Microscope and method for wavelength-selective and spatially high-resolution microscopy

Abstract

Method for wavelength-selective and spatially high-resolution fluorescence microscopy, wherein a) in a sample (2), fluorescence emitters are repeatedly excited to emit fluorescence radiation and individual images (16) are generated from the sample (2) using a microscope (1) having an imaging beam path (10) with an optical resolution, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image (16), and b) in the individual images (16) generated, the positions of the isolated fluorescent fluorescence emitters are localized with a spatial accuracy exceeding an optical resolution and a high-resolution overall image is generated therefrom, characterized in that c) the imaging beam path (10) of the microscope (1) has a spectrally selective element (11) which, when generating the individual images (16), imparts a spectrally dependent rotational asymmetry to a point image blurring function, so that the images (17, 18; 29, 30) of isolated fluorescent fluorescence emitters have a rotational asymmetry which depends on a wavelength at which the isolated fluorescent fluorescence emitters fluoresce, and d) in the individual images (16), the images (17, 18; 29, 30) of the isolated fluorescence emitters are analyzed with regard to their rotational asymmetry and an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived therefrom.

Description

[0001] The invention relates to a method for wavelength-selective and spatially high-resolution fluorescence microscopy, wherein fluorescence emitters in a sample are repeatedly excited to emit fluorescence radiation and individual images are generated of the sample using a microscope having an imaging beam path with an optical resolution, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image, the positions of the isolated fluorescent fluorescence emitters are localized in the individual images generated with a spatial accuracy exceeding an optical resolution and a high-resolution overall image is generated therefrom.

[0002] The invention further relates to a fluorescence microscope for wavelength-selective imaging of a sample with a spatial resolution increased above an optical resolution, which comprises an illumination device which is designed to repeatedly excite fluorescence emitters in the sample to emit fluorescence radiation, an imaging device comprising an imaging beam path with the optical resolution, which is designed to generate individual images of the sample with the optical resolution, a control device which is designed to control the illumination device and the imaging device such that several individual images of the sample are generated, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image, the control device is designed toto localize the positions of the isolated fluorescent fluorescence emitters in the generated individual images with a spatial accuracy exceeding optical resolution and to generate a high-resolution overall image from them.

[0003] Various methods have been developed in the prior art to overcome the diffraction limit in microscopy. A method abbreviated to PALM (photo-activated light microscopy) is known from WO 2006 / 127692 A3 or DE 10 2006 021 317 B3. This method uses a labeling substance to image a sample, which can be activated, for example, using optical radiation. Only in the activated state can the labeling substance emit specific fluorescence radiation. Non-activated molecules of the labeling substance emit no, or at least no noticeable, fluorescence radiation even after exposure to excitation radiation. The activation radiation is therefore generally referred to as a switching signal.In the PALM method, the switching signal is applied in such a way that at least a certain proportion of the activated marker molecules are spaced from neighboring activated molecules so that these marker molecules can be separated based on the optical resolution of the microscope or subsequently separated using image processing techniques. This is said to isolate a subset of the fluorescence emitters. After recording the fluorescence radiation, the center of their resolution-limited radiation distribution is then determined for these isolated emitters. This allows the position of the molecules to be determined mathematically with greater accuracy than the optical resolution actually allows. This process is referred to as localization. The increased resolution achieved through computational determination of the center of gravity of the diffraction distribution is also referred to as "super resolution" in the English technical literature.It requires that at least a subset of the activated marker molecules in the sample be distinguishable—i.e., isolated—with optical resolution. Then their location can be determined with greater precision, i.e., they can be localized.

[0004] To isolate individual marker molecules, the PALM principle exploits statistical effects. For a marker molecule that can be excited to fluorescence upon receiving a switching signal of a given intensity, adjusting the intensity of the switching signal can ensure that the probability of activating marker molecules present in a given area of the sample is so low that there are sufficient subregions in which only distinguishable marker molecules emit fluorescence within the optical resolution.

[0005] The PALM principle has been further developed with regard to the activation of the molecules to be detected. For example, for molecules that exhibit a long-lived non-fluorescent and a short-lived fluorescent state, separate activation with activation radiation that differs spectrally from the excitation radiation is not necessary. Instead, the sample is first activated with high-intensity illuminating radiation in such a way that the vast majority of the molecules are converted into the non-fluorescent, long-lived state (e.g., a triplet state). The remaining, still fluorescent molecules are thus isolated with respect to optical resolution.

[0006] It should also be noted that the PALM principle has since been given other abbreviations in the specialist literature, such as STORM. In this description, the abbreviation PALM is used for all microscopy images that achieve a spatial resolution beyond the optical resolution of the used equipment by first isolating and then localizing fluorescent molecules. The PALM method has the advantage that high spatial resolution is not required for the illumination. Simple wide-field illumination is possible.

[0007] The PALM principle requires the acquisition of many individual images of the sample, each containing subsets of isolated molecules. To image the sample in its entirety, the set of all individual images must ensure that, if possible, all molecules were included in a subset at least once. The PALM method therefore regularly requires a large number of individual images, which requires a certain amount of time to acquire a complete image. This involves considerable computational effort, as a large number of molecules must be computationally localized in each individual image. This generates large amounts of data.

[0008] There is now a need to capture high-resolution images not only in a color channel, but also to obtain color information, ie a wavelength specification, via the fluorescent emitters. The state of the art in fluorescence microscopy involves a microscope according to Fig. 8. The fluorescence microscope 100 shown therein comprises an illumination beam path 3 and an imaging beam path 4, which illuminate a sample 2 with excitation radiation via a common objective 5 and image the fluorescent sample 2. The illumination beam path 3 is combined with the imaging beam path 4 via a generally dichroic beam splitter 6, so that both illumination radiation from the illumination beam path 3 passes through the objective 5 to the sample 2, and the image of the sample is also created through the objective 5 and via the imaging beam path 4. The illumination beam path 3 usually has several spectral channels; in the illustration of the Fig. 8 shows two exemplary laser sources L1 and L2, the radiation of which is combined via a beam splitter 5. The illumination beam path 3 thus illuminates the sample 2 with radiation of at least two wavelengths, resulting in multi-color excitation of the sample 2 to produce fluorescence radiation. The sample 2 also emits multi-color fluorescence radiation (this could of course also be the case with monochromatic fluorescence excitation and different fluorescence molecules). In the imaging beam path 4, the image of the sample 2 is therefore split into three color channels via two beam splitters 7 and 8 and suitable optics (not further designated), i.e., directed to three cameras K1, K2, and K3. The splitting via the beam splitters 8 and 9 results in a spectrally selective splitting between the cameras K1 to K3. Alternatively or additionally, suitable color filters can also be used. This results in several color channels, one for each camera.A disadvantage of this setup, however, is that the camera systems used are very expensive due to the required high resolution. Furthermore, the installation space for the microscope 100 is large due to the necessary beam paths and color splitters. The cameras are also typically cooled and require a lot of space. Another problem is that the cameras K1, K2, and K3 must be precisely aligned to each other so that the images of the individual color channels are subsequently correctly positioned relative to each other. Any alignment error between the partial beam paths of the individual color channels would result in a color error in the overall image, in the sense of a chromatic aberration.

[0009] It would be conceivable that the microscope 100 of the Fig. 8 for the PALM principle, but then the amount of data and the computing effort would be multiplied by the number of color channels.

[0010] DE 11 2011 103 187 T5 discloses a generic method for 3D localization microscopy in which depth resolution is achieved by means of a phase modification device.

[0011] DE 10 2008 049 886 A1 provides for a phase mask in the detection beam path for PAL microscopy to achieve spectral splitting for a downstream spectrometer and thus color analysis. US 2009 / 0 059 360 A1 pursues the same goal, whereby image acquisition in PAL microscopy is accelerated by evaluating individual images in which fluorescence emitters are located with regard to various parameters, including the wavelength of the fluorophores. The use of a phase element for high resolution is also known in US 2009 / 0 263 002 A1.

[0012] DE 10 2009 043 744 A1 deals with three-dimensional resolution-enhanced microscopy and uses a microlens array for this purpose.

[0013] The invention is therefore based on the object of providing a microscope and a method for wavelength-selective, high-resolution fluorescence microscopy that avoids the disadvantages of the prior art described above. In particular, the size and variety of parts, adjustment effort, and data volume are to be reduced.

[0014] This object is achieved according to the invention with a method for wavelength-selective and spatially high-resolution fluorescence microscopy, wherein fluorescence emitters in a sample are repeatedly excited to emit fluorescence radiation and individual images are generated of the sample using a microscope having an imaging beam path with an optical resolution, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image, in the individual images generated, the positions of the isolated fluorescent fluorescence emitters are localized with a spatial accuracy exceeding an optical resolution and a high-resolution overall image is generated therefrom, the imaging beam path of the microscope has a spectrally selective element,which, when generating the individual images, imparts a spectrally dependent rotational asymmetry to a point spread function, so that the images of isolated fluorescent fluorescence emitters exhibit a rotational asymmetry that depends on a wavelength at which the isolated fluorescent fluorescence emitters fluoresce, and in the individual images, the images of the isolated fluorescence emitters are analyzed with regard to their rotational asymmetry, and from this, an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived.

[0015] This object is further achieved with a fluorescence microscope for wavelength-selective imaging of a sample with a spatial resolution increased above an optical resolution, which has an illumination device which is designed to repeatedly excite fluorescence emitters in the sample to emit fluorescence radiation, an imaging device comprising an imaging beam path with the optical resolution which is designed to generate individual images of the sample with the optical resolution, a control device which is designed to control the illumination device and the imaging device such that several individual images of the sample are generated, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image, the control device is designed toto locate the positions of the isolated fluorescent fluorescence emitters in the generated individual images with a spatial accuracy exceeding optical resolution and to generate a high-resolution overall image therefrom, the imaging beam path of the microscope has a spectrally selective element which imparts a spectrally dependent rotational asymmetry to a point image blurring function when generating the individual images, so that the images of isolated fluorescent fluorescence emitters have a rotational asymmetry that depends on a wavelength at which the isolated fluorescent fluorescence emitters fluoresce, and the control device is designed to analyze the images of the isolated fluorescence emitters in the individual images with regard to their rotational asymmetry and to derive therefrom an indication of the wavelength of the isolated fluorescent fluorescence emitters.

[0016] According to the invention, the limitation of multicolor wide-field detection for high resolution existing in the prior art is overcome by encoding the spectral information of the emitters in a rotational asymmetry of the point spread function and thus ultimately in its appearance on the camera. This is achieved by inserting a spectrally selective element in the imaging beam path, which makes the point spread function rotationally asymmetric in a spectrally dependent manner. This is possible for localization-based high resolution according to the PALM principle, in contrast to classical fluorescence microscopy, since the PALM principle deals with individual molecules due to isolation and localization. The disturbance of the point spread function, which is otherwise strictly avoided in normal microscopy, surprisingly leads to the advantage of simple wavelength selection for the PALM principle.

[0017] In the individual images, the images of the isolated fluorescence emitters are analyzed for their rotational asymmetry. The detected rotational asymmetry can be easily used to determine the wavelength of the detected radiation, since the spectrally dependent rotational symmetry perturbation introduced by the spectrally selective element is known.

[0018] The image of a fluorescence emitter refers to its usually diffraction-limited point image.

[0019] The rotational asymmetry can be analyzed particularly easily by determining an angle value for each image of one of the isolated fluorescence emitters. This angle value then provides information about the wavelength of the isolated fluorescent emitters, since the spectrally selective element converts individual wavelengths to different angular positions of the rotational asymmetry.

[0020] The method is particularly simple to implement when the spectrally selective element imparts a point-symmetric rotational asymmetry to the point-spread blur function. The rotationally asymmetric images of the individual fluorescence emitters then differ only in their rotational position, which depends on the color, i.e., the wavelength, of the individual fluorescence emitter. It is then relatively easy to derive the wavelength of the isolated fluorescent emitters by determining the rotational position of each isolated fluorescence emitter for each image.

[0021] Point-symmetric rotational asymmetry has the further advantage that the localization can be determined simply by referring to the center of gravity of the point-symmetric and rotationally asymmetric diffraction pattern of each isolated fluorescence emitter, regardless of wavelength. The position of the center of gravity provides the localization of the location.

[0022] The spectrally selective element can be designed in a variety of ways. A simple option is a plate made of a dispersive material whose thickness varies along a radius around an optical axis. Of course, instead of exploiting the dispersion of the material and thus varying the thickness, the transmission properties can also be varied in general.

[0023] A particularly advantageous design realizes the spectrally selective element as a plate consisting of several wedge-shaped sectors that differ in their spectral transmission properties. If sectors that are point-symmetric to each other are then given the same spectral transmission properties, the aforementioned advantageous rotationally symmetric image is obtained for each individual isolated fluorescent molecule. The center of symmetry is also the intersection point of the optical axis.

[0024] Localization can be facilitated if the spectrally selective element has a central region located on an optical axis, which has broadband spectral transmission properties and is surrounded by sectors that differ from each other and preferably also from the central region in terms of their spectral transmission properties. In the central region, the diffraction-limited point pattern of each fluorescence emitter is then spectrally independent, which enables easy localization. Due to the sectors, each diffraction-limited point pattern has at least one ear, preferably two symmetrically positioned ears, whose position encodes the color emission.

[0025] The optical design of the microscope is particularly simple if the spectrally selective element is arranged in a pupil or near a pupil of the imaging beam path.

[0026] A spectrally selective element with a point-symmetric variation in transmission properties results in diffraction-limited point patterns for the fluorescence emitters, which are shaped like a bow tie. The rotational position of the bow tie encodes the color at which an isolated emitter glows.

[0027] It is understood that the features mentioned above and those to be explained below can be used not only in the specified combinations, but also in other combinations or on their own, without departing from the scope of the present invention. To the extent that method features are mentioned in this description, they are implemented during operation of the microscope by a correspondingly designed control unit. Analogously, a disclosure of functional features of the control unit also counts as a description of corresponding method features, e.g., steps.

[0028] The invention is explained in more detail below, for example, with reference to the accompanying drawings, which also disclose essential features of the invention. They show: Fig. 1 a schematic representation of a microscope for wavelength-selective, high-resolution fluorescence microscopy, Fig. 2 a schematic plan view of a spectrally selective element in the microscope of the Fig. 1, Fig. 3 a section of a single image taken with the microscope of the Fig. 1, where the section shows exemplary diffraction-limited images of an isolated fluorescence emitter, Fig. 4 a single image 16 with several images according to the Fig. 3, Fig. 5 a representation similar to the Fig. 2 with a modified spectrally selective element, Fig. 6 a diagram illustrating the spectral properties of different areas of the spectrally selective element of the Fig. 5, Fig. 7 a representation similar to the Fig. 3 for the element of Fig. 5 and Fig. 8 a state-of-the-art fluorescence microscope with multiple color channels.

[0029] Fig. 1 schematically shows a fluorescence microscope 1, the operation of which is controlled by a control unit C. It is connected via connections not shown to the elements of the microscope 1, in particular to the laser sources / laser source and the camera. Elements or components that functionally and / or structurally correspond to elements or components that have already been described with reference to the microscope 100 of the Fig. 8, i.e., correspond to the state of the art in terms of their function or structure, are shown in the illustration of the Fig. 1 are provided with the same reference numerals. Their description therefore need not be repeated.

[0030] The microscope 1 of the Fig. 1 comprises, in addition to the illumination beam path 3, an imaging beam path 10, which, as will be explained below, does not require spectral division into multiple color channels. Therefore, only a single camera K1 is required. The image of the sample is guided through a spectrally selective element 11, which rotationally asymmetrically modifies the point-image blurring function of the microscope 1. Since the microscope 1 otherwise acquires diffraction-limited images of the isolated fluorescent molecules, these images are now asymmetrically modified by the spectrally selective element 1 with regard to their point-image blurring function.

[0031] Fig. Figure 2 shows schematically a simplified plan view of the spectrally selective element 11 along the optical axis, which is therefore in the representation of the Fig. 2 is perpendicular to the plane of the drawing. The spectrally selective element 11 comprises, by way of example, four sectors 12, 13, 14, and 15, which have different spectral transmission characteristics. The sectors differ in their spectral filter properties; in the example shown, opposite sectors have the same spectral filter properties, but neighboring sectors differ in their filter properties. By way of example and without limiting the principle of the invention, opposite sectors are equipped with the same transmission properties. Sectors 12 and 13 have the same spectral transmission properties. The same applies to sectors 14 and 15.

[0032] The effect of the spectrally selective element 11 on a point image of an isolated emitter is shown Fig. 3, which represents a section of a single image 16 generated with the microscope 1. The spectrally selective element 11 deforms the point spread function of a single illuminated fluorophore rotationally asymmetrically, namely in the form of the Fig. 3 depicted fly. Fig. Figure 3 shows a section of a single image 16, which is created with microscope 1 controlled by control unit C during the PALM procedure when an isolated fluorophore is illuminated. Depending on the wavelength at which a fluorophore primarily illuminates, the radiation from this fluorophore is transmitted either by sectors 12 and 13 or by sectors 14 and 15. Accordingly, a fly 17a or 17b is formed. The rotational position of the fly 17 around a center 18 encodes the color of the luminous fluorophore. Fig. Figure 3 schematically shows two flies 17a and 17b for different luminous colors. Naturally, the transmission properties of the sectors of the spectrally selective element 11 are selected such that only one of the two flies 17a or 17b is present for a given application. The direction of the fly's main axis is evaluated by the control unit C, which provides an indication of the wavelength at which the fluorophore glows. The center 18 is the starting point for the localization of the fluorophore, which follows the well-known PALM approaches already explained above using the term "super resolution."

[0033] The fly 17a is present when the emitter's radiation is such that it was transmitted through sectors 12 and 13 (and blocked by sectors 14 and 15). The fly 17b appears when the wavelength of the fluorescence emitter could only be transmitted through sectors 14 and 15. The flies 17a and 17b have a center 18 that corresponds to the actual position of the fluorescence emitter. By determining the center of gravity 18 of the fly 17a and 17b, the position of the fluorescence emitter can be specified with greater accuracy than spatial resolution. The angular position of the fly 17a and 17b provides the color information.

[0034] The use of the spectrally selective element with only one color channel in the imaging beam path has the advantage that only one of the comparatively expensive cameras is required. Furthermore, the construction effort and space requirements associated with multiple cameras are no longer necessary. Finally, the individual images 16 are also automatically aligned with respect to the color channels, since all emitters are recorded in a single image, regardless of their color channel. Chromatic aberration caused by inadequate alignment of the individual color channels is thus fundamentally avoided by this design. The resulting images are, by design, chromatically error-free in this respect.

[0035] The operation of the microscope 1 is controlled by a control unit C. To the extent that process features are described above or below, the control unit C ensures that the microscope 1 is put into a corresponding operation that implements these process features.

[0036] Of course, the formation of the spectrally selective element 11, as it is in Fig. 2 is purely exemplary. The spectrally selective element 11 does not necessarily have to be formed from sectors. The decisive factor is simply that its transmission properties are such that the point spread function of a single luminous fluorophore is asymmetrically distorted depending on the spectral range and / or the main wavelength, etc., at which the fluorophore luminesces. From this asymmetry, the control unit C can then simply derive a color output for the fluorophore.

[0037] A point-symmetric design of the spectral transmission properties of the spectrally selective element 11, as in Fig. 2, has the advantage that the center of each spot wash image of a single illuminated fluorophore can be easily used to localize the fluorophore.

[0038] The use of sectors that are largely constant in terms of their transmission properties and are clearly defined from one another has the further advantage that the control unit C only needs to distinguish between a discrete number of rotationally asymmetric distortions when executing the process. The price for this simplification is that only certain colors or spectral ranges can be distinguished.

[0039] It is also possible to design the transmission properties of the spectrally selective element such that the rotationally asymmetric spectral distortion varies in a multitude of fine steps or even continuously with wavelength. At the cost of requiring more precise determination of the rotational asymmetry of the point-sweep images in each individual image, a higher spectral resolution with respect to the color information of the fluorophores is then achieved.

[0040] Fig. Figure 4 shows an example of a single image 16 obtained with the microscope 1 under the control of the control unit C, which was generated with a spectrally selective element 11, which generates four different discrete rotational asymmetries for four different colors of luminous fluorescent emitters. Compared to the image in Fig. The element shown in Figure 2 has the spectrally selective element 11, with which the individual image 16 of the Fig. 4, not two opposing sectors, each paired in pairs, but four pairs—a total of eight sectors. The point-symmetric design of the spectrally selective element 11 ensures that the point-swept image of a single fluorophore is again a fly 17. In individual image 16, there are four different rotational positions encoding the four different transmission bands of the spectrally selective element. The centers 18, in turn, serve to localize the fluorophores.

[0041] Like the single image of the Fig. As Figure 4 clearly shows, all color channels are contained in a single image 16. The problem of chromatic aberration due to imperfect alignment of several independently recorded color channels thus does not arise. Furthermore, the number of individual images does not increase linearly with the number of color channels.

[0042] Fig. 5 shows a top view similar to the Fig. 2. However, the spectrally selective element 11 is not only formed by four pairs of sectors 19, 20, 21 and 22 (as they are for the individual image 16 of the Fig. 4 were used), the spectrally selective element 11 also includes a broadband transmitting central region 23. The spectral filter properties are exemplified in Fig. 6, which shows the transmission through the respective area as a function of wavelength λ. The transmission curve 24 is assigned to the sector pair 19, the transmission curve 25 to the sector pair 20, the transmission curve 26 to the sector pair 21 and the transmission curve 27 to the sector pair 22. The central area 23 transmits broadband in the spectral range 28. A single image of a fluorophore is then, as in Fig. 4 as a section of a single image 16, formed as a circular spot 29 with ears 30. The rotational position of the ears 30 encodes the color information. The central spot 29 facilitates the localization of the fluorophore in the single image 16, ie the generation of the so-called super resolution. The design of the spectrally selective element 11 is therefore different from the design principle of the spectrally selective element 11 of the Fig. 2 was modified to facilitate the localization of the fluorophores.

[0043] As is clear from the above description, the spectrally selective element causes a rotational asymmetry of the point spread function of a single illuminated fluorophore depending on the spectral composition of the fluorescence radiation. It is therefore advantageous to provide a switching mechanism for the spectrally selective element 11 in order to adapt the spectral transmission characteristics, for example, the transmission characteristics of individual sectors, to a microscopy task, in particular to the fluorophores used.

[0044] The point-symmetric, rotationally asymmetric point-spread blur functions described above facilitate the localization of the molecule. However, this is not mandatory. Thus, it is always possible to abandon the point symmetry of the transmission properties of the spectrally selective element 11, since localization can be easily performed using the spot 29. Using a central region 23, the point-spread image of a fluorophore would then have only one ear, not two ears 29. Such an approach increases the spectral resolution by a factor of 2. A non-point-symmetric configuration of the transmission properties of the spectrally selective element 11 can also be realized without a central region 23.It is essential that the basic structure of the spectrally dependent rotational asymmetry is known when evaluating the individual images 16, so that the fluorophore can be easily localized by detecting the rotationally asymmetric individual image. To do this, one only needs to know how the rotationally asymmetric point blur image is positioned relative to the respective center where the fluorophore is expected.

Claims

[1] Method for wavelength-selective and spatially high-resolution fluorescence microscopy, whereby a) in a sample (2), fluorescence emitters are repeatedly excited to emit fluorescence radiation and individual images (16) are generated from the sample (2) using a microscope (1) having an imaging beam path (10) with an optical resolution, wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image (16), and b) in the individual images (16) generated, the positions of the isolated fluorescent fluorescence emitters are localized with a spatial accuracy exceeding optical resolution and a high-resolution overall image is generated therefrom, characterized by , that c) the imaging beam path (10) of the microscope (1) has a spectrally selective element (11) which, when generating the individual images (16), imparts a spectrally dependent rotational asymmetry to a point image blurring function, so that the images (17, 18; 29, 30) of isolated fluorescent fluorescence emitters have a rotational asymmetry which depends on a wavelength at which the isolated fluorescent fluorescence emitters fluoresce, and d) in the individual images (16), the images (17, 18; 29, 30) of the isolated fluorescence emitters are analyzed with regard to their rotational asymmetry and an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived therefrom. [2] Method according to claim 1, characterized bythat in step d) an angle value is determined for each image (17, 18; 29, 30) of one of the isolated fluorescence emitters and the information about the wavelength of the isolated fluorescent fluorescence emitters is derived from the angle value. [3] Method according to claim 1, characterized by that in step c) the spectrally selective element imparts a point-symmetric rotational asymmetry to the point spread function. [4] Method according to claim 3, characterized by that in step d) for each image (17, 18; 29, 30) of one of the isolated fluorescence emitters a rotational position of the image is determined and from the rotational position the information about the wavelength of the isolated fluorescent fluorescence emitters is derived. [5] Method according to claim 3 or 4, characterized by that for each isolated fluorescence emitter its center of gravity is determined in the image (17, 29) and used in step b) to determine the position of the isolated fluorescence emitter. [6] Fluorescence microscope for wavelength-selective imaging of a sample (2) with a spatial resolution increased beyond an optical resolution, comprising: - an illumination device (L1, L2, 3, 5) designed to repeatedly excite fluorescence emitters in the sample to emit fluorescence radiation - an imaging device (5, 10, K1) comprising an imaging beam path (10) with the optical resolution, which is designed to generate individual images (16) of the sample with the optical resolution, - a control device (C) which is designed to control the illumination device (L1, 2, 5) and the imaging device (5, 10, K1) in such a way that a plurality of individual images (16) are generated of the sample (2), wherein the fluorescence emitters are excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters is isolated in each individual image (16), and - the control device (C) is designed to localise the positions of the isolated fluorescent fluorescence emitters in the generated individual images (16) with a spatial accuracy exceeding an optical resolution and to generate a high-resolution overall image therefrom, characterized by , that - the imaging beam path (10) of the microscope has a spectrally selective element (11) which, when generating the individual images (16), imparts a spectrally dependent rotational asymmetry to a point image blurring function, so that the images (17, 18; 29, 30) of isolated fluorescent fluorescence emitters have a rotational asymmetry which depends on a wavelength at which the isolated fluorescent fluorescence emitters fluoresce, and - the control device is designed to analyze the images (17, 18; 29, 30) of the isolated fluorescence emitters in the individual images (16) with regard to their rotational asymmetry and to derive therefrom an indication of the wavelength of the isolated fluorescent fluorescence emitters. [7] Fluorescence microscope according to claim 6, characterized by that the spectrally selective element (11) is designed as a plate made of a dispersive material, the thickness of which varies along a radius around an optical axis. [8] Fluorescence microscope according to claim 6 or 7, characterized by that the plate (11) is constructed from a plurality of wedge-shaped sectors (12-15; 19-22) which differ in their spectral transmission properties, wherein sectors which are preferably point-symmetrical to one another have the same spectral transmission properties. [9] Fluorescence microscope according to claim 6, characterized bythat the spectrally selective element (11) has a central region (23) lying on an optical axis, which is surrounded by the sectors (19-22) which differ from one another and preferably also from the central region (23) with regard to their spectral transmission properties. [10] Fluorescence microscope according to one of claims 6 to 9, characterized by that the spectrally selective element (11) is arranged in a pupil or near a pupil of the imaging beam path (10).

Images