Method and apparatus for recording nanoscale images of a multiply stained sample

Abstract

Positioning microscopic investigations of samples dyed with a plurality of dyes are disclosed. In the first case, excitation light of two different wavelengths (111, 112) is provided for exciting a first class of excitable isolated fluorophores (54), and the position (114) of the fluorophores (54) is obtained for each of the two wavelengths, or in the second case, a test excitation is first carried out in order to subsequently select (33) the wavelength of the excitation light used to position the isolated fluorophores. In the first case, the difference (61) of the positions obtained in this way of one or, preferably, more individual fluorophores is determined, and this difference is used to obtain the positions of the fluorophores (54, 55) of the other class and the first class in a common spatial reference system. The second case is advantageously applied to tracking structures. The advantages of the first case can also be achieved in the second case.

Description

[0001] Invention Technology This invention relates to localization microscopy, particularly MINFLUX microscopy. This invention enables improved nanoscale imaging of multiplexed samples (i.e., stained with multiple different fluorescent markers) and the tracking of individual molecules (i.e., fluorophores) within multiplexed samples.

[0002] Existing technology Positional microscopy allows for imaging of samples at resolutions below the Abbe diffraction limit. To image a sample, it is stained with markers that behave in a way that allows fluorescence for at least a certain period of time each time; that is, the markers are in a state where they can be excited to fluoresce for at least a certain time, and do not fluoresce for most of the time. This is often referred to as marker scintillation. For example, this could be due to the overall ensemble of markers being in equilibrium, with a small subset of markers being fluorescent and a large subset being non-fluorescent. The sample is prepared or processed in such a way that although some or even a large number of markers are evenly distributed across the sample and are fluorescent, the distance between the fluorescent markers is typically approximately equal to or greater than, for example, the diameter of the excitation spot, which is diffraction-limited. If excitation light is now applied to the sample, the light from each individual fluorescent fluorophore can be detected in a spatially resolved manner to obtain an image for each individual fluorophore, whose position can be determined with an uncertainty much smaller than the Abbe diffraction limit. Alternatively, an excitation beam, for example, having a series of structurally distributed excitation beams, can be applied to the sample. This allows the location of fluorescent markers to be determined based on the knowledge of the excitation and the detected fluorescence, with an uncertainty much smaller than the Abbe diffraction limit. Now, the locations of a large number of markers are determined chronologically, where multiple markers have been detected simultaneously using localization microscopy in a wide field of view or in parallel localization microscopy methods. Finally, an image of the sample is obtained from the set of locations determined in this way.

[0003] Various problems arise when attempting to obtain images of samples stained with different types of markers (e.g., for marking different structures) using localization microscopy. Even if each chosen marker category is suitable for localization microscopy, this often doesn't apply to paired categories or entire ensembles of markers. This is because if conditions are set to allow one marker category to achieve a suitable balance, under the same conditions, multiple or additional marker categories may either fluoresce too little, not at all, or fluoresce too frequently. Furthermore, the imaging conditions (i.e., wavelengths) for different colors are always slightly different, thus introducing uncertainty regarding the co-localization of different marker categories.

[0004] When the term colocalization is used in publications referring to data from non-super-resolution microscopy methods (e.g., simplified fluorescence microscopy or light-sheet fluorescence microscopy), it should generally be understood as the signals of colocalized fluorophores being indivisible in the image, i.e., these signals appear within the same image pixel (e.g., see "New Methods to Evaluate Colocalization of Fluorophores in Immunocytochemical Preparations as Exemplified by a Study on A2A and D2 Receptors in Chinese Hamster Ovary Cells", Luigi F. Agnati et al., J Histochem Cytochem 53:941-953, 2005). Regarding localization microscopy methods, the term is understood more broadly in the sense that colocalization of different types of fluorophores determines the spatial relationships in which different types of fluorophores are arranged. This also includes determining that the spatially inseparable positions of different types of fluorophores in the image.

[0005] The article “A guided tour into subcellular colocalization analysis in light microscopy,” referred to as a “tutorial review,” by S. Bolte and FP Cordelieres, *Journal of Microscopy*, Vol. 224, Pt 3, pp. 213-232 (2006), emphasizes that it is essential to avoid the fluorescence of one fluorophore class generating a signal in a detector channel associated with another fluorophore class; that is, crosstalk between detector channels should be avoided. Furthermore, excitation light intended to excite one fluorophore class should also be avoided to excite another fluorophore class; that is, crosstalk or crosstalk in fluorescence excitation should be avoided. In addition, it is recommended to perform image recording on different types of fluorophores sequentially rather than simultaneously. Both crosstalk and crosstalk between detector channels lead to mixing of spectral channels. If adverse interference occurs, methods for spectral separation can be applied. However, in principle, mixing of spectral channels is considered a form of interference.

[0006] The publication “Ultrahigh-Resolution Colocalization of Spectrally Separable Point-like Fluorescent Probes”, Xavier Michalet et al., METHODS 25, 87-102 (2001), https: / / doi.org / 10.1006 / meth.2001.1218, demonstrates a method for colocalizing different quasi-point fluorescent emitters. The emitters are excited using the same excitation source or wavelength in a confocal scanning array. In one of the arrays used, the detection path is divided into two spectral channels by means of a spectral beamsplitter, where detection in each of these channels is performed using a photon-counting detector. In the other array, prisms and cameras are used for spectral analysis of the detection. By ensuring that only a single point emitter exists within the resolution width of each spectral channel, the position of each emitter can be determined for each spectral channel with accuracy far exceeding the resolution limit, based on the point spread function recorded in the scan. Therefore, the distances between different emitters can be determined with very high resolution, which ultimately depends particularly on the number of photons detected. In other words, fluorescent emitters that are separable in the probe light but can be excited with the same excitation wavelength can be co-localized with super-resolution. As an advantage of using only one excitation wavelength, the authors point out that chromaticity errors in the excitation path are completely eliminated.

[0007] From the publication "The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy" by Leila Nahidiazar et al., Journal of Cell Science 128, pp. 3714-3719 (2015); https: / / doi.org / 10.1242 / jcs.171892, combined with the relevant "Supplementary Information," methods for two-color and three-color localization microscopy using wide-field fluorescence microscopy are known. Samples are stained with dyes Alexa Fluor 647, Alexa Fluor 488, and, if necessary, Alexa Fluor 532. A special buffer solution, which the authors call OXEA, is used to enable localization microscopy imaging with all three dyes, specifically a method known as GSDIM (Ground-State Depletion Individual Molecular Return) microscopy. For two-color imaging, imaging is first performed using dye Alexa Fluor 647, which is excited by a laser at a wavelength of 647 nm. Then, imaging is performed using dye Alexa Fluor 488, which is excited by a laser at a wavelength of 488 nm. According to the authors, the two dyes can be recorded independently without interference. For imaging in three colors, the dye Alexa Fluor 532 was also used. Here, the steps described above are performed first, where a bandpass filter is used in the detection path when using Alexa Fluor 488 to avoid detecting the fluorescence emitted by the third dye, Alexa Fluor 532. Finally, imaging is performed using Alexa Fluor 532, which is excited by a laser with a wavelength of 532 nm. The use of this third dye takes full advantage of the fact that, in the previously mentioned steps, Alexa Fluor 488 has been bleached to a sufficiently low level of interfering fluorescence. The image data obtained in this way is post-processed separately, then corrected for any drift that occurs, and then images are generated, finally corrected for any imaging chromatic aberrations in these images. To determine the distance to structures observed in different colors, the images are evaluated using knowledge of the shape of the imaged structures. In this way, the distance to the labeled structures is determined with a lower uncertainty than the uncertainty of locating two different fluorophores individually.As is known from the subsequent publication by Leila Nahidiazar et al., “Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution”, PLoS ONE 11(7):e0158884, (2016), https: / / doi.org / 10.1371 / journal.pone.0158884, the aforementioned corrections for the drift that occur are based on cross-correlation techniques or on imaging of dedicated reference markers integrated into the sample (i.e., fluorescent beads).

[0008] Another method for dual-color localization microscopy using a wide-field fluorescence microscope is known from the publication “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes”, Hari Shroff et al., Proceedings of the National Academy of Sciences, 104(51)20308-20313; (2007), https: / / doi.org / 10.1073 / pnas.0710517105. Two different photoactivated dyes are used, both of which can be activated with light at a wavelength of 405 nm, but are excited at different wavelengths and bleached if necessary. In the first step sequence, activation light and a long-wavelength excitation light are applied, and the fluorescent molecules are localized; this process is performed until a large number of fluorophores are localized. Here, the excited dyes are bleached. Subsequently, only the short-wavelength fluorophores that are subsequently in an excitable state are activated with a strong pulse of light at 488 nm (which is the shorter excitation wavelength), so that all short-wavelength fluorophores are subsequently in an inexcitable state. Then, as with the first fluorophore category, fluorophores that can be excited with shorter wavelengths are located. It is important to emphasize that these fluorophores exhibit very little crosstalk, meaning that the imaging in different channels has minimal mutual interference.

[0009] A method for localization microscopy of double-stained samples is known from the publication “A user-friendly two-color super-resolution localization microscope,” Teng Zhao et al., Opt. Express 23, 1879-1887 (2015). Samples were stained with two dyes, Alexa Fluor 647 and Alexa Fluor 750. A special buffer solution was used, adjusted to make the two dyes behave as similarly as possible in terms of switching properties important for localization microscopy. In TIRF wide-field mode (TIRF: Total Internal Reflectance Microscopy), the sample was simultaneously subjected to fluorescence excitation light for both dyes. In the detection path, the fluorescence of one dye was separated from that of the other. The fluorescence signals were detected separately in different halves of the same camera sensor. Calibration was performed to avoid color-related bias at certain locations of different fluorophores. For calibration, aggregates containing both classes of fluorophores were created, with an overall size below the Abbe diffraction limit. A large number of these aggregates are placed on a cover glass, and the cover glass with the aggregates is observed under a microscope to locate the aggregates. Since the aggregates contain two types of fluorophores, the fluorescence of each aggregate is detected in two sensor halves. Based on this measurement, the detector halves are mapped onto each other. A microscope calibrated in this way can be used continuously for several weeks, producing photographs without chromatic aberration.

[0010] In the publication "Multicolor Super-resolution Imaging with Photo-switchable Fluorescent Probes", W. Mark Bates et al., Science; 317(5845):1749pp, (2007), https: / / doi.org / 10.1126 / science.1146598, an alternative method for wide-field localization microscopy of multicolor stained samples was proposed. In each case, dye pairs were used instead of simple fluorophores. Here, one participant in the dye pair (called the activator) acts as the substance that determines the activation wavelength of the dye pair, and the other participant (called the indicator) is the substance that determines the excitation and emission wavelengths of the dye pair. By using different activator-indicator pairs, different pairs can be selectively activated, thus allowing for sequential localization of different pairs, or simultaneous activation and localization can be performed in different detection channels (i.e., when the emission wavelengths of the indicators are different). It has been demonstrated, i.e., based on experimentally obtained images, that the sequential method is based on different activator-indicator pairs at the activation wavelengths. Crosstalk between color channels was investigated. It was confirmed that crosstalk was low in the activator-indicator pair used.

[0011] In the publication "Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength," Ilaria Testa et al., Biophysical Journal, Volume 99, 2686-2694, (2010), https: / / doi.org / 10.1016 / j.bpj.2010.08.012, another GSDIM method for wide-field localization microscopy with multiple colors is introduced. To excite all dyes and to quench their ground states, the same laser, specifically a 488 nm cw laser, is used. The fluorescence signals of all fluorophores are split into two color channels in the detection path and detected separately in two sub-regions of the same camera sensor. Thus, fluorescence wavelengths above the threshold are detected primarily in one part of the sensor, while fluorescence wavelengths below the threshold are detected in the other. In this way, a pair of point images is obtained for each emitter in the sample. The ratio of fluorescence intensity signals from each individual fluorophore in the two sensor halves can be used to determine which fluorophore caused a specific pair of dot images. Then, not only can a location value be assigned to each dot image, but color information can also be assigned. This method is based on the fact that it is ensured or known that each individual spot is caused by a single molecule, meaning that no signal could possibly be generated by a mixture of different fluorescence emissions.

[0012] In the publication "Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species," Mariano Bossi et al., Nano Lett, 8, 8, 2463-2468, (2008), https: / / doi.org / 10.1021 / nl801471d, an alternative method for wide-field localization microscopy in multiple colors is introduced. Different fluorescent dyes were used, all of which could be switched from a non-fluorescent state to a fluorescent state by activation light at a wavelength of 375 nm. Furthermore, all the fluorescent dyes could be excited to emit fluorescence by excitation light of the same wavelength at 532 nm. Here, the fluorophores have very different emission spectra. Now, activation and excitation are performed separately in the wide field of view, and the resulting fluorescence is detected spatially in two channels with different spectral sensitivities. The emitter type is inferred from the ratio of the fluorescence detected by a single emitter, and localization is performed based on the local distribution of fluorescence. The relevant "supplementary information" clearly states that the position coordinates of the two spectral channels are mutually assigned by means of imaging the fluorescent beads emitted in the two spectral channels.

[0013] In the publication "Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging," Graham T Dempsey et al., Nat Methods 8, 1027-1036 (2011), https: / / doi.org / 10.1038 / nmeth.1768, a method for imaging samples stained with four different fluorescent dyes using STORM is described. Four dyes with significantly different excitation and emission spectra were selectively chosen. These dyes were individually excited with associated lasers, and the associated fluorescence was detected in associated spectral channels. The selection of dyes and excitation wavelengths ensured that no cross-excitation occurred, thus minimizing interference from the fluorescence of other dyes on each image. Before recording each individual image of the sample, the color channels were registered with each other by imaging the gold beads separately in all detection channels under wide-field illumination. The authors note that the uncertainty of co-registration is less than 20 nm. Drift occurring over time during recording was corrected using image correlation methods.

[0014] In the publication "Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging," Yongdeng Zhang et al., NatMethods 17, 225-231 (2020), https: / / doi.org / 10.1038 / s41592-019-0676-4, a method for locating fluorophores in samples stained with multiple fluorescent dyes is proposed, performed on a microscope equipped with a 4-PI detector. Excitation light is guided onto the sample via a dichroic element through the objective lens. On one hand, fluorescence emitted from fluorophores in the sample is guided via the same objective to a first detector with spatial resolution, where only the portion of fluorescence with wavelengths longer than the excitation wavelength passes through the dichroic element and reaches the first detector. On the other hand, in the opposite direction, fluorescence is guided via a mirror to the same detector through another dichroic element corresponding to the first dichroic element, and reflected by the other dichroic element as light with shorter wavelengths (particularly wavelengths lower than or slightly higher than the excitation wavelength). The reflected light is then guided through another filter (which just prevents the excitation wavelength from passing through) to another detector. Fluorophores are located based on the intensity or light quantity distribution measured for each fluorophore. To distinguish fluorophores, the bursts detected simultaneously on both detectors are assigned to each other, and the fluorophore type is inferred from the ratio of the measured intensity or light quantity. This fully utilizes the fact that the ratio of short-wavelength fluorescence (below a given excitation wavelength) to long-wavelength fluorescence (above a given excitation wavelength) varies considerably between different fluorophores. Here, the proportion of short wavelengths is usually so small that good localization based on the measured intensity or light quantity distribution is almost impossible. Therefore, localization is based solely on the intensity or light distribution of long-wavelength fluorescence, while measurements of short-wavelength fluorescence are used only to identify fluorophore categories.

[0015] In the publication "Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales," Yuting Guo et al., Cell (2018), https: / / doi.org / 10.1016 / j.cell.2018.09.057, a method for high-resolution imaging using structured illumination, which is not based on the localization of individual fluorophores, is described. Images based on three different fluorescent proteins are shown, in which biological structures labeled with different fluorophores are imaged at high resolution in their spatial relationships with each other. Furthermore, the method is demonstrated to be applicable to the detection of individual fluorophores as well.

[0016] MINFLUX nanotechnology is a method of localization microscopy. The localization of fluorophores is achieved using a structured excitation light distribution. A fundamental characteristic of MINFLUX nanotechnology is that fluorophore excitation is performed such that the fluorophore to be localized is always placed near or at the minimum of the excitation light distribution, ideally zero, where the excitation light distribution exhibits an intensity increase region near the minimum. This allows for better utilization of fluorescent photons to obtain information about the location of the corresponding emitted fluorophore. This is also applicable to applications where the movement of fluorophores over time needs to be tracked. Observing samples using the excitation minimum is fundamental to MINFLUX nanotechnology, as known from patents DE 10 2011 055 367 B4 (originally used only for tracking the movement of individual molecules in a sample) and DE 10 2013 114 860 B3. DE 10 2011 055 367 B4 also explores the possibility of simultaneously tracking two different fluorophores. For this, two different light sources are used, with rapid alternation between them.

[0017] Based on this, a series of refinement schemes for acquiring information were developed, which can locate fluorophores with uncertainties in the range of less than 2 nm. The magnitude of this uncertainty corresponds to the range of the fluorophore. For a detailed introduction to MINFLUX microscopy, see "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes," Francisco Balzarotti et al. arXiv:1611.03401[physics.optics] (2016). In principle, in order to locate a fluorophore using MINFLUX microscopy, the minimum or zero intensity point is placed at multiple locations relative to the fluorophore's position. For this, the fluorophore's position must be estimated with a first, relatively low precision during the preparation steps. This can be achieved, for example, by using conventional localization microscopy (PALM, STORM) or other known methods. A method described in the aforementioned publication involves scanning the sample with a Gaussian intensity distribution until fluorescence is detected at the scanned location, which has a probability of originating from an isolated molecule. The scan is then stopped, and the intensity distribution is subsequently localized at multiple locations around the scanned location, specifically four locations, at distances less than the wavelength of light. The position of the isolated emitter is estimated from the number of photons measured at each location, according to the method described in the publication. This estimation is essentially equivalent to a ratio assessment of the number of photons detected at different locations. Then, the intensity distribution of excitation light with a central minimum (e.g., an intensity distribution in the form of a donut, as known from STED microscopy) is placed at a known location, chosen such that the fluorophore is close to the minimum of the intensity distribution. The fluorescence response of the fluorophore is measured. The same operation is repeated at one or more other locations of the intensity distribution. The location of the fluorophore is determined with greater precision by means of a ratio evaluation of the measured intensity ratio or light intensity ratio. In principle, the farther the fluorophore is from the excitation minimum or the farther it shifts towards the region of increasing intensity, the higher the emission rate. This more precisely determined location can now be used as the initial location for repeating the above sequence of steps, where multiple locations can be more densely close to the estimated fluorophore location. In particular, in tracking the movement of the fluorophore, the change in emission rate as the fluorophore shifts towards the region of increasing intensity or to the minimum can also be used to estimate the displacement of the fluorophore with high precision. The closer the location of the minimum of the intensity distribution is to the actual location of the fluorophore, the fewer fluorescent photons are required to locate it with a given uncertainty or precision. For example, the MINFLUX method can also use so-called localized hollow beams (Bottle-Beams) for excitation in three-dimensional localization.

[0018] Patent publications relating to MINFLUX microscopy, particularly WO 2018 / 069 283 A1, US 2019 / 0235220 A1, US 2019 / 0234882 A1, and US 2019 / 0234879 A1, in which the U.S. patent applications mentioned are all subsequent applications of the aforementioned provisional international patent applications, and also disclose all the concepts subsequently referred to in the U.S. publications.

[0019] US 2019 / 0235220 A1 relates to a method of using a small or minimal number of locations to place minimum intensity values ​​at these locations in order to determine the location of a fluorophore, the minimum intensity values ​​being adjacent to the sides of an intensity-increasing region in each spatial direction in which the location of the fluorophore should be determined.

[0020] US 2019 / 0234882 A1 relates to the method further described above, wherein positional information obtained from the first MINFLUX step is used to place the minimum values ​​of the intensity light distribution closer to the fluorophore in subsequent steps, thereby deriving more accurate positional information.

[0021] US 2019 / 0234879 A1 discloses a method in which a minimum intensity is very rapidly and almost simultaneously placed at multiple locations around an estimated location of a fluorophore. Then, if an increase in emission rate is confirmed at a single location, that location is moved closer to the predicted minimum.

[0022] The publication “MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution,” Yvan Eilers et al., Proceedings of the National Academy of Sciences, 115(24) 6117-612, (2018); https: / / doi.org / 10.1073 / pnas.1801672115, specifically describes how MINFLUX can be used to track the motion of individual molecules with a motion resolution in the nanometer range. The outlook indicates that multicolor applications of MINFLUX technology are foreseeable, but no specific details are given.

[0023] The publication “MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells,” Klaus C. Gwosch et al., Nat Methods 17, 217-224 (2020), https: / / doi.org / 10.1038 / s41592-019-0688-0, relates to three-dimensional nanoscopy using a variety of different fluorophores. The publication also demonstrates that, compared to ratiometric localization with a fixed minimum position, a reduction in localization uncertainty is achieved by iteratively approximating the actual position of the relevant fluorophore to the minimum position. Furthermore, it is shown that three-dimensional isotropic resolution is achieved in this manner. It has been confirmed that MINFLUX nanoscopy can also be performed using photoactivated fluorescent proteins (specifically mMaple), particularly on living cells. To image using different fluorophores, dyes with large overlap in their spectral absorption curves and thus excitation wavelengths that can be used in each case are employed. Specifically, the three-dimensional nanotechnology was presented using two different fluorophores, with a total of two different pairs (A: CF660C and Alexa Fluor 647; B: CF680C and Alexa Fluor 647), where one participant in each pair was the dye Alexa Fluor 647. In MINFLUX recording, the same laser was used to excite both dyes in a pair. Furthermore, MINFLUX recording of live cell nuclear pores was also shown, using the fluorescence of Nup96-mMaple (i.e., only one dye) expressed by the correspondingly modified cells for MINFLUX recording. The experimental setup had two different lasers for excitation with an excitation ring: one at 642 nm for the aforementioned dye pair and one at 560 nm for NuP96-mMaple. The emission characteristics of the two dyes in this pair were different. A dichroic beam splitter was used to divide the detection path of the nanostructured array into spectral detection channels for light with wavelengths above 685 nm and spectral detection channels for light with wavelengths below 685 nm. During measurements, since the spectral emission profiles of all used fluorophores are significantly non-zero above and below the limiting wavelength, light was detected for all used fluorophores in both spectral channels. This signal was used for fluorophore localization. Because typically only one fluorophore fluoresces within the localized region, in addition to background fluorescence, and the spectral emission of different fluorophores in a pair differs, the category of fluorophore in the dye pair that caused the signal can be inferred from the signal ratio in different detection channels. Therefore, specific dyes can be assigned to each localization.Here, in the data analysis, potential misallocations are minimized by performing principal component analysis on the spectral fractions of all MINFLUX iterations. Since localization is based on the understanding of the location of the minimum value of the individual intensity distribution of the excitation light, there is no color-related offset between the localizations of different fluorophores, as these fluorophores are excited with the same excitation wavelength. As also mentioned in the publication, since the fluorophore is merely a marker of the biological structure to be imaged, the resolution of the imaging of the biological structure obtained using MINFLUX nanotechnology is ultimately determined by the size of the fluorophore used. Therefore, to fully utilize the potential of MINFLUX nanotechnology, the fluorescent markers must be optimized, taking into account both the size and orientation of the fluorophore. The advantage of MINFLUX nanotechnology is that fewer photons are required for precise localization, thus, in principle, a wider selection of fluorophores can be used for MINFLUX.

[0024] In the publication “Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOSproteins”, Jasmin Pape et al., PNAS, 117(34)20607-20614, (2020), https: / / doi.org / 10.1073 / pnas.2009364117, a method essentially the same as that described in the above publication is discussed for examining other samples.

[0025] In European publication EP 3 372 990 A1, a method similar to MINFLUX in one aspect is described, in addition to other MINFLUX methods. Similar to MINFLUX, an isolated molecule is applied with a focused intensity distribution having a central minimum (preferably zero) and a rising region around this minimum; also as with MINFLUX, this minimum is placed at multiple scan points around the predicted location of the isolated molecule, and fluorescence emission is probed for each scan point; the actual location can be estimated with high precision based on the intensity value or number of photons obtained. Unlike MINFLUX, the intensity distribution is a distribution of fluorescence suppression light (especially STED light). This distribution is applied together with the excitation light, where the intensity distribution of the excitation light does not have a central local minimum. In MINFLUX, fluorescence emission is higher when the isolated molecule is farther from the central minimum of the intensity distribution, but the opposite is true in this method. Like the MINFLUX method described in the literature, this method involves measuring multiple-stained nanostructures, where the staining is chosen to distinguish different fluorophores that are spatially closer than the resolution limit. This can be ensured, for example, by using fluorophores excited at different excitation wavelengths.

[0026] A variation of the MINFLUX-like localization method described above is presented in the publication “MINSTED fluorescence localization and nanoscopy”, Michael Weber et al., bioRxiv 2020.10.31.363424; doi: https: / / doi.org / 10.1101 / 2020.10.31.363424. To localize an isolated fluorophore, the intensity distribution of STED light with a central minimum intensity is superimposed with the Gaussian intensity distribution of the excitation light. At the start of localization, a low STED intensity is used, resulting in an effective excitation-diffusion function that is only slightly narrower than simple excitation. The superimposed intensity distribution is guided onto a circular orbit around the location of the isolated fluorophore, estimated according to pre-localization, while a detector is set to detect fluorescent photons emitted from the circular scanning region. Now, when a photon is detected, the center of the circular orbit is shifted towards the location of the center of the superimposed light distribution at photon emission by a certain value, which is a portion of the original circular orbit radius. Simultaneously, the STED intensity is increased, reducing the half-value width of the effective excitation-diffusion function. This method proceeds iteratively, but the increase in STED intensity terminates at a fixed value. Therefore, in subsequent steps, the center of the circular orbit is shifted if necessary, without simultaneously increasing the STED intensity. Thus, the center of the circular orbit gets closer and closer to the location of the fluorophore to be located. The iteration terminates when the isolated fluorophore stops emitting photons. Like MINFLUX, this method effectively utilizes the information contained in the detected photons. This publication claims that multiple color channels can be achieved by using spectrally shifted fluorophores. This method is particularly suitable for tracking emitters that move rapidly within a sample. The authors refer to the method described as MINSTED.

[0027] Using the MINFLUX nanotechnology, the position of a fluorophore can be experimentally determined in two spatial directions with an uncertainty of only 1 nm, meaning the precision of the position determination is comparable to the size of the fluorophore itself. Determining the position of a single fluorophore with a given measurement uncertainty requires significantly less time and, in particular, fewer fluorescent photons compared to determining the position of a single fluorophore using conventional localization microscopy. The same applies to the MINSTED nanotechnology.

[0028] All existing localization microscopy methods suffer from the drawback that imaging multiple-stained samples is only possible by specifically selecting fluorophore pairs according to the chosen imaging method. This makes it difficult to select fluorophores that best match the structure to be imaged.

[0029] Invention Task Therefore, the present invention is based on the objective of providing positioning microscopy methods and apparatus for studying samples stained with multiple dyes, which improve the imaging of samples stained with multiple dyes or the tracking of the movement of individual fluorescent molecules in samples stained with multiple dyes, because these methods and apparatus facilitate the selection of fluorophores that match the sample to be studied.

[0030] Solution The objective of this invention is achieved by a positioning microscope method for studying samples stained with multiple dyes, having the features of independent claim 1, and by another positioning microscope method for studying samples stained with multiple dyes, having the features of co-claim 21. Dependent claims 2 to 20 relate to preferred embodiments of the method according to claim 1, and dependent claims 22 to 31 relate to preferred embodiments of the method according to claim 21. Furthermore, this objective is achieved by a microscope having the features of claim 32, configured to perform the method according to the invention, particularly the preferred method having the features of independent claim 1 and dependent claim 6, or to perform the preferred method having the features of independent claim 21 and dependent claims 24 and 25. Dependent claims 33 and 34 relate to preferred embodiments of the microscope according to the invention.

[0031] Invention Description Certain terms are used within the scope of this application, the meaning of which is clarified at the outset. For the purpose of localization microscopy, it should be understood as any method that, at least based on the knowledge that the emitter exists in isolation, reveals its location. This includes methods typically performed in a wide field of view (e.g., PALM or STORM) and methods of structured illumination microscopy, provided these methods are performed on an isolated emitter. This also includes the MINFLUX and STED localization methods mentioned below, as well as methods that utilize the displacement of a focused excitation beam with a central intensity maximum within a small field of view.

[0032] Within the scope of this application, the MINFLUX method is understood to be one of the MINFLUX methods known in the art, which uses an excitation light distribution having a local, at least central (i.e., located on the optical axis) intensity minimum. For example, the excitation light distribution having a central minimum may move together with a fluorescence suppression light distribution having a central minimum, or the central minimum of the excitation light distribution may move in a region around the minimum of the fluorescence suppression light distribution.

[0033] Within the scope of this application, the STED localization method should be understood as those methods that use a fluorescence suppression light distribution for localization based on one of the MINFLUX methods known from the prior art or the MINSTED method, which is also known from the prior art, where the influence of the fluorescence suppression light distribution on localization is critical. For example, an excitation light distribution with a central maximum value may move together with a fluorescence suppression light distribution with a central minimum value, or the central minimum value of the fluorescence suppression light distribution may move within a region surrounding the maximum value of the excitation light distribution. In this application, the term MINSTED also applies to methods in which localization is terminated before an isolated emitter loses its excitability, or in methods where there is no threshold for the maximum STED intensity (beyond which the intensity of the STED light no longer increases), or in methods where the half-value width of the effective excitation-diffusion function is altered by measures other than increasing the STED intensity, and also to methods where the fluorescence suppression light is not STED light.

[0034] The basic objective of this invention is first achieved through a first positioning microscope method for studying samples stained with multiple dyes, which will be described below.

[0035] The method according to the invention first includes a first excitation and detection, wherein excitation light of a first wavelength is applied to a sub-region of a sample, and wherein fluorescence emitted by isolated molecules of a dye due to excitation with the first wavelength is detected from the sub-region of the sample, and a first detection signal is obtained. Here, the detection signal includes a numerical value representing the amount of fluorescence detected. This numerical value is subsequently referred to as a light intensity value. Here, excitation and detection can be performed in such a way that a first position of the excitable isolated fluorophore can be directly obtained. In any case, the method includes a first localization of the isolated molecule based on the first detection signal, and obtaining the first position.

[0036] Furthermore, the method includes a second excitation and detection, wherein excitation light of a second wavelength is applied to a sub-region of the sample, and wherein fluorescence emitted by isolated molecules of the dye due to excitation with the second wavelength is detected from the sub-region of the sample, and a second detection signal is obtained. This second detection signal includes a light intensity value. The second excitation and detection is performed in such a way that it is a second localization of the isolated molecule, and a second position is obtained based on the second detection signal. This second position is not necessarily obtained after the first position. It is called a second position because it is obtained using excitation light of a second wavelength.

[0037] Finally, the method includes determining the difference between the first and second positions. In the case of imaging samples stained with multiple dyes, the difference determined in this way can be used to achieve co-localization or co-registration of multiple dyes in a common spatial reference frame.

[0038] In a preferred embodiment, the sub-region of the sample is a small field-of-view region. A small field-of-view region should be understood as a region only slightly larger than the measure of separability defined by diffraction of the structure in the sample. For example, a small field-of-view region can have a range of less than 3 µm, preferably less than 2 µm, more preferably less than 1.5 µm, wherein the small field-of-view region has a corresponding range in one spatial direction (but preferably in two or three spatial directions), i.e., it relates to positioning in two or three spatial directions.

[0039] If the sub-region is a small field of view region, then it is preferable to use focused excitation light for excitation and detection separately. That is, both the first wavelength excitation light and the second wavelength excitation light are focused into the small field of view region.

[0040] In a preferred embodiment of the invention where the sub-region is a small field of view region, a first excitation and detection are performed using focused excitation light of a first wavelength, which has an intensity distribution in the focal region having a central minimum, particularly being substantially a circular distribution or a locally hollow beam distribution. The focal point coinciding with the center of the central minimum is located at a position within the sub-region. It is known from the prior art how to locate such a sub-region, i.e., a small field of view region with excitable isolated fluorophores. The preferred embodiment of the first method according to the invention described herein has the advantage that the use of an intensity distribution with a central minimum enables particularly good locating of small field-of-view regions with excitable isolated fluorophores and subsequent high-precision localization using the MINFLUX method. Therefore, it is advantageous for the search that neither a special light source nor a change in the intensity distribution is required. For example, for the search, the excitation light can be guided in a spiral path over the sample until a fluorescence amount indicating the presence of an excitable isolated fluorophore is detected in the sub-region of the sample at each time interval. The spiral scan can then be terminated at the relevant site. Then, as described above, the focal point is located at a position within the sub-region. Similarly, the light intensity value corresponding to the amount of fluorescence detected during the search can also be obtained directly.

[0041] A first position can be obtained by continuing excitation and associated detection with a first wavelength after the helical motion has stopped, while simultaneously detecting fluorescence with a detector having spatial resolution in a plane conjugate to (i.e., confocal) the plane of the focal point, or by simultaneously positioning the detection aperture at multiple locations around the optical axis in a plane perpendicular to the optical axis orientation. The signal detected in the confocal plane then depends on the position in that plane, where the position dependence depends on the position of the excitable isolated fluorophore relative to the excitation focal point. Therefore, the position of the fluorophore can be obtained. Although this has higher uncertainty compared to positioning according to the MINFLUX method, the achievable accuracy is sufficient to subsequently initiate, for example, MINFLUX positioning. The achievable accuracy is also sufficient to combine with other information to co-localize or co-register multiple dyes in a common spatial reference frame.

[0042] A suitable alternative for closely linking the search for a small field of view with an excitable isolated fluorophore to a first excitation with a first wavelength is that the intensity distribution of the focused light of the first wavelength has a central maximum, and is particularly substantially Gaussian; and that the first excitation and detection are performed by applying the first wavelength excitation light to a sub-region of the sample by focusing the excitation light of the first wavelength at a series of locations within the sub-region. As in the embodiments described above, a detection signal with a light intensity value is obtained. Based on the sub-detection signals obtained at a series of locations within the sub-region, a first position can be determined.

[0043] For example, the first excitation and detection can be terminated after a predetermined time span. Alternatively, the first excitation and detection can be terminated when the light intensity of the first detection signal reaches a threshold, i.e., when a minimum number of fluorescent photons or a minimum light intensity is detected. Other criteria can also be defined to terminate the first excitation and detection when these criteria are met. For example, during excitation, the detection signal can be studied while it is still fully recorded to determine whether a specific ratio to the background has been achieved. For example, an additional detection device can be used to measure the background.

[0044] Preferably, the intensity distribution of the focused light of the second wavelength has a central minimum, which is particularly essentially a circular distribution or a locally hollow beam distribution, both known from STED microscopy. Thus, preferably, the second excitation is performed by positioning the focus of the excitation light of the second wavelength at a series of nominal positions (nominelle Orte) within a sub-region (these nominal positions surround a first position serving as a reference point (i.e., the central position), where the surrounded region has a first extent). This series of positions can be a number of positions, or it can be a continuous sequence of positions. The region surrounding the first position also includes the first position being contained within the series of nominal positions. These positions are called nominal positions because, prior to performing the method, the potential shift in the focus of the excitation light distribution due to different excitation wavelengths is not entirely clear. Therefore, the apparatus for performing the method can, for example, be configured to prevent shifts, where, of course, existing calibration values ​​are taken into account. Thus, the actual focus in the sample differs from the nominal focus by approximately one offset value, which may be a value remaining even after considering the calibration value.

[0045] In another preferred embodiment of the final study of the small field-of-view region using the small field-of-view method, the following further steps are performed. A wavelength is selected based on one or both of the detection signals, and this wavelength is used to further locate the isolated fluorophore. Here, the selected wavelength can be chosen from either the first or second wavelength from which detection signals have been obtained, or, if the microscope provides more than two excitation wavelengths, another wavelength can be selected. Then, the sample is further excited in the small field-of-view region with light of the selected wavelength, wherein fluorescence emitted by isolated dye molecules due to excitation with the selected wavelength is detected from a sub-region of the sample, and a further detection signal is obtained. The further position of the isolated molecule is then obtained based on the further detection signal. For example, if the small field-of-view region is illuminated with focused light of a different wavelength residing at the focal point, and detected with a confocal detector (which locally resolves the diffraction pattern on the confocal plane), the position can be determined based on the detection signal. In the preferred method, excitation with a different wavelength is performed by focusing on a series of locations (which surround the location of the isolated fluorophore inferred from information obtained earlier in the method), that is, excitation and detection are performed as a whole as localization.

[0046] Here, the wavelength selection is preferably performed by evaluating a first detection signal, a second detection signal, or both detection signals. For example, this evaluation may aim to determine the dye class of an isolated fluorophore that has emitted detected fluorescence. For instance, based on the amount of light received at only one wavelength each time, it can be inferred whether the isolated fluorophore is strongly or weakly excited. One or more reference values ​​can also be stored, and one or both detection signals can be compared with these reference values. A preferred method is based on a comparison of the two detection signals, which in many cases is a good indicator of the fluorophore class. For example, if the amount of light received at one detection signal is many times greater than that at the other, a wavelength with a stronger signal can be selected even without the intermediate step of inferring the fluorophore type. The selected wavelength can be either a first wavelength or a second wavelength.

[0047] However, an additional wavelength, different from both the first and second wavelengths, can also be selected. This may be particularly meaningful when locating a class of fluorophores in a sample that cannot be located well enough with wavelengths capable of locating another class of fluorophores in the sample to achieve co-localization. If such an additional wavelength is selected, the method further includes determining the difference between the first position and a further position obtained with the additional wavelength, or determining the difference between the second position and the further position, or both. Preferably, the additional wavelength is focused light. This focused light further preferably has an intensity distribution in the focal region, which has a central minimum. Preferably, this intensity distribution can be a ring distribution or a locally hollow beam distribution. Further excitation and detection can then be performed in all the ways described above with respect to the second excitation and detection. Here, a reference point can be calculated based on the first position or the second position, or based on both the first and second positions, wherein the first position is only considered if it has already been obtained. Preferably, the reference point is determined based on the first position, or if the selected wavelength is the first wavelength, the reference point is directly given by the first position. Accordingly, the reference point is preferably determined based on the second position, or if the selected wavelength is the second wavelength, the reference point is directly given by the second position. If the selected wavelength is another wavelength between the first wavelength and the second wavelength, the reference point can be determined by interpolation from the first position and the second position, and if the selected wavelength is another wavelength greater than or less than the first wavelength and the second wavelength, the reference point can be determined by extrapolation from the first position and the second position.

[0048] It has been pointed out that the first excitation and detection are not necessarily related to the first localization. In particular, in such a case, the first localization is performed after the wavelength is selected. Then the selected wavelength is the first wavelength, and the further localization is the first localization. Then, preferably, further excitation and detection of the isolated fluorophore and further (i.e., the first) localization are performed continuously or iteratively until a further position with a maximum uncertainty of a predetermined value is obtained, and this further position is the first position. In this case, the second excitation and detection and the second localization are only performed after the further excitation and detection have been terminated.

[0049] To obtain images of multiple-stained samples or sample regions (e.g., specific biological structures), one of the methods performed in a small field of view can be repeatedly or in parallel on different isolated fluorophores. Here, if isolated fluorophores of multiple fluorophore categories are located as a whole, imaging where multiple staining contributes to obtaining information can be achieved. For this purpose, multiple small field-of-view regions are studied as a whole, but these small field-of-view regions preferably overlap, and if the structure under study is very small, they can also collectively form a small field-of-view region. Therefore, one of the small field-of-view methods is performed on a set of sub-regions (i.e., small field-of-view regions of the sample), wherein the set of sub-regions includes both sub-regions where fluorescence is emitted and detected by the corresponding isolated fluorophore of a first dye and sub-regions where fluorescence is emitted and detected by the corresponding isolated fluorophore of another dye.

[0050] The differences between the corresponding determined positions, i.e., the difference between the first and second positions, or the differences between the first and second positions and further positions, can be used to transfer all positions to a common reference frame, i.e., to obtain co-localization of isolated fluorophores of different categories. Preferably, for this purpose, a map with correction vectors for co-registering all positions in a common spatial reference frame can be determined based on the positions of the individual isolated fluorophores obtained with excitation light of different wavelengths and the differences between their positions. Preferably, for each position, information about the time at which excitation and detection for localization occur is stored. This information can then be included in the corresponding detection signal. Thus, the map with correction vectors can be a time-coordinate-dependent map. Here, the correction vectors can correspond to several differences or the average of multiple differences, respectively. If the sample or the imaging properties of the sample fluctuate over time, it is advantageous to form such an average of the differences obtained in a close temporal neighborhood. Therefore, in the case of spatial variability, the average can be calculated over a small spatial neighborhood. For example, methods known in the prior art can be used to correct for long-term drift in images (i.e., image stacks here). Active stabilization can be performed, and the relative drift between color channels can be corrected by means of the present invention, or post-processing can be performed. When applying the method according to the present invention, the post-processing is based on improved basic data.

[0051] In principle, all obtained positions and all determined differences can be used to obtain an image. Therefore, an image of the sample can be generated based on a first position and a second position and / or a first position and a further position and / or a second position and a further position, and optionally based on other positions obtained by excitation and detection with excitation light of a first wavelength or a second wavelength or one of other wavelengths.

[0052] In principle, even when using the small field-of-view method, the first excitation and detection and the second excitation and detection can be performed within the same or overlapping time periods. However, when using the small field-of-view method, it is preferable that they are performed within different time periods, which preferably do not overlap. Here, the first excitation and detection are performed temporally before the second excitation and detection. As mentioned above, this does not preclude the first positioning from being performed after the second excitation and detection.

[0053] The first method according to the invention can also be performed in a wide field of view. Thus, a sub-region of the sample is a wide field of view region, and detection involves imaging the sub-region onto a wide field of view detector, where fluorescence emitted by isolated molecules is detected on the wide field of view detector in a spatially resolved manner, thereby obtaining a spatially resolved detection signal. The wide field of view method advantageously enables the simultaneous localization of several or more isolated fluorophores in a relatively simple manner. Then, during the first excitation and detection and the second excitation and detection, fluorescence emitted by multiple isolated molecules due to excitation with excitation light of the corresponding wavelength is detected from the sub-region of the sample, and a first detection signal and a second detection signal with spatial resolution are obtained for each of the multiple isolated molecules. The multiple isolated molecules (i.e., fluorophores) are then firstly and secondly localized based on the corresponding first and second detection signals with spatial resolution, from which a first position and a second position are obtained, respectively. Those skilled in the art know how to perform localization based on wide field of view data. In the wide field of view method, localization ultimately involves the analysis of image data, whereas in some of the aforementioned small field of view methods, localization includes a scanning process performed in a specific manner. If the method according to the invention is performed in a wide field of view, then in each case at least the difference between the first position and the second position is determined.

[0054] Even with a wide-field-of-view approach, for multiple isolated molecules that have been detected emitting fluorescence, the dye to which the isolated molecule belongs can be determined separately based on the first detection signal and / or the second detection signal. When using a wide-field-of-view approach, it is preferable that the first excitation and detection, and the second excitation and detection, can be performed simultaneously. It is meaningful if detectors are available for detection in different spectral channels. Even in a wide-field-of-view approach, the first excitation and detection can be terminated after a predetermined time span. Further excitation and detection can also be performed using additional wavelengths. This is preferably performed from a predetermined set of at least two wavelengths. Subsequently, after selection, further excitation and detection are performed, wherein excitation light of the selected wavelength is applied to a sub-region of the sample, and wherein fluorescence emitted by multiple isolated molecules due to excitation with the selected wavelength is detected from the sub-region of the sample, and further detection signals are obtained separately. The multiple isolated molecules are then further localized based on the further detection signals, thereby obtaining the further positions of the multiple isolated fluorophores. It is not necessary to actually localize all excited isolated fluorophores.

[0055] When applying a wide-field-of-view method, the wavelength can preferably be selected by evaluating the set of first and second detection signals for the category of dye, where isolated molecules of the dye have emitted detected fluorescence that causes a large portion of the obtained set of detection signals. Selection can also be made even without analysis for the dye category, for example, by studying at which excitation wavelength more isolated fluorophores are observed to fluoresce. Similarly, the wavelength selected here can be not only the first wavelength or the second wavelength, but also other wavelengths different from the first and second wavelengths. Thus, the first method according to the invention includes determining, respectively, the difference between a corresponding first position and a corresponding further position and / or the difference between a corresponding second position and a corresponding further position.

[0056] Furthermore, the basic objective of this invention is achieved through a second positioning microscope method for studying samples stained with multiple dyes, which is now described below.

[0057] The second method includes test excitation and detection, wherein excitation light of a test wavelength is applied to a sub-region of the sample, and wherein fluorescence emitted by isolated fluorophores due to excitation with excitation light of the test wavelength is detected from the sub-region of the sample, and a test detection signal is obtained.

[0058] In an alternative embodiment, based on the test signal, and optionally based on other information (e.g., a second test signal), a first wavelength is selected from a predetermined set of at least two wavelengths for subsequent excitation of a sub-region of the sample. A first excitation and detection is performed using light of the selected first wavelength, wherein excitation light of the first wavelength is applied to the sub-region of the sample, and wherein fluorescence emitted by isolated molecules due to excitation with the first wavelength is detected from the sub-region of the sample, and a first detection signal is obtained. An excitable isolated fluorophore is located based on the first detection signal to obtain a first position. In a preferred embodiment, the first wavelength is selected based on two test detection signals. In this alternative embodiment, a second test excitation is performed before selection and after test excitation and detection, wherein excitation light of the second test wavelength is applied to the sub-region of the sample, and wherein fluorescence emitted by isolated molecules due to excitation with the second test wavelength is detected from the sub-region of the sample, and a second test detection signal is obtained.

[0059] In another alternative, a small field-of-view region is selected based on the test signal to perform subsequent excitation and detection of the sub-region. In this case, the wavelength used for subsequent excitation and detection can be predetermined. This wavelength then replaces the wavelength selected in the first alternative. However, these alternatives are not mutually exclusive. More precisely, either a small field-of-view region or a specific wavelength can be selected.

[0060] Whether one or two test detection signals are determined, each test detection signal can be a value representing the amount of fluorescence detected. When determining two test detection signals, the first wavelength can be selected based on the ratio of the first test signal to the second test signal. Alternatively, it can be based on a comparison of the first test signal, the second test signal, or the first and second test detection signals with one or more predetermined reference values. The test wavelengths can be wavelengths used for localization according to the method; that is, both the first and second test wavelengths can be wavelengths included in a predetermined set of wavelengths used for excitation.

[0061] The key feature of this method is not primarily based on determining the difference between two locations. Similarly, it is not particularly characterized by using a second wavelength for secondary localization. Even without these features, decisive advantages are achieved. For example, if tracking the movement of structures labeled with fluorophores in a multicolor stained sample, this method allows for the selection of wavelengths particularly suitable for tracking. Even when tracking structures, localization can be performed separately using the first wavelength, where new localizations are determined over time based on the same fluorophore.

[0062] However, in this second method according to the invention, after the test excitation and detection, a second wavelength can be selected from a predetermined set of at least two wavelengths for subsequent excitation of a sub-region of the sample and for second excitation and detection. Here, excitation light of the second wavelength is applied to the sub-region of the sample, and fluorescence emitted by isolated molecules due to excitation with the second wavelength is detected from the sub-region of the sample, thereby obtaining a second detection signal. Based on this detection signal, the isolated fluorophore is second-localized to obtain a second position. Finally, in the second method according to the invention, the difference between the first position and the second position can also be determined. Therefore, in the second method according to the invention, second excitation and detection with a second wavelength can also achieve most of the advantages of the first method.

[0063] Similar to the first method, the second method can be performed not only as a wide field-of-view method but also entirely as a small field-of-view method. Thus, the sub-region of the sample is a small field-of-view region from the outset, which may have a range of, for example, 3 µm or less than 2 µm. However, the second method can also preferably be performed by: conducting test excitation and probing with a wide field-of-view method (here, the first excitation and probing and the optional second excitation and probing are independent of each other), while the first localization or the second localization, or the first localization and the second localization, are performed respectively with a small field-of-view method. If this is the case, a small field-of-view region is selected after the test probing, and then the first localization or the first localization and the second localization are performed within that small field-of-view region.

[0064] Preferably, in the first and second methods according to the invention, the first localization is performed according to the MINFLUX method, the MINSTED method, or the STED localization method. Here, the MINSTED method is particularly advantageous when applying the first localization within the framework of tracking fluorescently labeled structures. Even if the localization is performed in a small field of view, the second method can be applied within the framework of imaging the structures in the sample. Then, the method is also performed on a set of sub-regions of the sample, such that the final set of sub-regions studied includes both sub-regions where fluorescence is emitted and detected by isolated molecules of the first dye and sub-regions where fluorescence is emitted and detected by isolated molecules of another dye.

[0065] Similar to the first method, in the second method, a map with correction vectors for co-registering all positions in a common spatial reference frame can also be determined based on the positions of individual isolated molecules obtained with excitation light of different wavelengths and the differences between those positions. This also applies to the fact that information about the time at which excitation and probing for localization occur can be stored for each position, and the map with correction vectors can be a time-coordinate-dependent map. Likewise, an image of the sample can be generated based on the first and second positions, and optionally other positions obtained by excitation with excitation light of either the first or second wavelength.

[0066] Finally, the basic objective of the invention is achieved by a microscope according to the invention. The microscope is configured to perform the method according to the invention, particularly for performing a wavelength selection method. The microscope has a laser unit for exciting fluorescence, which is configured to emit narrowband light of two wavelengths, or the microscope has two laser units for exciting fluorescence, which are jointly configured to emit narrowband light of two wavelengths. Furthermore, the microscope has a detection unit and a selection unit, which is configured to select the excitation wavelength based on a detection signal regarding the fluorescence emission of a single fluorophore. Additionally, the microscope has a calculation unit configured to determine the position and the position difference based on the two detection signals regarding the fluorescence emission of a single fluorophore. Preferably, the microscope can provide two wavelengths of excitation light, the difference between which is between 200 nm and 50 nm. More preferably, the microscope has a laser unit configured to emit narrowband light of a third wavelength. This does not preclude the microscope from having laser units configured to emit excitation light of wavelengths that collectively satisfy the above conditions. More preferably, the microscope has one or more STED lasers. Preferably, the microscope has a control unit, which is preferably configured to perform the MINFLUX method, the STED positioning method, or the MINSTED method. Further advantageous features of the microscope according to the invention are particularly apparent from the description relating to the methods.

[0067] Advantageous further aspects of the invention are derived from the claims, the description, and the drawings, as well as the related description of the drawings. Some of the drawings are flowcharts, according to which the method according to the invention is explained in great detail throughout.

[0068] The claims should not be construed as only those subject matter (apparatus or method) that possess all or none of the features of the dependent claims, in addition to those features of independent claims 1, 21, and 32, are possible further embodiments of the invention. Rather, other further embodiments can be derived from the features described in the specification and mentioned in the drawings and related expositions, which can produce effects individually or cumulatively. In particular, many features set forth in this submission in conjunction with only one of the two methods according to the invention as described in claim 1 or claim 21 are also features of advantageous further embodiments of the corresponding other method according to the invention. Brief description of the attached diagram Figure 1 The absorption and emission spectra of two fluorescent dyes suitable for performing the method according to the present invention are shown.

[0070] Figure 2 A flowchart of the first method according to the present invention is shown.

[0071] Figure 3 A flowchart illustrating a first embodiment of the method according to the invention is shown, wherein excitation and detection are performed in a small field-of-view region.

[0072] Figure 4 Another method for performing the first method according to the invention is shown, also... Figure 3 The flowchart of the implementation method is shown in the figure.

[0073] Figure 5 A flowchart of another embodiment of the first method according to the present invention is shown.

[0074] Figure 6 A flowchart is shown showing steps that can be performed in several embodiments of the two methods according to the present invention.

[0075] Figure 7 A schematic diagram is shown for selecting a wavelength from a set of wavelengths.

[0076] Figure 8 Each excitation and probe step is shown to consist of two elements: excitation and probe.

[0077] Figure 9 A flowchart of another embodiment of the first method according to the present invention is shown, wherein an additional wavelength is selected.

[0078] Figure 10 A flowchart of a first embodiment of the second method according to the present invention is shown.

[0079] Figure 11 A flowchart of a second embodiment of the second method according to the present invention is shown, wherein a small field of view region is selected.

[0080] Figure 12 A sub-region of a multicolor stained sample is shown, which has a small field of view with an excitable isolated fluorophore and other excitable fluorophores.

[0081] Figure 13 A flowchart illustrating an embodiment of the second method according to the invention is shown, wherein test excitation and detection are performed using two test wavelengths.

[0082] Figure 14 A flowchart of another embodiment of the second method according to the invention is shown, wherein test excitation and detection are performed with two test wavelengths and a small field of view is selected.

[0083] Figure 15 A flowchart of an embodiment of the second method according to the present invention is shown, wherein a first position and a second position are obtained and the difference between these positions is obtained by comparison.

[0084] Figure 16 A schematic diagram is shown illustrating how different types of fluorophores can be co-located in a common spatial reference frame.

[0085] Figure 17 Another schematic diagram is shown, illustrating how different types of fluorophores can be co-located in a common spatial reference frame.

[0086] Figure 18 Another schematic diagram is shown, illustrating how different types of fluorophores can be co-located in a common spatial reference frame.

[0087] Figure 19 A schematic diagram is shown regarding colocalization of more than two dye classes.

[0088] Figure 20 A microscope according to the present invention is shown schematically.

[0089] Description of the accompanying drawings and explanation of the invention based on the drawings. The present invention will be further illustrated and described below based on the embodiments shown in the accompanying drawings.

[0090] exist Figure 1The excitation spectra 6, 7 and emission spectra 8, 9 of two fluorescent dyes, Alexa Fluor 568 and Alexa Fluor 647, are shown in the figure. This pair of dyes is suitable for performing the method according to the invention. All excitation spectra 6, 7 and emission spectra 8, 9 have been normalized to their respective maximum values, and the values ​​on the vertical axis are percentages of the relevant maximum values. The values ​​on the horizontal axis represent wavelengths in nanometers. All the following descriptions of excitation spectra 6, 7 and emission spectra 8, 9 are derived from the figures, i.e., not from a table of values. This is because the exact values ​​are not important; what is important is only certain characteristics shown according to the figures. Thus, the excitation spectrum 6 of the fluorescent dye Alexa Fluor 568 has a value of approximately 30% at a wavelength of 525 nm, a maximum value at a wavelength of 568 nm, a value still slightly below 40% at a wavelength of 600 nm, approximately 5% at 625 nm, and a value close to zero (0) at a wavelength of 647 nm. The excitation spectrum 7 of Alexa Fluor 647 has its maximum value at 647 nm, approximately 40% at 625 nm, approximately 30% at 600 nm, approximately 10% at 568 nm, and less than 5% at 525 nm. Decisive to the availability of the dye pair is that, on the one hand, excitation spectra 6 and 7 show overlap, meaning some wavelengths of light can excite both dyes; on the other hand, some wavelengths of light can only excite one of the two dyes well, or can excite one dye significantly more intensely (e.g., ten times stronger than the other dye). For the dye pair shown, for example, dye Alexa Fluor 568 can be well excited at 525 nm, specifically with a maximum achievable excitation probability of approximately 30%, while dye Alexa Fluor 647 can only be excited with less than 5% of its maximum achievable excitation probability. Similar favorable ratios exist, for example, across the entire wavelength range from 525 nm to approximately 568 nm. In contrast, at a wavelength of 625 nm, dye Alexa Fluor 647 can be well excited with a relative excitation probability of about 40%, while dye Alexa Fluor 568 is hardly excited at this wavelength with a relative excitation probability of about 5%.

[0091] exist Figure 2The diagram shows a flowchart of a first method according to the present invention. This method is performed on a multicolor stained sample having excitable isolated fluorophores, or more precisely, on a sub-region 1 of a multicolor stained sample having excitable isolated fluorophores, wherein the sub-region 1 of the multicolor stained sample may also include the entire sample. Here, an excitable isolated fluorophore is a locatable fluorophore, wherein the terms locating 11, 21, 113, known from localization microscopy, refer to the process of obtaining the positions 12, 22, 114 of the fluorophore. Those skilled in the art know from the prior art how to ensure the presence of excitable isolated fluorophores in a sample or in a sub-region of the sample. In the left column, from top to bottom, the following method steps or method elements are given: first excitation and detection 10, first localization 11, first position 12. The first excitation and detection 10 is performed using excitation light of a first wavelength. Correspondingly, in the right column, from top to bottom, the following additional method steps or method elements are given: second excitation and detection 20, second localization 21, and second position 22. The second excitation and detection 20 is performed using excitation light of a second wavelength different from the first wavelength. The steps of first excitation and detection 10 and first positioning 11, as well as second excitation and detection 20 and second positioning 21, are connected by lines; that is, the steps of first excitation and detection 10 and first positioning 11 are closely related, and their type depends on the specific implementation or implementation method. In some implementations, first excitation and detection 10 and first positioning 11 are performed simultaneously, that is, the process of first positioning 11 is performed during first excitation and detection 10, or the result of detection 71 from first excitation and detection 10 is directly used to determine the first position 12. In other implementations, the result of detection 71 from first excitation and detection 10 is not directly used to determine the first position 12, or positioning 11 is not performed in direct relation to first excitation and detection 10, but is performed separately in relation to further excitation and detection 15. As a result of first positioning 11, the first position 12 is finally obtained. The above also applies in the same sense to the steps or elements of second excitation and detection 20, second positioning 21, and second positioning 22. However, typically, the second excitation and detection 20 and the second localization 21 are performed simultaneously; that is, the second localization 21 process is performed during the second excitation and detection 20, and the result of the detection 71 from the second excitation and detection 20 is directly used for the second localization 21. The steps in the left and right columns can be performed simultaneously. Therefore, at least two spectral channels are required for detection 71, where at least one channel is configured to detect the fluorescence of both dyes in the dye used and to localize the two dyes 11, 21, and 113. These steps can be performed sequentially in time, so a single spectral channel is sufficient for detection 71.The first excitation and detection 10 and the second excitation and detection 20 can be performed in a wide field of view (e.g., this is typically the case in the case of PALM or STORM), or they can each be performed in a small field of view region (e.g., in the case of MINFLUX). The first excitation and detection 10 and / or the second excitation and detection 20 can also be performed using the distribution of additional fluorescence suppression light, particularly by means of the STED localization method or the MINSTED method.

[0092] It is known in the prior art to perform localization microscopy methods in a wide field of view using two excitation wavelengths and two spectral channels. However, the combination of dye, excitation wavelength, and spectral channel used for detection is always selected such that, in each individual detection channel corresponding to the excitation wavelength, fluorescence of only one dye class is detected as much as possible. This is to avoid interference from the fluorescence of another dye class in the localization of a fluorophore. However, in the relevant prior art, this also excludes the possibility of localizing a single isolated fluorophore once using light with one excitation wavelength in a single spectral channel and then localizing it again using light with another excitation wavelength. In contrast, according to the present invention, a single isolated fluorophore or a group of single isolated fluorophores is localized once using light with a first excitation wavelength (wavelength A 111) and then localized again using light with another excitation wavelength (wavelength B 112). Therefore, the first position 12 in the left column is the position 114 of the same fluorophore, for which a second position 22 is also obtained. If the first excitation and detection 10 and the second excitation and detection 20 are performed in a wide field of view, multiple first positions 12 and multiple second positions 22 can be obtained simultaneously.

[0093] After obtaining one or more first positions 12 and one or more second positions 22, the one or more first positions 12 is compared with one or more second positions 22 so as to obtain, if necessary, the difference 61 between one or more first positions 12 and one or more second positions 22 in each case.

[0094] exist Figure 3The diagram shows a flowchart of an embodiment of the first method according to the invention. In this embodiment, excitation and detection 10, 15, 20 are performed in a small field-of-view region, which is the sub-region 1 to be studied of a multicolor stained sample. The method begins by finding the small field-of-view region 2. This can be done by the following steps: illuminating the sample or a region of the sample in a wide field of view, detecting fluorescence in a wide field of view in a manner that images the sample or the region of the sample, and evaluating the image to identify the small field-of-view region in which fluorescence originating from isolated fluorophores is detected. However, this can also be done in a scanning manner by scanning the sample or the region of the sample with focused excitation light 3 and detecting fluorescence emitted from the sample or the region of the sample. The small field-of-view region with excitable isolated fluorophores can then be identified based on the amount of fluorescence detected at the illumination position (e.g., according to two limit values), wherein the amount of fluorescence detected must exceed a lower limit value that can be selected based on the background signal and must be below an upper limit value. The first excitation and detection 10 is performed after or together with the search for the small field-of-view region 2, wherein focused excitation light 3 of a first wavelength is used. In the illustrated embodiment, the focused excitation light 3 has a central intensity maximum 4. The excitation 70 of the first excitation and detection 10 is performed within a small field of view. This means that during the first excitation and detection 10, the focus of the excitation light either does not shift at all or only places the focus within an area that expands by at most a few micrometers (e.g., a diameter of 3 µm or only 2 µm). In the illustrated embodiment, the first excitation and detection 10 is performed during a time span 40, after which the first excitation and detection 34 is terminated. As a result of the first excitation and detection 10, a first detection signal 51 is obtained, which specifically includes a light intensity value 50, a measure of the amount of fluorescence detected during the time span 40.

[0095] Furthermore, the first detection signal 51 is evaluated 32. For example, the light intensity value 50 of the first detection signal 51 can be evaluated by comparing it with a reference value 27 (not shown in the figure). For example, if a sample stained with Alexa Fluor 568 and Alexa Fluor 647 is excited with light at a wavelength of 640 nm during the first excitation and detection 10, a relatively large light intensity value 50 is obtained if the excitable isolated fluorophore is the fluorophore of dye Alexa Fluor 568, and a relatively small light intensity value 50 is obtained if the excitable isolated fluorophore is the fluorophore of dye Alexa Fluor 647. In addition to the light intensity value 50, other characteristics of the first detection signal 51 can also be evaluated if they are detected (e.g., variations in the share of the total light intensity value 50 obtained in various time periods of the time span 40 or the value of the share of the total light intensity value 50 detected in different spectral detection channels). In the example shown, wavelength 33 is selected according to evaluation 32 such that further excitation and detection 15 is subsequently performed using the first wavelength.

[0096] The further excitation and detection 15 is performed to obtain the first position 12. Accordingly, a first localization 11 is performed together with the further excitation and detection 15. The first localization 11 can be performed using the MINFLUX method. In this case, the further excitation and detection 15 is performed using a focused excitation light 3, the focal point of which has a central minimum intensity 5. In addition to the focused excitation light 3 of the first wavelength, even when using the MINFLUX method, an additional focused light as fluorescence suppression light can be applied to the small field of view, the intensity distribution of which has a central minimum intensity, wherein the focal points of the two light distributions coincide in each case, or wherein the fluorescence suppression light illuminates the sample in all cases, such that the focal point of the focused excitation light 3 is always located within the region of the minimum intensity of the fluorescence suppression light. Alternatively, the first localization 11 can be performed using the STED localization method or the MINSTED method, wherein in both cases, in addition to the excitation light of the first wavelength, a focused fluorescence suppression light with a central minimum intensity distribution is used. It is particularly advantageous when the dye with the smaller wavelength of the maximum excitation spectrum among the two dyes has a larger Stokes shift, so that both dyes can be excited essentially independently, but still with the same fluorescence suppression light. In the example shown, the first positioning 11 is performed together with further excitation and detection 15 until criterion 41 is met. In the specific case shown, criterion 41 is a pre-given positioning accuracy. Other possible criteria 41 are, for example, the number of MINFLUX iterations performed, or, in the case of applying MINSTED, the achieved fluorescence suppression light intensity. Furthermore, additional information from the first detection signal 51 can be used to obtain the first position 12.

[0097] If the excitable isolated fluorophore loses its excitability before reaching criterion 41, the method cannot be fully performed on that excitable isolated fluorophore. However, a first position 12 can be obtained, which can be used, for example, to acquire an image of the sample or a region of the sample.

[0098] Conversely, if criterion 41 is achieved, the excitable isolated fluorophore remains in an excitable state even after obtaining the first position 12. This is now utilized by further excitation and detection with a second wavelength, wherein the further excitation and detection is a second excitation and detection 20 from which the second position 22 is obtained. The applicable methods are the same for the second excitation and detection 20 with the second position 21 from which the second position 22 is obtained, and for the further excitation and detection 15 with the first position 11 from which the first position 12 is obtained, with the necessary modifications.

[0099] After obtaining the first position 12 and the second position 22, the first position 12 and the second position 22 are compared to obtain the difference 61 between the first position 12 and the second position 22.

[0100] exist Figure 4 In the middle, it is shown that... Figure 3 A flowchart of another implementation of the first method according to the same invention. This process differs because there are... Figure 3 The fluorophores of different dye classes shown in the figure Figure 3 The image shows an excitable, isolated fluorophore within the considered small field of view. Therefore, when selecting wavelength 33 based on the evaluation 32 of the first detection signal 51, a second wavelength is selected. In this case, after selection, a second excitation and detection 20 is performed together with a second positioning 21 to obtain a second position 22. Here, these steps are performed until criterion 41 is met, for example, until a second position 22 with predetermined accuracy is obtained. Subsequently, further excitation and detection 15 is performed together with the first positioning 11 to obtain a first position 12.

[0101] After obtaining the second position 22 and the first position 12 (here, the first position 12 is obtained after obtaining the second position 22), the first position 12 is compared with the second position 22 30 to obtain the difference 61 between the first position 12 and the second position 22.

[0102] exist Figure 5The diagram shows a flowchart of another embodiment of the first method according to the invention. In this embodiment, the search for the small field of view region 2 and the first positioning 11 from which the first position 12 is obtained are more closely linked. The search for the small field of view region 2 and the first positioning 11 are both performed using focused excitation light 3, which in the illustrated example has a minimum central intensity value 5. However, the search for the small field of view region 2 and the first positioning 11 can also be performed using focused excitation light 3 with a maximum central intensity value. Before searching for the small field of view region 2, a sub-region 1 of the multicolor stained sample is scanned with focused excitation light 3 until a fluorescence signal from an excitable isolated fluorophore is obtained. Here, the scan can be performed in a gridded manner, but it can also be performed on a spiral or circular track, each track shifting in one direction after completing one revolution. The region a few micrometers widening around the location where the fluorescence signal from the excitable isolated fluorophore is obtained is the small field of view region studied using the method according to the invention. For this purpose, the search for the small field of view region 2 transitions to the first excitation and detection 10 and thus the first positioning 11 to obtain the first position 12. Furthermore, in the first excitation and detection 10, a first detection signal 51, including a light intensity value 50, is obtained. For example, when the intensity distribution of the focused excitation light 3 is a ring distribution or a locally hollow beam distribution, the first excitation and detection 10 is performed by positioning the focal point of the focused excitation light 3 of the first wavelength within a small field of view region (i.e., sub-region 1 of the multicolor stained sample). Detection 71 includes detecting fluorescence emitted by isolated molecules (i.e., isolated fluorophores) as a result of excitation with the focused excitation light of the first wavelength. Fluorescence is detected with a detector having spatial resolution in a plane conjugate to the plane where the focal point is located, or the detection aperture is positioned at multiple locations around the optical axis in a plane perpendicular to the optical axis orientation. In both cases, detection with local resolution in the detection plane is achieved. Based on the local distribution of the fluorescence signal in the detection plane obtained in this way, a first position 12 can be obtained.

[0103] Subsequently, a second excitation and detection 20 is performed within the same sub-region 1 of the multicolor stained sample, wherein, based on the first position 12, a small field-of-view region 42 smaller than the sub-region 1 of the multicolor stained sample can be determined. A second positioning 21 is performed together with the second excitation and detection 20 to obtain a second position 22. In principle, this can be combined with information about... Figure 3 and Figure 4 Execute in the manner described.

[0104] After obtaining the second position 22 and the first position 12, the first position 12 is compared with the second position 22 to obtain the difference 61 between the first position 12 and the second position 22. Furthermore, the first detection signal 51 and the second detection signal 52 are evaluated 32, specifically their respective light intensity values ​​50. Based on the evaluation 32, information 62 is obtained regarding the dye category to which the excitable isolated fluorophore with its first position 12 and second position 22 belongs; typically, the dye category can be determined because it is usually known in advance which dye categories are used to stain the sample. The overall information 63 obtained according to this method includes the difference 61 between the first position 12 and the second position 22, and the information 62 regarding the dye category.

[0105] Figure 6 This is intended to illustrate an important aspect of the present invention. Figure 6A flowchart illustrating steps that can be performed in multiple embodiments of two methods according to the invention is shown. A key aspect of the invention is that an excitable isolated fluorophore of at least one fluorophore class stained in a sub-region 1 of a multicolor stained sample is located once using excitation light of a first wavelength (wavelength A 111 in this figure), and then once using excitation light of a second wavelength (wavelength B 112 in this figure), wherein wavelength A is different from wavelength B. If the sample is stained with more than two dye classes, the excitable isolated fluorophore can also be located once using light of wavelength A 111 and once using light of wavelength B 112 for fluorophores of two or all dye classes. In the left column, excitation and detection 110 and location 113 with wavelength A 111 and obtaining position 114 are schematically shown. Furthermore, a detection signal 115 including a light intensity value 50 is obtained. In the right column, the same situation with respect to wavelength B 112 is schematically shown. The process schematically shown in the two columns can be performed simultaneously by performing excitation and detection 110 in different spectral detection channels. If excitation and detection 110 are each performed in a small field of view, it may be advantageous to perform the steps in the left column (wavelength A 111) and the right column (wavelength B 112) sequentially. For example, these steps can be performed in such a way that positioning is performed according to the MINFLUX method, the MINSTED method, or the STED positioning method in each case. The steps in the right and left columns can also be performed alternately until the excitable isolated fluorophore loses its excitability. In this way, the position 114 determined from the set of positions or the set of multiple positions 114 can be obtained according to the steps performed on wavelength A 111. In addition, the total detection signal 115 obtained from the set of individual detection signals or the set of detection signals 115 can be obtained. The same applies to wavelength B 112. Then, the two positions 114 or the two sets of multiple positions 114 of wavelengths A 111 and B 112 are compared 30 to determine the difference between the positions of the two wavelengths. Furthermore, the two total detection signals 115, or two sets of detection signals 115, are evaluated to obtain information 62 regarding the dye class of the excitable isolated fluorophores located by light at two wavelengths, A 111 and B 112. The overall information 63 obtained according to this method includes the difference 61 of position 114 and the corresponding information 62 regarding the dye class.

[0106] Figure 7 The selection of wavelengths from a set of wavelengths is illustrated. Set 117 includes wavelengths A 111, B 112, and another wavelength 116, all of which are distinct from each other. Here, wavelength 33 (wavelength B 112) is selected based on two detection signals 115. Here, wavelength 33 is selected from... Figure 3 and Figure 4The evaluation step 32, shown separately, involves evaluating the first detection signal 51. The arrow from set 117 to the step of selecting wavelength 33 is intended to indicate that the elements in set 11 can be provided for use by selecting wavelength 33.

[0107] Figure 8 This illustrates that each excitation and detection step 110 includes two elements: excitation 70 and detection 71. In this application, the excitation and detection step 110 appears as a first excitation and detection 10, a second excitation and detection 20, a further excitation and detection 15, and generally as excitation and detection 110. In their respective cases, excitation and detection 10, 15, 20, and 110 all include the corresponding elements—excitation 70 and detection.

[0108] Figure 9 This is intended to illustrate an important aspect of the present invention. Figure 9 A flowchart illustrating steps that can be performed in multiple embodiments of two methods according to the present invention is shown. The upper part indicates steps already performed. Figure 6 The excitation and detection steps 110 shown (one for wavelength A 111, one for wavelength B 112) each have an associated positioning step 113 and associated elements—position 114 and detection signal 115, each detection signal 115 including a light intensity value 50. Figure 6 Other steps shown (comparison 30 and evaluation 32) and elements (positional differences 61 and information 62 regarding the dye class of excitable isolated fluorophores) are also included. Figure 9 However, this is additionally associated with the excitation and detection steps 110 performed at a different wavelength 116. Furthermore, the descriptions herein also apply to all [processes / processes]. Figure 6 The known steps. In this... Figure 9 The additional wavelength 116 shown may be the same as one of wavelengths A 111 and B 112, or it may be different from both. Figure 6The illustrated process is supplemented by selecting an additional wavelength 116 for further excitation and detection 110 at the point in time when at least one position 114 and at least one detection signal 115 have been obtained for both wavelengths A 111 and B 112. Here, selection 33 also includes an evaluation 32 shown separately elsewhere. For example, if at least one position 114 is to be obtained for each of wavelengths A 111 and B 112 for an excitable isolated fluorophore in each case, and furthermore, if the most accurate position 114 is desired overall, possibly additionally to minimize sample loading, one of wavelengths A 111 or B 112 is selected as an additional wavelength 116. Then, exactly two related positioning 113 are performed first, such that at least one position 114 is obtained for each of wavelengths A 111 and B 112.

[0109] When making selection 33, an additional wavelength 116 can now be selected, which excites the excitable isolated fluorophore as efficiently as possible. Thus, the excitable isolated fluorophore can be well localized with the lowest possible loading on the sample as a whole. For example, if only two wavelengths are available, one of the two wavelengths, A 111 or B 112, can be selected for which a larger light intensity value 50 is obtained in the associated excitation and detection 110, or a larger value derived from the light intensity value 50, taking into account, for example, the incident light intensity known to be part of the detection signal 115.

[0110] Another possibility, particularly when wavelengths different from A 111 and B 112 are available, is to infer the dye class to which the excitable isolated fluorophore belongs from the ratio of the light intensity value 50 or a value derived accordingly from that light intensity value, and to select, accordingly, a wavelength from the set of wavelengths 117 that best excites the associated dye class. For example, when studying a sample stained with at least Alexa Fluor 568 and Alexa Fluor 647 and studied at wavelengths A 111 at 525 nm and B at 625 nm, this comparison can conclude that the located excitable isolated fluorophore belongs to the dye class Alexa Fluor 647. Then, if a wavelength of 647 nm is selected as another wavelength 116, this wavelength can be selected for subsequent excitation and detection 110, at which the first excitation spectrum 7 is the excitation spectrum of the dye class Alexa Fluor 647. At this wavelength, not only is the excitability of dye Alexa Fluor 647 the highest, but the excitability of dye Alexa Fluor 568 is almost non-excitable. Therefore, when excited with another wavelength of 116 nm at 647 nm, the background fluorescence of dye Alexa Fluor 568 is hardly excited.

[0111] Then, a comparison 30 is performed at positions 114 to obtain a difference 61 at positions 114, and an evaluation 32 is performed on the probe signal 115 (specifically shown as light intensity value 50 in the figures) to obtain information 62 about the dye category or to confirm that information. If the selection 33 of another wavelength already includes an evaluation 32 about the dye category, the final evaluation 32 may also be omitted, wherein the information 62 about the dye category must be taken from the first evaluation 32 (not shown) for use in the overall information 63. The overall information 63 obtained according to this method then includes the difference 61 at positions 114 and the information 62 about the dye category.

[0112] exist Figure 10 The figure shows a flowchart of a first embodiment of the second method according to the invention. This method is also performed on a multicolor stained sample having excitable isolated fluorophores, or more precisely on a sub-region 1 of a multicolor stained sample having excitable isolated fluorophores, wherein the sub-region 1 of the multicolor stained sample may also include the entire sample. In this method, excitation and detection 110 is performed in a first step, which is performed test-wise with a test wavelength 25. In the test-wise excitation and detection 110, a detection signal 115 including a light intensity value 50 is obtained. In this first embodiment of the second method according to the invention, a first wavelength 33 is selected for performing the first positioning 11 based on the detection signal 115. Although in the first method, the wavelength used for performing the first positioning 11 is mandatory in the first performed excitation and detection 110 step, this is permissible but not required here. After selecting the first wavelength 33, the first excitation and detection 10 and the first positioning 11 are subsequently performed, thereby obtaining a first position 12. Several possibilities exist for selecting the first wavelength 33. Reference value 27 is shown in the figure. The dotted-line arrow indicating the first wavelength step (33) is to show that using reference value 27 is only one possibility. This possibility is particularly good when sub-region 1 of the multicolor stained sample is a small field-of-view region. Among other things, it is known from the prior art how to locate small field-of-view regions with excitable isolated fluorophores. If sub-region 1 of the multicolor stained sample is a wide field-of-view region, and further study of this sub-region 1 is to be conducted using methods performed in a wide field of view, then, for example, it can be determined based on the detection signal 115 whether and how many excitable isolated fluorophores are present in the image field, and, if necessary, how many exhibit weak and strong fluorescence emission. Then, based on this analysis, a wavelength can be selected that allows for the simultaneous localization of as many isolated fluorophores as possible at the relevant recording time points.

[0113] Figure 11The purpose is to illustrate the flow of another embodiment of the second method according to the invention. Similarly, in this embodiment of the second method according to the invention, excitation and detection 110 is performed in a first step, which is performed test-wise with a test wavelength 25. In the test-wise excitation and detection 110, a detection signal 115 including a light intensity value 50 is obtained. In principle, the test-wise excitation and detection 110 can be performed on a set of sub-regions of the sample as small field-of-view regions. However, preferably, in this embodiment, the test-wise excitation and detection 110 is performed in a wide field of view. In this other embodiment of the second method according to the invention, a small field-of-view region 42 (not shown in this figure) is selected based on the detection signal 115, in which a first positioning 11 is performed. Subsequently, as in the first embodiment of the method, the first excitation and detection 10 and the first positioning 11 are performed, resulting in obtaining a first position 12. Optionally, in addition to selecting the small field-of-view region 42, a wavelength 33 is also selected before the first excitation and detection 10. In principle, these two selections 33 and 43 can be performed in parallel or interdependently.

[0114] Figure 12 A subregion 1 of a multicolor stained sample with a small field of view 42 is shown, which has an excitable isolated fluorophore 44 and other excitable fluorophores 44, 45.

[0115] Now, it can be based on Figure 11 and Figure 12 Explanation Figure 11The possibilities for selecting a small field-of-view region 42 are shown in Figure 43. A wide-field image of sub-region 1 of the multicolor stained sample is recorded. In this image, fluorophores 44 and 45 in sub-region 1 of the multicolor stained sample can then be imaged. This imaging is comparable to the sample, except for measurement uncertainty and imaging error. The measurement uncertainty may be large because the test excitation and detection 110 at test wavelength 25 is not used to obtain precise locations, but only to locate the small field-of-view region 42, in which precise locations should be obtained during the study of the small field-of-view region 42. Based on the imaging, only the region containing excitable isolated fluorophores 44 can be selected as the small field-of-view region 42 to be studied. There are three excitable isolated fluorophores 44 in sub-region 1 of the multicolor stained sample, two of which emit strongly (represented by their larger radii) and one emits weakly (represented by their smaller radii). To the right of the sub-region, there are several fluorophores 45 with weak and strong emission, but they are not isolated. The small field-of-view region 42 can now be selected by studying the imaging of sub-region 1 of the multicolor stained sample. Now, based on imaging (not shown), a small field of view 42 can be defined around the imaging location of the strongly emitting fluorophore 44 (which is relatively far from the excitable non-isolated fluorophore 45). Due to measurement uncertainty, this small field of view 42 is slightly shifted relative to the actual position of the excitable isolated fluorophore 44. After selection, the test wavelength can be used as the first wavelength. If the small field of view 43 is selected such that the test wavelength can be used as the first wavelength, then in this respect, there is no step of selecting wavelength 33.

[0116] Another possibility is to select the region around the location of a single, excitable, isolated fluorophore 44 exhibiting weak emission as a small field-of-view region 42, since imaging studies have shown that particularly good localization of the fluorophore can be achieved by using a wavelength different from the test wavelength for the first localization 11. In this case, for the subsequent first excitation and detection 10, both the small field-of-view region 42 and the wavelength 33 used for the first excitation and detection 10 are selected.

[0117] exist Figure 13 The figure shows a flowchart of another embodiment of the second method according to the present invention. The figure includes... Figure 10 All elements shown. For a description of the relationships associated with these elements, please refer to [reference needed]. Figure 10 The explanation. Furthermore, in Figure 13Further test excitation and detection 110 is also shown, which is performed using a second test wavelength 26 different from the first test wavelength 25. If detection 71 is performed in multiple different spectral detection channels, this further test excitation and detection 110 can be performed simultaneously, and typically after test excitation and detection 110 with the first test wavelength 25. Further test excitation and detection 110 with the second test wavelength 26 also yields a detection signal 115 including a light intensity value 50. In the embodiment shown in the figure, a first wavelength 33 is selected for the first localization 11 based on the two detection signals 115 obtained for either the first test wavelength 25 or the second test wavelength 26. For example, the ratio of the two light intensity values ​​could be a characteristic of the dye class of the excitable isolated fluorophore, and then an optimally suitable wavelength is selected for the first excitation and detection 10 of that isolated fluorophore and the associated first localization 11. For example, when test excitation and detection 110 is performed in a wide field of view, it is possible to investigate which test wavelengths 25 and 26 contain more strongly emitting excitable isolated fluorophores when imaging a subregion 1 of a multicolor stained sample. These test wavelengths 25 and 26 can then be selected as the wavelengths for the first excitation and detection 10. In addition to these two detection signals 115, a reference value 27 can be used to select wavelength 33. The reference value 27, which does not necessarily have to be a scalar value, can include, for example, entries for a comparison of light intensity values ​​50 or a ratio of light intensity values ​​50, which are used to compare the two light intensity values ​​50 or their ratio to select the wavelength for the first excitation and detection 10.

[0118] exist Figure 14 An embodiment is shown, including the steps of further test excitation and detection 110 using a second test wavelength 26 and the selection of a small field-of-view region 42. In this embodiment, a small field-of-view region 42 (not shown in the figure) for which a first positioning 11 should be performed can be selected based on two detection signals 115 obtained once using a first test wavelength 25 and once using a second test wavelength 26. Subsequently, the first excitation and detection 10 and the first positioning are performed, resulting in a first position 12. Optionally, in addition to selecting the small field-of-view region 42, a wavelength 33 is also selected before the first excitation and detection 10. In principle, these two selections 33 and 43 are substantially based on... Figure 11 as well as Figure 11 and Figure 12 The relevant explanations can be carried out in parallel or interdependently.

[0119] exist Figure 15 One embodiment is illustrated, wherein the difference between the first position 12 and the second position 22 is ultimately obtained by comparing the first position 12 and the second position 22 of an excitable isolated fluorophore. The upper part of the figure is from... Figure 14 The known elements in. Figure 14 The group containing the elements of first excitation and detection 10, first positioning 11, and first position 12 is in Figure 15 The signal is supplemented by a detection signal of element 115, including an element with a light intensity value of 50 (downward on the left side of the attached figure), and the rest is the same as shown in the figure. Figure 14 The correlation between selecting the small field-of-view region 43 and selecting the element group with wavelength 33 is basically the same. Figure 15 In the diagram, these relationships, corresponding to optional steps or associations within the method flow, are shown using dashed lines. Relative to... Figure 14 A step to select the second wavelength 33' has been added to the upper part.

[0120] For also included Figure 14 For descriptions of elements within or for methods, steps, associations, and elements shown therein, first refer to the descriptions of elements within or for the methods, steps, associations, and elements shown therein. Figure 14 The description. In Figure 15 In the embodiment shown, a second positioning 21 is now added, which selects a second wavelength 33' and a second excitation and detection 20 and obtains a second position 22 therefrom. Additionally, in each case, a detection signal 115 with a light intensity value 50 is included, in addition to the first position 12 and the second position 22. Now, if the lower portion is compared with the elements of the first excitation and detection 10 and the second excitation and detection 20, and all the elements filled below them, it can be seen that this portion corresponds to... Figure 6 The difference lies in, Figure 6 In this context, specific elements such as wavelengths A111 and B112, and position 113 are typically referenced. Figure 15 In this context, specific elements such as the first positioning 11 and the second positioning 22 are referenced. Therefore, regarding the association of elements and the corresponding method flow, this section first refers to the specific elements. Figure 6 The description. In Figure 15 In the middle, an association, represented as optional, is filled between the left element block with the first excitation and detector 10 and the corresponding right block. This, although not in... Figure 6 The instruction manual states that, under certain conditions, the right block should be executed after the left block.

[0121] Therefore, the steps following the selection of wavelength 33 for the first detection 10 or the selection of small field-of-view region 42 (43) correspond to steps also known from the first method. When selecting the second wavelength 33' for the second excitation and detection 20, a wavelength different from that used for the first excitation and detection 10 is selected. Here, this wavelength can be selected such that it is particularly suitable for locating fluorophores of a different class than the class to which the fluorophore to be located in this step belongs. Simultaneously, this wavelength must be selected in such a way that the fluorophore to be located in this step can be located. How to understand this, from... Figure 1 As can be seen from the description and related information. In general, this is achieved by obtaining two positions 114 of an excitable isolated fluorophore, one of which can be highly accurate, while the other position 114 can have lower accuracy or higher uncertainty.

[0122] Figure 16 The possibility of co-locating fluorophores of multiple (specifically two) fluorescent dyes in a common reference frame is illustrated. Multiple fluorophores 54 of the first dye 53 are located by means of any method according to the invention, wherein, for each individual fluorophore, one or more positions L 56 are obtained at wavelength A 111 and one or more positions U 57 are obtained at wavelength B 112. Here, wavelength A 111 is a wavelength preferably suitable for locating the excitable isolated fluorophores 54 of the first dye 113. Here, wavelength A 111 can be a first wavelength depending on the method and specific method flow, that is, each individual position L 56 can be a first position 12, or wavelength A 111 can be a second wavelength, that is, each individual position L 56 can be a second position 22. Therefore, wavelength B 111 can be a wavelength less suitable for locating the excitable isolated fluorophores 54 of the first dye 113 but more suitable for locating the excitable isolated fluorophores 55 of the second dye 113. Similarly, wavelength B 112 can be either a first wavelength or a second wavelength depending on the method and specific process flow; that is, each individual position U 57 can be either a first position 12 or a second position 22. In general, multiple positions 58 will be obtained. This multiple can be a set of positions L 56, a subset, or a set derived from the set of positions L 56. For example, multiple positions L 56 can be identified as the positions of the same isolated fluorophore at different time points, and the individual positions of this fluorophore can be combined and included in the multiple positions 58. For positions L 56 and U 57, differences 61 are determined respectively. Based on the set of differences 61, an average difference 59 between positions is determined. This average difference 59 can be determined with higher precision (i.e., lower uncertainty than a single difference 61) based on the number of individual differences 61. Therefore, it is acceptable that the uncertainties of position U 57 included in the uncertainty of difference 61 are greater than the uncertainties of position L 57, which is generally due to the different applicability of wavelengths A111 and B 112 in locating the first dye 54. Here, the multiple positions L 56 and U 57 can be the result of locating 113 within a spatially confined area of ​​the sample, or the result of locating 113 within a confined time period. The latter is advantageous when the characteristics of the sample or the microscope used to study the sample fluctuate over time.

[0123] The excitable isolated fluorophore 55 of the second dye is located using wavelength B 112, which is well-suited for this purpose. Position L 56 is obtained. The average difference 59 generated by different wavelengths A 111 and B 112 during localization is obtained from the position of the first dye 54. The position L 56 of this isolated fluorophore 55 of the second dye is now included in the total position set 60 along with multiple positions 58 by means of this average difference 59, thereby achieving co-localization of fluorophores from different dyes. Preferably, the position L 56 and the average difference 59 of the fluorophore 55 of the second dye are determined from data acquired in a spatial or temporal neighborhood (preferably in both spatial and temporal neighborhoods).

[0124] It is immediately apparent that, in the same manner, the positions L 56 of several or more fluorophores 55 of the second dye can co-localize with the fluorophores 54 of the first dye. Similarly, it is immediately apparent that the fluorophores 54 of the first dye, which only obtained position L 56 using wavelength 111 but failed to obtain position U 57 using wavelength B 112, can also be co-localized in the same way. Therefore, in order to obtain an image of a multicolor stained sample or a subregion 1 of a multicolor stained sample (where fluorophores of multiple (e.g., two) dyes are co-localized, especially with high precision), it is sufficient to localize a subset of the fluorophores used to obtain the image using two wavelengths.

[0125] Figure 17 This illustrates another possibility for the co-location of fluorophores of multiple (specifically two) fluorescent dyes in a common reference frame. In this embodiment, to determine the position difference 61 for different wavelengths, it is not necessary to obtain position U 57 for the fluorophore of one dye using a wavelength particularly suitable for locating the fluorophore of the other dye. Instead, for the fluorophores 54, 55 of the two dyes, a common additional wavelength 116 is used to obtain their respective associated positions U 57. Here, position L 56 is obtained for the fluorophores 54 of the multiple 53 first dyes using wavelength A 111, and position L 56 is obtained for the fluorophores 55 of the multiple 53 second dyes using wavelength B 112. In this way, multiple positions 58 and an average difference 59 are obtained for the two dye categories. This average difference 59 is related to the common additional wavelength 116 for both dye categories in each case. Therefore, considering the two average differences 59, the multiple positions L 56 of the isolated fluorophore 55 of the second dye at positions 58 and L 56 of the isolated fluorophore 55 of the first dye are grouped together into the total position set 60, thereby achieving co-localization of fluorophores from different dyes. Based on the... Figure 16The same applies here, especially for each of the two dyes; it is not necessary to obtain position U 57 for all isolated fluorophores used for imaging multicolor stained samples. Instead, it is sufficient, for example, to obtain a set of isolated fluorophores that yields an average difference 59 with the desired accuracy in each case.

[0126] Figure 18 Another possibility for co-localization of fluorophores of multiple (specifically two) fluorescent dyes in a common reference frame is shown. In this embodiment, the fluorophore 54 of the first dye and the fluorophore 55 of the second dye are localized using light of wavelength A 111 and wavelength B 112, respectively. For the fluorophore 54 of the first dye, position L 56 is obtained using wavelength A 111 and position U 57 is obtained using wavelength B 112. Conversely, for the fluorophore 55 of the second dye, position L 56 is obtained using wavelength B 112 and position U 57 is obtained using wavelength A 112. This means that wavelengths A 111 and B 112 are chosen such that they well excite one dye and sufficiently excite the other, where the effect of the wavelength depends on the dye being considered. Now, taking into account two average differences 59 (from which, for example, a correctly signed average can be formed), the plurality of positions L 58 of each of the two dye categories are combined into a total set of positions 60, in which isolated fluorophores of the two dyes are co-localized. Here, as illustrated in the preceding figures, it is not necessary to obtain position U 57 for all isolated fluorophores used for imaging multicolor stained samples.

[0127] Fluoresceins from more than two dye classes can also co-localize. For example, this can be based on... Figure 18 The process shown in the diagram is used to complete this. For example... Figure 9 As shown, for each position 114, a detection signal 115, each including a light intensity value 50, can exist. Therefore, for example, dye molecules of both dye classes can be well localized using a common wavelength, and they can be distinguished based on the light intensity value 50. Advantageously, for both dye classes, the light intensity value 50 can also be recorded for all excitable isolated fluorophores using excitation light of a second wavelength, for example, by obtaining position U 57 for each excitable isolated fluorophore. For example, from... Figure 16 and Figure 17 Another possibility can be derived from the summary.

[0128] Here, according to Figure 19This paper describes another possibility for co-localizing fluorophores of more than two dye classes using the method of the present invention. As mentioned above, for co-localization, if the fluorophore of the second dye is localized using excitation light of one of the two wavelengths, then it is sufficient to localize one of the two dye classes using excitation light of both wavelengths. Now in Figure 19 The diagram illustrates the relationship between the fluorophores 54 of the first dye, 55 of the second dye, and 155 of another dye, and wavelengths A 111, B 112, and 116. Solid lines indicate that wavelength A 111 is well-suited for locating the fluorophore 54 of the first dye, wavelength B 112 is well-suited for locating the fluorophore 55 of the second dye, and wavelength 116 is well-suited for locating the fluorophore 155 of the other dye. Furthermore, wavelength A 111 is sufficiently suitable for locating the fluorophore 55 of the second dye, and wavelength B 112 is sufficiently suitable for locating the fluorophore 54 of the first dye to detect the difference 61 in position obtained using different wavelengths; this is indicated by dashed lines. Wavelength B 112 is sufficiently suitable for locating the fluorophore 155 of the other dye to detect the difference 61 in position obtained using different wavelengths (here, wavelength B 112 and wavelength 116). The difference 61 between the individual determined positions allows all positions to be combined into a total set 60. Here, for each position in the total set 60, information about the dye category can also exist, which in each case can be obtained from the detection signal 115 having a light intensity value 50, as described in the appropriate context.

[0129] Figure 20A microscope 80 according to the invention is schematically shown. In the illustrated embodiment, the microscope 80 has a control unit 89, into which a selection unit 87 and a calculation unit 88 are integrated. The selection unit 87 is configured to select the wavelength for excitation based on detection signals 51, 52, 115 regarding the fluorescence emission of a single fluorophore. The calculation unit is configured to determine the position and the difference between the positions based on the two detection signals 51, 52, 115 regarding the fluorescence emission of a single fluorophore. Selecting the wavelength based on the detection signals 51, 52, 115 includes evaluating further information such as a reference value 27 or a further detection signal 115 during selection 33. The selection unit 87 is associated via control lines with laser units 81 and 82, the laser unit 81 being configured to emit narrowband light of two wavelengths and the laser unit 82 being configured to emit narrowband light of a third wavelength different from the two wavelengths of the first laser unit 81. Furthermore, the microscope 80 has a detection unit 86 capable of detecting fluorescence emitted from a sample. The detection unit 86 is connected to the selection unit 87 via the control unit 89. Narrow-band lights 83, 84, and 85 are coupled into the optical path 93 via beam couplers 90 and 90', and the light can be guided onto or into the sample through the optical path 93. The basic structure of various microscopes is known to those skilled in the art. Therefore, descriptions of other components (e.g., the optical path 93) are omitted here. Fluorescence 92 emitted from the sample can be guided to the detection unit 86 via the optical path 93. In addition, the microscope 80 also has a calculation unit 88, which is configured to determine positions 12, 22 and position differences 61 based on two detection signals 51, 52, and 115 regarding the fluorescence emission of a single fluorophore. Here, the wavelengths of narrowband lights 83, 84, and 85 include two wavelengths, the difference between which is between 200 nm and 50 nm. For example, narrowband light 85 could be light with a wavelength of 488 nm or 525 nm, narrowband light 83 could be light with a wavelength of approximately 525 nm, and narrowband light 84 could be light with a wavelength of approximately 650 nm. Here, narrowband light refers to light with a spectral width in the range of a few nanometers. If narrowband lights 83, 84, and 85 are configured to excite fluorophores by two-photon absorption or, more generally, multi-photon absorption, the bandwidth of narrowband lights 83, 84, and 85 can reach 50 nm. If narrowband lights 83, 84, and 85 are configured to excite fluorophores by single-photon absorption, the narrowband lights typically have a bandwidth in the range of less than 20 nm, preferably less than 15 nm, or less than 10 nm.

[0130] Reference tag list 。

Claims

1. A positioning microscope method for studying samples stained with multiple dyes, the method comprising: First excitation and detection (10), wherein an excitation light of a first wavelength is applied to a sub-region (1) of the sample, and wherein fluorescence (92) emitted by isolated fluorophores of the dye due to excitation with the first wavelength is detected from the sub-region (1) of the sample, and a first detection signal (51) is obtained. The isolated fluorophore is first located (11) and the first position is obtained (12). The second excitation and detection (20) involves applying excitation light of a second wavelength to a sub-region (1) of the sample, and detecting fluorescence (92) emitted by isolated fluorophores of the dye due to excitation with the second wavelength from the sub-region (1) of the sample, and obtaining a second detection signal (52). The isolated fluorophore is then repositioned (21) and a second position is obtained (22). Determine the difference (61) between the first position (12) and the second position (22).

2. The method according to claim 1, characterized in that, The sub-region (1) of the sample is a small field of view (42) with a range of less than 3 µm, and the excitation light of the first wavelength and the excitation light of the second wavelength are focused light (3) with a focal point and intensity distribution.

3. The method according to claim 2, characterized in that, The intensity distribution of the focused light (3) of the first wavelength has a central minimum (5), and the first excitation is performed by positioning the focus of the excitation light of the first wavelength at a position within the sub-region (1). Further, the detection is characterized by detecting fluorescence (92) emitted by the isolated fluorophore due to excitation with the excitation light of the first wavelength, detecting the fluorescence (92) in a plane conjugate to the plane where the focus is located with a detector having spatial resolution, or positioning the detection aperture at multiple positions around the optical axis in a plane perpendicular to the orientation of the optical axis.

4. The method according to claim 2, characterized in that, The intensity distribution of the focused light (3) of the first wavelength has a central maximum value (4), and the first excitation and detection (10) is performed by positioning the focus of the excitation light of the first wavelength at a series of locations within the sub-region (1), wherein the excitation light of the first wavelength is applied to the sub-region (1) of the sample.

5. The method according to any one of claims 2 to 4, characterized in that, The intensity distribution of the focused light (3) of the second wavelength has a central minimum (5), and the second excitation is performed by positioning the focus of the excitation light of the second wavelength at a series of nominal positions surrounding the first position (12) within the sub-region (1), wherein the surrounded region has a first range.

6. The method according to claim 5, characterized in that, The method includes: To further excite a sub-region (1) of the sample, a wavelength (33) is selected from a predetermined set (117) of at least two wavelengths based on the first detection signal (51) and / or the second detection signal (52), such that the selected wavelength is obtained; and Following the selection (33), further excitation and detection (15) are performed, wherein excitation light of the selected wavelength is applied to a sub-region (1) of the sample, and wherein fluorescence (92) emitted by isolated fluorophores of the dye due to excitation with the selected wavelength is detected from the sub-region (1) of the sample, and a further detection signal is obtained. Along with the obtained further detection signal (115), the isolated fluorophore is further located (113) and its further position is obtained (114).

7. The method according to claim 6, characterized in that, The selection of the wavelength (33) is performed by evaluating (32) the first detection signal (51) and / or the second detection signal (52) for the dye, the isolated fluorophores of the dye having emitted detected fluorescence (92).

8. The method according to claim 6, characterized in that, The selected wavelength is either the first wavelength or the second wavelength.

9. The method according to claim 7, characterized in that, The selected wavelength is either the first wavelength or the second wavelength.

10. The method according to any one of claims 6 to 9, characterized in that, The selected wavelength excitation light is a focused light (3) with a focal point and an intensity distribution having a central minimum (5), and the further excitation and detection (110) is performed by positioning the focal point of the selected wavelength excitation light at a series of nominal positions within the sub-region, the series of nominal positions surrounding a central position calculated based on the first position (12) and / or the second position (22), wherein, within the sub-region, the enclosed region has a second range smaller than the first range, wherein, If the selected wavelength is the first wavelength, then the center position is the first position. If the selected wavelength is the second wavelength, then the center position is the second position. If the selected wavelength is another wavelength between the first wavelength and the second wavelength, the center position is determined by interpolation from the first position and the second position; and If the selected wavelength is greater than the first wavelength and the second wavelength or less than the first wavelength and the second wavelength, the center position is determined by extrapolation from the first position (12) and the second position (22).

11. The method according to any one of claims 2-4 and 6-9, characterized in that, The first excitation and detection (10) terminates after a predetermined time span (40) or when the light intensity value (50) of the first detection signal (51) reaches a threshold.

12. The method according to claim 6, characterized in that, The selected wavelength is the first wavelength, and the further excitation and detection (15) and the further localization are performed on the isolated fluorophore continuously or iteratively until a further position with a maximum of a predetermined uncertainty is obtained, wherein in this case the further position is the first position (12), and the second excitation and detection (20) and the second localization (21) are performed only after the further excitation and detection (15) is terminated.

13. The method according to claim 2, wherein, The sub-region (1) of the sample is a small field of view region (42) with a range of less than 2 µm.

14. The method according to claim 3, wherein, The intensity distribution of the focused light (3) of the first wavelength is basically a circular distribution or a local hollow beam distribution.

15. The method according to claim 4, wherein, The intensity distribution of the focused light (3) of the first wavelength is basically Gaussian.

16. The method according to claim 5, wherein, The intensity distribution of the focused light (3) of the second wavelength is basically a circular distribution or a local hollow beam distribution.

17. The method according to claim 10, wherein, The intensity distribution is essentially a circular distribution or a locally hollow beam distribution.

18. A positioning microscopy method for studying samples stained with multiple dyes. Its features are, For a set of sub-regions (1) of the sample, the method according to any one of claims 2 to 17 is performed, wherein the set of sub-regions (1) obtained in this way includes both the sub-region (1) where fluorescence (92) is emitted and detected by the corresponding isolated fluorophore of the first dye and the sub-region where fluorescence (92) is emitted and detected by the corresponding isolated fluorophore of the other dye.

19. The method according to claim 18, characterized in that, Based on the positions (12, 22, 56, 57, 114) of the isolated fluorophores obtained with excitation light of different wavelengths and the differences between these positions (61), a map with correction vectors is determined for co-registering all positions in a common spatial reference frame.

20. The method according to claim 19, characterized in that, For each of the locations (12, 22, 56, 57, 114), information about the time when the excitation and probing (10, 15, 20, 110) for localization occurs is stored, and the map with the correction vector is a time-coordinate-dependent map.

21. The method according to claim 19 or 20, characterized in that, An image of the sample is generated based on the first position (12) and the second position (22), and / or the first position and a further position, and / or the second position and a further position.

22. The method according to claim 19, characterized in that, The sub-region (1) of the sample is a wide field of view region, and the detection is to image the sub-region (1) onto a wide field of view detector, wherein the fluorescence emitted by the isolated fluorophores is detected on the wide field of view detector in a manner with spatial resolution, thereby obtaining a detection signal with spatial resolution, wherein during the first excitation and detection (10) and the second excitation and detection (20), the fluorescence emitted by multiple isolated fluorophores as a whole due to excitation with excitation light of the corresponding wavelength is detected from the sub-region (1) of the sample, and a first detection signal and a second detection signal (51, 52) with spatial resolution are obtained for each of the multiple isolated fluorophores.

23. The method according to claim 22, characterized in that, The method includes: Based on the corresponding first and second detection signals (51, 52) with spatial resolution, multiple isolated fluorophores were first and second located (11, 12), and the first and second positions (12, 22) were obtained respectively; and Determine the difference (61) between the corresponding first position (12) and the corresponding second position (22).

24. The method according to claim 22, characterized in that, The first excitation and detection (10) and the second excitation and detection (20) are performed simultaneously.

25. The method according to claim 23, characterized in that, The first excitation and detection (10) and the second excitation and detection (20) are performed simultaneously.

26. The method according to any one of claims 22-25, characterized in that, The method includes: To further excite the sub-region (1) of the sample, a wavelength (33) is selected from a predetermined set (117) of at least two wavelengths, based at least on the set of the first and second detection signals (51, 52), such that the selected wavelength is obtained; and Further excitation and detection (110) are performed after the selection (33), wherein excitation light of the selected wavelength is applied to a sub-region (1) of the sample, and wherein fluorescence (92) emitted by multiple isolated fluorophores due to excitation with the selected wavelength is detected from the sub-region (1) of the sample, and further detection signals (115) are obtained respectively. The multiple isolated fluorophores were further located (113) based on the further detection signal (115), and their further positions (114) were obtained respectively. The wavelength (33) is selected by evaluating (32) the set of the first and second detection signals (51, 52) for the category of the dye, the isolated fluorophore of the dye having emitted detected fluorescence (92), which caused most of the set of the obtained detection signals.

27. The method according to claim 26, characterized in that, The selected wavelength is either the first wavelength or the second wavelength.

28. The method according to claim 19 or 20, characterized in that, An image of the sample is generated based on the first position (12) and the second position (22), and / or the first position and a further position, and / or the second position and a further position, and other positions obtained by excitation and detection with excitation light of the first wavelength or the second wavelength or one of the other wavelengths.

29. A positioning microscopy method for studying samples stained with multiple dyes, the method comprising: Test excitation and detection (110), wherein excitation light of test wavelength (25) is applied to a sub-region (1) of the sample, and wherein fluorescence (92) emitted by isolated fluorophores due to excitation with the excitation light of test wavelength (25) is detected from the sub-region (1) of the sample, and a test detection signal is obtained. In order to subsequently excite the sub-region (1) of the sample, at least based on the test detection signal, a first wavelength is selected (33) from a predetermined set (117) of at least two wavelengths and / or a small field of view region (42) is selected (43) within the sub-region (1). First excitation and detection (10), wherein excitation light of the first wavelength is applied to a sub-region (1) of the sample or a small field-of-view region (42) within the sub-region (1) of the sample, and wherein fluorescence (92) emitted by the isolated fluorophore due to excitation with the first wavelength is detected from the sub-region (1) of the sample or from the small field-of-view region (42) within the sub-region (1) of the sample, and a first detection signal is obtained. The isolated fluorophore is first located (11) and the first position is obtained (12). (33) A second wavelength is selected from a predetermined set of at least two wavelengths to subsequently excite a subregion of the sample. The second excitation and detection (20) involves applying excitation light of the second wavelength to a sub-region (1) of the sample, and wherein fluorescence (92) emitted by the isolated fluorophore due to excitation with the second wavelength is detected from the sub-region (1) of the sample or a small field-of-view region (42) within the sub-region (1) of the sample, and a second detection signal (52) is obtained. The isolated fluorophore is second-localized (21) and a second position is obtained (22); and Determine the difference (61) between the first position (12) and the second position (22).

30. The method according to claim 29, characterized in that, The method includes: In order to subsequently excite the sub-region (1) of the sample, at least based on the test probe signal, a first wavelength (33) is selected from a predetermined set (117) of at least two wavelengths. Before selecting (33) the first wavelength from a predetermined set (117) of at least two wavelengths and after the test excitation and detection, a second test excitation is performed, wherein excitation light of the second test wavelength (26) is applied to a sub-region (1) of the sample or a small field-of-view region (42) within the sub-region (1) of the sample, and wherein fluorescence (92) emitted by an isolated fluorophore due to excitation with the second test wavelength (26) is detected from the sub-region (1) of the sample or from the small field-of-view region (42) within the sub-region (1) of the sample, and a second test detection signal is obtained, and the selection (33) of the first wavelength is based at least on the test detection signal and the second test detection signal, wherein the test detection signal and / or the second test detection signal are the light intensity values ​​(50) of the detected fluorescence (92).

31. The method according to claim 30, characterized in that, The selection (33) of the first wavelength is based on a comparison of the test detection signal and / or the second test detection signal with a predetermined reference value (27) or a plurality of predetermined reference values ​​(27), or is based on the ratio of the test detection signal to the second test detection signal.

32. The method according to any one of claims 29-31, characterized in that, The sub-region (1) of the sample is a small field of view region (42).

33. The method according to any one of claims 29-31, characterized in that, After the test excitation and detection, a small field of view region (42) is selected within the sub-region (1) (43).

34. The method according to claim 32, characterized in that, The first positioning (11) and / or the second positioning (21) are performed within the small field of view (42) according to the MINFLUX method.

35. The method according to claim 33, characterized in that, The first positioning (11) and / or the second positioning (21) are performed within the small field of view (42) according to the MINFLUX method.

36. The method according to claim 32, wherein, The small field of view region (42) has a range of less than 3 µm.

37. The method according to claim 36, wherein, The small field of view region (42) has a range of less than 2 µm.

38. The method according to claim 33, wherein, The small field of view region (42) has a range of less than 3 µm.

39. The method according to claim 38, wherein, The small field of view region (42) has a range of less than 2 µm.

40. A positioning microscopy method for studying samples stained with multiple dyes. Its features are, For the set of small field-of-view regions (42) of the sample, the method of any one of claims 32-39 is performed, wherein the set of small field-of-view regions (42) obtained in this way includes both the small field-of-view region (42) where fluorescence (92) is emitted and detected by the corresponding isolated fluorophore of the first dye and the small field-of-view region where fluorescence (92) is emitted and detected by the corresponding isolated fluorophore of the other dye.

41. The method according to claim 40, characterized in that, Based on the positions (12, 22, 56, 57, 114) of the isolated fluorophores obtained with excitation light of different wavelengths and the differences between these positions (61), a map with correction vectors is determined for co-registering all positions in a common spatial reference frame.

42. The method according to claim 41, characterized in that, For each of the locations, information about the time when the excitation and probing for localization occurred is stored, and the map with the correction vector is a time-coordinate-dependent map.

43. The method according to claim 41 or 42, characterized in that, An image of the sample is generated based on the first position and the second position, in which fluorophores of different dye classes are co-located.

44. The method according to claim 41 or 42, characterized in that, An image of the sample is generated based on the first position and the second position, as well as other positions obtained by excitation with excitation light of the first wavelength or the second wavelength, in which fluorophores of different dye classes are co-located.

45. The method according to claim 41 or 42, characterized in that, An image of the sample is generated based on the first position and the second position, as well as other positions obtained by excitation with excitation light of the first wavelength or the second wavelength or another wavelength, in which fluorophores of different dye classes are co-located.

46. ​​A microscope (80) for performing the method according to any one of claims 1-45, the microscope having: Laser units (81, 82) for exciting fluorescence, the laser units being configured to emit narrowband light of two wavelengths, or two laser units (81, 82) for exciting fluorescence (92), the laser units being configured together to emit narrowband light of two wavelengths (83, 84, 85). Detection unit (86) Selection unit (87), the selection unit being configured to select the wavelength for excitation based on a detection signal of fluorescence emission with respect to a single fluorophore. A calculation unit (88) is configured to determine the position and the difference between the positions (61) based on two detection signals of fluorescence emission of a single fluorophore.

47. The microscope (80) according to claim 46, characterized in that, The difference between the two wavelengths is between 200 nm and 50 nm.

48. The microscope (80) according to claim 46 or 47, characterized in that, The microscope has laser units (81, 82) configured to emit narrowband light of a third wavelength different from the two wavelengths.

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