determined.

[0018] According to patent documents EP 2 780 694 B1 and US 9,291,562 B2, the movement of particles in a sample is tracked by detecting light emitted from a region of spatially defined minimum values ​​of the excitation intensity distribution and by shifting the excitation intensity distribution in the sample, for example, by means of a beam scanner, so that the rate of photons emitted from the particles is kept to a minimum. According to the patent documents, by performing this method alternately for each particle individually, it should be possible to track two particles quasi-simultaneously. Similarly, if there are multiple beam shaping devices and multiple beam deflection devices, it should also be possible to track multiple particles simultaneously. Light beam sources of different wavelengths can also be used for simultaneous tracking. As a solution to ensure that the rate of photons emitted from particles remains at a minimum, it is recommended to experimentally shift the intensity distribution whenever the rate increases due to the movement of the particle. Particles can be tracked in this way because the minimum rate can only be achieved when the intensity distribution is shifted in the direction of the particle's movement. Alternatively, the movement can also be observed with the help of a camera, and the light beam can be tracked according to the movement determined in this way until the rate of photons (i.e., the measured brightness associated with the particle in the camera image) is minimized.

[0019] A fundamental disadvantage of this method is that it is almost impossible to track fast-moving fluorescent particles with high spatial resolution. If the rate of the photons is to be kept to a minimum by a trial shift of the intensity distribution, this trial shift should be carried out at a speed greater than the speed of movement of the particles, during which the rate of the photons is determined in such a way as to identify the position at which the rate of the photons is minimum. This makes it impossible to track fast-moving particles reliably with high spatial resolution. If two particles are to be tracked simultaneously or quasi-simultaneously at a distance less than the diffraction limit, a further problem arises, namely that the rates of the photons of the two particles can be kept to a minimum independently of each other only if crosstalk between the fluorescence of one fluorophore and the detection channel of the other fluorophore is completely (i.e. not just approximately) excluded.

[0020] Invention Task

[0021] It is now an object of the present invention to provide a solution which enables simultaneous tracking of two optically distinguishable luminescent particles or luminescent units in close proximity with high spatial and temporal resolution.

[0022] Solution

[0023] This object is achieved by a method according to claim 1 and a device according to claim 24. Advantageous embodiments of the method according to the invention are given in dependent claims 2 to 23, and advantageous embodiments of the device according to the invention are given in claim 25. These and other advantageous embodiments are described below.

[0024] definition

[0025] In this application, for the purposes of measurements according to the present invention, emitters are objects that can be considered as point sources of light when illuminated by excitation light. Light emitted from an object acting as a point source can, for example, be scattered light resulting from elastic scattering (such as Rayleigh scattering) or inelastic scattering (such as Raman scattering), or it can be luminescent light, particularly fluorescent light. For an emitter, it is crucial that it emits light immediately or after a short delay in response to illumination. The maximum time delay is related to the temporal resolution used to track the movement of the luminescent particle or unit and the speed at which the particle or unit moves through the sample. The time delay can be up to approximately 10 μs, but is typically in the tens of nanoseconds range, mostly in the 1 to 10 ns range. If the emitted light is scattered, the time delay is zero. In this application, the term "fluorescent emitter" is used to refer to an emitter that acts as a fluorescent point source. Emitters can be, for example, metal nanoparticles. More specifically, the term "fluorescent emitter" includes, for example, single fluorescent dye molecules or fluorescent chemical groups thereof. Such emitters, particularly such dyes, can be coupled to biomolecules, which can then be labeled. Multiple scattering emitters or dye molecules can also be coupled to a structure to be labeled (e.g., a biomolecule or a biological microstructure). In this case, two situations must be distinguished. If multiple optically indistinguishable or indistinguishable emitters (e.g., multiple identical dye molecules) are closely bound to a structure (e.g., within 1 nm or a few nanometers), these emitters can be considered as a single emitter. The same applies to the coupling of a small structure equipped with multiple indistinguishable emitters (e.g., identical fluorophores) to a target structure to be labeled. In another case, optically distinguishable emitters (e.g., different dyes) are used to label different regions of a structure (e.g., a biomolecule (e.g., a motor protein)). In this case, the different regions can have very small distances of a few nanometers from each other, with the range of a single emitter being smaller than these distances. Here, multiple fluorophores (i.e., fluorescent molecules or fluorescent groups of related molecules) can also present a fluorescent unit at each location on the structure to be distinguished, thereby collectively forming a single emitter, or multiple scattering objects can collectively form a single scattering emitter. Other fluorescent units (e.g., quantum dots (see prior art: "Differential Labeling of Myosin V Heads with Quantum Dots Allows Direct Visualization of Hand-Over-Hand Processivity")) or upconversion nanoparticles can also be used for labeling instead of dyes.

[0026] Therefore, in the context of this application, excitation light refers not only to fluorescence excitation light, but also generally to light that causes light to be emitted from an emitter.

[0027] In the present application, STED light is understood as light that suppresses fluorescence emission by stimulation of stimulated emission, as is generally the case. In the present application, STED light is a special form of emission-suppressing light.

[0028] In the present application, the MINFLUX method is understood to be a method for locating emitters in a sample by using an intensity distribution of excitation light having minima and regions of increased intensity adjacent to the minima multiple times, wherein the position of the emitter is inferred based on a plurality of different paths (Verlauf) of the regions of increased intensity in which the emitter is located and the measured values ​​of the emission assigned to these paths. For example, different distributions of regions of increased intensity in which emitters adjacent to the minima are located can be obtained by placing the minima of a specific intensity distribution at different positions so that the emitter is each time located at a different position within the region of increased intensity relative to the minima. Different distributions of regions of increased intensity can also be obtained by placing different intensity distributions having minima and regions of increased intensity adjacent to the minima in the sample so that the emitter is each time located within the region of increased intensity. Different paths to the regions of increased intensity can also be obtained by combining the two measures of placing the minima at different positions and using a plurality of different intensity distributions.

[0029] Therefore, methods that utilize local minima in the intensity distribution of the excitation light to locate emitters (wherein the emitter position is inferred based on multiple locations of local minima and the emission measurements associated with these locations) belong to the MINFLUX method. Here, the intensity distribution at each location of the minimum can vary. The expression "using local minima" means that the minimum in each dimension for which the emitter's position is to be determined is located at multiple locations that satisfy the following condition: the emitter is located within a region of increased intensity adjacent to the local minimum. However, this does not exclude the possibility of minima being located at locations that do not satisfy these conditions.

[0030] In the present application, the STED-MINFLUX method is understood to be a method corresponding to the MINFLUX method according to the above definition, with the difference that the intensity distribution of the excitation light is replaced by the intensity distribution of the emission suppression light overlapping with the excitation light.

[0031] Here, the intensity distribution of emission-suppressing light overlaps with the excitation light. This means that excitation light is applied to the sample in the region of the intensity minimum of the emission-suppressing light and the adjacent region of increased intensity (where the emitter is located) to stimulate emission from the emitter, or in the absence of emission-suppressing light, to stimulate emission from the emitter. The emission-suppressing light, depending on its intensity distribution, influences stimulated emission or excitation of emission, such that the measured emission of the emitter located within the intensity distribution of the emission-suppressing light decreases with increasing distance from the intensity minimum. If the emission-suppressing light is STED light and the emitter is a fluorescent emitter, this means that the spontaneous emission of the fluorescent emitter decreases with increasing distance from the intensity minimum of the STED light. The stimulated emission of the emitter has the same wavelength as the STED light and, as is common in STED microscopy, does not affect the measured emission, for example, by being blocked by a filter. Here, the intensity distribution of emission-suppressing light overlaps with the excitation light, but does not mean that the excitation light and the emission-suppressing light are introduced into the sample at the exact same time. For example, as is common in STED microscopy, a short excitation light pulse can be used, followed immediately by a STED light pulse as emission suppression light. The excitation light that overlaps with the intensity distribution of the emission suppression light can, for example, have an intensity distribution common in confocal microscopy, i.e., at least approximately corresponding to an Airy function or a Gaussian function, wherein the central maximum of the intensity distribution can coincide with the minimum of the emission suppression light. The intensity distribution can also correspond to a Tophat function. Continuous excitation in a wide field is also feasible.

[0032] The term STED-MINFLUX is used in a broad sense and should by no means be understood to mean that the emitted suppressive light must be STED light.

[0033] Two emitters are considered closely adjacent if they are at a distance from each other such that when a MINFLUX method is performed on one emitter, the other emitter necessarily enters the region of influence of the excitation light, or when a STED-MINFLUX method is performed on one emitter, the other emitter necessarily enters the region of influence of the emission suppression light. Therefore, when two emitters are simultaneously localized, the localizations should not be considered independent due to the distance. Specifically, two emitters are considered closely adjacent if the distance between them is less than the distance between the zeros closest to the maximum value in the Airy function corresponding to the maximum wavelength of the light used and the numerical aperture of the focus (in short, less than one Airy diameter). If the distance in these cases is less than half an Airy diameter, the emitters are also considered very closely adjacent. In the absence of focusing optics to generate an intensity distribution, emitters are particularly closely adjacent at a distance less than the longest wavelength of the light used. In this case, the emitters are also considered very closely adjacent if the distance between them is less than half the longest wavelength used.

[0034] In the context of this application, two emitters are considered optically distinguishable from one another if the emission can be assigned to one emitter or the other even when they are in close proximity. These assignments can be based, for example, on different optical properties, such as the excitability of the emission, or alternatively or additionally, on different characteristics of the emitters' emissions. In this case, the assignment does not have to be completely definitive. Thus, if two emitters are in close proximity but the emission cannot be assigned to a specific one of them, the emitters are considered optically indistinguishable from one another.

[0035] In the context of the present application, an emitter is said to be isolated if the distance between it and the nearest optically indistinguishable emitter is at least as great as the distance corresponding to the resolution of the optical arrangement used to detect the emission when performing the method.

[0036] In this application, a 3D minimum is understood to be a minimum that is surrounded by regions of increased intensity in all spatial directions. For example, a 3D minimum can be obtained by superimposing two beams, where in each pupil the wavefront of one beam is phase-modulated with the aid of a so-called annular phase plate and the wavefront of the other beam is phase-modulated with the aid of a vortex phase plate.

[0037] In this application, a 2D minimum is understood to be a minimum that is surrounded by regions of increased intensity in all spatial directions within a given plane and extends along an axis perpendicular to that plane. For example, a 2D minimum can be achieved in the beam path with the aid of a vortex phase plate. For example, the minimum can extend along the optical axis of the objective and be surrounded by regions of increased intensity in directions perpendicular to that axis. In principle, however, the plane in which the regions of increased intensity extend can also have a different spatial orientation.

[0038] In the present application, a 1D minimum is understood to be a minimum that is surrounded by regions of increased intensity in a spatial direction perpendicular to a given plane. Such a minimum can be achieved, for example, with the aid of a phase retarder plate that, in particular in the pupil, delays half of the wavefront by λ / 2 relative to the other half, where the wavefront is bisected along a straight line. An alternative method for generating a 1D minimum, which is mentioned here as a further example, is described in the publication “MINFLUX dissects the unimpeded Walking of kinesin-1” (Wolff et al.), which is referred to as prior art.

[0039] Description of the Invention

[0040] The invention relates to a method for simultaneously tracking the movement of a first emitter and a second emitter that are closely adjacent in a sample, the second emitter being optically distinguishable from the first emitter, by means of the MINFLUX method or the STED-MINFLUX method.

[0041] According to the present invention, an illumination and measurement step is performed, wherein the sample is illuminated with an intensity distribution of excitation light or emission suppression light (in particular STED light) overlapping with the excitation light, the intensity distribution having a local minimum and an intensity-increasing region adjacent to the minimum within a measurement range, wherein different paths of the intensity-increasing region from a set of predetermined paths of the intensity-increasing region are generated in a temporal sequence in the region of the presumed position of at least the first emitter. Furthermore, during the illumination and measurement step, the emission of the first emitter and, optionally, the second emitter is measured, and the measured values ​​of the emission are assigned to the corresponding paths of the intensity-increasing region.

[0042] Furthermore, according to the invention, a localization step is performed in which the position of the first emitter is determined based on the measured values ​​of the different paths and the assigned emission of the area of ​​increased intensity, or the position of the first emitter and the second emitter, or a common average position of the two emitters.

[0043] This method for determining the position of two closely adjacent emitters (specifically, two fluorophores) is generally known from Jasmin Pape's doctoral thesis, which was mentioned in the prior art section. However, regarding the simultaneous measurement of multiple fluorophores, this thesis only presents data from stationary subjects. Localization is performed during post-measurement evaluation.

[0044] In contrast, in the present invention, the positioning step is performed already during the data recording period, which allows tracking of mobile emitters over a greater range than the area monitored during the illumination and measurement steps. According to the present invention, based on the position of the first emitter determined in the positioning step, or the positions of two emitters determined in the positioning step, or the common average position of the two emitters determined in the positioning step, a corresponding new estimated position area is determined for each emitter, and a new set of paths is determined overall. Furthermore, according to the present invention, the illumination and measurement steps and the positioning step are repeated using this new set of paths.

[0045] According to one embodiment, the excitation light comprises a first excitation light for exciting a first emitter and a second excitation light for exciting a second emitter. That is, according to the present invention, both emitters are excited by the excitation light. This applies both when the excitation light forms an intensity distribution with a local minimum within the measurement range and when the emission suppression light forms an intensity distribution with a local minimum within the measurement range, and the excitation light overlaps with this intensity distribution. In principle, the first excitation light and the second excitation light can be identical. This has the advantage that no positional deviations associated with the excitation light occur. This advantage can be particularly important when the excitation light forms an intensity distribution with a local minimum (particularly as a focused intensity distribution). If, on the other hand, the first excitation light and the second excitation light are different, it may be advantageous to guide them into a common beam path, or, for example, to a section of a common optical fiber. These measures are intended to optimally adjust the intensity distributions of the two excitation lights in the sample to each other. Preferably, the beam paths can be "aligned by design," a term commonly used in STED microscopy for the alignment of STED light and excitation light.

[0046] Tracking the movement of a first and a second emitter that are in close proximity in a sample using a MINFLUX method or a STED-MINFLUX method does not necessarily mean that the MINFLUX method or the STED-MINFLUX method must be performed for each individual emitter according to the above definition. Rather, the MINFLUX method or the STED-MINFLUX method only needs to be performed for at least one emitter, and the movement of both emitters must be tracked using this method. Preferably, the MINFLUX method or the STED-MINFLUX method is performed at least for the first emitter.

[0047] Tracking the movement of closely adjacent emitters does not exclude that, before or after the movement is tracked by means of the method according to the invention, these emitters are no longer closely adjacent, but their movement is nevertheless tracked simultaneously. Nor does it exclude that, before or after the movement is tracked by means of the method according to the invention, the movement of only one of the two emitters is tracked until the emitter is in close proximity to the other emitter or after the emitter has moved away from the other emitter.

[0048] In preferred embodiments, the first excitation light and the second excitation light have different spectral compositions. Using different excitation light is one way to achieve optical separation of the two emitters. For example, the first excitation light may include a first spectral range, and the second excitation light may include a second spectral range that is different from the first spectral range and preferably does not overlap with the first spectral range. Preferably, both the first excitation light and the second excitation light are narrowband light, preferably laser light, for example, with a bandwidth of less than 10 nm, less than 5 nm, less than 1 nm, or less than 0.1 nm; both the first excitation light and the second excitation light may be narrowband laser light or monochromatic laser light. In preferred embodiments of the present invention, the distance between the spectral ranges or wavelengths may be greater than 50 nm, 100 nm, 150 nm, or 200 nm. For example, using spectrally different excitation light enables the use of emitters, such as fluorophores, with very different excitation spectra. This, in turn, enables substantially separate excitation of the first and second emitters, particularly first and second fluorescent emitters, i.e., cross-excitation with lower excitation, or even completely separate excitation of one of the emitters. Cross-excitation is understood to mean that one excitation light not only excites the emitter of interest, as desired, but also excites the other emitter. When unintended excitation is small relative to intended excitation, cross-excitation can be said to be low. Preferably, the spectral composition of the first and second excitation light, as well as the first and second emitters, are coordinated with one another such that excitation of the second emitter by the first excitation light is minimal relative to excitation of the first emitter by the first excitation light, or at the same excitation intensity is at most 10%, preferably at most 5%, more preferably at most 1%, even more preferably at most 0.1% of the excitation of the first emitter, and / or correspondingly, excitation of the first emitter by the second excitation light is minimal, or at the same excitation intensity is in particular at most 5%, preferably at most 1%, even more preferably at most 0.1% of the excitation of the second excitation light. Thus, in a preferred embodiment, the spectral composition of the first and second excitation light, as well as the first and second emitters, can be coordinated with one another such that cross-excitation of the first emitter by the second excitation light is particularly low, while cross-excitation of the second emitter by the first excitation light is high. This applies in particular to embodiments in which, in a localization step, the position of a first emitter is determined, and based on the position of the first emitter determined in the localization step, a corresponding new estimated position region is determined for each emitter and a new set of paths as a whole. Therefore, the corresponding position region can be estimated based on the position of the first emitter determined in the localization step because the emitters can be assumed to be closely adjacent in the sense defined in this application. Close proximity can be due to the fact that both emitters are coupled to the same biological structure (e.g., a motor protein). Therefore, knowledge of the position of the first emitter also provides knowledge of the position of the second emitter, although this knowledge is generally less precise.

[0049] In embodiments where the spectral composition of the first excitation light and the second excitation light differ, the illumination with the first excitation light and the illumination with the second excitation light can be temporally separated from each other during the illumination and measurement steps. This can be done by first recording all measurement data for the first excitation light or, alternatively, the second excitation light during the illumination and measurement steps, followed by recording all measurement data for the second excitation light or, alternatively, the first excitation light; or by alternating (e.g., "pulse interleaving") the use of the first excitation light and the second excitation light so that all associated measurement data are recorded approximately simultaneously. In conjunction with the difference in the spectral composition of the excitation light, the measured values ​​of the fluorescence emission detected in the illumination and measurement steps can be assigned not only to the location where the illumination light was directed or the corresponding path of the region of increased intensity generated in the sample, but also to the emitter from which the emission originated. This temporal separation can be achieved, for example, with the aid of optical switches (e.g., acousto-optic modulators). For example, these switches can switch which of multiple light sources is coupled into the optical path to illuminate the measurement range in the sample, or they can block light within the optical path or couple light out of it.

[0050] In principle, the method according to the present invention can be performed using multiple light sources that are continuous in time or one light source that is continuous in time (for example, using one or more CW lasers). As described above, this also applies to embodiments in which the irradiation of the first excitation light and the irradiation of the second excitation light are separated from each other in time. This applies regardless of whether the excitation light forms an intensity distribution having a minimum value within the measurement range or whether the excitation light overlaps with an intensity distribution having a minimum value within the measurement range. In this case, each beam of light can be a temporally continuous light.

[0051] In a preferred embodiment, the excitation light can be pulsed. This offers various advantages. For example, if two or more excitation light sources are used, the irradiation with a first excitation light source and a second excitation light source and / or further excitation light sources can be performed alternately, so that one excitation light pulse can be followed by another excitation light pulse. This does not preclude the possibility that one or more additional pulses, such as pulses emitting suppressor light, may occur between two consecutive pulses in this manner. Switching between the excitation light sources can be performed rapidly; for example, when both light sources operate at the same pulse frequency, the switching speed can be twice the pulse frequency of both lasers. For example, pulsed lasers with a pulse frequency of 80 MHz (e.g., diode lasers or fiber-reinforced diode lasers) are available and are suitable for performing these embodiments of the method according to the present invention. If two such light sources are used, pulsed irradiation can be performed at a total frequency of 160 MHz, with each individual excitation light source irradiating the sample at a frequency of 80 MHz. In this case, the time interval between the individual pulses is 6.25 ns, which is very small compared to the time scale of the motions typically tracked using the method according to the present invention. The irradiation of the different excitation light sources occurs almost simultaneously with respect to the process being observed. This also applies in many cases when using a pulsed light source with a relatively low pulse frequency (e.g., 60 MHz or 40 MHz). For example, a suitable pulsed light source can also be a light-emitting diode. Further advantages will be described elsewhere. The above description applies regardless of whether the excitation light forms an intensity distribution having a minimum value within the measurement range or whether the excitation light overlaps with an intensity distribution having a minimum value within the measurement range.

[0052] The pulse duration of the pulsed excitation light can be coordinated with the photophysical properties of the emitter used to label structures in the sample. Therefore, with respect to photobleaching of emitters (particularly fluorescent emitters), it may be advantageous to use longer pulses with a duration in the range of a few nanoseconds. In other cases, for example to determine the emission lifetime (e.g., fluorescence lifetime), or to assign emission measurements not only to the location where the illumination light is directed or the path of the region of increased intensity generated in the sample, but also to the emitter from which the emission originates, it may be advantageous to use pulses with a pulse duration that is shorter than the expected emission lifetime of the relevant emitter. Typical fluorescence lifetimes are generally in the range of about 1 ns to about 100 ns. In the method according to the present invention, the excitation light preferably has a pulse duration of a few nanoseconds or shorter, for example 3 ns, preferably less than 1 ns, more preferably less than 300 ps, ​​further preferably less than 150 ps, ​​and even more preferably less than 100 ps. In the apparatus according to the present invention, a pulsed light source is provided that is configured to generate pulses with a duration of less than 1 ns or 150 ps. This has the advantage that all samples labeled with fluorescent emitters, regardless of the specific fluorescent emitter present, can be excited using light pulses shorter than the fluorescence lifetime. The above description applies regardless of whether the excitation light forms an intensity distribution having a minimum value within the measurement range, or regardless of whether the excitation light overlaps with an intensity distribution having a minimum value within the measurement range.

[0053] If the intensity distribution with the minimum value is the intensity distribution of emission-suppressing light that overlaps with the excitation light, the emission-suppressing light can be either temporally continuous light or pulsed light. As described above, this also applies to the excitation light used in this case, whose distribution within the measurement range overlaps with the distribution of the emission-suppressing light. However, in the aforementioned case, the excitation light is preferably pulsed light. In this case, it is further preferred that the emission-suppressing light is pulsed light, and particularly preferred that both the excitation light and the emission-suppressing light are pulsed light. If the emitter used is a fluorescent emitter, or in particular a fluorophore, the emission-suppressing light is preferably STED light, in particular pulsed STED light.

[0054] Preferably, the emission is measured with one or more detectors which are arranged to detect individual photons and thereby count the photons. Such detectors include so-called photomultiplier tubes (PMTs), avalanche photodiodes (APDs) and hybrid photodetectors (HPDs). Particularly preferably, the emission is measured with the aid of an array of such detectors, wherein preferably the apertures of such an array are arranged in the image plane such that the distance between adjacent apertures in the plane is less than the diffraction limit of the imaging in the image plane, preferably less than half the diffraction limit, further preferably less than a quarter of the diffraction limit. For example, such an arrangement can be achieved using an APD array with individual elements of suitable size or by arranging the fiber ends of fiber bundles in the image plane, with a single fiber of each fiber bundle being guided to a single detector (e.g. a PMT).

[0055] If the excitation in the illumination and measurement steps is pulsed, the emission excited by the corresponding pulses is preferably measured in a time-resolved manner, in particular with a time resolution of better than 1 ns, more preferably better than 100 ps, ​​and even more preferably better than 30 ps. Time-resolved measurements performed in this manner can be used, for example, to monitor the path of the emission over time and, in the case of fluorescent emitters, to determine the fluorescence lifetime from the measurement results. In the subsequent data analysis, i.e., in the evaluation step after tracking the movement of the first and second emitters, the time-resolved measurement is particularly advantageous in combination with the determination of the trajectories of the first and second emitters.

[0056] The temporal resolution of the measurement can also be used generally to optimize the optical separation of the two emitters, for example, in particular to optimize the assignment of emission measurement values ​​to the emitter from which the emission originated during data recording and / or after the tracking of the movement of the first and second emitters. In particular, the different fluorescence lifetimes can also serve as the sole or primary criterion in an evaluation step performed after the data recording, in which the trajectories of the first and second emitters are determined based on the emission measurement values ​​obtained in the repeatedly performed illumination and measurement steps and the associated corresponding paths generated during the data recording. Optically distinguishable emitters can be physically separated based on the fluorescence lifetime, i.e., an emission (in particular a detected photon) can be assigned to the first emitter instead of the second emitter, or vice versa.

[0057] As an alternative or in addition to the highly time-resolved measurement of the emission, gated detection can also be performed. Gated detection can be used to detect the emission over time.

[0058] As already explained, the key to executing the method according to the present invention is the differentiation of emitters, that is, their optical separation. In embodiments where the positions of the first and second emitters are determined in corresponding positioning steps, this optical separation is already achieved during data recording. In other embodiments, this optical separation may not be achieved during data recording, but only after the tracking of the first and second emitters has been completed. Even if the positions of the first and second emitters are determined in the positioning step, this optical separation is not necessarily perfect; that is, a certain degree of error can be tolerated when assigning the measured values ​​of an emission to the emitter from which it originated. It is also possible that an emission is assigned to the first emitter during data recording, but in the evaluation step, as described above, the emission is assigned to the second emitter.

[0059] In particular with regard to the optical separation of the emitters from one another, in a preferred embodiment the measurement of the fluorescence emission is performed in two detection channels with different spectral sensitivities, or in more than two channels or with a high spectral resolution. In such embodiments, if the detection is performed in two channels, the spectral sensitivities of the detection channels are preferably coordinated with respect to the first emitter and the second emitter, so that the sensitivity of the first detection channel to the emission of the second emitter is minimized relative to the sensitivity to the emission of the first emitter. In general, the aim is to reduce crosstalk of the emission of one emitter into the detection channel of the other emitter. For example, two detection channels can be provided by placing a color splitter in the detection beam path (in which the emission is directed to the measuring device), the color splitting the detection beam path into two paths, wherein emission light with a wavelength below a critical wavelength is guided in one path and emission light with a wavelength above the critical wavelength is guided in the other path, and the emission light is respectively directed to the associated detectors. When using specific emitters, particularly specific fluorescent emitters, to mark structures in a sample, this critical wavelength can be selected, for example by selecting a corresponding dichroic mirror or adjusting a dichroic mirror with a variable critical wavelength, so that the sensitivity of the first detection channel to the emission of the second emitter is minimized relative to the sensitivity to the emission of the first emitter. Conversely, emitters used to mark structures in a sample can also be selected so that the above conditions are met when using a specific device with fixed, preset color channels. In general, in these embodiments, it may be advantageous to select emitters whose emission spectra are as distinct as possible and whose spectral detection channels are coordinated with their spectral emission characteristics to meet the above conditions. These conditions do not necessarily have to be strictly met. In a preferred embodiment, the spectral sensitivities of the emitters and the detection channels are coordinated such that the sensitivity of the first detection channel to the emission of the second emitter relative to the sensitivity to the emission of the first emitter is at most 10%, preferably at most 5%, and more preferably at most 1%, of the sensitivity to the emission of the first emitter; and / or the sensitivity of the second detection channel to the emission of the first emitter relative to the sensitivity to the emission of the second emitter is at most 10%, preferably at most 5%, and more preferably at most 1%, of the sensitivity to the emission of the second emitter. In a preferred embodiment, the spectral composition of the first and second excitation light and the first and second emitters are coordinated such that the crosstalk of the emission of the second emitter in the detection channel of the first emitter is particularly low, while a greater crosstalk of the emission of the first emitter in the detection channel of the second emitter is acceptable. This is particularly applicable to embodiments in which, in the localization step, the position of the first emitter is determined, and based on the position of the first emitter determined in the localization step, a corresponding new estimated position region is determined for each emitter and, overall, a new set of paths of intensity increase regions or a new set of positions of intensity minima is determined.Detection in two spectral channels can be carried out simultaneously in a simple manner with high efficiency, ie with low losses of the emitted light before detection.

[0060] In other preferred embodiments, the emission is measured in more than two spectrally distinct channels, for example, in three, five or more, or ten or more channels. The spectral channels may partially overlap or be spectrally separated. For example, the emission light may be split using a diffraction grating or a prism, or using corresponding devices known in the art of spectral measurement, and directed to an array, such as an array of detectors. However, it is also possible to cascade fixed or adjustable color splitters and direct the emission light to multiple individual detectors in this manner.

[0061] In a preferred embodiment, when measuring emission, the measured value of the emission of a first emitter is assigned to the first emitter. Here, only emissions that are highly likely to originate from the first emitter are assigned to the first emitter. This assignment can be achieved, for example, by the measures detailed above: the first excitation light and the second excitation light differ in their spectral composition; the fluorescence emission is measured in two detection channels with different spectral sensitivities, or in more than two channels; and the illumination with the first excitation light and the illumination with the second excitation light are temporally separated from each other during the illumination and measurement steps. These measures can, for example, act cumulatively by temporally separating illumination with the first excitation light and the spectrally different second excitation light, wherein the fluorescence emission is measured in two detection channels with different spectral sensitivities, or in more than two channels. As described above, the temporal resolution of the measurement can also be used to improve the assignment of measured values ​​emitted during data recording to the emitter from which the emission originated. However, this synergistic effect can be advantageous even if the illumination with the first excitation light and the spectrally different second excitation light are not temporally separated, for example, illumination with the first excitation light and the second excitation light and measurement of the fluorescence emission in two detection channels with different spectral sensitivities, or in more than two channels.

[0062] In embodiments where the measured value of the emission of a first emitter is assigned to the first emitter during emission measurement, the position of the first emitter can be determined based on the measured value of the emission assigned to the first emitter. If, for example, a fluorescent emitter is used as the emitter, where the emission spectrum of the first emitter has a maximum at a long wavelength and the emission spectrum of the second emitter has a maximum at a short wavelength, then the emission of the first emitter can be detected without significant loss within this spectral range. In other words, even if the second emitter is actually excited by the excitation light used for the first emitter, the emission of the second emitter is not detected within this spectral range, or at least only a negligible amount of the emission of the second emitter is detected. Therefore, if measurement is performed in this manner, the position of the first emitter can be determined in the positioning step as accurately as if the second emitter were not present in the measurement range.

[0063] In preferred embodiments, when measuring an emission, the measured values ​​of the emission are assigned to the emitter from which the emission originated. In this case, only emissions that are highly likely to originate from the relevant emitter are assigned to the relevant emitter. Several methods for achieving this assignment were explained above in connection with assigning the measured values ​​of the emission of a first emitter to the first emitter. In these embodiments, preferably, in the localization step, the positions of the first and second emitters are determined based on the different positions and the measured values ​​of the assigned emissions, and based on the positions of the two emitters determined in the localization step, a corresponding new estimated position area is determined for each emitter, and a new set of paths of intensity increase areas or a new set of positions of intensity minima is determined overall.

[0064] In a preferred embodiment, three spectral detection channels are used, two of which (herein referred to as channels A and B) are tuned to the emissions of a first emitter and a second emitter, such that in each of the two channels, channel A or B, only or almost only the emissions of the associated first or second emitter are detected, so that the emissions of the corresponding unassociated second or first emitter contribute less than 1%, or preferably less than 1‰, to the measured signal in the associated spectral channel A or B. Spectral channels A and B then have a spectral distance from each other within which no emissions are detected in either channel A or channel B. A third channel (herein referred to as channel C) is then provided to detect emissions in this spectral gap. Preferably, the spectral detection range of channel C fills the spectral gap. This detection arrangement is particularly advantageous because it allows the emissions detected in channels A and B to be assigned to the associated emitters with a high degree of certainty during data recording. This, in turn, enables the positions of the first and second emitters to be determined according to the MINFLUX method, without the emissions of the second emitter interfering with the location of the first emitter, and vice versa. It should be noted that in this manner, only a certain proportion of the total detected emission is initially used for localization, resulting in a high degree of uncertainty in the localization. It is crucial that the localization is free of deviations during the illumination and measurement steps. This is the case here. Then, in an evaluation step after data acquisition, the emission detected in the third channel can be used to determine the trajectories of the first and second emitters with even greater precision. For this purpose, the emission detected in the third channel is preferably classified, i.e., assigned to the first or second emitter. This can be done, for example, based on time-resolved detection in the third channel if the emitters have different lifetimes, or based on temporally separated excitation using spectrally distinct first and second excitation lights.

[0065] If emissions are detected in more than three spectral channels or with a higher spectral resolution, the same principle can be applied, i.e., only those emissions that can be unambiguously assigned to an emitter with high reliability are used for position determination during data recording by selecting the emissions from the corresponding spectral region.

[0066] In the evaluation step, for example, when determining the trajectory of the individual emitters, the measured emission and the path of the associated region of increased intensity or the location of the associated intensity minimum can be weighted according to the certainty with which the emission is assigned to the relevant emitter. For example, the corresponding weights can be obtained by evaluating the ratio of the overall recorded measurement values ​​or by evaluating the ratio of each portion of the overall recorded measurement values. It may also be appropriate to use a weighting that deviates from 1 only when determining the trajectory of one of the emitters (i.e., the first emitter or the second emitter) in the evaluation step.

[0067] For example, the method according to the present invention can be used to study the movement of structures (e.g., motor proteins) in a sample that are labeled with an emitter in at least two regions (e.g., at each end if the structure is elongated), that is, with a first emitter and a second emitter. In this case, the first and second emitters are connected to each other via the structure. The movement of such a doubly labeled structure, particularly a biological structure, can be tracked using the method according to the present invention, typically with a range below the diffraction limit in at least one direction. To track the movement of such a structure, the position of the first emitter must be determined in a localization step to track its movement and, therefore, the movement of the first and second emitters. Based solely on this position, a corresponding new estimated position region can be determined for each emitter, and a new set of positions can be determined overall. This applies even if the structure is not rigid but deforms during tracking, as long as the distance between the emitters is essentially limited by the structure. Although no separate trajectory is recorded during tracking, the trajectory of the second emitter can be recorded in a subsequent evaluation step, i.e., in the data analysis after the tracking movement has been completed. This is possible because, during the illumination and measurement steps, due to the close proximity of the two emitters, the emission of the second emitter is also excited and measured each time, with the measured value for each emission being assigned to the location where the illumination light was located. Since the extent of the structure is below the diffraction limit, the second emitter is necessarily located in the region illuminated by the excitation light. A corresponding new estimated position region can now be determined for each emitter. For the first emitter, a new estimated position region around a specific location can be determined based on the expected localization uncertainty or the localization uncertainty determined based on the measured values ​​(particularly the number of detected photons and the size and type of the predetermined set of positions). For the second emitter, a new estimated position region can be determined based on the estimated position region of the first emitter, for example, based on the size of the structure connecting the emitters, or generally based on a priori knowledge of the structures to which the emitters are coupled. Based on the newly estimated position regions of the first and second emitters, a new set of paths of intensity increases or new sets of positions of intensity minima is now determined overall. This set is preferably selected so that the movement of the first emitter can be tracked with high speed and precision, while the trajectory of the second emitter can be determined with high precision in the data analysis after the tracking movement is completed.

[0068] In preferred embodiments, a common average position of the two emitters can be determined during the localization step based on the different positions and the measured values ​​of the assigned emissions. In these embodiments, based on the common average position of the two emitters determined during the localization step, a new estimated position region is determined for each emitter, and a new position set is determined overall. The estimated position regions for the first and second emitters can be identical or different. For example, if the emitters contribute to the measurement signal in different quantities, different estimated position regions will result. In this case, the estimated position region of the emitter with the greater contribution may be smaller than the estimated position region of the emitter with the smaller contribution. For example, the knowledge of the different quantitative contributions can be based on prior knowledge of the emitters or on an evaluation of measurement data.

[0069] Preferably, the common average position of the first and second emitters is determined based on the total amount of emission assigned to the relevant paths. This also applies, for example, if the emission of the first and second emitters is measured in a plurality of different spectral channels. The measured values ​​of the plurality of spectral channels used to determine the common average position of the two emitters are then combined into a data set representing the respective positions of the respective paths or intensity minima and the associated emission. In general, this means that the total amount of emission assigned to the relevant paths is used to determine the common average position, rather than assigning the emission to the first or second emitter, as will be used herein. This has the advantage, for example, that the method is less sensitive to the influence of background emission that does not originate from either the first or second emitter.

[0070] When determining the estimated position area and determining a new set of paths or positions, it is now preferred to consider that the emitters are at a certain distance from one another, and that the estimated position of either the first or second emitter is less accurate than would be achievable if all emissions originated from a single emitter. In cases where different paths are obtained by placing minima of a specific intensity distribution at different locations, or where placing minima of the intensity distribution from different locations is part of the process of obtaining different paths, the distances between emitters and the increased uncertainty in the position of a single emitter can be taken into account by expanding the estimated position area in accordance with the increased uncertainty and determining a set of paths or positions that corresponds to the expanded estimated position area. The same applies if the estimated position area and set of paths are determined based on the position of the first emitter rather than the second emitter. In this case, while the uncertainty in the position of the first emitter can, in favorable cases, be as low as it would be without the second emitter, it is generally increased. Furthermore, the position of the second emitter is less certain in all cases.

[0071] In many cases, the distance between the two emitters, for example the maximum distance, average distance or typical distance of the emitters, may be known before tracking is performed, because a specific study of a substantially known structure is usually performed or because a sample may have already been imaged for tracking the emitters. Therefore, in both of the above-described variants (determining the common average position of the two emitters and determining the position of the first emitter), the previously known distance may preferably be taken into account in determining the estimated position region and in subsequent steps, for example by determining that the estimated position region around a specific common average position or position of the first emitter has an extent that is at least a multiple of this distance, for example at least four times, ten times or fifty times the distance between the two emitters.

[0072] In general, in embodiments where different paths of the intensity increase region are obtained by placing the intensity minima of the intensity distribution at different locations and a common average position of the two emitters is determined, it is preferred that the set of predetermined positions include a first position and a second position whose distance from each other is at least four, ten, or fifty times the distance between the two emitters. In particular, if the distance between the emitters is not known in advance, it is preferred that the set of predetermined positions include a first position and a second position whose distance from each other is at least four, ten, or fifty times the distance between the two emitters, or at least 50 nm, at least 100 nm, or at least 200 nm. The same applies to new sets of positions. In other embodiments where a common average position of the two emitters is not determined, it is preferred that the set of predetermined positions include a first and a second position whose distance from each other meets the aforementioned specifications.

[0073] For example, the method according to the invention makes it possible to track the movement of two emitters, wherein the first emitter and the second emitter are at a distance of less than 100 nm or less than 50 nm or less than 20 nm or less than 10 nm or even less than 5 nm from each other.

[0074] If the distance between the emitters is less than 50 nm or less than 20 nm or in particular less than 10 nm or even less than 5 nm, in particular if the emitters are coupled to a structure such that the distance between the emitters changes only slightly or not at all during the measurement, embodiments of the invention in which, in a localization step, the position of the first emitter is determined separately and based on this position, a new estimated position area for the first and second emitter is determined, as well as embodiments in which a common average position of the two emitters is determined separately and based on this position, a new estimated position area for the first and second emitter is determined, can be used particularly advantageously.

[0075] In methods using a predetermined set of positions for intensity minima, it may be preferable that the predetermined set of positions include a first predetermined subset of positions (positions arranged around the estimated position region of the first emitter) and a second predetermined subset of positions (positions arranged around the estimated position region of the second emitter). These subsets may be randomly or intentionally identical. In many cases, it is preferable that the first and second predetermined subsets of positions are different from each other. The same applies to the new set of positions.

[0076] If the predetermined position set includes two subsets, in particular, if it includes two different subsets, it may be preferred that: in the irradiation and measurement steps, the local minimum of the first excitation light or the local minimum of the emission suppression light overlapping with the excitation light is located at the position of the first predetermined position subset, and the local minimum of the second excitation light or the local minimum of the emission suppression light overlapping with the second excitation light is located at the position of the second predetermined position subset. If a new position set also has a corresponding subset, the same situation applies to the new position set.

[0077] If the predetermined path set includes two subsets, particularly if it includes two different subsets, then based on the above-described circumstances, it may be preferable that, in the irradiation and measurement steps, the path of the first excitation light or the path of the emission suppression light overlapping with the first excitation light is generated according to the first predetermined path subset, and the path of the second excitation light or the path of the emission suppression light overlapping with the second excitation light is generated according to the second predetermined path subset. If the new path set also has a corresponding subset, the same applies to that path set.

[0078] In both of the above cases, i.e., the general case with two path subsets or the more specific case with two position subsets, it may be preferable that: during the illumination and measurement steps, the path of the first excitation light or the path of the emission suppression light overlapping with the excitation light is first generated according to the first predetermined path subset, and then the path of the second excitation light or the path of the emission suppression light overlapping with the second excitation light is generated according to the second predetermined path subset; or, during the illumination and measurement steps, the local minimum of the first excitation light or the local minimum of the emission suppression light overlapping with the first excitation light is first located at a position in the first predetermined position subset, and then the local minimum of the second excitation light or the focus of the second excitation light and the emission suppression light are located at a position in the second predetermined position subset. In summary, in these embodiments, the first position subset with relevant light is processed first, followed by the second position subset. It should be noted that in some embodiments, certain light can be explicitly or implicitly assigned to the first subset and the second subset, for example, long-wave excitation light is assigned to the first subset and short-wave excitation light is assigned to the second subset. With respect to these embodiments, it should be noted that in many cases the order in which the subsets are processed is not important; rather, the second subset with correlated light may be processed first, followed by the first subset with correlated light.

[0079] In a method for sequentially processing subsets, particularly if the method determines the positions of a first emitter and a second emitter in a localization step, preferably, after generating a path of the first excitation light or a path of emission suppression light overlapping with the first excitation light according to the first predetermined path subset, a localization step is initiated based on different paths from the first predetermined path subset and the associated emission measurement values ​​during the period of continued illumination and measurement using the second excitation light, wherein the position of the first emitter is determined, and based on the position of the first emitter determined in the localization step, a new estimated position region and a new first subset of a new path set are determined for the first emitter, wherein the new first path subset includes paths that match the new estimated position region of the first emitter. In many cases, the order is also unimportant. Instead, it is important that both subsets are processed in such a way that the localization step is already initiated during data recording, i.e., during the period of acquiring measurement data for a particular emitter, while the illumination and measurement steps are being performed, to determine the position of the other emitter. Preferably, this portion of the positioning step, i.e., determining the position of the first emitter from a chronological perspective, is completed during the duration of the illumination and measurement steps, and during the illumination and measurement steps, a new first path subset is determined based on the position of the first emitter, which matches the already determined position of the first emitter. In this case, the subsequent illumination and measurement steps can be performed immediately after the previous illumination and measurement step is completed, i.e., the illumination and measurement steps can be repeated before the illumination and measurement step is completed. Of course, if different paths are generated by placing intensity minima at different locations, the description herein also applies to the locations of the intensity minima involved.

[0080] In the above case, it is further preferred that, after generating the paths of the second excitation light or the emission suppression light overlapping with the second excitation light according to the second predetermined subset, a localization step is performed based on the different paths of the second predetermined path subset and the assigned emission measurements within a time period starting with the repeated illumination and measurement steps with the first excitation light, wherein the position of the second emitter is determined, and based on the position of the second emitter determined in the localization step, a new estimated position region and a new second subset of the set of paths are determined for the second emitter, wherein the new second subset includes paths that match the new estimated position region of the second emitter. Preferably, the new second subset is determined before the portion of the repeated illumination and measurement steps processing the first new subset is completed. This measure also serves to accelerate data recording, or more precisely, to improve the temporal resolution of emitter tracking.

[0081] When performing the preferred method, the local minimum of the intensity distribution of the illumination light, having a local minimum, is temporally located at different locations in the predetermined set of locations within the measurement range. This can also mean that, within a time period, the area surrounding a location in the predetermined set of locations is covered. This also applies to new sets of locations. Similarly, in confocal microscopy, for example, we speak of scanning at scanning positions, even if the scanner actually moves the scanning beam continuously. The same applies to sets of paths in the region of increased intensity. Here, the path may also change during the time period associated with a path in the set, so that the path in the set is equivalent to the average of the actual paths during that time period. This is clearly true if the different paths in the region of increased intensity are generated by placing the intensity minima of the intensity distribution at different locations. However, the same applies if the different paths are generated by other means, such as by differentially modulating the phase of the pupil or a beam near the pupil, or by interfering with phase-shifted sub-beams.

[0082] Here, locating local minima at different positions of a predetermined set of positions around the presumed position area does not mean that the predetermined set of positions only contains positions around the presumed position area. On the contrary, such a set of positions may also include positions within the presumed position area, such as the center of the presumed position area, or these positions may fill the presumed position area in a grid-like manner. It is important for carrying out these preferred embodiments of the invention that local minima are also located at predetermined positions around the presumed position area in any case. Furthermore, this does not exclude, for example, placing minima along a continuous route, wherein the emitted measurement values ​​are respectively assigned to the positions of the continuous route on which the irradiating light is located. The route may surround or cover the presumed position area. The same applies to each new set of positions, i.e., the repetition of the irradiation and measurement steps. Therefore, a predetermined path set or a new path set does not necessarily only contain those paths that are located within the presumed position area of ​​one of the emitters.

[0083] It is generally preferred that the local minimum of the intensity distribution of the excitation light or the emission suppression light is a zero point.

[0084] A local minimum can be a 3D minimum, a 2D minimum, or a 1D minimum. The meaning of each of these terms has been explained above in conjunction with the definitions of the terms. If a 3D minimum, a 2D minimum, or a 1D minimum is used, the associated region of increased intensity is also used. This will not be explicitly mentioned below. When tracking an emitter, different types of minima and minima with different orientations can be used, particularly 1D minima with different orientations. For example, during an illumination and measurement step, a first 1D minimum oriented in a second direction can be used, and the position of the emitter along a first direction perpendicular to the second direction can be determined based on the measurement data. A second 1D minimum oriented perpendicular to the first 1D minimum can be used either in the same illumination and measurement step or in a subsequent illumination and measurement step. If the second 1D minimum is placed in the same illumination and measurement step, this is done according to a predetermined set of positions associated with that illumination and measurement step. Conversely, if the second 1D minimum is placed in a subsequent illumination and measurement step, i.e., during a repeated illumination and measurement step, this can be done according to a new set of paths or positions derived from the illumination and measurement step. When placing the second ID minimum, the position obtained using the first minimum may be relevant, a property of minima that has not been discussed in detail but is familiar to those skilled in the art.

[0085] In a preferred embodiment, the 1D minimum and / or the 2D minimum and / or the 3D minimum are generated by modulating and focusing the light. The intensity distribution then fills the focal volume. Typically, the intensity distribution in the focal volume where the above-mentioned region of increased intensity is formed is of particular interest. Along the direction of the 1D minimum, the characteristics of the region of increased intensity decrease with increasing distance from the focus (i.e., the center of the focal volume). Therefore, the goal is to place the 1D minimum so that the region of increased intensity used begins at the focus of the intensity distribution. As is well known to those skilled in the art, similar situations also apply to the 2D minimum.

[0086] Therefore, a 2D minimum can also be used in conjunction with a 1D minimum to determine the position along the axis of the 2D minimum in the same and consecutive illumination and measurement steps. In this case, for example, a minimum generated by an annular phase plate can be used instead of a true 1D minimum, which corresponds approximately to a 1D minimum near the focus. Therefore, in this application, minima generated with the aid of an annular phase plate (and without a vortex phase plate) are categorized under the term "1D minimum."

[0087] Preferably, a 2D minimum can be used in conjunction with a corresponding 1D minimum, such that a plurality or a number of illumination and measurement steps are performed using the 2D minimum, while the 1D minimum is occasionally used, for example, after a fixed time interval or when motion of the emitter is tracked beyond a predetermined range. This approach is suitable for many biological samples because the motion to be observed in these samples typically occurs in a plane substantially parallel to the sample surface, with little motion perpendicular to the sample surface. In these cases, the use of the 1D minimum is particularly useful for ensuring that the focus of the intensity distribution forming the 2D minimum is appropriately positioned along the axial direction, particularly to improve the positioning of the axis perpendicular to the 2D minimum.

[0088] In other cases, the movement of the first and second emitters can be aligned with structures in the sample. For example, a motor protein (e.g., kinesin) labeled with the first and second emitters can be moved along microtubules. Prior to tracking, these microtubules can be imaged, for example, using MINFLUX, to determine their orientation in the sample. The order in which the 2D and 1D minima are used can then be selected based on the known locations of the structures along which the emitters are moving.

[0089] The structure along which the structures labeled with the first and second emitters are moved is typically elongated, i.e., the emitters move along a fixed path. This results in the two emitters moving at least approximately along a straight line during the time period of performing a plurality of illumination and measurement steps and associated further steps. In this case, it may be advantageous to use multiple 1D minima oriented such that the straight line is perpendicular to the plane along which the minimum of the intensity distribution extends. Since these structures are often aligned parallel to the sample surface, this can be achieved by using a device for rotating the linear polarization, a corresponding phase modulator, and focusing the light forming the minima into the sample. Preferably, after the emitters have moved a predetermined distance or after a fixed time period, a measurement is performed by using another 1D minimum oriented preferably perpendicular to the first 1D minimum, or by using multiple such 1D minima (e.g., one of which is used to determine the position along the optical axis), or by using a 2D minimum or a 3D minimum to ensure that the emitters are within the focus range during the further illumination and measurement steps, i.e., during subsequent repetitions of the illumination and measurement steps, and to check the direction of movement or determine a changed direction of movement. In subsequent illumination and measurement steps, i.e., during repeated illumination and measurement steps, the orientation of the 1D minimum can be adapted to the new direction of movement. This method of using the minimum oriented relative to the direction of the structure offers the advantage of being particularly fast, since in the correlation step, the position along only one direction must be inferred from the emission.

[0090] This method using a 1D minimum can be designed as a MINFLUX method and a STED-MINFLUX method.

[0091] This method using a 1D minimum can be performed using only one excitation light or using two (in particular different) excitation lights.

[0092] This method using 1D minima can also be used for one of the two emitters, ie the first emitter and / or the second emitter, while the other emitter is tracked with the aid of a different type of minimum.

[0093] This method using ID minima can be performed in such a way that the position of the first emitter is determined in the localization step, or the positions of the first emitter and the second emitter are determined in the localization step, or a common average position of the two emitters is determined in the localization step.

[0094] If different types of minima are used in the illumination and measurement steps, they can be used sequentially or interleaved in time, i.e., the different types of minima are rapidly alternating so that the measurement information corresponding to the different minima is obtained almost simultaneously. In combination with the use of pulsed light, the various types of minima can be used in a "pulse-interleaved" manner.

[0095] In a preferred embodiment, the first emitter, the second emitter, or both emitters are fluorescent emitters. In this case, the first emitter, the second emitter, or both emitters are fluorophores or units composed of fluorophores. Using units composed of multiple fluorophores has the advantage of achieving a larger measurement signal. In contrast, using a single fluorophore as an emitter has the advantage that the area of ​​fluorescence emission is limited to a minimum, which has a favorable effect on the resolution that can be achieved in principle. Both the first emitter and the second emitter can be emitters that can be excited by multiphoton processes or, preferably, by single-photon processes.

[0096] Particularly preferably, a single fluorophore can be used as the first emitter, and a unit consisting of multiple fluorophores can be used as the second emitter. It is particularly advantageous if, in the localization step, the position of the first emitter (i.e., in this case, a single fluorophore) is determined based on its emission, wherein a corresponding new estimated position region is determined for the emitter based on this position, and a new set of paths of intensity increase regions or a new set of positions of intensity minima are determined overall. The large measurement signal that can then be obtained when measuring the emission of the second emitter can be combined, in particular, with spatially resolved detection of the emission in the detection plane, which resolves the Airy diameter of the point spread function of the image of the point source, and wherein the detector detects an area larger than one Airy diameter (e.g., 1.5 Airy diameters, or preferably 2 or more Airy diameters), in order to also determine the position of emitters with high resolution that were detected by the excitation light during the illumination and measurement steps but were not regularly located within the intensity increase region. This can occur, for example, if both emitters are excited by excitation light with the same spectral distribution (e.g., the same narrowband or monochromatic laser light) and the emitters are relatively far apart from each other (e.g., at a distance equal to or greater than the diffraction limit for optical imaging), so that if the first emitter is located in a region of increased intensity, the second emitter may be located in the region of a maximum of the excitation light distribution. Here, too, the detection is preferably spectrally separated, so that the detection of the fluorescence of the first emitter is suppressed on the aforementioned detector with spatial resolution. The emission from the second emitter can then be used to determine the position of the second emitter during data detection based on the position of its impact on the detector with spatial resolution. However, these measured values ​​are particularly suitable for determining the trajectory of the second emitter in an evaluation step after data detection.

[0097] If the distance to the element (e.g., a biological structure) marked with the emitter or to the element marked with the emitter (e.g., a biological structure) is expected to fluctuate significantly during the tracking of the emitter, using a single fluorophore as the first emitter and a unit consisting of multiple fluorophores as the second emitter may also be particularly advantageous. If the distance between the emitters is small, separate excitation light can be used for the first and second emitters, each of which is localized according to the MINFLUX method. However, if the distance increases to the point where the second emitter enters the region of influence of the intensity maximum of the first emitter's excitation light, the excitation light of the second emitter is discarded. This exploits the fact that, due to the high intensity of the first emitter's excitation light, low cross-excitation is sufficient to significantly excite the second emitter, allowing its position to be determined from its measurement signal. In this case, dispensing with the excitation light of the second emitter (whose intensity increase region matches the second emitter's position) ensures that the first emitter does not enter the region of maximum of the second emitter's excitation light. In order to increase the measurement signal of the second emitter at the aforementioned greater distance from the first emitter, the intensity distribution of the excitation light of the second emitter can also be used, which is placed specifically according to the position of the first emitter, i.e., the intensity distribution has a minimum at the presumed position of the first emitter.

[0098] In another embodiment, the first emitter, the second emitter, or both emitters can be scattering emitters. If only one emitter is scattering, the other emitter can be fluorescent. This allows the two emitters to be separated, for example, based on the time interval between emission measurement and excitation, since scattering has no time delay. This can be particularly advantageous if excitation is pulsed. If both emitters are scattering, the scattering properties of the two emitters must depend differently on the wavelength of the excitation light. This can be achieved, for example, by selecting suitable nanoparticles. This requires the use of two spectrally distinct excitation lights. However, if a scattering emitter is used, it is preferred that the other emitter be fluorescent. Furthermore, it is preferred that the scattering emitter selectively scatters the excitation light within a narrow wavelength range. The scattering and fluorescent emitters can then be excited separately—that is, temporally separated—which enables particularly good separation of the emissions and, accordingly, their assignment to the relevant emitters. This, in turn, facilitates the method according to the present invention in which the positions of the first and second emitters are determined in the localization step based on the different paths of the intensity increase region and the measured values ​​of the assigned emissions.

[0099] In a preferred embodiment, during the illumination and measurement steps, the sample can be illuminated with focused excitation light and an intensity distribution of emission-suppressing light having a local minimum within the measurement range, the emission-suppressing light being particularly STED light. Various embodiments are possible. For example, a first excitation light that overlaps with the intensity distribution of the first emission-suppressing light (wherein the first excitation light and the emission-suppressing light are coordinated with the first emitter) can be combined with a second excitation light that is coordinated with the second emitter and forms an intensity minimum in the sample. In this case, the position of the first emitter can be determined using the MINFLUX method, and the position of the second emitter can be determined using the STED-MINFLUX method. If the emitter is a fluorescent emitter and the fluorescence-suppressing light is STED light, the first emitter (or, more generally, the emitter whose position is determined using the STED-MINFLUX method) is an emitter with a longer-wavelength absorption maximum, i.e., the first emitter is excited by longer-wavelength light; whereas the second emitter (or, more generally, the emitter whose position is determined using the MINFLUX method) is an emitter with a shorter-wavelength absorption maximum, i.e., the second emitter is excited by shorter-wavelength light. In this way, an emitter whose position is determined by means of MINFLUX is prevented from being excited and / or bleached by the STED light of another emitter.

[0100] In a preferred embodiment, the emission suppressing light may include a wavelength that suppresses the emission of the two emitters. For example, the emission suppressing light may be a narrowband light or a monochromatic light that suppresses the emission of the two emitters, in particular a laser. Even so, the first illumination light may be an excitation light that excites the light emission of the two emitters, that is, the measurement of the emission of the first emitter and the second emitter may be based on a pair of common excitation light and emission suppressing light (preferably based on a pair of common excitation light and STED light). Alternatively, in the case where the emission suppressing light includes a wavelength that suppresses the emission of the two emitters, preferably, a first excitation light may be used that is coordinated with the first emitter, i.e., preferably excites the first emitter but does not excite or only slightly excites the other emitter, and a second excitation light may be used that is coordinated with the second emitter, i.e., preferably excites the second emitter but does not excite or only slightly excites the other emitter.

[0101] The use of emission-suppressing light (including a wavelength that suppresses the emission of both emitters), in particular corresponding narrowband or monochromatic laser light, and in particular STED light, has the following advantages: regardless of whether one or two excitation lights are used, the positions of the two emitters are determined based on the intensity path of a single light source, i.e., there are no deviations in the position determination due to misalignment between the two lights. The use of two different excitation lights that are coordinated with the respective emitters has the following advantages: improved separation of the two emitters, i.e., the emission can be more reliably assigned to the relevant emitters. However, it should be noted that this STED-MINFLUX method can also be used as a method for determining the position of the first emitter (instead of the second emitter) in the localization step, or for determining the common average position of the first and second emitters. Only the combination of the MINFLUX method and the STED-MINFLUX method described above is unsuitable for performing the method according to the present invention by determining the common average position of the first and second emitters.

[0102] Preferably, after tracking the movements of the first and second emitters is completed, an evaluation step is performed in which the trajectories of the first and second emitters are determined based on the emission measurements obtained in the repeated illumination and measurement steps and the associated respective positions at which the illumination light was located. This evaluation step ensures particularly high spatiotemporal resolution. In principle, the presence of two closely adjacent emitters reduces the spatiotemporal resolution with which each emitter can be tracked in real time, as the emission of one emitter increases the uncertainty in determining the position of the other emitter. In the method according to the present invention, in which the average position of both emitters is determined, or in which the position of only one emitter is determined in the localization step and then a new estimated position range for both emitters is derived from this position, the range in which at least the second emitter's position is sufficiently reliable is obviously larger than the range of positions determined using MINFLUX when only a single emitter is present under otherwise identical conditions. In this embodiment according to the present invention, utilization is made of the fact that the trajectories do not need to be finally determined when performing data recording. Instead, during data recording, it is only necessary to ensure that the emission of both emitters is measured, which can be assigned to a specific path or location of an intensity minimum. In some embodiments, the emission can also be initially assigned to the individual emitters in the evaluation step. In embodiments in which emissions have already been assigned to emitters during data recording, such assignments can be changed in an evaluation step.

[0103] In such an evaluation step, when determining the trajectory, the distance-dependent influence of the proximity of an emitter on the emission of another emitter can be taken into account. In particular, the influence of Förster Resonance Energy Transfer (FRET) between a first fluorescent emitter and a second fluorescent emitter, in particular between a first fluorophore and a second fluorophore, can be taken into account. This applies in particular if, during the illumination and measurement steps, the emission of the fluorescent emitters is detected in multiple spectral channels or in a spectrally resolved manner, and if excitation light is used that strongly excites the first emitter, acting as the donor, but does not or only slightly excites the second emitter, acting as the acceptor; or if a first excitation light and a second excitation light spectrally separated from the first are used, wherein the excitation light is coordinated with the donor and the acceptor such that the acceptor is minimally excited by the donor's excitation light and, conversely, the donor is not or only slightly excited by the acceptor's excitation light. This latter condition is easily met for many FRET pairs because, for FRET pairs consisting of two fluorophores, the maximum of the acceptor's excitation spectrum is typically at a wavelength where the absorption of the donor is very low; in particular, the wavelength of this excitation maximum is longer than the wavelength of the donor's emission maximum. When FRET occurs, if only the excitation light from the donor is used for excitation, the closer the distance, the more fluorescence from the acceptor will be detected. Conversely, the closer the distance, the less fluorescence from the donor will be detected.

[0104] Furthermore, in some cases, the proximity of two fluorophores can affect the fluorescence lifetime, for example, when FRET occurs, which affects the fluorescence lifetime of the donor. Therefore, if the emission of at least one emitter is measured in a time-resolved manner during the determination, the effects of proximity can be taken into account under appropriate conditions.

[0105] If two closely adjacent emitters form a FRET pair, in particular when the two emitters are bound to a structure such that FRET occurs during the entire measurement time period of interest, it may be sufficient to use excitation light that excites the acceptor but not the donor. In such an embodiment, it is particularly preferred to determine a common average position of the two emitters during the localization step. In this case, the estimated position area determined for the first emitter can be identical to the estimated position area determined for the second emitter. However, it is also advantageous to use two excitation lights to track the individual emitters of the FRET pair, the emission light and the emitters being coordinated with each other as described above. This is particularly advantageous when the distance between the emitters is so great at certain times during the measurement time period of interest that no FRET occurs. The estimated position area can then be determined separately for each emitter in the corresponding localization step during the entire measurement time period.

[0106] In the evaluation step, the trajectories of the first and second emitters can preferably be determined by means of a maximum likelihood method or a least squares method. These methods are known to those skilled in the art and are mentioned in the prior art cited in this application and explained in the known prior art.

[0107] In the evaluation step, the measured values ​​of the emission can be assigned to the respective emitters based on an evaluation of the ratios of the measured values ​​of the emission obtained in the repeatedly performed irradiation and measurement steps.

[0108] In the evaluation step, the trajectories of the first and second emitters can be determined, preferably based on a model, or by a calibration method based on simulations. Preferably, a combination of these two approaches can also be employed, i.e., a model calibrated based on simulations can be used. The simulation itself can be based on measurement data, for example, measurement data for detecting the intensity distribution in the sample.

[0109] The invention further relates to a device, in particular a microscope, which is configured to carry out the method according to the invention.

[0110] The device comprises at least one light source. The light source is preferably a laser, more preferably a pulsed laser. The light source is preferably configured to generate broadband light, wherein: the light source or the device is preferably further configured to select different spectral ranges, so as to provide different excitation light and / or excitation light and emission suppression light (preferably STED light) and / or different emission suppression light (preferably STED light), and / or the device comprises multiple light sources, preferably lasers, more preferably pulsed lasers, more preferably pulsed lasers and cw lasers, so that different excitation light and / or excitation light and emission suppression light (preferably STED light) and / or different emission suppression light (preferably STED light) are provided in the device. The presence of both one or more cw lasers and one or more pulsed lasers is advantageous, as the MINFLUX method is more advantageously performed using cw light, and the STED method, and therefore the STED-MINFLUX method, is more advantageously performed using pulsed light. Therefore, the presence of two laser types makes it possible to perform the MINFLUX method and the STED-MINFLUX method using the most appropriate light type (pulsed or cw).

[0111] An imaging objective is not necessarily required to perform the MINFLUX method or the STED-MINFLUX method, since high spatial resolution does not result from high spatial resolution imaging of the light emitted from the sample, but rather from the type of illumination. Therefore, the apparatus must be configured to actually generate the intensity distribution or path to be generated when performing the method according to the present invention and to detect the light emitted from the sample.

[0112] The device according to the invention therefore has an illumination device which is arranged to influence the light of the light source such that the light guided into the sample forms in the sample an intensity distribution having a local minimum and adjacent regions of increased intensity within the measurement range.

[0113] Accordingly, the device according to the invention further comprises light-influencing means, which are configured to generate different paths of regions of increased intensity in the sample. In particular, the light-influencing means can be configured to shift the local minimum relative to the sample. The light-influencing means can be an integral part of the illumination device.

[0114] Furthermore, the apparatus according to the present invention comprises a measuring device configured to detect emission from a range (i.e., a measurement range) within which the first and second emitters of the sample are excited (wherein the first and second emitters are in close proximity). Furthermore, the measuring device is preferably configured to detect emission in multiple spectral channels or in a spectrally resolved manner. For this purpose, the measuring device preferably comprises a color separator, more preferably a variable color separator or a cascade of multiple variable color separators.

[0115] Preferably, the device has an objective lens that is arranged to direct light from the light source into the sample and to collect light emitted from the sample. This enables the device to also be used to perform other methods, such as confocal microscopy or STED microscopy.

[0116] Preferably, the measuring device is arranged to measure light emitted from the sample and collected by the objective lens.Preferably, the objective lens is located in the light path of the illumination device and the measuring device.

[0117] Preferably, the light-affecting means comprises a displacement means.Preferably, the displacement means comprises one or more scanners, such as a galvanometer scanner or an electro-optical scanner.

[0118] Furthermore, the device according to the invention has a memory unit which is configured to store the position of the local minimum and the associated measured value.

[0119] Furthermore, the device according to the invention has an evaluation unit which is configured to evaluate the measured values ​​of the positions of the paths or local minima assigned to the areas of increased intensity, wherein based on the different paths or positions and the measured values ​​of the assigned fluorescence emission one of the following is determined: i) the position of the first emitter, ii) the position of the first emitter and the second emitter, iii) the common average position of the two emitters, and i) based on the determined position of the first emitter, or ii) based on the determined positions of the two emitters, or iii) based on the determined common average position of the two emitters a corresponding position area is determined for each emitter and again on this basis a set of paths of the areas of increased intensity or a set of positions of the local minima is determined.

[0120] The device according to the invention has a control unit which is arranged to control the light-influencing device, wherein different paths of areas of increased intensity are generated in the sample according to the set of paths, in particular wherein local minima are shifted to positions of the determined set of positions.

[0121] Furthermore, the device according to the invention has a control unit which is configured to control the device with which the local minimum can be shifted relative to the sample.

[0122] Further advantageous elements can be incorporated into the description of the method. The technical measures and elements mentioned there can advantageously be integrated into the microscope according to the invention.

Claims

1. A method for simultaneously tracking the movement of an isolated first emitter and an isolated second emitter in a sample by means of a MINFLUX method or a STED-MINFLUX method, the first emitter being optically distinguishable from the second emitter, wherein the first emitter and the second emitter are in close proximity, the method comprising: a) an illumination and measurement step, wherein the sample is illuminated with an intensity distribution of in particular focused excitation light or an intensity distribution of in particular focused emission suppression light overlapping with the excitation light, the emission suppression light in particular STED light, the intensity distribution having a local minimum in a measuring range and an area of ​​increased intensity adjacent to the minimum, the local minimum in particular being a zero point, wherein different paths of an area of ​​increased intensity from a set of predetermined paths of areas of increased intensity are generated in a chronological order in at least the area of ​​the presumed position of the first emitter and optionally in the area of ​​the presumed position of the second emitter, and the emission of the first emitter and optionally the emission of the second emitter are measured, wherein measured values ​​of the emission are assigned to the respective paths of the areas of increased intensity, and b) a localization step, in which, based on the different paths of the area of ​​increased intensity and the measured values ​​of the assigned emissions, i) the position of the first emitter, or ii) the position of the first emitter and the second emitter, or iii) the common average position of both emitters is determined, c) wherein, based on i) the position of the first emitter determined in the positioning step, or ii) the positions of the two emitters determined in the positioning step, or iii) the common average position of the two emitters determined in the positioning step, a corresponding new estimated position area is determined for each emitter and a new path set is determined as a whole, and d) wherein the irradiating and measuring steps and the positioning step are repeated using a new set of paths of regions of increased intensity.

2. The method according to claim 1, characterized in that The excitation light of the irradiating and measuring steps includes a first excitation light for exciting the first emitter and a second excitation light for exciting the second emitter.

3. The method according to claim 2, characterized in that The spectral compositions of the first excitation light and the second excitation light are different, in particular wherein, in the irradiation and measurement steps, irradiation with the first excitation light and irradiation with the second excitation light are performed temporally separated from each other.

4. The method according to any one of the preceding claims, characterized in that The local minimum is a 3D minimum, or the local minimum is a 2D minimum, or the local minimum is a 1D minimum, in particular wherein, in the irradiation and measurement steps, different types of minima and / or minima with different orientations are used alternately, and / or different types of minima and / or minima with different orientations are used alternately to perform and repeat the irradiation and measurement steps.

5. The method according to any one of the preceding claims, characterized in that The excitation light is pulsed, in particular wherein the emission excited by the respective pulse is measured in a time-resolved manner, further in particular with a time resolution better than 1 ns, more preferably better than 100 ps, ​​even more preferably better than 30 ps.

6. The method according to claim 2 or any one of claims 3 to 5 when dependent on claim 2, characterized in that In the positioning step, the positions of the first emitter and the second emitter are determined based on the different paths and the measured values ​​of the assigned transmissions, and based on the positions of the two emitters determined in the positioning step, a corresponding new estimated position area is determined for each emitter and a new path set is determined as a whole.

7. The method according to claim 6, characterized in that The predetermined path set includes: a first predetermined path subset, which is adapted to the inferred position area of ​​the first emitter; and a second predetermined path subset, which is adapted to the inferred position area of ​​the second emitter, in particular, wherein the first predetermined path subset and the second predetermined path subset are different from each other, and further in particular, wherein, in the irradiation and measurement steps, according to the first predetermined path subset, the path of the first excitation light or the path of the emission suppression light overlapping with the first excitation light is generated, and according to the second predetermined position subset, the path of the second excitation light or the path of the emission suppression light overlapping with the second excitation light is generated.

8. The method according to claim 7, characterized in that In the irradiation and measurement steps, first, according to the first predetermined path subset, the path of the first excitation light or the path of the emission suppression light overlapping with the excitation light is generated, and then according to the second predetermined path subset, the path of the second excitation light or the path of the emission suppression light overlapping with the second excitation light is generated.

9. The method according to claim 8, characterized in that After generating the path of the first excitation light or the path of the emission suppression light overlapping with the first excitation light according to the first predetermined path subset, the positioning step is started based on the different paths of the first predetermined path subset and the assigned emission measurement values ​​during the time period of continuing the irradiation and measurement steps using the second excitation light, wherein the position of the first emitter is determined, and based on the position of the first emitter determined in the positioning step, a new inferred position area and a new first subset of the new path set are determined for the first emitter, wherein the paths of the new first subset include paths that are adapted to the new inferred position area of ​​the first emitter.

10. The method according to claim 9, characterized in that After generating the path of the second excitation light or the path of the emission suppression light overlapping with the second excitation light according to the second predetermined subset, the positioning step is completed based on the different paths of the second predetermined path subset and the assigned emission measurement values ​​within a time period starting and preferably completing the repetition of the irradiation and measurement steps using the first excitation light, wherein the position of the second emitter is determined, and based on the position of the second emitter determined in the positioning step, a new inferred position area and a new second subset of the new path set are determined for the second emitter, wherein the new second subset includes paths adapted to the new inferred position area of ​​the second emitter.

11. The method according to any one of claims 1 to 5, characterized in that In the positioning step, the position of the first emitter is determined based on the measured values ​​of the different paths and the assigned transmissions, and based on the position of the first emitter determined in the positioning step, corresponding new estimated position areas are determined for the first emitter and the second emitter and a new set of paths as a whole.

12. The method according to claim 11, characterized in that The first emitter and the second emitter are connected to each other via a structure, in particular a biological structure, to which the first emitter and the second emitter are coupled as markers.

13. The method according to claim 12, characterized in that Based on the position of the first emitter determined in the locating step and based on a priori knowledge of the structure to which the emitter is coupled, a new presumed position area of ​​the second emitter is determined.

14. The method according to any one of claims 1 to 5, characterized in that In the positioning step, based on the measured values ​​of the different paths and the assigned emissions, a common average position of the two emitters is determined, and based on the common average position of the two emitters determined in the positioning step, a corresponding new inferred position area and a new set of paths as a whole are determined, in particular wherein the distance between the first emitter and the second emitter is less than 100 nm or less than 50 nm or less than 20 nm or less than 10 nm or less than 5 nm.

15. The method according to claim 14, characterized in that Based on the sum of the measured values ​​of the emissions of the first emitter and the second emitter of the illuminating and measuring steps, the common average position of the two emitters is determined.

16. The method according to any one of the preceding claims, characterized in that The different paths of the intensity increased area are obtained by placing the minimum of the intensity distribution at different positions, so that the set of paths corresponds to the set of positions of the minimum, in particular wherein the set of positions comprises a first position and a second position, the distance between the first position and the second position being at least four times, ten times or fifty times the distance between the two emitters, or at least 50 nm, at least 100 nm or at least 200 nm.

17. A method according to any one of the preceding claims, characterised in that The first emitter and / or the second emitter is a fluorescent emitter, in particular wherein the first fluorescent emitter and / or the second fluorescent emitter is a fluorophore or a fluorescent unit consisting of a fluorophore.

18. A method according to any one of the preceding claims, characterised in that The first emitter and / or the second emitter are scattering emitters.

19. The method according to claim 3 or according to claim 3 and any one of claims 4 to 18, characterized in that The spectral composition of the first excitation light and the second excitation light are coordinated with the first emitter and the second emitter, so that the excitation of the second emitter by the first excitation light is minimal relative to the excitation of the first emitter by the first excitation light, or at the same excitation intensity, is at most 10% of the excitation of the first emitter, preferably at most 5%, more preferably at most 1%, even more preferably at most 0.1%, and / or the spectral composition of the first excitation light and the second excitation light and the first emitter and the second emitter are coordinated with each other, so that the excitation of the first emitter by the second excitation light is minimal, or, in particular at the same excitation intensity, is at most 5%, preferably at most 1%, more preferably at most 0.1% of the excitation of the second emitter.

20. The method according to any one of the preceding claims, characterized in that Fluorescence emission is measured in two detection channels with different spectral sensitivities, in particular wherein the spectral sensitivities of the detection channels are coordinated with the first emitter and the second emitter such that the sensitivity of the first detection channel to the emission of the second emitter relative to the sensitivity to the emission of the first emitter is minimal, or is at most 10% of the sensitivity to the emission of the first emitter, preferably at most 5%, more preferably at most 1%, and / or wherein the spectral sensitivities of the detection channels are coordinated with the first emitter and the emitter such that the sensitivity of the second detection channel to the emission of the first emitter relative to the sensitivity to the emission of the second emitter is minimal, or is at most 10% of the sensitivity to the emission of the second emitter, preferably at most 5%, more preferably at most 1%.

21. The method according to any one of the preceding claims, characterized in that After the tracking of the movements of the first and second emitter has been completed, in an evaluation step the trajectories of the first and second emitter are determined based on the measured values ​​of the emission obtained in the repeatedly performed irradiation and measurement steps and the corresponding assigned paths that have been generated.

22. The method according to claim 21, characterized in that When determining the trajectory, if an emitter and a further emitter are present, distance-dependent influences of the proximity of the emitter on the emission of the other emitter, in particular the Forster resonance energy transfer, are taken into account.

23. A method according to any one of the preceding claims, characterised in that In the irradiation and measurement steps, the sample is irradiated with focused excitation light and an intensity distribution of emission suppression light, in particular STED light, having an intensity distribution having a local minimum within a measurement range, wherein the emission suppression light includes a wavelength that suppresses emission of two emitters.

24. An apparatus for performing the method according to any one of the preceding claims, comprising: light source, an illumination device, which is arranged to influence the light of the light source in such a way that the light guided into the sample forms an intensity distribution having a local minimum in the measurement range in the sample, light-affecting means, in particular shifting means, arranged to generate different paths of regions of increased intensity in the sample, in particular to shift the local minimum relative to the sample, wherein the light-affecting means can be a component of the illumination means, a measuring device arranged to detect emissions from said measuring range, a storage unit arranged to store the location of the local minimum and the assigned measurement value, an evaluation unit, which is arranged to evaluate the measured values ​​of the paths assigned to the areas of increased intensity or the positions of the local minima, wherein based on the different paths or positions and the measured values ​​of the assigned fluorescence emission one of the following is determined: i) the position of the first emitter, ii) the position of the first emitter and the second emitter, iii) the common average position of the two emitters, and i) based on the determined position of the first emitter, or ii) based on the determined positions of the two emitters, or iii) based on the determined common average position of the two emitters a corresponding position area is determined for each emitter and on this basis a set of paths of the areas of increased intensity or a set of positions of the local minima is determined, A control unit is arranged to control the light-affecting means, wherein different paths of areas of increased intensity are generated in the sample according to the set of paths, in particular wherein the local minima are shifted to positions of the determined set of positions.

25. The device according to claim 24, characterized in that The device has an objective lens in the optical path of both the illumination device and the measurement device, the objective lens being arranged to direct the light of the light source into the sample and to collect the light emitted from the sample.