Methods, apparatus, and computer programs for locating emitters in a sample

Abstract

The invention relates to a method for localizing an emitter (F) in a sample (S), the method comprising illuminating the sample (S) with a stationary circular excitation beam (E), acquiring fluorescent photons; and estimating the position of the emitter (F) in the sample (S) from the acquired fluorescent photons. The invention also relates to a device (1) for localizing an emitter (F) in a sample (S), the device comprising an illumination means (10), an acquisition means (20) and a processing means (30), and to a computer program comprising instructions for causing the device (1) to perform the method for localizing an emitter (F).

Description

Technical Field

[0001] This invention relates to a method, apparatus, and computer program for locating emitters in a sample. In particular, the method can be applied to pre-locate emitters in MINFLUX positioning, wherein the position estimate obtained by the method according to the invention can be used as a starting point for MINFLUX positioning.

[0002] Existing technology

[0003] In the prior art, for example in patent applications DE 10 2011 055 367 A1 , WO 2015 / 052186 A1 Patent Publication DE 10 2013 114 860 B3 and Balzarotti F, Eilers Y, Gwosch KC, , "Nanometer resolution imaging and tracking offluorescent molecules with minimal photon fluxes" by A. WestphalV, Stefani F, Elf J, Hell SW ( arXiv:1611.03401 A localization and single-molecule tracking method based on the MINFLUX process has been described in the publication [physics.optics] (2016).

[0004] Essentially, samples containing fluorophores are detected by the intensity distribution of excitation light that excites the fluorophore, triggering the emission of fluorescent photons. This intensity distribution has local minima (particularly near-zero intensity at the center of the distribution) and maxima on either side. Such distributions are known, for example, from STED microscopy, where an intensity distribution of depletion light (e.g., 2D or 3D donuts) forms with a central minimum to deplete fluorescence signals from fluorophores off-center, thus increasing effective resolution beyond the diffraction limit.

[0005] However, MINFLUX nanomicroscopy utilizes the fact that if the fluorophore of interest is located precisely at the minimum of the excitation light distribution, a minimum fluorescence signal (ideally zero) is obtained, while the fluorescence signal increases as the distribution moves away from the minimum. In MINFLUX localization, the excitation light distribution is sequentially located at several positions close to the desired location of the fluorophore (forming a so-called "targeted coordinate pattern" (TCP), also known as the "targeted coordinate set" (STC)). The fluorescence signal is measured at each of these locations, and the fluorophore's location is estimated based on the measured fluorescence signal and the corresponding position of the excitation light distribution, for example, using a least mean square estimator.

[0006] This process is typically iterated, i.e., repeated by placing the minimum of the excitation light distribution at a location around the position estimate obtained in the previous step. In particular, the minimum is placed within a radius around the current position estimate, where the radius decreases in each step, resulting in an improved effective resolution in each step, converging to a value as low as 1 nm depending on the sample and measurement parameters.

[0007] Furthermore, MINFLUX positioning has high photon efficiency because as the location of the excitation minimum gets closer to the actual location of the fluorophore, the amount of light emitted by the fluorophore decreases.

[0008] By using the MINFLUX scheme to sequentially determine the positions of several single fluorophores in a sample, fluorescence images of samples with a resolution in the range of 1 nm can be constructed.

[0009] In a related technique called MINFLUX tracking, the trajectory of a single fluorophore is tracked over time by sequential MINFLUX localization optimized for velocity rather than accuracy.

[0010] In both methods, the excitation beam is preferentially moved to the TCP position relative to the sample by a fast beam deflection device such as an electro-optic deflector to reduce measurement time.

[0011] To locate or track fluorescent molecules using MINFLUX, it is necessary to identify individual fluorophores in the sample and obtain initial positional information about the identified individual molecules. The TCP (transient fluorophore) can then be placed near the initial position estimate, and the minimum values ​​of the excitation light distribution can be sequentially moved to the TCP positions as described above.

[0012] For example, the initial location of a fluorophore can be determined by wide-field illumination of the sample and low-resolution fluorescence detection by a camera, for example, in a manner similar to PALM / STORM microscopy.

[0013] This requires a microscope system capable of rapidly switching between wide-field illumination and confocal illumination with a circular excitation beam, which is technically demanding, especially considering that the switching needs to occur before each localization of an individual fluorophore, potentially slowing down the localization process significantly. Furthermore, high photon counting is necessary for camera detection of fluorescence, thus requiring the fluorophore to be exposed to the excitation light for extended periods or at high intensities. This either slows down the localization process or exposes the fluorophore to the risks of photobleaching and phototoxicity (in the case of live samples).

[0014] Among the alternative methods known according to the prior art (e.g., in Balzarotti F, Eilers Y, Gwosch KC, ...), , "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes" by A. Westphal V, Stefani F, Elf J, Hell SW ( arXiv: 1611.03401 As disclosed in [physics.optics] (2016), a Gaussian excitation beam is scanned over the region of interest (ROI) of a sample, and fluorescent photons are detected by a confocal detector until the photon count reaches a predetermined threshold. Scanning stops at the ROI where the threshold photon count is obtained. Subsequently, the Gaussian excitation beam is placed at several locations around the ROI, and the corresponding photon counts are determined by a point detector. Due to the predetermined shape of the Gaussian excitation beam and the corresponding shape of the emission (detection) point spread function, the approximate location of the fluorophore can be determined, for example, by a so-called modified least mean square estimator, based on the position of the Gaussian excitation beam and the associated photon count. This process is often combined with photoactivation of the fluorophore by an activation laser beam centered on the scanned ROI to maximize the chance of detecting the fluorescent emitter in the ROI.

[0015] While this method offers reliable fluorophore pre-positioning and reduces the risk of photobleaching compared to camera detection, it requires beam-generating optics for two different excitation beam shapes (Gaussian beam and ring beam), rapid switching between beam shapes, and the potential for rapid and high-precision displacement of the two beams (ideally using an electro-optic deflector). Furthermore, the excitation beam must be precisely aligned with the activation beam (if applicable).

[0016] This resulted in the highly complex and expensive MINFLUX system.

[0017] question

[0018] Therefore, the basic objective of this invention is to provide a method for locating an emitter in a sample, wherein the obtained position estimate is suitable as an initial position estimate for MINFLUX positioning or MINFLUX tracking, which minimizes the complexity and cost of the optical instruments used.

[0019] Solution

[0020] This objective is achieved through the subject matter of the claims and the description, particularly the methods, apparatus, and computer programs according to the independent claims. Embodiments of the invention are set forth in the dependent claims and described below.

[0021] Invention Description

[0022] A first aspect of the invention relates to a method for locating an emitter in a sample, the method comprising irradiating the sample with a stationary annular excitation beam to acquire fluorescent photons; and estimating the position of the emitter in the sample based on the acquired fluorescent photons.

[0023] As used herein, the term "emitter" describes a molecule or particle that emits light (particularly luminescence, more particularly fluorescence) when illuminated with excitation light. Specifically, an emitter can be activated, wherein activation transitions the emitter from an inactive state where it does not emit fluorescence in response to excitation light to an activated state where it does emit fluorescence in response to excitation light. This activation can be achieved by illuminating the emitter with activation light of appropriate wavelength (which may be the same wavelength as the excitation light or a different wavelength compared to the excitation light) and intensity. Alternatively, an emitter can spontaneously enter the activated state, i.e., for example, due to thermal fluctuations in the absence of activation light. However, alternatively, an emitter can be inactive and always remain in the activated state.

[0024] The excitation beam or excitation beam is stationary, meaning it does not move relative to the sample during the current acquisition of fluorescence photons. This has the advantage that the measurement is not directly affected by the shape of the annular excitation beam.

[0025] In the context of this specification, the term "ring-shaped excitation beam" means that the excitation beam includes a local intensity minimum at a center coinciding with the optical axis (z-direction) along which the excitation beam propagates, flanked by intensity maximums in at least one direction, particularly in at least two directions in space. This definition includes a local minimum in the focal plane surrounded by a ring-shaped maximum value, which manifests as two maximum values ​​flanking the central minimum in a cross-section parallel to the optical axis. Specifically, the ring-shaped excitation beam can be shaped as a 2D ring or a 3D ring. As used herein, a "2D ring" includes, in a plane perpendicular to the optical axis (i.e., in the x and y directions), particularly in the focal plane, a local intensity minimum flanked by intensity maximums. A "3D ring," which may also be designated as a "locally hollow bottle beam," includes, in all three dimensions (i.e., along the optical axis and in a plane perpendicular to the optical axis), a local intensity minimum flanked by intensity maximums.

[0026] Fluorescent photons can be acquired by an acquisition device comprising at least one detector configured for single-photon counting. Specifically, the at least one detector may be a point detector, such as, for example, a photomultiplier tube, an avalanche photodiode, or a hybrid detector combining the characteristics of both photomultiplier tubes and avalanche photodiodes. In particular, the at least one detector is arranged in a confocal plane relative to the focal plane in the sample.

[0027] The inventors have surprisingly discovered that the annular excitation beam used in MINFLUX localization and MINFLUX tracking according to existing technology can be used to identify and pre-locate emitters in regions of interest of a sample without moving the excitation beam relative to the sample, and with sufficient accuracy for subsequent MINFLUX localization with high photon efficiency.

[0028] In existing methods, pre-positioning is typically performed by scanning a Gaussian excitation beam across the sample. Applying a ring-shaped beam with local minima for this purpose seems counterintuitive, as the excitation intensity at the beam center is close to zero, making it undetectable when the emitter is located at or near the center of the ring. However, when the sample is coarsely scanned for the emitter, the probability of precisely placing the center of the ring at the emitter's location is practically very low. Furthermore, the inventors have found a method for positioning the emitter at a fixed location on the excitation beam.

[0029] Unlike estimating the emitter's position during a MINFLUX localization or tracking sequence, the emitter's position is not estimated based on fluorescence photon counts associated with different positions of the excitation beam relative to the sample. Instead, the emitter's position is specifically estimated based on multiple fluorescence photon counts detected in a position-specific manner, while keeping the excitation beam's position relative to the sample constant. Specifically, the position-specificity of detection can be achieved, for example, by sampling or measuring the spatial distribution of the light emitted by the emitter. This can be achieved, for example, by altering the optical path between the detector pinhole and the sample, thereby effectively changing the position of the pinhole projection in the focal plane of the sample. Alternatively, the spatial distribution of the emitted light in the detector plane can be measured directly, for example, by an array detector comprising several detector elements arranged in a two-dimensional pattern in the detector plane. The emitter's position can be determined, for example, by a least mean square estimator based on the fluorescence photon counts and the corresponding configuration of the detection beam path.

[0030] The described solution has the advantage of improving the efficiency of the emitter search process. Unlike previous MINFLUX implementations, this positioning mode does not resort to regularly focused beams, such as Gaussian-shaped excitation beams. Instead, the same beam shape can be applied to both the pre-positioning step and the MINFLUX positioning or tracking sequence. This significantly simplifies setup because it makes dedicated hardware for microsecond-level switching between different focal intensity distributions (e.g., between Gaussian and toroidal shapes) redundant. Spatial light modulators typically used for switching between Gaussian and toroidal shapes are often too slow to switch between shapes during measurement routines. While the capture range (in other words, the size of the region in which a single emitter can be detected) depends on several parameters, the toroidal shape helps extend the capture range or region beyond that of the Gaussian shape.

[0031] In some embodiments, fluorescent photons are acquired in a location-specific manner. Specifically, location-specific detection of fluorescent photons is performed in an image plane that is confocally arranged relative to the plane of the excitation focus of the excitation beam (i.e., the focal plane). For example, when the excitation beam is stationary relative to the sample, location-dependent acquisition can be achieved by projecting images of detector pinholes at different locations on the focal plane within the sample. Alternatively, for example, an array detector comprising a plurality of detector elements arranged in a two-dimensional array in a plane perpendicular to the optical axis can be used for location-specific detection by directly measuring the spatial distribution of the emitted light (while the excitation beam remains stationary relative to the sample).

[0032] In some embodiments, the method includes projecting a pinhole onto first detection locations (specifically, first detection locations within a first target coordinate pattern), specifically by sequentially projecting the pinhole onto these first detection locations. In particular, fluorescent photons are acquired for the projection onto the first detection locations or for the center of the pinhole located at the first detection locations. In other words, the pinhole is arranged in the detector optical path between the detector of the acquisition device and the sample, and images of the pinhole are generated at several locations in the focal plane of the sample. This method is also referred to below as "pinhole trajectory scanning." Typically, the pinhole itself remains stationary as the image of the pinhole is moved within the focal plane. Specifically, the change of the pinhole's projection position is specifically achieved by altering the detector optical path using a scanner (such as a galvanometric scanner). By altering the detector optical path (e.g., by rotating the mirror of the galvanometric scanner), the de-scanning of the emitted light is changed, such that different emitted light intensities or fluorescent photon counts are obtained for different de-scanning configurations depending on the position of the emitter in the sample. Based on this information, the position of the emitter can be estimated, for example, using a least mean square estimator. When the same scanner is used to scan the excitation beam and descan the emission beam on a sample, as is the case in many confocal microscopy setups known in the art, if the descan optical path is changed, the scanning optical path will also change, which will cause the excitation beam to deflect relative to the sample. To compensate for this deflection and ensure that the excitation beam remains stationary relative to the sample, an additional beam deflection device, as described below, can be applied.

[0033] In some embodiments, the pinhole is moved along a circular trajectory around a center (partially in the focal plane) (i.e., the image of the pinhole is moved), wherein, in particular, the local minimum of the annular excitation beam is located at the center of the circular trajectory. The term "circular trajectory" refers to a trajectory on an arc, which includes partial and complete circles. Specifically, the pinhole is moved gradually or stepwise along the circular trajectory. The circular trajectory has the advantage that the distance between the pinhole center and the minimum of the annular excitation beam remains constant at different locations of the pinhole projection.

[0034] In some embodiments, the annular excitation beam includes a maximum-to-maximum distance, wherein the diameter of the circular trajectory is 50% to 120% of this maximum-to-maximum distance, particularly 60% to 100%, and even more particularly 70% to 90%. Herein, the term maximum-to-maximum distance is defined, particularly in the focal plane, as the distance between the maximum values ​​on either side of the local intensity minimum of the annular excitation beam, especially the minimum distance (in the case of light intensity distribution, where the distance between the maximum values ​​is not constant). A diameter within this range increases the chance of identifying and locating the emitter in a region sufficiently close to the annular excitation beam to obtain a sufficient emission signal against the background, particularly in the absence of prior information about the emitter's location, for example, if no focused activation light is provided to activate the switchable emitter, but the emitter is always active in the sample or (e.g., due to thermal fluctuations) randomly enters an active state. Typical values ​​for the diameter of the circular trajectory are 250 nm to 400 nm, particularly 270 nm to 350 nm. When a circular trajectory of this size in the focal plane is realized, an electro-optic deflector can be used to correct the position of the annular excitation, while another scanning device (such as a galvanometer scanner) moves the pinhole projection.

[0035] In some embodiments, the diameter of the circular trajectory is 50% to 100%, particularly 60% to 80%, of the full-width at half-maximum (FWHM) of the detection point spread function (PSF) of the emitted light during confocal detection (i.e., when the pinhole projection is centered on the local minimum of the annular excitation beam), especially when an activation beam is provided to activate emitters at the activation focus of the activation beam, where more specifically, the activation focus is located at the local minimum of the annular excitation beam. In particular, the maximum intensity of the annular excitation beam is located at the center of the circular trajectory. In the above embodiments, for emitters, pinhole trajectory scanning preferentially searches for sample regions already exposed to the activation light, where emitters activated are thus identified with increased probability. By adapting the diameter of the circular trajectory to the FWHM of the detection PSF, emitters in the sample search region are exposed to a steep gradient during pinhole trajectory scanning, which improves localization.

[0036] In some embodiments, the pinholes are sequentially placed at at least three locations on a circular track. In particular, the at least three locations are evenly spaced on the circular track.

[0037] Advantageously, the aforementioned embodiments associated with circular trajectories provide a particularly time-saving method for searching for emitters across a relatively large region of interest.

[0038] In some embodiments, a synergistic first and second scanning device, i.e., mutually canceling first and second scanning devices, is used to keep the excitation beam stationary. Specifically, the first scanning device alters the descanning optical path to move the pinhole (i.e., the image of the pinhole in the focal plane) between detection positions, while the second scanning device moves the excitation beam to compensate for the alteration of the descanning optical path, such that the excitation beam remains stationary relative to the sample.

[0039] In some embodiments, the first scanning device is a galvanometer scanner. In some embodiments, the second scanning device is an electro-optic scanner. In some embodiments, the first scanning device is a galvanometer scanner and the second scanning device is an electro-optic scanner. Specifically, the galvanometer scanner alters the descanning optical path to move the pinhole (i.e., the image of the pinhole in the focal plane) between detection positions, while the electro-optic scanner moves the excitation beam to compensate for the alteration of the descanning optical path, such that the excitation beam remains stationary relative to the sample.

[0040] In some embodiments, the third scanning device includes a deformable mirror. In some embodiments, the third scanning device is configured to shift the excitation beam along the optical axis (in the z-direction).

[0041] In some embodiments, the following steps are repeated: illuminating the sample with a stationary annular excitation beam and acquiring fluorescent photons, wherein in each repetition the center of the annular excitation beam is positioned at a corresponding scan position, wherein, in particular, the scan positions are arranged on a grid, more specifically on a hexagonal grid. In other words, a search and pre-positioning for combinations of emitters is performed sequentially at multiple scan positions. In this way, a larger region of interest can be searched for a single emitter in the sample, and their preliminary position estimates can be determined for subsequent high-accuracy MINFLUX localization. Once an emitter has been found and a sufficiently accurate position estimate has been determined, the scanning process can be aborted and a MINFLUX localization or tracking sequence can be initiated. Specifically, if no emitter is detected at the current position, the excitation beam is moved to the next scan position, and the search and localization process is repeated.

[0042] In some embodiments, the distance between the scanning positions is between 10% and 50% of the excitation wavelength of the excitation beam.

[0043] In some embodiments, fluorescent photons are acquired by an array detector comprising an array of detector elements. Such an array detector can be used to measure the spatial distribution of light emitted by a single emitter, thereby obtaining location-specific light intensities on different detector elements, and estimating the emitter's position based on the detected light intensities or photon counts and the positions of the corresponding detector elements. Advantageously, this can be done without physically altering the descanning optical path and without correcting the position of the excitation beam, further reducing the complexity of the measurement setup and increasing the speed of the measurement.

[0044] In some embodiments, the detector element is a photon counting avalanche diode (or a single-photon avalanche photodiode, SPAD).

[0045] In some embodiments, fluorescent photons are acquired by an array detector in a first step, wherein a signal intensity distribution generated by the fluorescent photons on the detector elements of the array detector is determined, and wherein the optical path of the emitted light from the sample to the array detector is adjusted based on the determined signal intensity distribution, and wherein in a second step following the first step, the array detector uses the adjusted optical path to acquire additional fluorescent photons from the emitter. Specifically, the position of the emitter in the sample is estimated based on the fluorescent photons acquired in the first step, the second step, or both the first and second steps. Specifically, the optical path is adjusted such that the maximum value of the signal intensity distribution or the signal-weighted positional average of the signal intensity distribution, particularly the photon-weighted positional average pwpa, is closer to the center of the array detector in the second step than in the first step. The signal-weighted or photon-weighted positional average is the sum of position vectors assigned to the individual detector elements, wherein each vector is multiplied by a weight defined as the signal intensity or photon count detected by the corresponding detector element divided by the sum of the signal intensity or photon counts detected by all detector elements of the array detector. The center of the array detector may be the centroid of the array of detector elements. The center can involve a single detector element (e.g., in the case of a non-even number of detector elements in a row or column) or multiple detector elements. In the latter case, the maximum value of the signal intensity distribution is defined as the average signal intensity of the detector elements forming the array center, particularly the signal-weighted positional average, and more particularly the photon-weighted positional average. The purpose of this embodiment is an arrangement in which the emitters in the sample are arranged confocally with the center of the array detector. In this way, more emitted light can be collected, especially when there are relatively few detector elements.

[0046] In some embodiments, when fluorescent photons are acquired by an array detector including an array of detector elements, the excitation beam is sequentially positioned at multiple scan positions, fluorescent photons are acquired by multiple detector elements of the array detector at each scan position, while the excitation beam remains stationary relative to the sample, and the position of the emitter is estimated based on the fluorescent photons acquired by the detector elements at the current scan position.

[0047] In some embodiments, the estimated position of the emitter is determined in a pre-positioning step, followed by a MINFLUX positioning step (i.e., a MINFLUX positioning sequence). Specifically, the pre-positioning step is a lateral pre-positioning step, which estimates the position of the emitter in a plane (particularly the focal plane) perpendicular to the optical axis along which the excitation beam propagates. More specifically, an additional axial pre-positioning step is performed before the MINFLUX positioning, and even more specifically between the lateral and MINFLUX positioning steps, to estimate the position of the emitter in the axial direction along the optical axis.

[0048] In some embodiments, the MINFLUX positioning step includes illuminating the sample with a circular excitation beam, positioning the circular excitation beam at a plurality of second detection positions on a second target coordinate pattern based on the estimated position determined in the pre-positioning step, acquiring fluorescence photons for each of the second detection positions, and estimating the emitter's position in the sample with a higher resolution than in the pre-positioning step based on the acquired fluorescence photons for the second detection positions. In the MINFLUX positioning step, the circular excitation beam is moved relative to the sample, compared to the pre-positioning step. Applying the method according to the invention to the pre-positioning step, which is followed by the MINFLUX positioning step, has the advantage that both steps can be performed using the same excitation source and the same beam-shaping optics. This reduces the complexity of the apparatus according to the invention and increases the measurement speed because it is no longer necessary to switch between light sources or change the phase pattern on the spatial light modulator to switch between the Gaussian excitation beam and the circular excitation beam.

[0049] The second aspect of the invention relates to an apparatus (particularly a microscope or a controller for a microscope) for locating an emitter in a sample, particularly by means of the method according to the first aspect, wherein the apparatus comprises: an illumination device configured to illuminate the sample with a stationary annular excitation beam; an acquisition device configured to acquire fluorescent photons, particularly in a position-specific manner; and a processing device configured to estimate the position of the emitter in the sample based on the acquired fluorescent photons.

[0050] In some embodiments, the illumination device includes a light source (particularly a laser source) configured to generate excitation light capable of exciting emitters in the sample.

[0051] In some embodiments, the illumination device further includes a beam shaping device configured to generate a circularly abbreviated excitation beam from the excitation light. Specifically, the beam shaping device includes a phase modulator configured to modulate the phase of the excitation light. Specifically, the device includes an objective lens with a pupil plane, wherein the phase modulator is disposed at or near a plane conjugate to the pupil plane. Specifically, the phase modulator is a phase plate or a spatial light modulator (e.g., a reflective, liquid crystal-based spatial light modulator). Specifically, the device also includes a polarization modification element (e.g., a quarter-wave plate) configured to circularly polarize the phase-modulated excitation light to generate a circularly abbreviated excitation beam. To generate an excitation beam in the shape of a 2D annulus, a vortex-shaped phase pattern (i.e., increasing from zero to a maximum value, particularly 2D, in the circumferential direction relative to the optical axis) is used. The phase pattern can be set on the phase modulator, and the phase-modulated excitation beam can be circularly polarized by a polarization modification element. Alternatively, to generate a 3D ring, a phase difference of... The phase pattern of the concentric rings can be set on the phase modulator. In this case, in particular, no polarization modification element is required.

[0052] In some embodiments, the illumination device includes an activation light source configured to illuminate the sample with an activation beam, the activation beam being configured to activate emitters in the sample. Specifically, the illumination light source is configured to focus the activation light onto the sample.

[0053] In some embodiments, the device includes a projection device (particularly including a first scanning device) configured to project (particularly sequentially project) a pinhole (i.e., an image of the pinhole) onto a detection location, particularly a detection location of a target coordinate pattern, in the focal plane of the sample.

[0054] In some embodiments, the device includes a sample holder configured to hold a sample, a detector optical path between the sample holder and a detector of the acquisition device, a pinhole arranged in the detection optical path, and a projection device including at least one scanning device configured to change the detection optical path such that the pinhole is projected (particularly sequentially projected) onto a detection position, particularly in the focal plane of the sample, and particularly the detection position of a target coordinate pattern. Hereinafter, the term pinhole describes an aperture configured to adjust the diameter of a detection beam from the sample.

[0055] In some embodiments, the pinholes are arranged in a conjugate plane relative to the focal plane in the sample.

[0056] By projecting a pinhole onto the detection location, location-specific information is obtained, which can be used to determine the position estimate of the transmitter.

[0057] In some embodiments, the projection device is configured to move the projection of the pinhole on a circular trajectory around a center, or to sequentially place the pinhole at at least three locations on the circular trajectory, wherein, in particular, these locations are evenly spaced on the circular trajectory. In some embodiments, the projection device is configured to move the projection of the pinhole on a circular trajectory around a center, wherein the diameter of the trajectory is 50% to 120%, particularly 60% to 100%, and more particularly 70% to 90% of the distance from the maximum value to the maximum value of the annular excitation beam. In some embodiments, the projection device is configured to move the projection of the pinhole on a circular trajectory around a center, wherein the diameter of the trajectory is 50% to 100%, particularly 60% to 80%, of the full width at half maximum (FWHM) of the detection point spread function of the emitted light.

[0058] In some embodiments, the processing apparatus is configured to estimate the location of the emitter in the sample using a least mean square estimator based on the acquired fluorescent photons.

[0059] In some embodiments, the acquisition device is configured to acquire fluorescent photons in a location-specific manner, wherein, in particular, the acquisition device is configured to perform location-specific detection of fluorescent photons in an image plane that is confocally arranged relative to the excitation focus of the excitation beam.

[0060] In some embodiments, the device (particularly a projection device) includes a first scanning device, wherein a second scanning device of the device is configured to work in concert to keep the excitation beam stationary, particularly while projecting (particularly sequentially) the pinhole onto the detection location.

[0061] In some embodiments, the first scanning device is a galvanometer scanner, and / or the second scanning device is an electro-optic scanner. In some embodiments, the device includes a third scanning device (particularly a deformable mirror) configured to scan the excitation beam relative to the sample along the optical axis (in the z-direction).

[0062] In some embodiments, the device is configured to repeatedly perform the following steps: irradiating a sample with a stationary annular excitation beam and acquiring fluorescence photons, wherein in each repetition the center of the annular excitation beam is positioned at a corresponding scanning position, wherein, in particular, the scanning positions are arranged on a grid, more particularly on a hexagonal grid. Specifically, the mutual distance between the scanning positions is between 10% and 50% of the excitation wavelength of the excitation beam.

[0063] In some embodiments, the acquisition device includes an array detector comprising an array of detector elements. To acquire location-specific information, measured fluorescence photons from multiple detector elements of the array detector can be analyzed individually to obtain location-specific information that can be used to determine an emitter position estimate. Specifically, the detector elements of the array detector are arranged in a confocal plane relative to the focal plane in the sample.

[0064] In some embodiments, the detector element is a photon-counting avalanche diode.

[0065] In some embodiments, the array detector is configured to acquire fluorescent photons in a first step, wherein the processing means is configured to determine the signal intensity distribution generated by the fluorescent photons on the detector elements of the array detector, and wherein the projection means (in particular a first scanning device) is configured to adjust the optical path of the emitted light from the sample to the array detector based on the determined signal intensity distribution, and wherein the array detector is configured to acquire additional fluorescent photons from the emitter using the adjusted optical path in a second step after the first step, wherein, in particular, the processing means is configured to estimate the position of the emitter in the sample based on the fluorescent photons acquired in the first step and / or the second step, wherein, more particularly, the projection means is configured to adjust the optical path such that the maximum value of the signal intensity distribution or the signal-weighted positional average value of the signal intensity distribution, in particular the photon-weighted positional average value, is closer to the center of the array detector in the second step than in the first step.

[0066] In some embodiments, the device (particularly a processing device) is configured to determine the estimated position of the emitter in a pre-positioning step following a MINFLUX positioning step. Specifically, in the MINFLUX positioning step, the illumination device is configured to illuminate the sample with a circular excitation beam and position the circular excitation beam at a plurality of second detection positions on a second target coordinate pattern based on the estimated position determined in the pre-positioning step. In the MINFLUX positioning step, the acquisition device is configured to acquire fluorescent photons for each of the second detection positions. In the MINFLUX positioning step, the processing device is configured to estimate the position of the emitter in the sample with a higher resolution than in the pre-positioning step, based on the acquired fluorescent photons, particularly by means of a least mean square estimator.

[0067] In some embodiments, the second scanning device (particularly an electro-optic deflector) is configured to position the annular excitation beam at multiple second detection locations on the second target coordinate pattern.

[0068] In some embodiments, the processing device is an integrated circuit or includes an integrated circuit, such as a field-programmable gate array (FPGA) or an application-specific integrated circuit (ASIC). This has the advantage that position estimation can be performed very quickly in an automated process, including pre-positioning steps and MINFLUX positioning steps performed in an automated manner. Alternatively, for example, the processing device may be a computer including software configured to estimate the position of the transmitter. In any case, the processing device may or may not be part of the same integrated circuit or computer as the controller that controls components of the device, such as illumination devices, beam shaping devices, and / or first scanning devices and / or second scanning devices.

[0069] A third aspect of the invention relates to a computer program comprising instructions, which, when executed by at least one processor, cause the apparatus according to the second aspect to perform the following steps for locating an emitter in a sample: illuminating the sample with a stationary annular excitation beam, particularly acquiring fluorescent photons in a position-specific manner; and estimating the position of the emitter in the sample based on the acquired fluorescent photons. Specifically, the instructions of the computer program cause an illumination device of the apparatus according to the second aspect to illuminate the sample with a stationary annular excitation beam, and the instructions of the computer program cause an acquisition device of the apparatus according to the second aspect to acquire fluorescent photons. Then, the instructions of the computer program cause at least one processor, particularly a processing device of the apparatus according to the second aspect, to estimate the position of the emitter based on the acquired fluorescent photons.

[0070] The terms “computer” and “computer program” as used in this article should be interpreted broadly. In other words, a computer can also be an integrated circuit, such as an FPGA or ASIC, and a computer program can be embodied in hardware and / or software components.

[0071] In some embodiments, the computer program is configured to determine the estimated position of the emitter in a pre-positioning step following the MINFLUX positioning step. Specifically, the pre-positioning step is a lateral pre-positioning step, which estimates the position of the emitter in a plane perpendicular to the optical axis along which the excitation beam propagates. More specifically, an additional axial pre-positioning step is performed before the MINFLUX positioning, and even more specifically between the lateral and MINFLUX positioning steps, to estimate the position of the emitter in the axial direction along the optical axis.

[0072] In some embodiments, during the MINFLUX positioning step, the computer program instructs the device (particularly an illumination device) according to the second aspect to illuminate the sample with an annular excitation beam and position the annular excitation beam at a plurality of second detection positions on a second target coordinate pattern based on the estimated position determined in the prepositioning step. The computer program instructs the device (particularly an acquisition device) to acquire fluorescent photons for each of the second detection positions, and the computer program instructs at least one processor, particularly a processing device according to the second aspect, to estimate the position of the emitter in the sample at a higher resolution than in the prepositioning step based on the acquired fluorescent photons.

[0073] The fourth aspect of the invention—which may be applied independently of or in combination with the first aspect—relates to a method for estimating the position of an excitable emitter, particularly the position of an excitable fluorophore, using an excitation ring (i.e., a ring-shaped excitation beam as described above), wherein the lateral position of the excitable fluorophore is estimated based on spatially resolved detection of fluorescence emission in a first spatial direction and a second spatial direction.

[0074] All embodiments described in conjunction with the first, second, and third aspects can also be freely combined with the embodiments described in the fourth aspect and below.

[0075] Specifically, spatially resolved detection of fluorescence emission involves detecting Airy disks or point images in an image plane, where, more specifically, the image plane is confocal with respect to the excitation focus.

[0076] In some embodiments, the method is used for lateral pre-positioning of the excitable fluorophore (i.e., positioning in a plane perpendicular to the optical axis), followed by MINFLUX positioning of the excitable fluorophore, wherein MINFLUX positioning is performed specifically based on the estimated position.

[0077] In some embodiments, additional axial prepositioning (i.e., positioning along the optical axis) of the excitable fluorophore is performed between lateral prepositioning and MINFLUX positioning.

[0078] In some embodiments, the excitation ring is a 2D ring or a 3D ring.

[0079] In some embodiments, spatially resolved detection of fluorescence emission is performed by an array detector, which in particular includes an avalanche photodiode, especially a single-photon avalanche photodiode (SPAD).

[0080] In some embodiments, spatially resolved detection of fluorescence emission is performed by scanning an Airy disk or point image with a pinhole in the detection plane. Specifically, the projection of the pinhole is guided around its center on a circular trajectory, particularly on a pinhole track. Specifically, the projection of the pinhole is positioned such that its center is on the circular trajectory, at least three locations, for example, six locations, wherein more particularly, at least three locations are evenly spaced on the circular trajectory. Specifically, the center is the center of an imaginary point light source located precisely at the center of the excitation ring.

[0081] In some embodiments, the size of the pinhole is set such that the image of the pinhole in the sample always includes the center of the excitation ring.

[0082] In some embodiments, the excitation ring is deflected by a second scanning device, particularly an electro-optic scanner, and a first scanning device, particularly a galvanometer scanner. Specifically, the first scanning device is positioned in the detection beam path, while the second scanning device is arranged outside the detection beam path.

[0083] In some embodiments, the projections of pinholes arranged in the detection beam path are sequentially placed at selected positions on the pinhole track by means of a first scanning device, particularly a galvanometer scanner, while a second scanning device, particularly an electro-optic scanner, induces the reverse movement of the excitation light, so that the excitation ring remains stationary in the sample, thereby scanning the pinhole track while fixing the excitation position of the excitation ring.

[0084] Because the excitation ring remains stationary during this position estimation, regardless of how it is achieved, the excitable fluorophore is always exposed to the same excitation intensity during positioning. Therefore, the shape of the intensity distribution of the excitation ring does not immediately affect the quality of the lateral prepositioning, but rather indirectly by varying the signal-to-background ratio depending on the actual position of the excitable fluorophore relative to the center of the excitation ring.

[0085] The fifth aspect of the invention relates to a microscope including a control device configured to control the microscope according to the method of the fourth aspect. All embodiments described above in conjunction with the fourth aspect can be applied to the microscope according to the fifth aspect with necessary modifications.

[0086] In some embodiments, the microscope includes an illumination device configured to illuminate an excitation ring with an excitation ring that can excite fluorophores.

[0087] In some embodiments, the microscope includes an acquisition device configured to detect fluorescence emission of an excitable fluorophore in a spatially resolved manner.

[0088] In some embodiments, the microscope includes processing means configured to estimate the lateral position of an excitable fluorophore in a first spatial direction and a second spatial direction based on spatially resolved detection of fluorescence emission.

[0089] In some embodiments, the microscope includes a first scanning device (in particular a galvanometer scanner) configured to deflect an excitation ring, particularly perpendicular to the optical axis, and more particularly wherein the first scanning device is positioned in the detection beam path of the microscope.

[0090] In some embodiments, the microscope includes a second scanning device (in particular an electro-optic scanner) configured to deflect an excitation ring, particularly perpendicular to the optical axis, and more particularly, wherein the second scanning device is positioned outside the detection beam path of the microscope.

[0091] In some embodiments, the microscope includes a third scanning device (particularly a deformable mirror) configured to deflect an excitation ring along the optical axis, and more particularly, the third scanning device is positioned outside the detection beam path of the microscope.

[0092] In some embodiments, the microscope includes a pinhole arranged in the detection beam path of the microscope.

[0093] In some embodiments, the controller is configured to control a first scanning device and a second scanning device (and optionally a third scanning device) such that the projection of a pinhole arranged in the detection beam path is sequentially placed on a circular track (pinhole track) by means of the first scanning device, while the second scanning device, particularly an electro-optic scanner (and optionally a third scanning device), induces a reverse motion of the excitation light, so that the excitation ring remains stationary in the sample.

[0094] In some embodiments, the second scanning device (and optionally a third scanning device) is configured to excite the ring to a second detection position of the second target coordinate pattern during MINFLUX positioning after a predetermined position is obtained by scanning through the pinhole track.

[0095] In some embodiments, the acquisition device includes an array detector comprising a plurality of detector elements arranged in an array.

[0096] The sixth aspect of the invention relates to a computer program comprising instructions which, when executed by at least one processor, cause a microscope according to the fifth aspect to perform the steps of the method according to the fourth aspect.

[0097] Further embodiments of the invention can be derived from the claims, description, and drawings. In this document, the claims should not be interpreted in such a way that only subjects, devices, or methods that include, or do not include, all the features of the dependent claims in addition to those features set forth in the independent claims and aspects described herein can be possible embodiments of the invention. Rather, further embodiments can be derived from the features set forth in the description or from the drawings, which can be applied individually or cumulatively. Brief description of the attached diagram

[0099] The invention is further illustrated and described below with reference to exemplary embodiments shown in the accompanying drawings. These embodiments are non-limiting examples and are not intended to limit the scope of the invention.

[0100] Figure 1 The diagram illustrates the first detection position corresponding to the annular excitation beam and the pinhole projection in an embodiment of the method according to the present invention;

[0101] Figure 2 A grid of sequential scanning positions of an annular excitation beam and a corresponding circular trajectory of the first detection position are shown in an embodiment of the method according to the present invention.

[0102] Figure 3 An array detector comprising a plurality of detector elements arranged in an array is schematically shown in another embodiment of the method according to the invention, the array detector being used for the location-specific detection of fluorescent photons;

[0103] Figure 4 An example of the light intensity distribution of the annular excitation beam used by the method according to the invention is shown;

[0104] Figure 5 The diagram schematically illustrates the MINFLUX positioning sequence after pre-positioning of the transmitter using the method according to the invention.

[0105] Figure 6 An embodiment of the device (MINFLUX microscope) according to the invention is shown, which is configured to perform the method according to the invention.

[0106] Detailed description of the attached figures

[0107] Figure 1 An embodiment of the method according to the invention, also known as pinhole track scanning, is illustrated schematically, wherein the sample S is illuminated by a stationary annular excitation beam E (see also...). Figure 6 ), and an image of pinhole 21 (see also) Figure 6The first detection positions 101 are sequentially projected onto the focal plane of the excitation focus EF in the sample S. The coordinates of the focal plane are indicated by the axes x and y, and the focal plane is perpendicular to the optical axis along which the excitation beam E propagates. The center C of the annular excitation beam E is schematically shown as a rhombus, and the positions of the emitters F, particularly fluorescent dye molecules, which are positioned (or pre-positioned) by the method according to the invention, are depicted as stars. The plurality of first detection positions 101 form a first target coordinate pattern (TCP) 100.

[0108] Before excitation by the annular excitation beam E, the emitter F can be activated, i.e., transitioned from an inactive state where the emitter F does not emit fluorescence in response to the excitation light to an activated state where the emitter F emits fluorescence in response to the excitation light. This can be achieved, for example, by irradiating the sample S with an activation light of a suitable wavelength. In some applications, an activatable fluorophore is required to enable the localization of individual fluorophores. Alternatively, an emitter that spontaneously cycles between inactive and activated states, for example, due to thermal fluctuations (i.e., without irradiation with activation light), can also be used.

[0109] Pinhole 21 (see Figure 6 The illustration shows the detection optical path of the device 1 according to the invention arranged in a confocal plane (relative to the focal plane of the sample S) between the sample S and the acquisition device 20, which includes a detector configured to detect fluorescent photons from the emitter F. In a manner similar to confocal microscopy known in the art, a pinhole 21 is used to exclude defocused fluorescence, i.e., fluorescence from regions of the sample S above and below the focal plane.

[0110] In an embodiment of the method according to the invention, the image of the pinhole 21 is sequentially projected onto a detection position 101 arranged around the center of the annular excitation beam E in the focal plane. Specifically, this is achieved by changing the position of the descanning detection light (fluorescence emission from the sample) relative to the detector of the acquisition device 20, while simultaneously moving the position of the excitation beam E relative to the sample S, such that the excitation beam E remains stationary and always illuminates the same portion of the sample S. This can be achieved, for example, by a first scanning device 16 arranged in the detection beam path (see...). Figure 6 The position of the detection beam E is controlled, and the position of the excitation beam E relative to the sample S is adjusted by a second scanning device 12 arranged outside the path of the detection beam.

[0111] If it is possible to scan the excitation beam E along the optical axis (i.e., in the z-direction), the method according to the invention can also be used to obtain a 3D position estimate of the emitter F. In this case, the first detection position 101 of the first TCP 100 is specifically arranged in a volume surrounding the center of the excitation beam E, more specifically on a sphere, the first scanning device 16 can be configured to scan the excitation beam E in three dimensions, and an additional third scanning device can be used to compensate for the deflection of the excitation focus in the z-direction along the optical axis.

[0112] exist Figure 1 In the depicted example, the pinhole 21 is sequentially projected onto three first detection positions 101 arranged on a circular trajectory T having a diameter d around a center C defined by the center of the annular excitation beam E. Importantly, the circle indicating the first detection position 101 marks the center of the pinhole projection, and in particular, the circle does not indicate the size of the typically much larger pinhole 21. Specifically, the radius of the circular trajectory T is chosen such that the annular excitation beam E always overlaps with the pinhole projection at each first detection position 101. The first detection positions 101 may, but need not, cover the entire circumference of the circular trajectory T (i.e., the entire circle). Instead, the first detection positions 101 may be arranged on a partial circle (arc), such as... Figure 1 The situation described in the text.

[0113] At each first detection position 101, fluorescent photons emitted by the emitter F are acquired by the acquisition device 20 (including the detector). The acquired photon count or signal depends on the relative position of the corresponding pinhole projection and the actual position of the emitter F in the focal plane. In particular, if the emitter F is closer to the center of the pinhole projection, the intensity of the fluorescence signal or the fluorescence photon count will be greater. In this way, position-specific information can be obtained without moving the annular excitation beam E relative to the sample S. Surprisingly, only a few (especially three or more) pinhole projections are needed to obtain position information sufficient to identify and pre-locate the emitter F with sufficient accuracy for subsequent MINFLUX localization. Furthermore, due to pinhole orbit scanning, a relatively large sample area can be searched for the emitter in a short time period.

[0114] In principle, the larger diameter of the circular trajectory T—the first detection position 101 of the first TCP 100 (i.e., the center position of the pinhole 21 projection)—is arranged on this circular trajectory T, which facilitates a faster search of a larger area of ​​the sample S for the emitter F and determines the estimated position of the emitter F. However, due to the size of the annular excitation beam E, there is an upper bound on this diameter, because the emitters F will only be excited by the excitation light if they are arranged within a specific radius around the center of the annulus, and a sufficient amount of emitted fluorescence must reach the detector of the acquisition device 20 with a sufficient signal-to-noise ratio in order to identify the emitter F and determine its position. To achieve this, in the absence of a focused activation light to activate the emitters in a predefined volume, a diameter of approximately 50% to 120%, particularly 60% to 100%, and even more particularly 70% to 90% of the maximum value distance of the annular excitation beam E may be advantageous.

[0115] When additional activation light is provided, such as a Gaussian activation beam centered at a local minimum of the annular excitation beam E, orbital scanning can be adapted to scan a region of the sample in which the emitter is expected to be activated with a high probability. For this purpose, for example, the diameter of the circular trajectory T can be set to a value of approximately 50% to 100%, particularly 60% to 80%, of the FWHM of the detection PSF of the emitted light.

[0116] Figure 2 A grid G ​​is depicted at the scanning position SP, on which the center C of the annular excitation beam E can be sequentially placed during the search for a single emitter F in the sample S. The center position of the annular excitation beam E is marked by a black rhombus, and the associated circular trajectory T on which pinhole track scanning is performed is depicted as a circle around the corresponding rhombus.

[0117] The entire region of sample S can be densely covered by the pinhole track trajectory T, such as Figure 2 As shown. In each step, the annular excitation beam E is moved so that its center is moved to the scanning position SP, and the pinhole 21 is sequentially projected so that its center is projected onto the first detection position 101, which is arranged on a corresponding circular trajectory T around the corresponding scanning position SP. Subsequently, the excitation beam E is moved to the next scanning position SP, and the process is repeated. In this way, the entire grid G ​​can be searched sequentially for the emitter E. In particular, if a fluorescence photon count higher than a preselected threshold is obtained, which indicates the presence of the emitter F, the scanning process can be stopped, and the position estimate of the emitter F can be determined based on the obtained fluorescence photon count.

[0118] Although the hexagonal grid G ​​at the scan position SP is Figure 2 As shown, but the scope of the invention includes any suitable type of grid pattern, such as rectangular and irregular arrangements.

[0119] In particular, when the sample S is activated by confocal light, the method according to the invention can also be selectively performed at the location where the activated beam has been focused. This can be done at a selected scan position SP of the grid G ​​(such as the grid G ​​described above), or without using the grid G ​​at the scan position SP.

[0120] Furthermore, a grid G ​​or an activation-based approach can be used to define specific regions of interest in the sample S and selectively search for the emitter F. Information about these regions of interest can be obtained from previous measurements of the sample S, i.e., for example, by confocal microscopy or wide-field microscopy.

[0121] Specifically, the acquisition device 20 used during the described pinhole track scanning is a point detector (e.g., photomultiplier tube, hybrid detector, or avalanche photodiode) configured to count individual photons.

[0122] As an alternative to pinhole track scanning, array detector 22 can be used to obtain location-specific information to identify and locate individual emitters F in sample S. In this case, pinhole 21 is not required in the detection path. When pinhole 21 is provided, it is typically fully open, allowing all detector elements 23 of array detector 22 to be illuminated by detection light.

[0123] Figure 3 This array detector 22 is shown in a simplified manner. As part of the acquisition device 20, the array detector 22 includes an active region (particularly an active region arranged perpendicular to the optical axis of the detection beam path), wherein the active region includes a plurality of detector elements 23 configured to individually detect a single photon emitted by the emitter F. In the depicted example, the detector elements 23 are arranged in a hexagonal grid, but other configurations (such as rectangular arrangements, random arrangements, etc.) are of course within the scope of the invention.

[0124] According to an embodiment of the method using array detector 22, the center of the annular excitation beam E is placed at the scanning position SP in the sample S, and the fluorescence photon count is acquired by a plurality of detector elements 23 of array detector 22. Here, the amount of light received by each detector element 23 depends on the position of the projection of the respective detector element 23 onto the focal plane in the sample S relative to the actual position of the emitter F. Depending on the relative arrangement of the detector elements 23 and the actual positions of the emitter F, the detected photon count will be different for different detector elements 23. In other words, the amount of light received by each detector element 23 depends on the position of the projection of the respective detector element 23 onto the focal plane in the sample S relative to the actual position of the emitter. Based on these photon counts, the position of the emitter F is estimated by processing device 30.

[0125] Figure 4 An exemplary light intensity curve is shown on a cross-section of a circular excitation beam E in the focal plane. Here, intensity I is plotted relative to a position within the circular excitation beam E along the x-axis (perpendicular to the optical axis along which the excitation beam propagates). The intensity curve shows a central local minimum 3, which is surrounded on all sides by an intensity-increasing region 4 and a relative maximum 5, which has a maximum-to-maximum distance 6 along the x-axis. Specifically, Figure 4 The curve shown depicts a cross-section of a 2D annulus. A similar intensity curve can be produced through a 2D cross-section of a 3D annulus. In particular, the 3D annulus also includes local minima in the focal plane, specifically points of zero (or near-zero) intensity, surrounded by substantially annular maxima. Furthermore, the 3D annulus includes an increase in intensity near the intensity minima along the optical axis (z-direction).

[0126] The advantage of using this annular excitation beam E to excite the emitter F in the method according to the invention is that the same excitation beam E can be used for subsequent MINFLUX positioning of the emitter (which requires an excitation light intensity distribution with local minimums) without switching optical components, such as beam-shaping phase plates or spatial light modulators. This increases the speed of measurement and reduces the complexity of the device 1 according to the invention.

[0127] Figure 5 An example of a MINFLUX positioning sequence for transmitter F is shown, which can be executed immediately after pre-positioning of transmitter F according to the method of the invention. Figure 5 In the diagram, the black empty circle indicates the second probe position 201 forming the second TCP 200. In multiple steps ( Figure 5During each step of the five steps illustrated in the diagram, the center of the annular excitation beam E is sequentially placed at the second detection position 201 of the corresponding second target coordinate pattern 200, and fluorescent photons from the emitter F are acquired at each of the second detection positions 201. The acquired fluorescence intensity or photon count varies depending on the relative position of the emitter F and the corresponding second detection position 201. If the emitter F is exactly at the center of the annular excitation beam E, the excitation light intensity at the center is zero or close to zero, and no fluorescence signal or photon count is acquired above the background. However, as the distance from the center increases, the emitter F is exposed to a higher excitation light intensity (see [reference needed]). Figure 4 Therefore, a higher number of fluorescent photons are emitted per unit time. Based on the obtained photon count and the associated second detection position 201, the position of the emitter F can be estimated, for example, using a least mean square estimator, as described in the prior art.

[0128] exist Figure 5 The example shown illustrates a second TCP 200 comprising seven second detection positions 201 arranged in the focal plane. One of the second detection positions 201 is located at the center of a circle of radius L, and the other six second detection positions 201 are evenly spaced around the circumference of this circle. In this document, at a given step, the center of the circle corresponds to the estimated position of the emitter F. For Figure 5 The first step shown (labeled I) can use the position estimate obtained in the pre-positioning step according to the invention. In prior art MINFLUX methods, this pre-positioning has been performed, for example, by camera imaging (similar to PALM / STORM microscopes) or by excitation with a Gaussian excitation beam at several locations near the expected emitter position. However, here, the same excitation beam shape can be used in the pre-positioning step and the subsequent high-precision MINFLUX positioning, thus avoiding the need to switch optical components.

[0129] After the first step (I), a series of additional MINFLUX steps are performed (three steps marked II in the subsequence and another final step marked III), in which the position estimate of the transmitter F determined in the previous step is used as the new center of the second TCP 200, that is, the second TCP 200 is re-centered using the previously obtained position information in each step.

[0130] Specifically, the radius L of the circle constrained by the second TCP decreases in each step or subset of steps. According to Figure 5Between step I and the first step of subsequence II, the radius L remains the same, but decreases in each subsequent step. In this way, the position estimation in each iteration becomes increasingly accurate. As the circle defining the second TCP 200 becomes smaller, the center of the excitation beam E tends to be closer to the actual position of the emitter F, thus reducing the average value of the obtained fluorescence photon count. To compensate for this effect, the intensity of the excitation light can be increased as the MINFLUX sequence proceeds to obtain a higher photon count, thereby improving the signal-to-noise ratio.

[0131] The final step (III) is the final localization of the emitter F, where the circle defining the second TCP 200 is so small that the fluorescence signal has almost disappeared into the background; that is, according to the experimental boundary conditions, the location estimation is converging to a value with maximum accuracy. In typical MINFLUX applications, this accuracy can be in the range of 1 nm–2 nm.

[0132] Of course, the second TCP 200 shown is merely an example and can be replaced with any other suitable second TCP 200. Furthermore, although... Figure 5 A 2D MINFLUX sequence is shown, in which the second probe position 201 is arranged in the focal plane, but the method according to the invention can also be applied to a 3D MINFLUX sequence. In this case, the annular excitation beam E is typically a 3D annular or locally hollow beam (with the lowest intensity at the center and regions on either side where the intensity increases in all directions in space), and the second TCP 200 includes the second probe position 201 outside the focal plane (above and below).

[0133] Figure 6 A simplified version of device 1 (particularly the MINFLUX microscope) according to an embodiment of the present invention is shown.

[0134] The device 1 includes: an illumination device 10 configured to illuminate a sample S with an annular excitation beam E, the illumination device 10 including a light source 11 (e.g., a laser source) configured to generate an excitation beam capable of exciting an emitter F in the sample S; a phase modulator 14 (e.g., a spatial light modulator or a phase plate) configured to shape the excitation beam into an annular shape; a microscope objective 18 configured to focus the annular excitation beam E onto the sample S; a first scanning device 16 (particularly a galvanometer scanner); a second scanning device 12 (particularly including electro-optic scanners 12a and 12b) and a third scanning device 19 (particularly including a deformable mirror), the second scanning device 12 and the third scanning device 19 configured to shift the annular excitation beam E; a dichroic beam splitter 15 configured to separate the excitation beam and the detection beam (fluorescence emitted by the emitter F); and mirrors 13 and 17.

[0135] Specifically, the first scanning device 16 and the second scanning device 12 are configured to shift the excitation beam E in a first direction x and a second direction y perpendicular to the optical axis along which the excitation beam E propagates (the first direction x is perpendicular to the second direction y). For this purpose, the first scanning device 16 may include at least a first rotatable mirror and a second rotatable mirror (not shown), each rotatable mirror coupled to a corresponding driver (e.g., a galvanometer driver). Figure 6 As shown, the second scanning device 12 may include a first electro-optic modulator 12a configured to shift the excitation beam in a first direction x and a second electro-optic modulator 12b configured to shift the excitation beam in a second direction y, wherein the first electro-optic modulator 12a and the second electro-optic modulator 12b are arranged in series in the beam path of the excitation beam E.

[0136] Specifically, the third scanning device 19 is configured to shift the excitation focus EF of the excitation beam E in the third direction z along the optical axis. For this purpose, the third scanning device 19 may include a deformable mirror and a refractive element. The deformable mirror is configured to shift the excitation focus EF by deformation of its surface when the excitation beam E is reflected from the surface, and the refractive element is configured to guide the excitation beam E to the deformable mirror such that the excitation beam E is reflected on the deformable mirror and the reflected excitation beam E is coupled back into the beam path.

[0137] In addition, device 1 includes: an acquisition device 20, i.e., a point detector or array detector, configured to detect fluorescent photons emitted by an emitter F in sample S; a pinhole 21 arranged in an image plane IP, the image plane IP being confocal with respect to the focal plane of the excitation focus EF of the annular laser beam E; a processing device 30 configured to estimate the position of the emitter E in sample S based on the acquired fluorescent photons; and a control device 40 configured to control a first scanning device 16 and a second scanning device 12.

[0138] The excitation beam E generated by the light source 11 passes through the second scanning device 12 and is reflected by the mirror 13 onto the phase modulator 14. The phase modulator 14 is depicted as... Figure 6 The reflective element generates a reflected phase-modulated excitation beam E, which is reflected by a dichroic beam splitter 15, passes through a first scanning device 16 (e.g., a galvanometer scanner), and is reflected by a mirror 17 to an objective lens 18, which is used to focus the excitation beam E onto the sample S. The dichroic beam splitter 15 reflects light of the excitation wavelength but transmits light of the fluorescence emission wavelength.

[0139] The phase pattern introduced into the excitation beam E by the phase modulator 14 results in a ring-shaped light intensity distribution at the excitation focus EF. The phase modulator 14 is specifically arranged in a plane conjugate to the pupil plane of the objective lens 18, or near such a plane. In particular, the excitation beam E, which is shaped like a 2D ring, can be combined with a polarization modification element (not shown in the image) to generate circularly polarized light through a vortex phase pattern. Figure 6 As shown in the figure, for example a quarter-wave plate (not shown), is used to generate a vortex phase pattern that gradually increases from a phase value of 0 to... The value of , where n is a natural number in the circumferential direction relative to the optical axis (where specifically n=1). In particular, a 3D ring (also known as a locally hollow beam) can be obtained by phase modulating the excitation beam E with a phase pattern comprising concentric rings with a phase difference of π.

[0140] Despite Figure 6 The diagram shows a reflective phase modulator 14, such as a spatial light modulator, but a transmissive phase modulator 14 (e.g., a phase plate) may be used instead in device 1.

[0141] The emitted light (detection beam D) from the emitter F in sample S is picked up by objective lens 18 and reflected by mirror 17 onto first scanning device 16, which descans the detection beam D. The descanned detection beam D passes through dichroic beam splitter 15, confocal pinhole 21 to remove defocused light, and is detected by acquisition device 20, particularly by point detectors including photomultiplier tubes, hybrid detectors, or avalanche photodiodes, or arrays of these detectors.

[0142] Based on the above description of the "pinhole track scanning" embodiment, when using Figure 6 In the device 1 shown, the annular excitation beam E is first moved to the scanning position SP in the sample S using the first scanning device 16. Then, the image of the pinhole 21 is sequentially projected onto the first detection position 101 of the first target coordinate pattern 100 using the first scanning device 16, while the second scanning device 12 (and optionally the third scanning device 19 in the case of 3D scanning) deflects the excitation beam E without affecting the detection beam path to compensate for the displacement of the first scanning device 16, thereby keeping the annular excitation beam E stationary relative to the sample S. In this document, the first scanning device 16 and the second scanning device 12 (and optionally the third scanning device 19) receive control signals from the control device 40. For each first detection position 101, fluorescent photons are acquired by the acquisition device 20.

[0143] According to another embodiment, the acquisition device 20 includes an array detector 22, which includes an array of detector elements 23 (see [link to previous embodiment]). Figure 3 Each of the detector elements is configured to independently detect a single photon emitted by the emitter F. The array detector 22 includes a center 24, which is based on... Figure 3 The example shown is a single detector element 23. If the array detector 22 is used for photon detection, a pinhole 21 is not required in the detection beam path of device 1. However, if a pinhole 21 is arranged in the detection beam path, it is typically fully open to maximize the amount of light illuminating the array detector 22. In this embodiment, the excitation focus EF of the excitation beam E (particularly through the first scanning device 16) is moved to the scanning position SP in the sample S, while the center of the array detector is confocally arranged relative to the center of the annular excitation beam E. Fluorescent photons are then captured by the plurality of detector elements 23 of the array detector 22.

[0144] According to the two embodiments described above, the acquisition device 20 converts the received fluorescent photons into signals processed by the processing device 30. The processing device 30 can also receive input signals from the first scanning device 16, and in particular the second scanning device 12 and / or the third scanning device 19, wherein the input signals contain positional information regarding the current position of the excitation beam E, particularly the positional information of the pinhole 21 image, allowing the processing device 30 to determine the position estimate of the emitter F based on the acquired fluorescent photons and positional information, for example, using a least mean square estimator. In the case of array detection, the processing device 30 can receive from the acquisition device 20 a signal indicating the position of the detector element 23 that has received a corresponding photon count.

[0145] After determining the estimated position of the emitter F, MINFLUX localization can be performed by device 1. For this purpose, the annular excitation beam E is sequentially moved around its center to the second detection position 201 of the second TCP 200 using the second scanning device 12 (see...). Figure 5 The second TCP 200 centers on a position estimate determined by the method according to the invention (e.g., pinhole track scanning or array detection using a stationary annular excitation beam E), and for each second detection position 201, the acquisition device 20 acquires fluorescent photons. The processing device 30 then determines a new position estimate based on the acquired fluorescent photons and the second detection position 201. Next, the second TCP 200 iteratively repeats the MINFLUX sequence using the new position estimate as the center, within a decreasing radius L around the current position estimate. Specifically, this process continues until the position estimate converges, or alternatively, until the emitter F is deactivated or bleached. In particular, in each iteration, the intensity of the excitation light can be increased to compensate for the reduced emission signal.

[0146] Reference tag list

[0147] 1. Device for locating emitters in a sample

[0148] 3 minimum values

[0149] 4. Areas with increased intensity

[0150] 5 maximum value

[0151] 6. Distance between maximum values

[0152] 10 lighting fixtures

[0153] 11 Light Sources

[0154] 12 Second scanning device

[0155] 12a First Electro-Optical Modulator

[0156] 12b Second Electro-Optical Modulator

[0157] 13 reflectors

[0158] 14-phase modulator

[0159] 15-beam splitter

[0160] 16 First Scanning Device

[0161] 17 reflectors

[0162] 18 objectives

[0163] 19 Third Scanning Device

[0164] 20 Acquisition Device

[0165] 21 pinholes

[0166] 22-array detector

[0167] 23 detector elements

[0168] 24 array detector center

[0169] 30 processing units

[0170] 40 Controllers

[0171] 100 First target coordinate pattern (TCP)

[0172] 101 First Detection Position

[0173] 200 Second target coordinate pattern (TCP)

[0174] 201 Second Detection Position

[0175] The center of the C trajectory

[0176] diameter of the d-trajectory

[0177] D detection beam

[0178] E-excitation beam

[0179] EF stimulates focus

[0180] F-projectile

[0181] G grid

[0182] IP Image Plane

[0183] L radius

[0184] SP scan location

[0185] T-trajectory.

Claims

1. A method for locating an emitter (F) in a sample (S), comprising: The sample (S) is irradiated with a stationary annular excitation beam (E). Acquiring fluorescent photons; as well as The position of the emitter (F) in the sample (S) is estimated based on the acquired fluorescent photons. The method includes sequentially projecting pinholes (21) onto a first detection position (101), the pinholes (21) being arranged in the detection optical path of a device (1) in a confocal plane between the sample (S) and the acquisition device (20), and wherein the excitation beam (E) is kept stationary using a first scanning device (16) and a second scanning device (12) working in concert, the first scanning device (16) sequentially placing the projections of the pinholes (21) at selected positions on the pinhole track, the second scanning device (12) inducing a reverse motion of the excitation light such that the excitation ring remains stationary in the sample (S), and wherein the spatial distribution of fluorescent photons emitted by the emitter (F) is acquired, and the position of the emitter (F) is estimated based on the detected light intensity or photon count and the position of the corresponding detector element.

2. The method according to claim 1, characterized in that, The fluorescent photons are acquired in a location-specific manner.

3. The method according to claim 2, characterized in that, The acquisition of the location-specific fluorescent photons is performed in an image plane (IP) that is confocally arranged relative to the plane of the excitation focus (EF) of the excitation beam (E).

4. The method according to claim 1, characterized in that, The pinhole (21) is moved on a circular trajectory (T) around the center (C), or is placed sequentially on at least three of the first detection positions (101) on the circular trajectory (T).

5. The method according to claim 4, characterized in that, The first detection position (101) is evenly spaced on the circular trajectory (T).

6. The method according to claim 4, characterized in that, The annular excitation beam (E) includes a maximum-to-maximum distance (6), wherein the diameter (d) of the circular trajectory (T) is 50% to 120% of the maximum-to-maximum distance (6).

7. The method according to claim 4, characterized in that, The diameter (d) of the circular trajectory (T) is 50% to 100% of the full width at half maximum (FWHM) of the detection point spread function of the emitted light.

8. The method according to any one of claims 1 to 7, characterized in that, The first scanning device (16) is a galvanometer scanner and / or the second scanning device (12) is an electro-optic scanner.

9. The method according to any one of claims 1 to 7, characterized in that, Repeat the following steps: irradiate the sample with the stationary annular excitation beam (E) and acquire the fluorescent photons, wherein in each repetition the center of the annular excitation beam (E) is positioned at the corresponding scan position (SP).

10. The method according to claim 8, characterized in that, Repeat the following steps: irradiate the sample with the stationary annular excitation beam (E) and acquire the fluorescent photons, wherein in each repetition the center of the annular excitation beam (E) is positioned at the corresponding scan position (SP).

11. The method according to claim 10, characterized in that, The scan positions (SP) are arranged on a grid (G).

12. The method according to claim 11, characterized in that, The mutual distance between the scanning positions (SP) is between 10% and 50% of the excitation wavelength of the excitation beam (E).

13. The method according to any one of claims 1 to 7 or 10 to 12, characterized in that, The estimated position of the emitter (F) is determined in a pre-positioning step of the subsequent MINFLUX positioning step, wherein the MINFLUX positioning step includes illuminating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated position determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitter (F) in the sample (S) at a higher resolution than in the pre-positioning step based on the acquired fluorescent photons.

14. The method according to claim 8, characterized in that, The estimated position of the emitter (F) is determined in a pre-positioning step of the subsequent MINFLUX positioning step, wherein the MINFLUX positioning step includes illuminating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated position determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitter (F) in the sample (S) at a higher resolution than in the pre-positioning step based on the acquired fluorescent photons.

15. The method according to claim 9, characterized in that, The estimated position of the emitter (F) is determined in a pre-positioning step of the subsequent MINFLUX positioning step, wherein the MINFLUX positioning step includes illuminating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated position determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitter (F) in the sample (S) at a higher resolution than in the pre-positioning step based on the acquired fluorescent photons.

16. A device (1) for locating an emitter (F) in a sample (S), the device (1) comprising: An illumination device (10) is configured to illuminate the sample (S) with a stationary annular excitation beam (E). Acquisition device (20), the acquisition device (20) being configured to acquire fluorescent photons; Processing device (30), the processing device (30) being configured to estimate the position of the emitter (F) in the sample (S) based on the acquired fluorescent photons, and A projection device configured to sequentially project pinholes (21) onto a first detection position (101), the pinholes (21) being arranged in the detection optical path of the device (1) in a confocal plane between the sample (S) and the acquisition device (20), wherein the device (1) includes a first scanning device (16) and a second scanning device (12) configured to work in concert to keep the excitation beam (E) stationary, the first scanning device (16) sequentially placing the projections of the pinholes (21) at selected positions on the pinhole track, the second scanning device (12) inducing a reverse motion of the excitation light such that the excitation ring remains stationary in the sample (S), and wherein the spatial distribution of fluorescent photons emitted by the emitter (F) is acquired, and the position of the emitter (F) is estimated based on the detected light intensity or photon count and the position of the corresponding detector element.

17. A computer program comprising instructions, which, when executed by at least one processor, cause the device (1) according to claim 16 to perform the following steps for locating an emitter (F) in a sample (S): The sample (S) is irradiated with a stationary annular excitation beam (E). Acquiring fluorescent photons; and The position of the emitter (F) in the sample (S) is estimated based on the acquired fluorescent photons.

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