Methods for locating individual dye molecules in a sample and methods for generating high-resolution images of the structure in the sample.

Abstract

This invention relates to methods for generating high-resolution images of structures in a sample or for locating individual molecules of fluorescent dyes in a sample, and the use of fluorescent dyes in such methods. The method according to the invention is characterized in that a fluorescent dye is first formed from a protected non-fluorescent form of the dye in a photoactivation reaction prior to scanning the fluorescent dye with excitation and fluorescence suppression light, the photoactivation reaction comprising at least two reaction steps, and the protected non-fluorescent form of the dye is inert to both excitation and fluorescence suppression light.

Description

Technical Field

[0001] This invention relates to methods for generating high-resolution images of structures and methods for locating individual molecules of fluorescent dyes in a sample. For this purpose, the sample or a fragment of the sample is scanned with an intensity distribution having local minima by an excitation light and a fluorescence suppression light, the fluorescence suppression light preventing, reducing, or completely suppressing fluorescence emission from the fluorescent dye. Compared to related methods known in the prior art, the method according to the invention is characterized in that, prior to scanning the fluorophore with excitation light and fluorescence suppression light, a fluorophore is first formed from the protected non-fluorescent form of the dye in a photoactivation reaction comprising at least two reaction steps, and the protected non-fluorescent form of the dye is inert to both the excitation light and the fluorescence suppression light. Existing technology

[0002] STED microscopy is understood as a method of laser scanning microscopy that enables high-resolution spatial imaging of samples labeled with fluorescent dyes. Here, the sample is scanned with focused excitation and stimulation light, wherein the intensity distributions of the excitation and stimulation light are substantially complementary, and the intensity distribution of the stimulation light has a zero point at the location of the maximum intensity of the excitation light. At high-intensity locations, the stimulation light inhibits fluorescence emission from the fluorescent dye, thus confining fluorescence emission to a small area around the zero point of the stimulation light intensity distribution and reducing the size of the effective point spread function.

[0003] As an improvement to STED microscopy, DE 10 2013 100 172 A1 discloses a method for STED microscopy in which, before illumination with excitation and stimulation light, the sample in the measurement region is additionally (like the stimulation light) illuminated with an intensity distribution of fluorescence suppression light having local minima. This fluorescence suppression light causes the fluorophore to transition from a fluorescent state to a protective state, in which the fluorophore is protected from excitation by the excitation and stimulation light photoelectrons. Here, the primary objective of this method is not to improve the resolution of the microscope by superimposing the intensity distributions of the stimulation and fluorescence suppression light; rather, it aims to reduce photobleaching of the fluorophore by the excitation and stimulation light, particularly in the high-intensity regions of the stimulation light, by transforming the fluorophore, especially in these regions, into an unexciteable protective state, thereby protecting it from the bleaching effects of the excitation and stimulation light. A prerequisite for using this method is the availability of a suitable fluorophore that can temporarily transition to a protective state, i.e., photo-switching. For example, the requirements for switching contrast are not as stringent as those for RESOLFT microscopes, but the protective state must be (essentially) inert to excitation and stimulation light—a requirement that is not met or only insufficiently met in practice for many photoswitching fluorophores.

[0004] Under the general term "localization microscopy," other related methods for high-resolution fluorescence microscopy are known. These methods are based on the high-precision spatial localization of individual fluorescent dye molecules and the reconstruction of high-resolution images from the localization of these individual molecules. A prerequisite for using these methods is that the fluorescent dye molecules exist in the sample in an individual form at any given time point, i.e., spatially separated from each other, and thus appear as individual objects in the camera image. While spatially separated fluorescent dye molecules can be achieved, for example, by using a correspondingly high dye dilution when labeling the sample, to generate high-resolution images of structures that are as spatially continuous as possible, it is necessary to label the structure with many fluorescent dye molecules arranged at a high density and to determine the location of a sufficiently large fraction (typically thousands) of these fluorescent dye molecules. The labeling density of the fluorescent dye molecules must be high, meaning that during the localization of the dye molecules, only a small fraction of all dye molecules are always allowed to exist in a fluorescent state in order to satisfy the requirement that the fluorescent molecules exist individually and spatially isolated. Therefore, photoswitched fluorescent dyes are used in positioning microscopes. These dyes possess a fluorescent state, in which they can be excited to fluorescence by excitation light of a suitable wavelength, and a dark state, in which they cannot be excited to fluorescence. Here, the dye can be photoactivated at least once, i.e., transitioning from the dark state to the fluorescent state. Photoactivation is typically lichtinduziert, i.e., by irradiating the sample with photoactivating light of a suitable wavelength (typically in the blue-violet spectral range), thereby allowing for precise adjustment and control of the proportion of photoactivated dye molecules. Alternatively, dye molecules can also spontaneously transition to the activated state. Depending on the type of fluorescent dye, activation can also be reversible, i.e., undergoing multiple activation-deactivation-switching cycles, where deactivation or transition to a non-fluorescent state can also occur photoinduced or spontaneously. A typical switching mechanism is a (reversible) transition from the fluorescent state to a transient dark state (e.g., to a triplet state). In particular, the switching kinetics of these transitions can be adapted to specific requirements in the sample by adjusting the solvent composition, adding redox reagents, and / or controlling the oxygen concentration.

[0005] The positioning accuracy achievable in a positioning microscope depends on multiple factors, and, for example, as demonstrated by RE Thompson et al. Biophys. J. 82, 2775 (2002) gives:

[0006]

[0007] in, The width (standard deviation) of the PSF, which is an approximation of the microscope as a Gaussian function. The number of fluorescent photons detected in the dye molecule. The edge length of the detector pixel and The background signal consists of background fluorescence and detector noise.

[0008] As long as the positioning accuracy is not affected by the size of the detector pixels Or background signal The obvious limitation, equation (1) shows, is that at a smaller width Under (effective) PSF, for a given number of photons This can achieve higher positioning accuracy or lower positioning uncertainty. Or use a lower number of photons Ideal positioning accuracy can be achieved. For example, a narrower effective PSF (with a smaller value) can achieve this. This can be achieved by irradiating the sample not with a uniformly distributed excitation light planarly, but rather, similar to STED microscopy, with focused excitation and stimulation light that scans the image field using a scanning device. Here, the intensity distribution of the stimulation light, with local minimums, is superimposed on the Gaussian excitation focus, which suppresses fluorescence emission in the edge regions of the excitation focus, thereby reducing the width of the effective PSF. Since scanning the entire image field is naturally slower than planar wide-field illumination, in some cases the recording scheme must be adjusted so that the image field is not scanned as a whole, but in segments, for example, segments are selected such that each segment contains a fluorescent dye molecule.

[0009] A newer MINFLUX nanomirror, first described by F. Balzarotti et al. in arXiv 1611.03401 [physics.optics], combines features of the aforementioned methods. Here, a spatially isolated fluorescent molecule is irradiated at a series of different locations with an intensity distribution of excitation light having an intensity minimum. For each irradiation location, fluorescence emission excited by the excitation light is recorded, and the location of the fluorescent molecule is inferred from the amount of the recorded fluorescence intensity value. Of course, this location determination has uncertainty, which can be reduced by iteratively applying the method. To this end, the irradiation location is adjusted before each iteration step, i.e., closer to the corresponding assumed location of the molecule. Simultaneously, the intensity of the excitation light is increased, such that the intensity gradient increases near the intensity minimum. Alternatively, the measurement duration can be increased, which is equivalent to an increase in the excitation light intensity calculated in terms of effective light quantity. Using the adjusted parameters, the molecule is sequentially irradiated at each adjusted irradiation location, and the intensity of fluorescence emission is recorded. Based on the dependence of the fluorescence signal on the location of the intensity minimum, the location of the molecule can now be determined with lower uncertainty than before. These steps can be repeated until the location is determined and converges or until another interruption criterion is reached, such as a pre-determined maximum acceptable uncertainty. The MINFLUX method achieves a localization accuracy of approximately 1 nm, and based on current technology, it represents the most accurate commercially available fluorescent molecule localization method.

[0010] Document WO 2015 / 097000 A1 further discloses a (high-resolution) image of the molecular distribution in a sample obtained from the positional data of individual molecules (“MINFLUX imaging”). This method corresponds to methods known from localization microscopy for generating high-resolution images from determinations of multiple positions of a single fluorescent molecule, but in the case of MINFLUX nanomirrors, the spatial resolution of the image is further improved to 5 nm or higher.

[0011] A modified version of the MINFLUX method is described in DE 10 2017 104 736 B3, in which the scanning of a solitary fluorescent dye molecule is not performed by irradiation with an intensity distribution of excitation light having local minimums, but rather by two substantially complementary intensity distributions of excitation and fluorescence suppression light. Here, the intensity distribution of the excitation light has local maxima, while the intensity distribution of the fluorescence suppression light has local minimums at the same locations. Specifically, the fluorescence suppression light can be an irradiation light that prevents excited fluorescent dye molecules from emitting fluorescent photons in the edge regions of the excitation light's intensity distribution by triggering stimulated emission. Thus, the excitation and fluorescence suppression lights are superimposed with such intensity distributions, as is also done in RESOLFT and STED microscopy. The intensity of fluorescence recorded for a given fluorescent dye molecule depends on its distance from the local minimum of the fluorescence suppression light, and the position of the fluorescent dye molecule can be determined with high precision by using fluorescence intensities recorded from multiple locations corresponding to the minimum of the fluorescence suppression light. In this modified version of the MINFLUX method, local minimum intensity can also be located at several sites within the sample, and the intensity of the recorded fluorescence can be evaluated using the same principles as in the MINFLUX nanomirror. However, the difference lies in the fact that in the MINFLUX nanomirror, the intensity of fluorescence from the fluorescent label increases with increasing distance from its location to the local minimum intensity, while in this modified version where the light source is fluorescence-blocking light, the fluorescence intensity decreases with increasing distance.

[0012] In summary, several variations of high-resolution fluorescence microscopy are known from the prior art, in which a stimulus light is used in addition to the excitation light to achieve high spatial resolution in imaging. These methods also require the use of photoactivated or photoswitched fluorescent dyes to label the sample. In practice, the applicability of these methods is limited because the photoactivated fluorescent dyes with photosensitive protective groups commonly used are not inert or not sufficiently inert, especially when irradiated with high-intensity stimulus light. This is particularly true if the stimulus light is used in the form of short laser pulses with very high peak intensity (preferred in STED microscopy). Then, because the photosensitive protective group also increases multiphoton absorption, the fluorescent dye can be photoactivated in an undesirable manner.

[0013] Invention Task

[0014] Therefore, the object of the present invention is to provide a method for high-resolution fluorescence microscopy, wherein a photoactivated fluorescent dye can be exposed to high-intensity stimulating light or other fluorescence-inhibiting light, and the photoactivated fluorescent dye is not activated by the stimulating light or fluorescence-inhibiting light in a manner that hinders the method, but rather is inert to the stimulating light or fluorescence-inhibiting light.

[0015] Solution

[0016] The object of the invention is achieved by the method according to independent claims 1, 14, and 20. Dependent claims 2 to 13, 15 to 19, and 21 to 33 relate to preferred embodiments of the method. Application claim 34 relates to the use of a photoactivated fluorescent dye in the method according to the invention.

[0017] The methods according to the invention are based on methods of high-resolution fluorescence microscopy known from the prior art, in which, in order to achieve high spatial resolution in imaging, fluorescence-blocking light or stimulation light is used in addition to excitation light. However, they decisively improve upon known methods by using photoactivation with a fluorescent dye, which can greatly reduce or completely suppress unintentional and interfering photoactivation caused by stimulation light or fluorescence-blocking light. Thus, the scope of application is greatly expanded compared to known methods, or the application of this method is realized first.

[0018] This invention is based on the applicant's understanding that protected non-fluorescent fluorescent dyes typically have a small absorption cross-section at the wavelength of the stimulating light. However, given that the intensity of the fluorescence-blocking light or stimulating light is sometimes very high, this small absorption cross-section is still sufficient to lead to undesirable photoactivation. Furthermore, two-photon or multi-photon absorption of the fluorescence-blocking light or stimulating light may occur, which can also lead to photoactivation. Some photoinstantaneous protecting groups used to prepare photoactivated fluorescent dyes have even been optimized for multi-photon absorption.

[0019] The stability of photoactivated fluorescent dyes relative to fluorescence-inhibiting or stimulating light can now be significantly improved by designing the photoactivation of protected, non-fluorescent dyes so that activation occurs not in a single reaction step, but in multiple reaction steps. Here, if each individual reaction step is photoinduced, and fluorescence is generated only after the last reaction step, photoactivation therefore requires the absorption of two (or more) photons, resulting in a nonlinear (i.e., quadratic, cubic, ...) dependence of the photoactivation rate on light intensity, similar to that of two / multiphoton fluorescence. As is known from two-photon fluorescence excitation, two-photon absorption is only meaningful in practice when using very short light pulses; this also applies to photoactivation when transitioning from single-photon absorption to two-photon absorption. Therefore, undesirable photoactivation can be suppressed, and incidentally, this also applies to the case of multiphoton absorption, where it becomes (or even) a higher-order absorption process.

[0020] While using dedicated activation light may reduce the probability of photoactivation, sufficient intensity is usually available, and in particular, it can be quantitatively supplied independently of the stimulus light. Due to the high order of the photoactivation process, photoactivation is confined to a small volume, and thus even allows for more precise spatial control of photoactivation. Invention Description

[0021] This invention relates to three methods linked by the same inventive concept for generating high-resolution images of structures in a sample, or for locating individual molecules of fluorescent dyes in a sample, and the use of fluorescent dyes in these methods. All three methods share the common feature of selecting photoactivated fluorescent dyes, wherein photoactivation comprises at least two corresponding photoinduced reaction steps. Furthermore, according to the invention, in all methods, the photoactivation of the fluorescent dye takes the form of an activation reaction comprising at least two photoinduced reaction steps, wherein a fluorophore is formed only after the final reaction step, and the dye acquires its fluorescent properties. Finally, in all methods according to the invention, the sample or a fragment of the sample is scanned with an intensity distribution having a minimum intensity value of excitation light and / or fluorescence suppression light.

[0022] The first method according to the present invention includes the following steps:

[0023] 1. A fluorescent dye is selected, the obtained fluorescent dye initially existing in a protected non-fluorescent form, and photoactivated by irradiating the fluorescent dye with activating light, thereby converting it from the protected non-fluorescent form to an activated fluorescent form. Fluorescence is understood herein as the ability of a dye to emit fluorescence when excited by light of a suitable wavelength, which is impossible in its non-fluorescent form. According to the invention, the fluorescent dye is selected such that photoactivation comprises at least two corresponding photoinduced reaction steps.

[0024] 2. Label the structure of interest in the sample with the selected dye. For this purpose, fluorescent labeling methods known in the art, including immunofluorescence labeling techniques, can be used.

[0025] Other steps of the method may be performed once or more, and include:

[0026] 3. By irradiating the sample with activating light, a (small) subset of the fluorescent dye is photoactivated from its protected non-fluorescent form to its activated fluorescent form. Here, this subset can be spatially defined and, for example, comprised of molecules of the fluorescent dye within a fragment or plane of the sample; however, it can also be statistically defined and include a random selection of all molecules. In particular, the subset can comprise individual molecules that are spatially separated from each other.

[0027] According to the invention, photoactivation is carried out in the form of an activation reaction comprising at least two photoinduced reaction steps, wherein the fluorophore is formed only after the final reaction step, and the dye acquires its fluorescent properties. Photoactivation significantly increases the fluorescence quantum yield of the dye, i.e., the probability that an electronically excited dye molecule releases its excitation energy in the form of fluorescent photons, rather than through a nonradiative process. However, since the fluorescence quantum yield of the protected form is typically very small, but not exactly zero, the fluorescence contrast obtained by photoactivation is practically limited. Therefore, as a fluorescence contrast, a ratio of at least 1:10 is generally sought, preferably at least 1:100, and ideally at least 1:1000.

[0028] Here, the design scheme of the photoactivation process in two or more reaction steps can be achieved through different reaction schemes. One possible implementation scheme of the photoactivation process is to combine multiple photoinstantaneous protecting groups with the functional groups of the fluorescent dye, such that the fluorophore can only be formed after all protecting groups (in parallel or sequential) have undergone photolytic cleavage. Potential binding sites are particularly hydroxyl, amino, carbonyl, and carboxyl groups contained in the fluorophore. For these functional groups, many photoinstantaneous protecting groups are known, such as o-nitrobenzyl, benzoin, benzoylmethyl, coumaric acyl, and arylmethyl groups, benzoin, arylsulfonamides, and substituted variants of these groups. For a comprehensive overview, see P. Klán et al. in their commentary " Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy "in Chem. Rev. As mentioned in 113, 119 (2013). Protecting groups of this type are also suitable for preparing photoactivated fluorescent dyes. Examples of protected rhodamine and fluorescein dyes are known to those skilled in the art from the prior art [see, for example, " Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells “S. Hauke” et al. , Chem. Sci. 8,559 (2017)】.

[0029] While most photoinstable protecting groups can be cleaved by ultraviolet light, an increasing number of protecting groups can be cleaved in the visible spectrum. These specifically include coumarin acyl protecting groups, which can be cleaved by blue light (400-500 nm), and dipyrrole methylene boron (BODIPY)-derived protecting groups, which can be cleaved by green light (>500 nm) (see, for example, "..."). BODIPY- Derived Photoremovable Protecting Groups Unmasked with Green Light "), PPGoswami et al., J. Am. Chem. Soc. 137, 3783 (2015). The wide spectral range of currently available protecting groups also allows for the selective cleavage of different protecting groups from the molecule; see " Wavelength-Selective Cleavage ofPhotoprotecting Groups: Strategies and Applications in Dynamic Systems “MJ Hansen et al., Chem. Soc. Rev. 44,3358 (2015).

[0030] The cleavage of protecting groups can be carried out using monochromatic activation light. In particular, if the photoinstantaneous protecting groups are chemically identical or at least cleavable by light of the same wavelength, the method can be implemented using only one activation source. This activation source can be a continuous source or a pulsed source. In the latter case, especially when using an ultrashort pulse laser as the activation source, photoactivation can also be initiated by two-photon or multi-photon absorption. However, alternatively, if the protecting groups are different and have different spectral absorption characteristics, the activation light can also include multiple wavelengths, particularly multiple wavelengths from multiple sources.

[0031] The high variability of protecting groups allows the design of photoactivated fluorescent dyes to be tailored to individual cases. Optimization can be achieved by minimizing photoactivation under either fluorescence suppression or stimulation light, while simultaneously providing the largest possible absorption cross-section for the activation light. In this regard, it is advantageous for protecting groups to differ chemically and spectrally in absorption characteristics. In this case, the activation light can also have multiple wavelength components, enabling the fragmentation of chemically different protecting groups that absorb in different spectral ranges. Here, activation light of different wavelengths can be emitted simultaneously or sequentially.

[0032] In the aforementioned variant, the cleavage of multiple protecting groups can occur simultaneously, in parallel time, or sequentially. A strictly sequential series of two photoinduced reaction steps also occurs in another advantageous variant of the method, where a so-called tandem reaction is used (which does not necessarily lead to the cleavage of the protecting groups). A tandem reaction is understood as a sequence of reactions consisting of multiple independent reaction stages (but not mechanical steps) proceeding in sequence. While tandem reactions are generally understood as reactions that occur spontaneously one after another, in the context of this document, the term also explicitly includes reactions whose steps are initiated individually and sequentially (particularly by the action of light). According to the invention, a fluorescent state is generated only after the second (or further) photoinduced stage of the tandem reaction. A. Paul et al. "o- Hydroxycinnamate for Sequential Photouncaging of Two Different Functional Groups and its Application in Releasing Cosmeceuticals”, Org. Biomol. Chem. 33 (2019) provides an example of such a cascade reaction:

[0033] .

[0034] Each stage of the cascade reaction can also be reversible, so that if subsequent reaction stages are not triggered, the initial or intermediate state of photoactivation can be restored.

[0035] 4. An intensity distribution of excitation light and fluorescence suppression light is formed in the sample, wherein the intensity distribution of fluorescence suppression light has local minimum intensity values. Preferably, as is common in STED microscopy, the intensity distribution of excitation light is formed in the form of a diffraction-limited focused spot, the (central) maximum intensity of which spatially superimposes with the minimum intensity of fluorescence suppression light. However, in principle, other intensity distributions of excitation light are also possible and suitable for implementing this method, such as a uniform distribution of excitation light in the sample.

[0036] The term "fluorescence suppression light" should be understood as any type of light suitable for blocking, reducing, or completely suppressing the fluorescence of activated fluorescent dye molecules when irradiated with excitation light. Specifically, fluorescence suppression light can be the stimulating light known from STED microscopy, which induces stimulated emission of electronically excited dye molecules, thereby causing the dye molecules to (return) to their electronic ground state and suppressing spontaneous fluorescence emission. As known from RESOLFT microscopy, fluorescence suppression light can also trigger photoinduced chemical reactions, particularly isomerization and cyclization / reversal reactions, which are accompanied by modulation of fluorescence emission.

[0037] The superposition of excitation light and fluorescence suppression light narrows the region where fluorescent dye molecules illuminated by the excitation light can emit fluorescence, thus reducing the size of the effective photoluminescence field (PSF). Here, according to common practice, analytical power... This can be understood as the distance between two identical point objects, for which the two point objects can still be separated and imaged as individual objects. The analytical capabilities obtained during scanning depend on the intensity of the fluorescence suppression light, and as is known to those skilled in the art from STED and RESOLFT microscopes, are given by the following formula:

[0038]

[0039] in The wavelength of the fluorescence blocking light. The refractive index of the optical material that forms the intensity distribution of the fluorescence-suppressing light, The half-angle of the optical component used to illuminate the sample. The intensity of the fluorescent suppressor light or stimulus light at the point of maximum intensity distribution, and A specific saturation strength for the dye. This saturation strength is typically defined by using an intensity of... The number or intensity of the fluorescence emitted by the dye is reduced by a factor of 2.

[0040] Here, the formation of the intensity distribution should not be understood as a completed step after the intensity distribution is canceled; rather, it should be understood as the intensity distribution being formed during subsequent scanning steps.

[0041] 5. The sample or a fragment of the sample is scanned at a series of scanning locations using a previously formed intensity distribution of fluorescence suppression light with local minimum intensity values, wherein the intensity or number of fluorescence at each scanning location is detected and correlated with the scanning location. Here, scanning along a regular, particularly Cartesian or hexagonal grid is preferred; however, this is not mandatory. In particular, scanning can also be performed adaptively, for example, by scanning the sample or a fragment of the sample along the grid with relatively large step sizes, and only scanning with smaller step sizes, if necessary, matching the structure, in those regions where fluorescence emission is detected. If regions with such structures can be pre-identified (e.g., from fluorescence images of the sample or a fragment of the sample), then scanning outside these regions can also be completely abandoned.

[0042] 6. A high-resolution rasterized image of the structure is generated based on the interrelated photon counts or intensities of fluorescence and the scan positions, wherein brightness values ​​are assigned to each image pixel of the rasterized image. In the simplest case, there is a direct one-to-one relationship between the scan positions and the image pixels of the rasterized image; that is, the photon count or (digitized) intensity value detected at the corresponding scan position is directly assigned as a brightness value to each image pixel. However, the number of scan positions and the number of image pixels do not need to be strictly consistent; it is possible to combine the photon counts or intensities detected at multiple scan positions into a single image point, just as it is possible to interpolate image pixels from the photon counts or intensities of adjacent scan positions.

[0043] To improve the dynamic range of a grid image, to highlight weakly fluorescent structures, or to match the brightness value to the display characteristics of a display device or the perceptual characteristics of the human eye, the brightness value may optionally be a (monotonic) function of the photon number or intensity value, for example, in the form of a logarithmic function or a gamma function.

[0044] In a preferred embodiment of the method, the fluorescence suppressor light specifically has a ring-shaped intensity distribution with local intensity minima in the form of a central zero point, while the excitation light forms a typically diffraction-limited Gaussian focal point, such that the minimum intensity of the fluorescence suppressor light and the maximum intensity of the excitation light spatially coincide. Methods for forming such a ring-shaped intensity distribution are known to those skilled in the art from the prior art; exemplarily, only the arrangement of a spiral phase plate (vortex phase plate) in the fluorescence suppressor light beam is mentioned here. In this embodiment, the intensity distributions of the excitation light and the fluorescence suppressor light are substantially complementary; that is, the intensity of the fluorescence suppressor light is low where the excitation light has high intensity, and vice versa.

[0045] In a parallel variant of this implementation, alternatively, multiple, and if necessary, even a very large number, corresponding intensity distributions of excitation and fluorescence suppression beams can be generated by pairwise interference of four excitation and fluorescence suppression beams, forming two mutually orthogonal standing waves [see "Nanoscopy with more 100,000' doughnuts", A. Chmyrov et al.]. Nature Meth. [10,737 (2013)]. Here, the standing waves of the excitation light and the fluorescence suppression light are phase-shifted to each other, thus generating essentially complementary intensity distributions.

[0046] To position the intensity distribution of the excitation and suppression fluorescence at the scanning location, a beam deflection device arranged in the optical path can be used, for example, by utilizing a galvanometer. For higher scanning speeds and especially for irregularly arranged scanning positions, electro-optic or acousto-optic deflectors are alternatively suitable, as they operate without moving parts and allow for particularly rapid beam deflection.

[0047] Here, fluorescence detection can be performed using a point detector, detector array, or camera, depending on the intensity distribution of the excitation light and the fluorescence suppression light. Due to their sensitivity, avalanche photodiodes or avalanche photodiode arrays operating in photon counting mode, as well as (hybrid) photomultiplier tubes or integrating detectors such as CCD and sCMOS cameras, are particularly suitable. In the case of point illumination using excitation light and detection using a point detector, the point detector is preferably positioned behind a confocal pinhole aperture to suppress scattered and background light. However, in the case of point illumination, a detector array can also advantageously be used to record fluorescence.

[0048] If needed, the sample can be repeatedly irradiated with excitation and fluorescence suppression light, and fluorescence at the scan position can be detected, for example, to improve the signal-to-noise ratio or record a time series. Optionally, additional dye molecules can be photoactivated before rescanning, for example, to replace the dye that faded during the scan. Alternatively, it may be desirable to activate a completely different subset of fluorescent dyes. In this case, the (remaining) fluorescent dye molecules must first be deactivated again, which in the simplest case can be achieved by fading with strong excitation light.

[0049] The present invention also relates to a method for locating individual molecules of fluorescent dyes in a sample. The method includes photoactivating individual molecules of fluorescent dyes in an activation reaction comprising two reaction steps, scanning these molecules with an intensity distribution of excitation light and an intensity distribution of fluorescence suppression light, and probing fluorescence at each scan location. During this scan, one of the two intensity distributions may remain stationary, and at least one of the two intensity distributions may be sequentially located at multiple scan locations. A new position estimate of the fluorescent molecule is calculated from the fluorescence intensity and scan location relative to a previous position estimate. If a stationary fluorescent dye molecule is located, the new position estimate is an improved position estimate, i.e., one that estimates the actual position of the relevant fluorescent dye molecule with lower uncertainty. If a moving fluorescent dye molecule is observed, it may be a value that estimates a new, altered position of the relevant fluorescent dye molecule, respectively. The steps of the method are as follows:

[0050] 1. A fluorescent dye is selected, the obtained fluorescent dye initially existing in a protected non-fluorescent form, and photoactivated by irradiating the fluorescent dye with activating light, thereby converting it from the protected non-fluorescent form to an activated fluorescent form. According to the invention, the fluorescent dye is selected such that photoactivation comprises at least two corresponding photoinduced reaction steps.

[0051] The method also includes a first set of method steps:

[0052] 2. By irradiating the sample with activation light, the fluorescent dyes are spaced as far apart as possible. One or more molecules are photoactivated from a protected non-fluorescent form to an activated fluorescent form. According to the invention, as with the first method according to the invention, photoactivation is carried out via an activation reaction comprising at least two photoinduced reaction steps, wherein the fluorophore is formed only after the final reaction step, and the dye acquires its fluorescent properties. The requirements and design of the photoactivation process are the same as those described in the specification of the first method according to the invention.

[0053] To ensure the minimum distance between multiple activated dye molecules Precise control of the spatial density of activated dye molecules during photoactivation is essential. To this end, the irradiation parameters of the activation light (i.e., particularly the irradiation duration and intensity) can be determined one-time through trial and error, ensuring the presence of spatially isolated dye molecules for a given sample and under given recording conditions. If the sample can be continuously observed during photoactivation, for example by exposing it to excitation light where fluorescence is detected, photoactivation can also be performed under direct control, and irradiation can be stopped when a molecular density limit is reached. If activation occurs in a locally confined region, activation can be stopped if the fluorescence signal exceeds a threshold (which can also be 0) during observation. Controlled activation is particularly advantageous during data recording, where the total amount of dye decreases due to photobleaching and the irradiation duration or intensity must be increased to maintain a constant density of activated dye molecules or to activate dye molecules in locally confined regions. Alternatively or additionally, the distance between activated dye molecules can be controlled by irradiating the sample not with a large and uniform area of ​​activation light, but with an irradiation pattern, for example, irradiation points in a grid.

[0054] 3. Determine initial position estimates for one or more activated dye molecules as starting points for subsequent scanning and probing steps of the method. Determine initial position estimates such that their uncertainty is at most [value missing]. This allows us to definitively determine the initial position estimate and the position that is at least a distance away from each other. It is associated with the activated dye molecules.

[0055] The initial position estimate can, in principle, be determined in different ways. In particular, the position estimate can be determined by locating the activated dye molecules from a fluorescence image of the sample or sample fragment. Fluorescence images recorded using a camera or with the aid of confocal laser scanning are particularly suitable for this. In a more specific variation of this method, the initial position estimate can also be determined using an STED microscope, which can, if necessary, achieve a higher spatial density of the activated dye molecules.

[0056] The initial position estimate determined using confocal or STED microscopy can also be modified. Instead of scanning the entire image field to (subsequently) identify and locate individual dye molecules in these images, the sample is scanned until a single activated dye molecule is detected, and its position can be approximated from the corresponding scan position. Subsequent scanning and detection steps can then follow immediately after the detection of dye molecules; therefore, these steps do not necessarily have to be performed strictly separately but can be alternately. Similarly, photoactivation can be combined with scanning. For this purpose, the sample can be scanned point-by-point and line-by-line with activation and excitation light (simultaneously or as a rapid sequence) until a single activated dye molecule is detected. Scanning and detection steps can again follow immediately after the detection of dye molecules.

[0057] In particular, if activation is performed in a locally narrow region using an activation beam with diffraction-limited focusing, the initial position estimate can also be determined directly from the position of the known focal point of the activation beam; this position is then used as the initial position estimate.

[0058] 4. Establish intensity distributions for both excitation and fluorescence suppression light in the sample. Excitation light is chosen based on its wavelength to excite an activated fluorescent dye to emit fluorescence, while the term "fluorescence suppression light" should be understood as any type of light suitable for blocking, reducing, or completely suppressing the fluorescence of activated fluorescent dye molecules when irradiated with excitation light. Specifically, fluorescence suppression light can be stimulating light that induces stimulated emission of electronically excited dye molecules, causing them to (return to) their electronic ground state and preventing them from spontaneously fluorescing. Typically, both intensity distributions (at least the excitation light intensity distribution) are also maintained during subsequent scanning steps.

[0059] According to the invention, at least the fluorescence-suppressing light has an intensity distribution with local intensity minima, and prevents or suppresses fluorescence emission in regions outside these local intensity minima. Here, the focus is not, as in STED microscopy, on reducing the volume of the effective PSF in the sense of improving resolution, but on suppressing undesirable fluorescence from dye molecules in the focal edge region or other planes of the sample. Thus, fluorescence detection can be limited to fluorescent dye molecules located near the local intensity minima, and undesirable contributions from other fluorescent dye molecules (especially from the focal edge region or other planes of the sample) can be suppressed. Therefore, unlike in STED microscopy, given the specific implementation of the subsequent scanning and positioning steps, it can even be explicitly desired that the intensity minima be designed to be as wide as possible, and that the intensity increase near the intensity minima be as flat as possible, so that shifting the intensity minima only slightly affects the fluorescence emission of fluorescent dye molecules located near the intensity minima. For example, such an intensity distribution with wide zeros can be generated by phase modulation of the fluorescence-suppressing beam using a high-order vortex phase plate whose phase delay does not change from 0 to 0 with rotation angle. (as is commonly seen in STED microscopes), but rather changes from 0 to or Higher magnification. However, despite the above, in various embodiments of the method, improving resolution by fluorescence suppression in the sense of STED microscopy can also be advantageous and desirable.

[0060] According to corresponding embodiments of this method, the intensity distribution of the excitation light can be formed in different ways; however, in particular, the intensity distribution of the excitation light can also have local minimum intensity values. Preferred embodiments of this method include a combination of a point-focused excitation light and a ring-shaped intensity distribution of fluorescence suppression light, as well as a combination of two ring-shaped intensity distributions of the excitation light and the fluorescence suppression light.

[0061] The method concludes with a second set of method steps:

[0062] 5. Scan the sample or a fragment of the sample at a series of scan locations using one of the previously formed intensity distributions with minimum intensity values, wherein at each scan location, the photon number or fluorescence intensity is detected and assigned to the scan location. These values, formed by the photon number or fluorescence intensity and the scan location, form the data basis for the subsequent localization step. Select scan locations such that there are at least two locations surrounding the estimated position of each dye molecule to be localized that are less than [a certain value]. The scanning position is determined by an intensity distribution having local minimum intensity values—ideally, zero intensity. This can be the intensity distribution of excitation light or fluorescence suppression light. In either case, the sample or a fragment of the sample is loaded with excitation light in each scanning step. If scanning is performed with fluorescence suppression light, the intensity distribution of the excitation light can be fixed in position. If the fluorescence suppression light is the stimulating light, the sample or a fragment of the sample is loaded with fluorescence suppression light, i.e., the stimulating light, in each scanning step. If the fluorescence suppression light is the switching light that switches the fluorophore to a stable non-fluorescent state, it is sufficient to load the sample with fluorescence suppression light once at the start of the scan. In this regard, the first step of the second group of method steps is not strictly separated from the last step of the first group. Especially when using high intensity light that forms the minimum intensity value, a steep intensity gradient is generated from the minimum intensity value, causing the fluorescence emission of dye molecules located near the minimum intensity value to be strongly altered even with a small shift in the scanning position. In this process, fluorescence emission can increase (when scanning with excitation light) or decrease (when scanning with fluorescence suppression light) as the distance between the dye molecule and the minimum intensity increases. This strong dependence of fluorescence emission on the scanning position, regardless of its orientation, lays the foundation for improving position estimation in the localization step.

[0063] In a preferred embodiment, both the excitation light and the fluorescence suppression light have intensity distributions with spatially superimposed minimum intensity values, for example, in the form of a ring-shaped intensity distribution. In this embodiment, it is further preferred to use the intensity distribution of the excitation light for scanning, while the position of the intensity distribution of the fluorescence suppression light is fixed during scanning at the scanning positions associated with the activated fluorescent dye molecules, and therefore has no effect on variations in the fluorescence signal at different scanning positions. However, the intensity distribution of the fluorescence suppression light can also shift together during scanning, transitioning from one dye molecule to another. In particular, the intensity distribution is suitable for the fluorescence suppression light, exhibiting only small intensity variations within the scanning range of the dye molecules and can be generated, for example, using a high-order vortex phase plate (see above). In this embodiment, the purpose of the fluorescence suppression light is solely to suppress the contribution of dye molecules outside the minimum intensity value to the detected fluorescence, and thus enables localization of the scanned molecules, or more precise localization by eliminating interference. In principle, scanning using two intensity distributions is also possible, but in this case, it must be considered that the fluorescence of the dye molecules is affected by the two intensity distributions during scanning. In particular, advantages can be obtained when fluorescence excitation and suppression are performed using stimulation light and fluorescence detection is performed using time gating, especially when fluorescence detection is limited to a very short time window, for example, limited to less than half, preferably less than a quarter, and more preferably less than a tenth of the fluorescence lifetime after the time point when the fluorescence emitted from the observation volume reaches its maximum value at the excitation pulse.

[0064] In another preferred embodiment, a combination of a Gaussian focused spot of excitation light known from STED microscopy and a ring-shaped intensity distribution of fluorescence suppression light is used. Here, only the fluorescence suppression light has an intensity distribution with minimum intensity values, and scanning is performed accordingly using the fluorescence suppression light, while the position of the excitation light intensity distribution is preferably fixed during scanning at the respective scanning positions associated with the activated fluorescent dye molecules, and therefore has no effect on variations in the fluorescence signal at different scanning positions. The excitation light intensity distribution is introduced during scanning only during the transition from one dye molecule to another or during tracking the movement of dye molecules in the sample.

[0065] The minimum number of two scan positions per dye molecule allows for high-precision localization in one dimension. Conversely, if two-dimensional localization is required, the minimum number of scan positions increases to three per dye molecule. In this case, the arrangement of the scan positions is advantageous, with the dye molecule situated within a triangle formed by the three scan positions; however, this is not strictly necessary. For three-dimensional localization, the minimum number of scan positions per dye molecule increases to four. Here, the arrangement of the scan positions is also advantageous (but not mandatory), with the dye molecule situated within a tetrahedron spanned by the four scan positions. In practice, increasing the number of scan positions beyond these minimums may be meaningful to further improve localization accuracy. It is particularly advantageous to use the position estimates determined prior to the corresponding execution of the second set as the scan positions. This achieves the goal of obtaining the position estimates explicitly from the recorded fluorescence signal, i.e., the measured fluorescence signal cannot be ambiguous.

[0066] 6. In the final localization step, a refined position estimate of the activated dye molecule is determined by the interrelated photon counts or intensities and scan positions. In the simplest case, this is done by forming a position vector sum of scan positions weighted by photon counts or fluorescence intensity. Other common, more precise methods use, for example... 最大似然估计器 ( Maximum Likelihood Estimators , MLE) and known from existing techniques used in MINFLUX nanoscience.

[0067] According to the present invention, select This ensures that the initial position estimate can be explicitly associated with the activated fluorescent dye molecule, and that at each scan position, the detected fluorescence originates only from the activated molecule of a single fluorescent dye. On the one hand, this means that the minimum distance between activated dye molecules must not be less than the optical analytical capability of the method used to determine the initial position estimate, i.e., it is usually predetermined due to limitations in optical diffraction. Only when the initial position estimate is determined using a method with already higher resolution (e.g., via STED microscopy) should this be chosen. The value of can be chosen, and smaller values ​​can be selected if necessary. However, it should also be considered that the minimum distance between activated dye molecules is also limited to a small value, that is, when scanning a sample or a fragment of a sample using a light distribution with minimum intensity, only a single activated molecule of the fluorescent dye is allowed to contribute to the detected fluorescence signal. Therefore, it is only possible to obtain in special variations of this method (e.g., when scanning using a combination of a Gaussian focused spot of excitation light and a ring-shaped intensity distribution of fluorescence suppression light). The value drops below 250nm.

[0068] In implementing the method according to the invention, the series of scanning positions need not necessarily be fully determined before the scan begins, but can be added sequentially. In a particularly advantageous manner, the sequential determination of scanning positions associated with the dye molecule can continue, taking into account one or more previous determination steps. Because the uncertainty in position estimation is significantly reduced through the localization step, the scanning positions can then be arranged very densely around the dye molecule to be localized. Scanning at these newly determined scanning points, along with a (further) increase in the intensity of the light used for scanning, leads to an improvement in position estimation in the next determination step. Repeated application of these steps allows the localization of the dye molecule to a range of several nanometers.

[0069] In an improved embodiment of the method according to the invention, the determination of position estimates for individual activated dye molecules is performed multiple times, for example at fixed intervals. If the method is performed to track the movement of dye molecules in a sample, the scan positions for the determination step are determined based on the position estimates from the preceding determination steps, as can also be done in the case of locating stationary dye molecules. When tracking molecules, it is advantageous to match the distance between the scan positions to the speed and type of movement of the dye molecules, particularly as long as they are known from the preceding determination steps. If the movement is rapid and random, a large scan position distance is chosen so that the molecules are reliably located within the range determined by the scan positions in each case, within which the position of the molecules can be estimated. Conversely, if the movement is, for example, slow and directional, the distance between the scan points is smaller, but again, it is chosen so that the molecules are reliably located within the regions determined by the scan positions, where the position of the molecules can be estimated. In both cases, the center of the set of scan positions is moved to the expected position of the molecules during the next sequence of scan steps. In the case of random movement, this is the position corresponding to the last determined position estimate. Based on the continuously determined position estimates, the trajectories of the dye molecules can be reconstructed, visualized, and, if necessary, further analyzed. When dyes are used as markers for biomolecules such as proteins or lipids, this tracing method is well-suited for studying dynamic cellular processes involving the labeled biomolecules. In addition to its high spatial resolution, the method of this invention allows for the determination of the location of individual molecules more quickly than methods known in the prior art, and thus expands the applicability of single-molecule tracking to fast dynamic processes.

[0070] The entire process of tracking a single dye molecule can be repeated for more dye molecules. Such repetitions can begin with activation. If, after tracking one molecule, other molecules are activated, then repeating only the second set of method steps (now at another dye molecule) is sufficient. Finally, a high-resolution image of the structure in the sample can also be reconstructed from the set of trajectories. For example, the values ​​of the diffusion constant can be determined locally. The spatial distribution of these values ​​can then map a structure.

[0071] In another improvement to the method according to the invention, high-resolution images, for example in the form of two-dimensional histograms, are located for reconstructing structures labeled with fluorescent dyes in a sample. This type of image reconstruction is known from STORM and PALM microscopy. However, in order to generate high-resolution, spatially as continuous structural images as possible, the positions of a sufficient number (typically several thousand) of fluorescent dye molecules must be determined. For this purpose, the method steps of localization from photoactivation to activated dye molecules are used multiple times to locate the desired high number of fluorescent dye molecules. In this case, the active dye molecules must be transformed into a non-fluorescent state between repetitive sequences. In the simplest case, this can be achieved by irreversibly bleaching the active molecules with strong excitation light. If photoactivation is reversible, the activated molecules can also be restored to a non-fluorescent state using light of a suitable wavelength.

[0072] Finally, the present invention relates to another method for locating individual molecules of fluorescent dyes in a sample, wherein the method combines features of the first two methods according to the present invention. The specific steps of this method are as follows:

[0073] 1. A fluorescent dye is selected, the obtained fluorescent dye initially existing in a protected non-fluorescent form, and photoactivated by irradiating the fluorescent dye with activating light, thereby converting it from the protected non-fluorescent form to an activated fluorescent form. According to the invention, the fluorescent dye is selected such that photoactivation comprises at least two corresponding photoinduced reaction steps.

[0074] The method also includes a set of method steps:

[0075] 2. By irradiating the sample with activation light, the fluorescent dyes are spaced as far apart as possible. One or more molecules are photoactivated from a protected non-fluorescent form to an activated fluorescent form, wherein the photoactivation is carried out through an activation reaction comprising at least two photoinduced reaction steps.

[0076] 3. An intensity distribution of excitation light and an intensity distribution of fluorescence suppression light are formed in the sample, wherein the intensity distribution of fluorescence suppression light has a local minimum. Typically, both intensity distributions (at least the intensity distribution of excitation light) are also maintained during subsequent scanning steps.

[0077] 4. Scan the sample or a fragment of the sample at a series of scanning positions using an intensity distribution with localized minimum intensity of fluorescent suppressor light, with the scanning positions spaced no more than [value missing]. At each scanning position, the intensity or number of fluorescence photons is detected and correlated with the scanning position. Preferably, scanning is performed along a regular, particularly Cartesian or hexagonal, grid; however, this is not mandatory. In particular, scanning can also be adaptive, for example, by scanning the sample or a fragment of the sample along the grid with relatively large step sizes, and only scanning with smaller step sizes, if necessary, matching the structure, in those regions where fluorescence emission is detected. If the regions with the structure can be pre-identified (e.g., from fluorescence images of the sample or a fragment of the sample), then scanning outside these regions can be completely omitted.

[0078] 5. Activated dye molecules are localized based on the associated photon number or intensity of fluorescence and the scanning position, wherein the uncertainty of this localization in at least one spatial direction is at most [missing information]. ,

[0079] According to the present invention, select The value of is such that the fluorescence detected at each scan position comes only from a single activated molecule of the fluorescent dye, respectively.

[0080] Further advantageous improvements of the invention will be apparent from the claims, description, and drawings. The features and advantages of combinations of features described in the description are merely exemplary and can be implemented alternatively or cumulatively, and are not necessarily achieved through embodiments according to the invention. Without altering the subject matter of the appended claims, the following applies to the disclosure of the original application and patent: further features can be found in the drawings. Combinations of features from different embodiments of the invention or from different claims may also deviate from the reference relationships chosen in the claims and are hereby proposed. This also applies to features shown in separate figures or mentioned in their description. These features may also be combined with features from different claims. Similarly, features of other embodiments of the invention listed in the claims may be omitted.

[0081] The indefinite article “a” used for a feature in the claims and description should be understood to mean exactly one or more instances of that feature in terms of quantity, without requiring the explicit use of the adverb “at least”. A feature listed in a claim may be supplemented by other features if necessary, or may be the only feature possessed by the respective method. Brief description of the attached diagram

[0082] Figure 1 A portion of the method according to the invention is shown in flowchart form.

[0083] Figure 2The photoactivation step and the scanning step in the method according to the present invention are shown.

[0084] Figure 3 A fluorescent dye used in the method according to the invention is shown.

[0085] Figure 4 An embodiment of the method is illustrated, wherein the scanning position is along a grid and an image is generated directly.

[0086] Figure 5 The illustration shows one embodiment of the method in which a single fluorescent dye molecule is located to generate an image.

[0087] Figure 6 A table overview of various embodiments of the method according to the present invention is shown.

[0088] Attached Figure Description

[0089] exist Figure 1 The following diagram illustrates a portion of the method according to the invention, in flowchart form. First, in the photoactivation step S1, a portion of a fluorescent dye (used to stain structures in the sample) is converted from a protected, initially non-fluorescent form to an activated fluorescent form by irradiation with activation light. According to the invention, this photoactivation is carried out through at least two photoinduced reaction steps. In the subsequent scanning and detection step S2, excitation light that excites the fluorescent dye to emit fluorescence and fluorescence suppression light that blocks, reduces, or completely inhibits fluorescence emitted by the fluorescent dye are positioned at a series of scanning locations in the sample (positioning sub-step S2.1). At each scanning location where the sample is irradiated, the number or intensity of fluorescence photons is detected (scanning and detection sub-step S2.2). The detected number or intensity of fluorescence photons, along with the corresponding scanning location, is stored in data memory 1 for subsequent processing. Optionally, scanning can be repeated at all or selected scanning locations. Also optionally, all method steps (i.e., photoactivation step S1 and scanning and detection step S2) are repeated to activate and scan another portion or other portion of the fluorescent dye used to label the sample.

[0090] exist Figure 1 In the embodiment shown, a high-resolution image 2 is finally generated from the number or intensity of fluorescence photons detected at the scanned locations. Image 2 is, for example, a grid image whose image pixels reproduce the fluorescence signal detected at each scanned location, thus representing a spatial representation of the structure labeled with the dye. If the photoactivation is controlled such that individual spatially separated molecules of the fluorescent dye are activated, the number or intensity of fluorescence photons now associated with each molecule can instead be used in an intermediate step (not shown) to locate these molecules of the fluorescent dye, and image 2 can be reconstructed from the positions of the located dye molecules.

[0091] Figure 2 Fragment 3 of a sample containing structure 4 is shown. Structure 4 is labeled with fluorescent dye 5, which initially exists in a protected non-fluorescent form 6. In the initial photoactivation step S1, fragment 3 of the sample is irradiated with activation light 7, which converts a small portion of the fluorescent dye 5 into the activated fluorescent form 8. Photoactivation 9 is carried out in a two-step reaction 10, here by reaction step 11 in the form of cleavage of two photoinstantaneous protecting groups 13 and 14, thereby forming the activated fluorescent dyes 5 and 8. After photoactivation 9, fragment 3 of the sample is scanned at a series of scan positions 19 with excitation light 16 (here in the form of an intensity distribution 15 with local maxima) and an intensity distribution 17 with local minima of fluorescence suppression light 18, wherein, exemplarily, only the first two scan positions are shown here. Since the photoactivation 9 is designed as a two-step reaction 10 according to the present invention, the fluorescent dye 5 in the protected form 6 is inert to the excitation light 16 and the fluorescence suppression light 18. Therefore, the fluorescent dye 5 in the protected non-fluorescent form 6 will not be activated when irradiated during the scanning and detection step S2 with the excitation light 16 and the fluorescence suppression light 18.

[0092] Figure 3 A fluorescent dye 5 derived from caged Q-Rhodamin known in the prior art is shown, having a so-called Carbopyronin backbone and two photoinstantaneous protecting groups 13 and 14 used in the method according to the invention. The fluorescent dye can be incorporated into the sample structure via a linker L. Protecting groups 13 and 14 prevent the formation of fluorescent Carbopyronin fluorophores at two different positions in molecule 24: while the ortho-nitroveratroloxycarbonyl group (NVOC) 13 blocks one of the amino groups, the azide group (Az) 14 fixes the (non-fluorescent) helix of molecule 24. Therefore, the fluorescent dye 5 exists in a protected non-fluorescent form 6 until the two protecting groups 13 and 14 are cleaved. The protecting group can be cleaved using UV light with a wavelength in the range of 320 nm to 360 nm. In reaction step 11, the NVOC group 14 is decomposed by decarboxylation (CO2 cleavage 12), while the azide protecting group 14 is removed in further reaction step 11 by nitrogen molecule N2 cleavage 12 and downstream Wolf rearrangement, thereby forming activated fluorescent dyes 5 and 8.

[0093] Figure 4A preferred embodiment of one of the methods according to the invention is shown, wherein the scanning positions 19 are arranged on grid points 20 of a regular (here, Cartesian) grid 21. As is common in laser scanning microscopy, the sample is scanned line by line with excitation light and fluorescence suppression light in the scanning and detection step S2, and the number or intensity of fluorescence photons detected at each scanning position 19 is assigned as a brightness value 23 to the image pixel 22 in the image 2 corresponding to the respective scanning position 19. In this embodiment, the scanning and detection step S2 and the image generation step S3 do not need to be separated in time; rather, it is advantageous that the image 2 is generated during the scanning of the sample and displayed on a display device.

[0094] Unactivated fluorescent dyes 5 and 6 are inert to excitation and fluorescence suppression light, and therefore neither photoactivate nor fade during sample scanning. By repeatedly performing the photoactivation step S1, the scanning and detection step S2, and the image generation step S3, the method according to the invention also enables multiple image recordings of structure 4, even if the photoactivated fluorescent dyes 5 and 8 fade during scanning with excitation and fluorescence suppression light, which is impossible with conventional image recording methods. The images obtained in the repetitions can be stored individually, for example, to measure changes in the sample over time. These images can be combined cumulatively to image the structure in the sample using a larger number of existing fluorophores or essentially all fluorophores, thereby generating optimal sequential imaging of the structure. Relative displacements or drifts between structures can also be determined from the sequentially recorded images, compensated for, and subsequently imaged cumulatively. Furthermore, if sufficiently high-quality structural images have already been obtained in the sample or in a portion of the sample, the method can be terminated entirely or continued only in areas where the image quality is not yet satisfactory.

[0095] Figure 5 Another preferred embodiment of the method according to the invention is shown, wherein only individual, spatially separated molecules 24 of the fluorescent dye 5 are converted from the protected non-fluorescent form 6 to the activated fluorescent form 8. The photoactivation step (not shown here) is controlled such that the activated molecules 8, 24 of the fluorescent dye 5 are spaced 25 apart from each other, a distance not less than a minimum distance. Here, the minimum distance is measured according to the resolution of the method used for pre-locating the activated fluorescent dye molecules 5, 8, 24 (see below) or alternatively, according to the degree to which the position of the activated fluorescent dye molecule is known solely based on knowledge of the activation position and the resolution achieved when scanning with excitation light 16 and fluorescence suppression light 18. Furthermore, the minimum distance must not be less than either of the two relevant values.

[0096] and Figure 3 The method variation shown differs in that the number or intensity of fluorescence photons obtained in scanning and detection step S2 is not used here to directly generate image 2 in the sense of a direct spatial representation of the detected brightness value, but is first used to precisely locate the individual photoactivated molecules 8, 24 of the fluorescent dye 5 in the positioning sub-step S3.1. For this purpose, the scanning positions 19 are not arranged globally on a regular grid, but rather around each photoactivated fluorescent dye molecule 5, 8, 24, initially arranged in groups of at least three scanning positions 19. For this purpose, the required position estimate 26 of the photoactivated fluorescent dye molecules 5, 8, 24 can be obtained from the activation itself, or by fluorescence microscopy methods known to those skilled in the art, which will not be described in detail here. In the simplest case, this can be achieved, for example, by recording an epifluorescence image; alternatively, the sample can be scanned with excitation light 16 to locate individual activated dye molecules 5, 8, 24 in the sample and to make a preliminary position estimate 26.

[0097] In the subsequent scanning and detection step S2, starting from at least three scanning positions 19 for each molecule 24, the activated dye molecules 5, 8, and 24 are illuminated with an intensity distribution of excitation light and fluorescence suppression light, and the number of photons or intensity of fluorescence is detected at each scanning position 19. From the fluorescence detected at each of the at least three scanning positions 19, the improved position estimate of the activated dye molecules 5, 8, and 24 relative to the initial position estimate 26 is now calculated using a triangulation method. For clarity, the scanning positions at the positions of the initial position estimate 26 are not shown. Such scanning positions can be used to reliably avoid ambiguity in the improved position estimate of the activated dye molecules 5, 8, and 24. Based on this improved position estimate, additional scanning positions 19 can now be determined for each activated dye molecule 5, 8, and 24, and scanning continues as the maximum intensity of the fluorescence suppression light increases. Since the scanning positions 19 are increasingly closer to the actual positions of the activated dye molecules 5, 8, and 24, the scanning positions are approximately located on the helical path 28. The scanning and probing step S2 ends after a predetermined number of scan points for each activated dye molecule 5, 8, 24, or when the error is below the maximum acceptable error of the position estimate.

[0098] To generate image 2 within the scope of image generation step S3, firstly in the localization sub-step S3.1, the final coordinates 29 of the activated dye molecules 5, 8, and 24 are determined from the photon count or intensity of the correlated fluorescence and the scan position 19. Then, in the display sub-step S3.2, the coordinates 29 of the localized molecules 24 are displayed, for example, in the form of a two-dimensional histogram with an appropriate grid width. To obtain a high-resolution image of the structure in the sample in this way, the method steps (including photoactivation) are repeated until the histogram includes so many coordinates 29 of the localized dye molecules 5 and 24 that the structure 4 labeled with dye 5 is represented by the localized dye molecules 5 and 24 throughout the process.

[0099] exist Figure 6 In this document, six different embodiments A to F of the method according to the present invention are listed in tabular form. This list is exemplary and does not represent a final list of all embodiments of the method according to the present invention.

[0100] The common feature of the illustrated embodiments is that the fluorescent dye is initially present in a protected non-fluorescent form, and a portion of the fluorescent dye is converted into an activated fluorescent form by irradiation with activating light in a reaction comprising at least two photoinduced reaction steps. In the table, features distinguishing the illustrated embodiments are indicated by symbols. The second column of the table indicates whether, in the corresponding embodiment, photoactivation 9 is provided for a single molecule 24 or for an aggregate of molecules 30 (i.e., multiple molecules within a probe volume). The third column of the table indicates the scanning scheme 36, which specifies whether, in the corresponding embodiment, a regular scan 31 is performed, particularly along a regular grid 21, or an adaptive scan 32 is performed, in which the scan position is determined taking into account the fluorescence signal detected in previous scan steps. The fourth column symbolically shows the intensity distribution of the excitation light 16, distinguishing between an intensity distribution 15 with a central maximum intensity, a uniform intensity distribution 33, and an intensity distribution 17 with a central minimum intensity. The fifth column shows whether the intensity distribution of the excitation light 16 is scanned 35, or whether the intensity distribution of the excitation light 16 occupies a fixed position 34. The sixth and seventh columns show the corresponding characteristics of the fluorescence suppressor light 18.

[0101] The particularly preferred embodiment shown in line A corresponds to a combination of focused excitation light 16 and fluorescence suppression light 18, or a ring-shaped intensity distribution 17 of excitation light, as known from STED microscopy. Both intensity distributions scan together and synchronously over the sample or a fragment of the sample along a regular, typically Cartesian, grid. To generate a grid image, the fluorescence detected at each scan point is assigned as a brightness value to the corresponding image pixel. The variant shown in line B differs from embodiment A in that the excitation light 16 radiates in the form of a uniform intensity distribution 33 over the scanned area. Scanning 35 is performed only with the intensity distribution 17 of the fluorescence suppression light 18, which has the minimum intensity value. In embodiment B, the increased contribution of fluorescence from areas outside the current scan position due to the illumination of the entire sample (or at least the fragment of interest) with excitation light 16 makes this embodiment less preferred. In embodiments C through F, photoactivation 9 is performed in the form of individual, spatially separated molecules 24, such that the fluorescence of only one activated dye molecule is detected at a time when scanning the sample or a fragment of the sample. In a particularly preferred embodiment C, two intensity distributions 17, each with a minimum intensity value, are superimposed, wherein at least the sample or a fragment of the sample is scanned with the excitation light 16. During each scan of the activated dye molecule 24, if the fluorescence suppression light 18 remains at a fixed position 34, the fluorescence suppression light 18 is primarily used solely to suppress fluorescence contributions from regions outside the minimum intensity value; for this purpose, an intensity distribution with the widest possible minimum intensity value is particularly advantageous. Based on the fluorescence signal detected at the scan position, the previously determined position estimate of the activated dye molecule can be improved, i.e., the activated dye molecule can be located more precisely. Preferably, the scan is performed adaptively, i.e., the scan point is determined taking into account the fluorescence signal detected at the previous scan position, wherein the total intensity of the excitation light 16 is simultaneously increased. In this way, the dye molecule can be located with an uncertainty of a few nanometers. In embodiment C, optionally, the excitation light 16 and the fluorescence suppression light 18 can be scanned together; in this case, the fluorescence emission at the scanning positions of the excitation light and the fluorescence suppression light is modulated, which makes it difficult to calculate the improved position estimate. Embodiments D and E differ from embodiment C in that the fluorescence suppression light 18 is used not only to suppress fluorescence contributions from regions outside the minimum intensity, but also primarily to scan the sample or a fragment of the sample at the scanning position and modulate the fluorescence emission of the scanned dye molecule 24 to calculate the improved position estimate. In these embodiments, the excitation light can be uniformly distributed or structured, for example, in the form of a Gaussian focal spot, and can be fixed in position or move together with the fluorescence suppression light 18 during scanning.

[0102] Finally, Embodiment F combines features of Embodiment A on the one hand, and features of Embodiments C to E on the other. Similarly, individual, spatially separated dye molecules are activated, but the scanning 35 of these individual molecules is the same as in Embodiment A, i.e., scanning is performed using a superimposed intensity distribution of excitation light 16 (with maximum intensity) and fluorescence suppression light 18 (with minimum intensity), wherein scanning 35 is performed along a regular grid 21. Due to the photoactivation 9 of the separated dye molecules 24, the position of each molecule 24 can be estimated from the number or intensity of fluorescence photons detected at the scanning position along the grid 21, with uncertainty far lower than the distance of the scanning position.

[0103] Reference tag list

[0104]

[0105]

[0106] .

Claims

1. A method for locating a single molecule (24) of a fluorescent dye (5) in a sample, comprising the following method steps: Select a fluorescent dye (5) and irradiate the fluorescent dye (5) with activation light (7) to enable the fluorescent dye (5) to change from a protected non-fluorescent form (6) to an activated fluorescent form (8). The first set of method steps includes the following steps: By irradiating with activation light (7), the fluorescent dyes (5) are made to be spaced apart to a minimum. One or more molecules (24) are photoactivated from the protected non-fluorescent form (6) to the activated fluorescent form (8). Determine the initial position estimate (26) of one or more activated molecules (24) of the fluorescent dye (5), wherein the uncertainty of the initial position estimate is no greater than d. , An intensity distribution of excitation light (16) and an intensity distribution of fluorescence suppression light (18) are formed in the sample, wherein at least the intensity distribution of fluorescence suppression light (18) has a local intensity minimum, and the intensity distribution of excitation light (16) can also have an intensity minimum. The second set of method steps includes the following steps: The sample or a fragment (3) of the sample is scanned at a series of scan positions (19) using one of the intensity distributions having a minimum intensity value, the series comprising subsets each having at least two scan positions (19), the at least two scan positions being around the estimated position (26) of the activated molecule (24) of the fluorescent dye (5) associated with the subset at a value less than [value missing]. Arranged at a distance of (25), At each scan position (19) in the series, the number or intensity of fluorescence photons is detected, and the number or intensity of photons is associated with the corresponding scan position (19). A new position estimate (26) of each of the activated molecules (24) of the fluorescent dye (5) associated with the subset is determined from the associated photon number or intensity of the fluorescence and the scan position (19). in, d The value is such that the initial position estimate (26) can be clearly associated with the activated molecule (24) of the fluorescent dye (5), and the fluorescence detected at each scan position (19) in each case comes only from the single activated molecule (24) of the fluorescent dye (5). Its features are, The fluorescent dye (5) is selected such that the photoactivation includes at least two corresponding photoinduced reaction steps (11).

2. The method according to claim 1, characterized in that, 。 3. The method according to claim 1 or 2, characterized in that, The intensity distribution of the excitation light (16) has a local minimum intensity, and the sample or a segment (3) of the sample is scanned with the intensity distribution of the excitation light (16).

4. The method according to claim 1 or 2, characterized in that, The intensity distribution of the excitation light (16) has a local intensity maximum, wherein the intensity distribution of the fluorescence suppression light (18) and the intensity distribution of the excitation light (16) are substantially complementary to each other.

5. The method according to claim 1, characterized in that, The scan was performed using two intensity distributions.

6. The method according to claim 1, characterized in that, The scanning position (19) in the series of scanning positions is arranged on a circular path, a spiral path (28) or a spherical shell.

7. The method according to claim 1, characterized in that, Repeat the steps of the second group of methods.

8. The method according to claim 7, characterized in that, Between the repetitions, the total intensity of the intensity distribution of the excitation light (16) and / or the fluorescence suppression light (18) with local intensity minimum values ​​increases, and the scan position (19) of the subset moves toward the corresponding current position estimate (26) of the associated activated molecule (24) of the fluorescent dye (5).

9. The method according to claim 7 or 8, characterized in that, The uncertainty of the final determined position estimate (26) in at least one spatial direction is at most [missing information]. .

10. The method according to any one of claims 1-2 and 5-8, characterized in that, Track the movement of individual molecules (24) of the fluorescent dye (5) in the sample.

11. The method according to claim 10, characterized in that, Between two repetitions, the total intensity of the intensity distribution of the excitation light (16) and / or the fluorescence suppression light (18) with local intensity minimum decreases, and the scan position (19) of the subset moves toward the position estimate (26) of the associated activated molecule (24) of the fluorescent dye (5) determined by time extrapolation.

12. The method according to any one of claims 1-2 and 5-8, characterized in that, The first set of method steps and the second set of method steps are repeated in general, wherein between the repetitions, the corresponding activated molecule (24) of the fluorescent dye (5) is converted to a non-fluorescent state.

13. The method according to claim 12, characterized in that, From the localization of individual molecules (24) of the fluorescent dye (5), a high-resolution spatial image of structure (4) in the sample is reconstructed.

14. A method for locating a single molecule (24) of a fluorescent dye (5) in a sample, comprising the following method steps: Select a fluorescent dye (5) and irradiate the fluorescent dye (5) with activation light (7) to enable the fluorescent dye (5) to change from a protected non-fluorescent form (6) to an activated fluorescent form (8). And a set of method steps, including the following steps: By irradiating with activation light (7), the fluorescent dyes (5) are made to be spaced apart to a minimum. d One or more molecules (24) are photoactivated from the protected non-fluorescent form (6) to the activated fluorescent form (8). An intensity distribution of excitation light (16) and an intensity distribution of fluorescence suppression light (18) are formed in the sample, wherein the intensity distribution of fluorescence suppression light (18) has a local intensity minimum. The sample or a fragment (3) of the sample is scanned at a series of scanning positions (19) using the intensity distribution of the fluorescence suppression light (18) with the minimum intensity value, the scanning positions being spaced apart by a distance not greater than [missing value]. ; At each scan position (19) in the series, the number or intensity of fluorescence photons is detected, and the number or intensity of photons is associated with the corresponding scan position (19). The activated molecule (24) of the fluorescent dye (5) is located based on the associated photon number or intensity of the fluorescence and the scanning position (19), the uncertainty of which the location is in at least one spatial direction is at most [missing information]. , in, The value is such that the fluorescence detected at each scan position (19) comes only from a single activated molecule (24) of the fluorescent dye (5). Its features are, The fluorescent dye (5) is selected such that the photoactivation includes at least two corresponding photoinduced reaction steps (11).

15. The method according to claim 14, characterized in that, 。 16. The method according to claim 14 or 15, characterized in that, The intensity distribution of the excitation light (16) has a local intensity maximum, wherein the intensity distribution of the fluorescence suppression light (18) and the intensity distribution of the excitation light (16) are substantially complementary to each other.

17. The method according to claim 14 or 15, characterized in that, The scan position (19) is arranged on a regular grid (21).

18. The method according to claim 14 or 15, characterized in that, The set of method steps is repeated, wherein between the repetitions, the corresponding activated molecule (24) of the fluorescent dye (5) is converted to a non-fluorescent state.

19. The method according to claim 18, characterized in that, A high-resolution spatial image of structure (4) in the sample is reconstructed from the location of the activated molecule (24) of the fluorescent dye (5) determined by positioning.

20. A method for generating a spatially high-resolution image of a structure (4) in a sample, comprising the following method steps: Select a fluorescent dye (5) and irradiate the fluorescent dye (5) with activation light (7) to enable the fluorescent dye (5) to change from a protected non-fluorescent form (6) to an activated fluorescent form (8). The structure (4) is labeled with the fluorescent dye (5). And the following method steps, performed once or repeatedly: By irradiating with activation light (7), a subset of the fluorescent dye (5) is photoactivated from the protected non-fluorescent form (6) to the activated fluorescent form (8). An intensity distribution of excitation light (16) and an intensity distribution of fluorescence suppression light (18) are formed in the sample, wherein the intensity distribution of fluorescence suppression light (18) has a local intensity minimum. The sample or a fragment of the sample (3) is scanned at a series of scanning positions (19) using the intensity distribution of the fluorescence suppressor light (18) with the minimum intensity value. The number or intensity of fluorescence photons is detected at each scan position (19), and the number or intensity of photons is correlated with the corresponding scan position (19). A high-resolution grid image of the structure (4) is generated from the associated photon number or intensity of the fluorescence and the scan position (19) by associating brightness values ​​(23) with each image pixel (22) of the grid image, wherein the brightness values ​​are monotonic functions of the photon number or intensity of the fluorescence detected at the corresponding scan position (19) or the corresponding set of scan positions. Its features are, The fluorescent dye (5) is selected such that the photoactivation includes at least two corresponding photoinduced reaction steps (11).

21. The method according to claim 20, characterized in that, The excitation light (16) forms an intensity distribution with local intensity maxima that is substantially complementary to the intensity distribution of the fluorescence suppression light (18), and the scanning is performed together with the excitation light (16) and the fluorescence suppression light (18).

22. The method according to claim 20 or 21, characterized in that, The scan position (19) is arranged on a regular grid (21).

23. The method according to any one of claims 1-2, 5-8, 11, 13-15, and 19-21, characterized in that, Multiple irradiation points are formed in the sample using the activation light (7).

24. The method according to claim 23, characterized in that, The irradiation points are arranged on a regular grid (21).

25. The method according to any one of claims 1-2, 5-8, 11, 13-15, 19-21, and 24, characterized in that, The photoinduced reaction step (11) is induced by multiphoton absorption.

26. The method according to any one of claims 1-2, 5-8, 11, 13-15, 19-21, and 24, characterized in that, All photoinduced reaction steps were induced by activation light of the same wavelength (7).

27. The method according to any one of claims 1-2, 5-8, 11, 13-15, 19-21, and 24, characterized in that, One of the photoinduced reaction steps (11) is induced by activation light (7) of a different wavelength than that of the other photoinduced reaction step (11).

28. The method according to any one of claims 1-2, 5-8, 11, 13-15, 19-21, and 24, characterized in that, At least one of the photoinduced reaction steps in the photoinduced reaction step (11) is the photolysis of the photoinstability protecting group.

29. The method according to claim 28, characterized in that, The photoinstability protecting group (13) is selected from the following unsubstituted or substituted groups: nitrobenzyl, nitrophenylethyl, nitroindolinyl, dinitroindolinyl, nitroveratrol, arylcarbonylmethyl, alkylbenzoylmethyl, hydroxybenzoylmethyl, benzoin, hydroxycinnamate, o-nitro-2-phenylethoxycarbonyl, nitroaniline, coumarinyl, aminocoumarinyl, methoxycoumarylmethyl, anthraquinone-2-ylmethoxycarbonyl, (2-naphthyl)methyl, (anthraquinone-9-yl)methyl, (pyrene-1-yl)methyl, (peryl-3-yl)methyl, (phenanthrene-9-yl)methyl, o-hydroxyarylmethyl, azide compounds, dipyrrole methylene boron.

30. The method according to claim 28, characterized in that, The photoinduced reaction step (11) is the same photolytic cleavage reaction of the photoinstability protecting group.

31. The method according to claim 29, characterized in that, The photoinduced reaction step (11) is the same photolytic cleavage reaction of the photoinstability protecting group.

32. The method according to claim 28, characterized in that, The photoinduced reaction step (11) is a photolytic cleavage reaction of different photoinstantaneous protective groups.

33. The method according to claim 29, characterized in that, The photoinduced reaction step (11) is a photolytic cleavage reaction of different photoinstantaneous protective groups.

34. The method according to any one of claims 1-2, 5-8, 11, 13-15, 19-21, 24, and 29-33, characterized in that, At least two of the photoinduced reaction steps in the photoinduced reaction step (11) are tandem reaction steps.

35. The method according to claim 34, characterized in that, The steps of the cascade reaction are reversible.

36. Use of a fluorescent dye (5) in the method according to any one of claims 1 to 35, characterized in that, The fluorescent dye (5) is irradiated with activation light (7) to enable the fluorescent dye (5) to be converted from a protected non-fluorescent form (6) to an activated fluorescent form (8), and the conversion of the dye (5) to the fluorescent form (8) includes at least two corresponding photoinduced reaction steps (11).

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