Single particle localization microscope

Abstract

To provide a microscope capable of localizing single particle.SOLUTION: A single particle localization microscope 100 comprises: an optical system 102 using sequence of light pattern having spatially different distribution of illumination light adapted to cause single particle positioned in a sample region to emit detection light to illuminate the sample region; a detector 116 detecting sequence of intensity of detection light emitted from the sample region in response to sequence of the illumination light pattern; and a processor 132 determining, on the basis of the sequence of the intensity of the detection light, the arrangement of potential position for localizing the particle in the sample region. The processor causes the optical system to illuminate the sample region with subsequent light pattern having specific spatial distribution, and the detector to detect subsequent intensity of detection light emitted from the sample region in response to the subsequent light pattern, and on the basis of subsequent intensity of the detection light, determines which one of a plurality of potential positions indicates a real position of the particle in the sample region.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a single particle localization microscope and method for locating a single particle within a sample area. [Background technology]

[0002] In the field of optical microscopy, methods are now available that allow the localization of individual particles with precision below the diffraction resolution limit. These methods can be used for single-particle tracking and super-resolution imaging of diffusing particles.

[0003] For example, the technique of super-resolution fluorescence imaging is based on conical diffraction, which occurs when a polarized light beam is diffracted through a biaxial crystal. Methods based on this technique are known by abbreviations such as Conical Diffraction Microscopy (CODIM), Super-Resolution using Conical Diffraction (SRCD) with Projected Sequence of Intensities with Various Topologies (PSIT), and Position Dependent Optical Semaphore (PDOS). According to these methods, a single fluorescent emitter can be localized by sequentially illuminating the emitter with a sequence of light patterns, measuring the intensity of the light emitted during illumination with each light pattern, and calculating the emitter position that fits the set of measured light intensities.

[0004] The methods described above are disclosed in WO 2012 / 049381, WO 2013 / 153294 and WO 2019 / 043458. Further reference is made to the following publications: Julien Caron et al., Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging, Cell Adhesion & Migration (2014), pages 430-439, DOI:10.4161 / cam.29358; Clement Fallet et al., Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy, Nanoimaging and Nanospectroscopy II (2014), Proc. of SPIE Vol.9169, 916905; and Francisco Balzarotti et al., Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science (2016), DOI 10.1126 / science.aak9913. As described in the latter publication, tracking particles with minimal photon fluxes is often referred to as dark tracking. This category is also commonly known as MINFLUX and includes the approaches disclosed in WO 2013 / 072273, WO 2018 / 069283, WO 2015 / 097000 and WO 2017 / 153430.

[0005] The above-mentioned methods suffer from ambiguity uncertainty regarding the localization of particle positions, which has not previously been considered. Thus, when calculating an emitter position based on a sequence of light intensities measured in response to a sequence of illumination light patterns, for symmetry reasons, multiple potential emitter positions that equally match the measured light intensities are determined, and it is unclear which of these positions represents the actual emitter position.

[0006] To date, no efficient solution exists to this ambiguity problem. One approach would be to simply repeat a localization measurement sequence at one of multiple potential locations to determine whether this location is the actual emitter location. However, such a solution is time-consuming due to the complex repetition of the measurement sequence. In particular, it requires slowly moving an optical scanning device, such as a scanning mirror, to redirect the illumination light to the selected potential location. The mechanical movement of such a scanning device requires a significant amount of time, especially compared to the short time required to change the illumination light pattern by using electro-optical or acousto-optical effects, such as conical diffraction. Furthermore, mechanical tolerances can affect measurement accuracy. Furthermore, this simple solution is particularly inefficient if an incorrect potential location is selected by tampering to repeat the measurement sequence. Finally, the particle must emit enough light to complete the measurement sequence, which is unfavorable in terms of photon budget. Summary of the Invention [Problem to be solved by the invention]

[0007] It is therefore an object of the present invention to provide a microscope and method that allows unambiguous and efficient localization of single particles in a sample area. [Means for solving the problem]

[0008] The above-mentioned object is achieved by the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following description.

[0009] The single particle localization microscope comprises an optical system configured to illuminate a sample region with a sequence of light patterns having spatially distinct distributions of illumination light adapted to cause single particles located within the sample region to emit detection light. The single particle localization microscope comprises a detector configured to detect a sequence of intensities of detection light emerging from the sample region in response to the sequence of illumination light patterns. The single particle localization microscope comprises a processor configured to determine an arrangement of potential locations for localizing the particle within the sample region based on the sequence of intensities of the detection light. The processor is further configured to cause the optical system to illuminate the sample region with at least one subsequent light pattern having a spatial distribution of illumination light that is asymmetric with respect to the arrangement of the plurality of potential locations. The processor is configured to cause the detector to detect at least one subsequent intensity of detection light emerging from the sample region in response to the at least one subsequent light pattern. The processor is configured to determine which of the plurality of potential locations represents the actual location of the particle within the sample region based on the at least one subsequent intensity of detection light.

[0010] The microscope proposed herein is adapted to distinguish between otherwise indistinguishable particle locations due to the symmetry of the illumination light pattern applied in a sequence of intensity measurements, thereby distinguishing between multiple particle locations. Symmetry in the illumination light pattern arises in some super-resolution localization approaches, such as, but not limited to, the conical diffraction-based method described above. The MINFLUX method described above can also be operated using optically symmetric patterns to localize single particles. In all of these methods, the symmetry of the light patterns sequentially illuminating the sample results in multiple locations that are equally consistent with the sequence of detected light intensities measured in response to the sequence of symmetric light patterns. Distinguishing between multiple potential particle locations can be achieved by illuminating the sample with at least one additional illumination light pattern used as a test pattern to resolve localization ambiguities. For this purpose, the spatial distribution of the test pattern is selected so that the pattern has an asymmetric shape relative to the arrangement resulting from the previously calculated particle locations.

[0011] The proposed solution can be realized with only one additional measurement and does not require any mechanical repositioning of the scanning device, such as the scanning mirror, typically used in laser scanning microscopy, resulting in fast and light-efficient localization.

[0012] For example, the optical system can include a movable mechanical scanning device, such as the scanning mirror described above, to scan the illumination light over the sample. This allows individual points in the sample to be scanned and imaged. Each point corresponds to a specific sample area, which includes a single emitter that coincides with the focal area of ​​the optical system and is located by the microscope. In such a configuration, the processor can cause the optical system to illuminate the specific sample area with at least one subsequent light pattern that forms the asymmetric test pattern without using the scanning device for this purpose, thereby eliminating the need for mechanical repositioning.

[0013] In particular, undesirable solutions involving physical, and therefore slow, movement of the scanning device and repetition of multiple measurement sequences for a particular potential location are particularly inefficient when that particular location does not reflect a true particle location but a tampering situation. In contrast, the proposed solution allows a clear distinction between verification and tampering situations.

[0014] Different light patterns may result in similar ambiguities regarding potential particle positions to be distinguished. The solution of the present invention can be applied to any kind of light pattern that causes ambiguities due to its symmetry. This allows asymmetric test patterns to be adopted for specific situations.

[0015] Additional intensity measurements using test patterns can also be used to improve the accuracy of the localization process, in other words, the additional measurements are not limited to identifying ambiguities.

[0016] Furthermore, the additional intensity measurement based on the test pattern can be performed in less time and / or with a reduced illumination intensity of the test pattern compared to the measurement preceding the test measurement, with the only condition being that the detection signal provided by the test measurement is high enough to allow a clear distinction between correct and incorrect particle positions.

[0017] In the following, single particles are understood in a broad sense to include, for example, simple molecules, proteins, especially fluorescent molecules and proteins, as well as more complex particles such as nanoparticles, vesicles, lipid droplets, etc., but are not limited to these.

[0018] Preferably, the optical system includes an electro-optical or acousto-optical device configured to generate both the sequence of light patterns for illuminating the sample area and at least one subsequent light pattern. For example, a beam shaping unit operating according to the principle of conical diffraction can be used to sequentially shape the illumination light into light patterns with a predetermined spatial distribution. This applies both to the symmetric light pattern that forms the basis for calculating potential particle positions and to the subsequently illuminated test pattern, which has an asymmetric shape that only allows for distinguishing true particle positions from false positions. Such electro-optical or acousto-optical devices can operate at higher speeds than scanning devices that require mechanical repositioning of components such as scanning mirrors. Therefore, if the optical system includes a scanning device that is moved to scan the illumination light over the sample, it is preferable that the electro-optical or acousto-optical device, rather than a mechanical scanning device, generate all of the illumination light patterns, or at least the test pattern, if possible.

[0019] According to a preferred embodiment, the optical system is configured to form a light distribution from the illumination light at the sample area and to move the light distribution to generate a sequence of light patterns at the sample area. Fast electro-optical movement of the light distribution without the need for mechanical adjustment can be achieved, for example, by conical diffraction as described above.

[0020] For example, the optical system may be configured to sequentially shift the light distribution laterally to two different shift positions relative to the light propagation direction and / or sequentially rotate the light distribution about a central axis parallel to the light propagation direction to different rotational positions to generate a sequence of light patterns within the sample region.

[0021] The light distribution can be spiral or doughnut shaped, which can be achieved for example in localization using the MINFLUX approach.

[0022] Preferably, the light distribution comprises at least two intensity maxima symmetrically opposite from a central axis of symmetry extending between said intensity maxima, and an intensity minimum along the central axis of symmetry, said intensity minimum preferably being 0. Such a configuration results in at least two potential particle positions that can be reliably distinguished between true and false when applying the asymmetric illumination test pattern proposed herein.

[0023] The processor may be configured to determine, based on the sequence of intensities of the detected light, a spatial probability distribution of a plurality of extrema representing a plurality of potential positions for locating the particle within the sample region, which may be used in an efficient manner to first examine a set of potential particle positions, which are then classified as correct or incorrect by the asymmetric test pattern.

[0024] Preferably, the processor may be configured to determine the spatial probability distribution from a pre-calculated table depending on the sequence of detected intensities, which allows a particularly fast and accurate determination of the particle position.

[0025] The processor may be configured to determine a line of symmetry along which the plurality of potential locations lie based on the spatial probability distribution, and to define the spatial distribution of the subsequent light pattern to be asymmetric with respect to the line of symmetry. By referencing such a line of symmetry, it becomes easy and efficient to find a suitable asymmetric light distribution for the test pattern.

[0026] According to a preferred embodiment, the processor is configured to estimate subsequent intensities of the detected light based on the sequence of detected intensities prior to illuminating the sample area with a subsequent light pattern, and to compare the estimated subsequent intensities with the detected subsequent intensities to determine the actual position of the particle within the sample area. For example, estimated intensity values ​​may be pre-calculated for each potential particle position. The estimated intensity values ​​can then be readily compared to detected light intensities measured in response to illumination with subsequent light patterns forming the test pattern.

[0027] Preferably, the detector is configured to count single photons to detect both the sequence and subsequent intensities of the detected light. The use of a single photon counter allows for a highly accurate measurement of the detected light intensity.

[0028] According to another aspect, a method for locating a single particle within a sample region is provided, the method comprising: illuminating the sample region with a sequence of light patterns having spatially distinct distributions of illumination light adapted to cause particles located within the sample region to emit detection light; detecting a sequence of intensities of detection light emerging from the sample region in response to the sequence of illumination light patterns; and determining an arrangement of potential locations for locating the particle within the sample region based on the sequence of intensities of detection light. The sample region is illuminated with at least one subsequent light pattern having a spatial distribution of illumination light that is asymmetric with respect to the arrangement of the plurality of potential locations. In response to the at least one subsequent light pattern, at least one subsequent intensity of detection light emerging from the sample region is detected. A determination is made based on the at least one subsequent intensity of detection light that represents an actual location of the particle within the sample region.

[0029] Preferably, the sample volume is prepared to satisfy the single particle localization condition, where only one particle is located within the sample volume, which defines a singularization that ensures that the average distance between particles contained in the sample is greater than the diffraction-limited spatial resolution of the optical system.

[0030] The single particle localization condition can be met, for example, by selecting a particle concentration that is low enough to achieve the intended specialization.

[0031] Alternatively, the single particle localization condition can be met by photoactivation or photodeactivation of particles to keep the majority of particles in a non-emissive state.

[0032] Specific embodiments will be described below with reference to the drawings. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a block diagram illustrating a single particle localization microscope according to one embodiment. [Figure 2] FIG. 10 illustrates a sequence of three intensity measurements based on light patterns with different spatial distributions for localizing a single fluorescent emitter, according to one embodiment. [Figure 3] FIG. 3 illustrates the symmetry of the three light patterns shown in FIG. 2. [Figure 4] FIG. 10 illustrates a spatial probability distribution showing multiple potential emitter locations arranged along a line of symmetry. [Figure 5] FIG. 10 illustrates a fourth intensity measurement based on a light pattern having an asymmetric spatial distribution for multiple potential emitter positions, according to one embodiment. [Figure 6] FIG. 10 illustrates the determination of the correct emitter position based on a fourth measurement. [Figure 7] FIG. 1 is a flow diagram illustrating a method for localizing a single fluorescent emitter according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0034] FIG. 1 is a block diagram illustrating a single particle localization microscope 100 according to one embodiment. The microscope 100 can be realized as, but is not limited to, a laser scanning confocal microscope (LSCM)-based configuration. FIG. 1 illustrates only those components of the microscope 100 that are useful for understanding the mode of operation of the solution presented herein. Of course, the microscope 100 can include additional components not explicitly shown in the block diagram of FIG. 1. For example, although not shown in FIG. 1, the microscope 100 can include optical lenses, such as a tube lens and an objective lens, used for both illumination and detection, as well as a pinhole that serves to reduce the amount of out-of-focus light in image formation according to a confocal setup.

[0035] The microscope 100 includes an optical system 102 that illuminates a sample 104 disposed on a sample carrier 106. The optical system 102 includes a laser light source 108 configured to generate illumination light 110 in a wavelength range adapted to excite fluorophores contained in the sample 104 and cause them to emit fluorescent radiation as detection light 112. The optical system 102 may include a dichroic beam splitter 114 configured to reflect the illumination light 110 onto the sample 104 and transmit the detection light 112 toward a detector 116. The optical system 102 may further include a scanning device 118, such as a movable mirror assembly, for scanning the illumination light 110 over the sample 104, thereby enabling individual points within the sample 104 to be scanned and imaged. Each point corresponds to a specific sample region 120, which includes a single fluorescent emitter 122 that coincides with the focal region of the optical system 102 and is located by the microscope 100. Because the detected light 112 is returned to the scanning device 118 before being captured by the detector 116, the embodiment shown in FIG. 1 is a so-called descanned configuration.

[0036] The optical system 102 is configured to illuminate the sample area 120 with a sequence of light patterns 124 having spatially distinct light distributions. To this end, the optical system 102 may comprise an electro-optical or acousto-optical device configured to perform sequential beam shaping of the illumination light 110 emitted from the light source 108. According to the specific embodiment shown in FIG. 1, the optical system 102 comprises a beam shaping unit 126 that operates according to the principle of conical diffraction to sequentially shape the illumination light 110 into predetermined light patterns, as described, for example, in the above-mentioned publication by Carot et al. Thus, the beam shaping unit 126 can include a biaxial crystal, a polarization state generator (PSG), and a polarization state analyzer (PSA). The PSG consists of a linear polarizer followed by a pair of Pockels cells, which are arranged at the entrance 128 and exit 130 of the biaxial crystal, respectively. The PSA is similarly arranged in reverse order. All Pockels cells are electronically controlled, making it possible to control the polarization without moving any mechanical parts in the beam shaping unit 126. The optical setup is adapted to switch from one light pattern to another light with a different light distribution in a very short time. It should be noted in particular that the switching time of the beam shaping unit 126 is several orders of magnitude shorter than the time it takes, for example, for the scanning device 118 to affect the illumination light 110 in a corresponding manner by its mechanical movement.

[0037] The microscope 100 further includes a processor 132 that may be used to control microscope operation as a whole. According to this embodiment, the processor 132 specifically controls the light source 108, the beam shaping unit 126, the scanning device 118, and the detector 116, as described below, to enable the single emitter 122 to be unambiguously located within the sample region 120.

[0038] In particular, the processor 132 can cause the beam shaping unit 126 to illuminate the sample area 120 with a sequence of light patterns having different topologies as shown in FIG. 2. According to this embodiment, a first light pattern 124-1, a second light pattern 124-2, and a third light pattern 124-3 are sequentially generated, as shown from top to bottom in FIG. 2. When the sample area 120 is sequentially illuminated with the light patterns 124-1, 124-2, and 124-3, the detector 116 captures the detected light 112 emerging from the sample area 120 with a corresponding sequence of light intensities I1, I2, and I3. For this purpose, the detector 116 can be configured to count single photons when detecting the intensity sequence I1, I2, and I3. Thus, one sequence of three intensity measurements for detecting intensities I1, I2, and I3 results in triple integers (m1, m2, m3) representing the respective photon counts.

[0039] 2, each of the three light patterns 124-1, 124-2, and 124-3 is generated as a light distribution formed from a double peak. More specifically, each light pattern 124-1, 124-2, and 124-3 includes a first intensity maximum (peak) 236-1, 236-2, and 236-3 and a second intensity maximum (peak) 238-1, 238-2, and 238-3, the first and second intensity maxima being symmetrically opposed from an intensity minimum 240-1, 240-2, and 240-3 that forms a center of symmetry for the respective light pattern 124-1, 124-2, and 124-3. The intensity minimums 240-1, 240-2, and 240-3 are preferably zero. 2, the centers of symmetry of the three light patterns 124-1, 124-2, and 124-3 given by the intensity minima 240-1, 240-2, and 240-3 coincide with each other, in other words, the centers of symmetry do not change when the light patterns 124-1, 124-2, and 124-3 are switched from one pattern to another in the sequence of three intensity measurements.

[0040] It should be noted that Figure 2 is a plan view illustrating each light pattern 124-1, 124-2, 124-3 in a cross section perpendicular to the direction of propagation of the illumination light 110. Accordingly, the intensity minima 240-1, 240-2, 240-3 of each light pattern 124-1, 124-2, 124-3 define an axis of symmetry extending along the light propagation direction. It should further be noted that Figure 2 is a simplified diagram illustrating the spatial variation of the light intensity of each light pattern 124-1, 124-2, 124-3. Thus, although FIG. 2 shows each light pattern 124-1, 124-2, 124-3 with its two intensity maxima 236-1 / 238-1, 236-2 / 238-2, 236-3 / 238-3 sharply separated from the intensity minimum between them, each intensity peak is actually characterized by a continuous decrease in intensity from its maximum to its minimum at the respective symmetry center 240-1, 240-2, 240-3.

[0041] Overall, the process of generating the sequence of light patterns 124-1, 124-2, 124-3 by the beam shaping unit 126 can be viewed as providing a structured light distribution and shifting this light distribution within the sample region 120 without changing its shape. According to the embodiment shown in FIG. 2 , the structured light distribution formed by two peaks and an intensity minimum between them is rotated as a whole by an angle of 120° around a center of symmetry coinciding with the intensity minimum. However, such a configuration should be understood as an example only. For example, a structured light distribution, such as a spiral-shaped or doughnut-shaped distribution, may be shifted as a whole in position to generate a sequence of different light patterns in the sample region 120. Furthermore, other types of structured illumination light distributions and / or different or more complex sequences may be used to generate the sequence of light patterns.

[0042] To enable localization of a single fluorescent emitter based on a sequence of light patterns, such as that shown in FIG. 2, a specificity condition must be met. Therefore, when performing the super-resolution localization process, it is necessary to ensure that only one particle is present within the sample region 120 of the optical system 102, which coincides with the focal region of the optical system 102. The specificity condition can be met by applying various methods to properly prepare the sample 104. For example, the sample 104 may be prepared by selecting an appropriate concentration of fluorescent emitters to be included in the sample 104. Alternatively, depending on the nature of the particles involved, photoactivation or photoinactivation may be applied to keep the majority of the particles in a non-emitting state. In either case, the concentration of the fluorescent emitters should be low enough to achieve a sparse emitter distribution within the sample 104, ensuring that the average distance between emitters is greater than the diffraction-limited spatial resolution of the optical system 102. When such emitter specificity is achieved, it becomes possible to locate a single emitter 122 within the sample region 120 with spatial precision much greater than the spatial dimensions of the light patterns 124-1, 124-2, 124-3 determined by the diffraction-limited spatial resolution of the optical system 102.

[0043] In the following, it is assumed that the fluorescence emitter 122 is localized within the sample area 120 as described above. Then, based on the sequence of intensities I1, I2, I3 detected in response to the sequence of light patterns 124-1, 124-2, 124-3 illuminating the sample area 120, it is possible to attempt to localize the fluorescence emitter 122 within the sample area 120. This can be done with precise knowledge of the spatial distribution of the light patterns 124-1, 124-2, 124-3, which is controlled by the beam shaping unit 126. Assuming that the intensities I1, I2, I3 are measured noise-free and that the light patterns 124-1, 124-2, 124-3 are known without tolerances, it can be concluded that the position of the fluorescence emitter 122 can be calculated with infinite precision. In other words, the localization accuracy can be considered limited only by the noise ΔI1, ΔI2, and ΔI3 that occurs when measuring the intensities I1, I2, and I3, respectively, and by imperfections in the light patterns 124-1, 124-2, and 124-3. However, as will be explained below with reference to Figures 3-6, ambiguities arise regarding the emitter positions that can be calculated based on a priori knowledge of the measured intensities I1, I2, and I3 and the light patterns 124-1, 124-2, and 124-3.

[0044] As shown in Figures 3 and 4, the symmetry of the spatial light distribution of the light patterns 124-1, 124-2, and 124-3 therefore creates an ambiguity in that multiple potential positions of the fluorescent emitter 122 are equally matched to the detected intensity triple (I1, I2, I3). According to this example, each light pattern 124-1, 124-2, and 124-3 exhibits mirror symmetry with respect to an axis M extending between two peaks, i.e., the intensity maxima 236-1 / 238-1, 236-2 / 238-2, and 236-3 / 238-3, of the light pattern (see Figure 2). For simplicity, the mirror axis M is shown only for the first light pattern 124-1 at the top of Figure 3. The mirror symmetry of each light pattern 124-1, 124-2, and 124-3 results in a line of symmetry 342 defined by two points, as shown in Figure 3. The first of these points is given by the center of symmetry of the light patterns 124-1, 124-2, 124-3, i.e., the intensity minimum 240-1, 240-2, 240-3 between two peaks of the light patterns, and the second point is given by the true position of the fluorescent emitter 122.

[0045] FIG. 4 illustrates a map representing a spatial probability distribution 444 calculated by processor 132 based on intensities I1±ΔI1, I2±ΔI2, and I3±ΔI3, taking into account measurement errors ΔI1, ΔI2, and ΔI3 and the spatial distribution of the previously known light patterns 124-1, 124-2, and 124-3. Spatial probability distribution 444 includes multiple regions representing potential emitter locations that fit the triple measurements (I1±ΔI1, I2±ΔI2, I3±ΔI3). As can be seen from FIG. 4, there are two potential emitter locations 446a and 446b, which are located on a line of symmetry 342 symmetrically opposite the aforementioned center of symmetry (first point), indicated by C in FIG. 4. Thus, potential emitter locations 446a and 446b are mirror-symmetric with respect to center C of line of symmetry 342. The mirror symmetry of potential emitter locations 446a, 446b reflects the mirror symmetry of light patterns 124-1, 124-2, 124-3. In the example of Figure 4, two potential emitter locations 446a, 446b are assumed to be located within an Airy disk 450.

[0046] 4, there are two additional emitter locations 448a, 448b located outside of Airy disk 450 on line of symmetry 342 that are symmetrically opposite from that aforementioned center of symmetry. Potential emitter locations 448a, 448b are also mirror symmetric about center C of line of symmetry 342, and the mirror symmetry of potential emitter locations 448a, 448b similarly reflects the mirror symmetry of light patterns 124-1, 124-2, 124-3.

[0047] Thus, according to the example shown in FIG. 4, two pairs of potential emitter positions 446a / 446b and 448a / 448b are calculated, with each pair of emitter positions corresponding equally to the measured intensity triple (I1±ΔI1, I2±ΔI2, I3±ΔI3). Depending on the specific circumstances of the experiment, it may be possible to determine which of the two position pairs 446a / 446b and 448a / 448b contains the actual position of emitter 122. For example, suppression of detected light by a pinhole in the detection path can be used to rule out pair 448a / 448b as the correct position in the example of FIG. 4. Note that, as will become clear below, it is not even necessary to distinguish between position pairs 446a / 446b and 448a / 448b.

[0048] In either case, it is not possible to determine which of the potential locations 446a, 446b, 448a, 448b represents the actual emitter location based solely on spatial probability distribution 444. Therefore, due to the symmetry of light patterns 124-1, 124-2, and 124-3, the measured intensity triple (I1 ± ΔI1, I2 ± ΔI2, I3 ± ΔI3) is not sufficient to unambiguously identify the location of emitter 122 within sample region 120.

[0049] To solve the problems described above, it is proposed herein to illuminate the sample area 104 with at least one subsequent light pattern that is used as a test pattern for performing final measurements to resolve ambiguities regarding multiple potential emitter locations. More specifically, the test pattern is generated with a predetermined spatial distribution that is asymmetric with respect to the arrangement of potential locations 446a, 446b, 448a, 448b previously calculated based on the intensity triples (I1±ΔI1, I2±ΔI2, I3±ΔI3). Subsequent detected light intensities are detected in response to the test pattern and can be used to determine which of the multiple potential locations 446a, 446b, 448a, 448b is the correct emitter location.

[0050] According to this embodiment, the shape and / or orientation of the subsequent light pattern may be determined according to the estimated probability distribution 444 shown in FIG. 4. Preferably, an asymmetric light pattern is used that maximizes the difference in detected light intensity between the correct emitter position and the incorrect position. The lower part of FIG. 5 illustrates a suitable asymmetric light pattern 524 following the example shown in FIGS. 2-4. As can be seen from FIG. 5, the subsequent light pattern 524 has a significantly different spatial light distribution than the previous light patterns 124-1, 124-2, and 124-3. For example, the light pattern 524 has only one intensity maximum (peak) 538, which is laterally displaced from the line of symmetry 342 along which all of the potential emitter positions 446a, 446b, 448a, and 448b lie. This ensures that the spatial distribution of the light pattern 524 is asymmetric with respect to the overall arrangement of the previously calculated emitter positions 446a, 446b, 448a, and 448b. In this regard, it should also be noted that Figure 5 is a simplified illustration of the spatial intensity variation of light pattern 524. Similar to light patterns 124-1, 124-2, 124-3, the intensity of light pattern 524 is actually characterized by a continuous decrease in intensity from its maximum value to a minimum value, which is preferably zero.

[0051] 5, the subsequent light pattern 524 has its intensity maximum located near the fluorescent emitter 122, i.e., near the location 466a that was found to be the correct emitter location. Therefore, the subsequent (fourth) intensity I4±ΔI4 measured by the detector 116 will be relatively high. Based on the subsequent intensity I4±ΔI4, the processor 132 can determine that the emitter location 446a is correct, while the other locations, such as 446b, 448a, and 448b, are incorrect.

[0052] To make the appropriate determination, the processor 132 can be configured to estimate the intensity of the subsequent light intensity I4±ΔI4 detected in response to illumination of the sample area 120 with the subsequent light pattern 524 based on the intensity triple (I1±ΔI1, I2±ΔI2, I3±ΔI3) previously measured before illuminating the sample area 120 with the light pattern 524. For example, in this embodiment, the processor 132 can calculate estimated intensity values ​​that would be expected to be detected if the fluorescent emitter 122 were located at potential location 446a or any of the other potential locations 446b, 448a, 448b. By comparing these estimated intensity values ​​to the intensities I4±ΔI4 actually measured in response to the light pattern 524, the processor 132 can determine which of the potential locations is the correct emitter location. In the example shown in FIGS. 2-6, the estimated intensity value for location 446a is significantly higher than the estimated intensity values ​​for the other locations 446b, 448a, 448b. The actually measured intensity I4±ΔI4 corresponds to a relatively high estimated intensity value, resulting in location 446a being recognized as the correct emitter location. In other words, the other potential emitter locations 446b, 448a, and 448b can be reliably eliminated as erroneous locations, as indicated by the cross symbols in FIG. 6.

[0053] FIG. 7 is a flow diagram illustrating a method for locating a fluorescence emitter 122 using a microscope 100 according to one embodiment.

[0054] The method begins with step S1, in which the sample 104 is prepared to satisfy the above-mentioned characterization conditions, thus ensuring that only one particle is located within the sample area 120 to be examined.

[0055] In step S2, the processor 132 controls the light source 108 and the scanning device 118 to direct the illumination light 110 to a specific point on the sample 104. This point coincides with the focal region of the optical system 102 and forms a sample region 120 containing the differentiated emitters 122 to be located.

[0056] In steps S3 to S5, the processor 132 controls the beam shaping unit 126 to generate a sequence of light patterns 124-1, 124-2, 124-3 having spatially different light distributions, as shown as an example in FIGS.

[0057] More specifically, in step S3, a first measurement is performed by illuminating sample area 120 with a first double-peak light pattern 124-1. In response, detector 116, configured as a single-photon counter, detects a first number m1 of photons emitted from fluorescence emitter 122 illuminated with light pattern 124-1. According to FIG. 2, it is assumed that fluorescence emitter 122 is located at the periphery of the peak formed by intensity maximum 236-1 of light pattern 124-1, and that an exemplary number of 13 photons is counted at intensity I1 (i.e., m1 = I1 = 13).

[0058] Subsequently, in step S4, a second measurement is performed by illuminating the sample area 120 with the second double-peak light pattern 124-2. In response, the detector 116 detects a second number of photons m emitted from the fluorescence emitter 122 illuminated with the second light pattern 124-2. According to Figures 2 and 3, the fluorescence emitter 122 is assumed to be located slightly closer to one of the symmetric intensity maxima formed by the second light pattern 124-2 compared to the first measurement, and as a result, a larger number of photons, such as 19, is counted as the intensity I (i.e., m = I = 19).

[0059] Subsequently, in step S5, a third measurement is performed by illuminating the sample area 120 with a third double-peak light pattern 124-3. In response, the detector 116 detects a third number m3 of photons emitted from the fluorescence emitter 122 illuminated with the third light pattern 124-3. According to Figures 2 and 3, the fluorescence emitter 122 is assumed to be located farther from the maximum of the two peaks formed by the third light pattern 124-3 than in the first and second measurements. Therefore, a smaller number of photons, such as 1, is assumed to be counted (i.e., m3 = I3 = 1).

[0060] In step S6, processor 132 can determine a spatial probability distribution 444 from the triple intensity (m1, m2, m3), which in this example is equal to (13, 19, 1). Spatial probability distribution 444 exhibits multiple extrema representing potential emitter locations 446a, 446b, 448a, 448b, as shown in FIG. 4. When calculating spatial probability distribution 444, processor 132 takes into account the specific spatial distributions of light patterns 124-1, 124-2, 124-4, which are known in advance. For example, spatial probability distribution 444 can be determined based on a precalculated table.

[0061] In step S7, processor 132 may determine, based on spatial probability distribution 444, a line of symmetry 342 along which all potential emitter positions 446a, 446b, 448a, 448b lie, as shown in Figure 4. Processor 132 may then define an appropriate illumination light distribution that is asymmetric with respect to the arrangement of potential emitter positions 446a, 446b, 448a, 448b along line of symmetry 342.

[0062] In step S8, processor 132 may estimate the expected detected light intensity in response to illuminating sample area 120 with the subsequent fourth light pattern, the asymmetric light distribution being determined in step S7. Specifically, processor 132 may calculate a plurality of estimated intensity values ​​in the form of photon counts associated with a plurality of potential locations determined in step S6.

[0063] In step S9, processor 132 causes beam shaping unit 126 to form an asymmetric light distribution from illumination light 110 determined in step S7 to perform a fourth intensity measurement. Sample area 120 is illuminated with light pattern 524, as shown in the lower part of Figure 5. In response, detector 116 detects a fourth number of photons m4 emitted from fluorescence emitter 122 illuminated with light pattern 524.

[0064] In step S10, processor 132 compares photon number m4 with the estimated photon number calculated in step S8. In this example, photon number m4 is assumed to be close to the photon number previously estimated for emitter position 446a. In contrast, m4 differs significantly from the photon numbers estimated for the other potential positions 446b, 448a, and 448b. As a result, processor 132 determines that fluorescence emitter 122 is located at position 446a.

[0065] It should be noted that subsequent intensity measurements, as described above, to resolve ambiguities regarding potential emitter locations can be performed very quickly. Thus, a test pattern, such as light pattern 524 shown in FIG. 5, can be generated by beam shaping unit 126, which can be realized by, but is not limited to, an electro-optical or acousto-optical device. Such a device does not require any physical movement of mechanical parts, unlike other types of beam-influencing components, such as scanning device 118 shown in FIG. 1. Furthermore, because the light distribution of the test pattern is asymmetric with respect to the arrangement of potential emitter locations, it may be sufficient to perform only one additional test measurement to resolve the ambiguity, rather than repeating the complete sequence of measurements for one of the potential emitter locations.

[0066] The ambiguity check proposed herein is not limited to the above-described embodiment. For example, the light patterns 124-1, 124-2, 124-3 and the subsequent light pattern 524 shown in FIG. 5 are generated by applying conical diffraction. Furthermore, the light patterns are generated in the form of structured light distributions that include two intensity peaks symmetrically opposed to an intensity minimum located at the center of the pattern. However, other types of light distributions may also be realized, such as shifted spiral or doughnut-shaped patterns, as realized in MINFLUX.

[0067] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0068] While some aspects have been described in the context of an apparatus, it will be apparent that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a step or feature of a step, and similarly, aspects described in the context of a step also represent a description of a corresponding block or item or feature of a corresponding apparatus. [Explanation of symbols]

[0069] 100 Single Particle Localization Microscopy 102 Optical System 104 samples 106 Sample Carrier 108 Light source 110 Illumination 112 Detection light 114 Dichroic Beam Splitter 116 detector 118 Scanning Device 120 Sample Area 122 Fluorescent Emitter 124 Light Pattern Sequences 124-1 Light Pattern 124-2 Light Pattern 124-3 Light Pattern 126 Beam shaping unit 128 Entrance to biaxial crystals 130 Biaxial Crystal Exit 132 processors 236-1 Maximum intensity of light pattern 236-2 Maximum intensity of light pattern 236-3 Maximum intensity of light pattern 238-1 Maximum intensity of light pattern 238-2 Maximum intensity of light pattern 238-3 Maximum intensity of light pattern 240-1 Light pattern intensity minimum 240-2 Light pattern intensity minimum 240-3 Minimum intensity of light pattern 342 Line of Symmetry 444 Spatial Probability Distribution 446a Potential location 446b Potential location 448a Potential location 448b Potential location 450 Airy Disc 524 trailing light patterns

Claims

1. A single particle localization microscope (100), comprising: an optical system (102) configured to illuminate a sample region (120) with a sequence of light patterns (124-1, 124-2, 124-3) having spatially distinct distributions of illumination light (110) adapted to cause single particles (122) located within the sample region (120) to emit detection light (112); a detector (116) configured to detect a sequence (I1, I2, I3) of intensities of detection light (112) emerging from said sample region (120) in response to a sequence (124-1, 124-2, 124-3) of illumination light patterns; a processor (132) configured to determine an arrangement of potential locations (446a, 446b, 448a, 448b) for locating a particle (122) within the sample region (120) based on the sequence (I1, I2, I3) of intensities of the detected light (112); Equipped with The processor (132) causing the optical system (102) to illuminate the sample area (120) with at least one subsequent light pattern (524) having a spatial distribution of illumination light (110) that is asymmetric with respect to the arrangement of the plurality of potential positions (446 a, 446 b, 448 a, 448 b); causing the detector (116) to detect at least one subsequent intensity (I4) of detected light (112) emerging from the sample region (120) in response to the at least one subsequent light pattern (524); determining which of the plurality of potential locations (446a, 446b, 448a, 448b) represents an actual location of a particle (122) within the sample region (120) based on at least one subsequent intensity (14) of the detected light (112); and further configured to: Single particle localization microscope (100).

2. the optical system (102) comprises an electro-optical or acousto-optical device (126) configured to generate both the sequence of light patterns (124-1, 124-2, 124-3) and the at least one subsequent light pattern (524) for illuminating the sample area (120); The single particle localization microscope (100) of claim 1.

3. the optical system (102) is configured to form a light distribution from the illumination light (110) in the sample area (120) and to move the light distribution to generate the sequence of light patterns (124-1, 124-2, 124-3; 524) in the sample area (120); The single particle localization microscope (100) according to claim 1 or 2.

4. the optical system (102) is configured to sequentially shift the light distribution laterally to different shift positions relative to a light propagation direction and / or sequentially rotate the light distribution about a central axis parallel to the light propagation direction to different rotation positions in order to generate the sequence of light patterns (124-1, 124-2, 124-3) within the sample region (120). The single particle localization microscope (100) of claim 3.

5. The light distribution is spiral or doughnut shaped. The single particle localization microscope (100) of claim 3.

6. said light distribution comprises at least two intensity maxima (236-1, 238-1; 236-2, 238-2; 236-3, 238-3) symmetrically opposite from a central axis of symmetry extending between said intensity maxima (236-1, 238-1; 236-2, 238-2; 236-3, 238-3), and intensity minima (240-1, 240-2, 240-3) along said central axis of symmetry, said intensity minima (240-1, 240-2, 240-3) preferably being zero; The single particle localization microscope (100) of claim 3.

7. the processor (132) is configured to determine, based on the sequence (I1, I2, I3) of intensities of the detected light (112), a spatial probability distribution (444) having a plurality of extrema representing a plurality of potential positions (446a, 446b, 448a, 448b) for localizing the particle (122) within the sample region (120). The single particle localization microscope (100) according to claim 1 or 2.

8. the processor (132) is configured to determine the spatial probability distribution (444) from a pre-calculated table in response to the detected sequence of intensities (I1, I2, I3). The single particle localization microscope (100) of claim 7.

9. the processor (132) is configured to determine a line of symmetry (342) that includes at least two of the plurality of potential locations (446 a, 446 b, 448 a, 448 b) based on the spatial probability distribution (444), and define a spatial distribution of the subsequent light pattern (524) to be asymmetric with respect to the line of symmetry (342). The single particle localization microscope (100) of claim 7.

10. the processor (132) is configured to estimate a subsequent intensity (I4) of the detected light (112) based on the detected intensities sequence (I1, I2, I3) before illuminating the sample area (120) with the subsequent light pattern (524), and compare the estimated subsequent intensity with the detected subsequent intensity (I4) to determine an actual position of the particle (122) within the sample area (120). The single particle localization microscope (100) according to claim 1 or 2.

11. the detector (116) is configured to count single photons to detect both the sequence of intensities (I1, I2, I3) of the detected light (112) and the subsequent intensity (I4); The single particle localization microscope (100) according to claim 1 or 2.

12. 1. A method for localizing a single particle (122) within a sample region (120), the method comprising: illuminating the sample area (120) with a sequence of light patterns (124-1, 124-2, 124-3) having spatially distinct distributions of illumination light (110) adapted to cause particles (122) located within the sample area (120) to emit detection light (112); detecting a sequence (I1, I2, I3) of intensities of detection light (112) emerging from said sample area (120) in response to a sequence (124-1, 124-2, 124-3) of illumination light patterns; determining an arrangement of potential locations (446a, 446b, 448a, 448b) for locating a particle (122) within the sample region (120) based on the sequence (I1, I2, I3) of intensities of the detected light (112); Including, the sample area (120) is illuminated with at least one subsequent light pattern (524) having a spatial distribution of illumination light (110) that is asymmetric with respect to the arrangement of the plurality of potential locations (446 a, 446 b, 448 a, 448 b); detecting at least one subsequent intensity (I4) of the detection light (112) emerging from the sample area (120) in response to the at least one subsequent light pattern (524); determining, based on at least one subsequent intensity (I4) of the detected light (112), which of the plurality of potential locations (446a, 446b, 448a, 448b) represents an actual location of the particle (122) within the sample region (120); method.

13. The sample region (120) is prepared to satisfy a single particle localization condition in which only one particle (122) is located within the sample region (120).

13. The method of claim 12.

14. The single particle localization condition is met by selecting an appropriate particle concentration.

14. The method of claim 13.

15. the single particle localization condition is satisfied by photoactivation or photoinactivation of particles to keep the majority of particles in a non-emissive state; 14. The method of claim 13.

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