Wavelength-selective and spatially high-resolution fluorescence 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 of 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 for a subset of the fluorescence emitters, their images are isolated in each individual image (16), and wherein the sample is imaged onto a detector with the fluorescent fluorescence emitters, b) in the individual images (16) generated, the positions of the isolated fluorescent fluorescence emitters in the sample are localized with a spatial accuracy exceeding optical resolution and a high-resolution overall image is generated therefrom, c) the imaging beam path (10) of the microscope (1) has a diffractive element (11) whose diffraction properties are known and which, during imaging, diffracts the image of the sample with the isolated fluorescent fluorescence emitters into a first diffraction order, so that each individual image contains the images of the isolated fluorescent fluorescence emitters in the first diffraction order, d) in the individual images (16), a parameter of the images of the isolated fluorescent fluorescence emitters is evaluated in the first diffraction order and an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived therefrom, characterized in that fluorescence emitters are used which each consist of emitter pairs formed by one of a plurality of first emitters and a second emitter, the second emitter being present in each emitter pair, whereby in the individual images the image of each isolated fluorescent fluorescence emitter consists of a diffraction image of the first emitter and a diffraction image of the second emitter separate therefrom, and in that in step b) the positions of the isolated fluorescent fluorescence emitters in the sample are determined from the positions of the diffraction images of the second emitters, taking into account the known diffraction properties of the spectrally selective element (11).

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 / 127 692 A2 and 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 specialist 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 an overall image. This involves considerable computational effort, as a large number of molecules must be mathematically localized in each individual image. Large amounts of data are generated. DE 10 2009 043 744 A1 further develops the PALM method with regard to 3D resolution. Phase manipulation is used for this purpose.

[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 falls through the objective 5 to the sample 2, and the image of the sample is also produced 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] Another possibility for color discrimination using the PALM principle is known from the subsequently published WO 2012 / 039 636 A2. The generic DE 10 2008 049 886 A1 uses phase manipulation in a pupil for color resolution.

[0011] 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.

[0012] The invention is defined in claims 1 and 7.

[0013] In particular, a method for wavelength-selective and spatially high-resolution fluorescence microscopy is provided, wherein a) in a sample, fluorescence emitters 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 for a subset of the fluorescence emitters, their images are isolated in each individual image, and wherein the sample is imaged onto a detector with the fluorescent fluorescence emitters, b) in the individual images generated, the positions of the isolated fluorescent fluorescence emitters in the sample are localized with a spatial accuracy exceeding optical resolution and a high-resolution overall image is generated from this, c) the imaging beam path of the microscope has a spectrally selective element whose diffraction properties are known and which, during imaging, diffracts the image of the sample with the isolated fluorescent fluorescence emitters into a first diffraction order, so that each individual image contains the images of the isolated fluorescent fluorescence emitters in the first diffraction order, d) in the individual images, a parameter of the images of the isolated fluorescent fluorescence emitters is evaluated in the first diffraction order and from this an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived.

[0014] Furthermore, a fluorescence microscope is provided for wavelength-selective imaging of a sample with a spatial resolution increased beyond an optical resolution, which has: - an illumination device 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 and a detector, which is designed to image the sample with the optical resolution onto the detector and thus generate individual images, - a control device which is designed to control the illumination device and the imaging device in such a way that a plurality of individual images are generated of the sample, 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, wherein - the control device is designed to localise 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 whose diffraction properties are known and which, during imaging, diffracts the image of the sample with the isolated fluorescent fluorescence emitters into a first diffraction order, so that each individual image contains the images of the isolated fluorescent fluorescence emitters in the first diffraction order, - the control device is designed to evaluate the images of the isolated fluorescence emitters in the first diffraction order in the individual images and to derive therefrom an indication of the wavelength of the isolated fluorescent fluorescence emitters.

[0015] The invention overcomes the limitations of classical multicolor widefield detection for PALM-based high resolution by using the diffractive element, which images fluorescence emitters in diffraction patterns onto the detector. While conventional widefield microscopy strictly avoids such diffraction patterns and images color channels onto different detectors or separate detector areas, the invention images the fluorescent sample such that the sample image on the detector contains the first diffraction order of the diffractive element. Since, in the PALM principle, the individual fluorescence emitters flash stochastically throughout the generation of the individual images and neither position nor color are known a priori, a corresponding parameter of the images of the isolated fluorescent fluorescence emitters is evaluated in the first diffraction order.The parameter includes location and / or diffraction-induced distortion of the first-order diffraction images.

[0016] This parameter is evaluated based on the diffraction properties of the diffractive element. This allows the location and / or diffraction-induced distortion of the first-order diffraction images to be used to generate the wavelength information. Various approaches are possible for using this parameter to determine both the location and color of an isolated fluorescent emitter.

[0017] To enable the assignment of color to detected locations, the first emitter used for marking a sample is coupled to a second emitter whose spectral imaging properties in the diffractive element are known, thus having a known spectral luminescence behavior. Each first emitter comes from an emitter group. The emitters of the emitter group have different spectral fluorescence behavior (hereinafter referred to as "chromaticity"). Each emitter pair always contains the same second emitter and a first emitter from the emitter group. The first emitters color different sample structures differently. It is advisable to use an optical switch that is as bright as possible, i.e., photoefficient, for the second emitter, for example a so-called quantum dot.The sample is then marked with the coupled emitter pairs, and in each individual image the location is derived from the position of the second emitter, for which the diffraction behavior is determined.

[0018] The first emitter in the emitter pair provides the color marking. It is imaged to a different location on the detector according to its emission wavelength by the effect of the diffractive element. Therefore, both emitters emit independently of each other, and this is not a FRET pair.

[0019] To distinguish the chromaticity of the first emitter belonging to a second emitter, two different ways are possible, which can be combined to increase accuracy: 1. The distance between the location of the first emitter and the location of the second emitter can be measured. Since the spectral influence of the position of the second emitter, whose wavelength behavior is known, is known, the distance, as an absolute measure, encodes the chromaticity of the first emitter. The distance can be positive or negative, depending on whether the first emitter fluoresces at a shorter or longer wavelength than the second emitter. Due to the properties of the diffractive element in the image, the image of the first emitter is offset in a known spatial direction from the image of the second emitter; the measure and direction distinguish the chromaticities. The shift corresponds to half the spectral difference. 2. Due to the diffraction properties of the diffractive element, the image of the first emitter is rotationally asymmetrically distorted compared to the image of the second emitter. The diffractive element's first order of diffraction causes a chromatic aberration, which leads to a blurring of the point distribution that inevitably occurs in the imaging system. Alternatively or in addition to evaluating the distance, it is therefore also possible to evaluate the rotational asymmetry, as this is wavelength-dependent. The greater the wavelength difference between the first and second emitters, the more rotationally asymmetric the image of the first emitter. The degree of rotational asymmetry encodes the chromaticity of the first emitter.

[0020] The variants mentioned under numbers 1 and 2 are particularly easy to implement if the diffractive element used is a component that diffracts the majority of the incident radiation into the first diffraction order. One such component is a blazed grating or a so-called DOE. High imaging quality is achieved if this component is designed so that the image of the second emitter in the first diffraction order is as unsmeared as possible. With a blazed grating, the emission wavelength of this second emitter is selected as the blaze wavelength. This wavelength is de facto efficiently retroreflected. Tilting such a grating about its non-diffracting axis (so-called off-axis Littrow configuration) produces the desired image in which the second emitters, from which the spatial information is obtained, are imaged as unchanged as possible in the first diffraction order.

[0021] Since most fluorescence emitters have illuminated at least once at the end of a PALM measurement, chromaticity assignment can be performed in variants 1 and 2 after processing the entire data set by searching for emitter pairs with known distances in a known direction and / or with known rotational asymmetry. The assignment process can be further improved by deliberately using fluorescence emitters with different emission bandwidths for the first emitter.

[0022] The assignment of first and second emitters can be further simplified, and misassignments minimized, by selecting different photon statistics for the second emitters and the first emitters, or by influencing them so that they are different. This can be achieved by adjusting the excitation power and / or the chemical environment, as is known, for example, from the publication by van de Linde et al., Appl. Physics, B93, page 725, 2008. If different photon statistics are set for the second emitters than for the first emitters, the identification of the second emitters is simplified. For example, the second emitters can be selected or adjusted to be as bright as possible so that the photon yield of these second emitters occurs within one or two camera frames, whereas the emission of the first emitters occurs correspondingly more slowly.

[0023] Of course, instead of a pair of emitters, you can also use a group of three (hereinafter referred to as a “pair of three”), with there always being a second emitter in each pair of three.

[0024] For localization, i.e., to generate the position information, a third variant can be used instead of a pair of emitters, a single emitter, which also comes from an emitter group of different chromaticities, if the zeroth and first diffraction orders of the diffractive element are imaged simultaneously onto the detector. This results in a zeroth and first-order image for each fluorescence emitter. The distance between these two images encodes the chromaticity, and the position of the zeroth order encodes the position information. Here, too, rotational asymmetry, i.e., the smearing of the diffraction image in the first diffraction order, can be used alternatively or in addition to the distance to determine the chromaticity.

[0025] The inventive approach has the fundamental advantage that no alignment of color channels is required, since imaging occurs on a single detector. Complex readjustments over the lifetime of a microscope are thus unnecessary.

[0026] Furthermore, the data volume is significantly reduced because, despite multi-color measurements, only one camera needs to be read out and only the data from one camera needs to be processed and stored.

[0027] There is greater flexibility regarding the fluorescence emitters that can be used. The only limitation here is ultimately the spectral resolution of the system. This is particularly advantageous when tracking different molecules simultaneously.

[0028] Since only one camera is required, the construction effort and thus also the cost factor are reduced, since the high-resolution cameras are one of the most complex and therefore most expensive elements of a PALM system.

[0029] In the PALM system, the sample is typically attached to the fluorescence emitters. The connecting molecules required for this should be as short as possible (preferably between 22 nm). However, in the first and second variants, this requirement is only relevant for the second emitter, since only its images provide the spatial information. The first emitters, whose diffraction patterns only provide the color information, can also be connected to the sample and the second molecule at comparatively greater distances, as long as these distances do not change significantly (in which case, determining the color information might be difficult).

[0030] In the third variant, the diffraction images in the zeroth diffraction order are used for localization, i.e., for obtaining the position information. This increases the effective photon yield, since each photon is evaluated for both spatial and color information. Ultimately, the localization accuracy increases. However, the diffraction properties in the imaging beam path generated by the diffractive element must be precisely known. In this case, it is advantageous to perform a calibration measurement using calibrated, colored fluorescence emitters.

[0031] It is understood that the features mentioned above and those to be explained below can be used not only in the combinations indicated, but also in other combinations or on their own, without departing from the scope of the present invention.

[0032] 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 implementing the PALM principle with simultaneous color resolution, Fig. 2 a schematic representation of a diffraction pattern obtained in the microscope of Fig. 1 according to a first embodiment variant, Fig. 3 a schematic representation of a sample image after determining location information and before assigning color information, Fig. 4 a representation similar to the Fig. 3, but after the assignment of the color information, Fig. 5 a schematic representation similar to the Fig. 2 for determining the color information according to a second variant, Fig. 6 a representation similar to the Fig. 5 for a combination of the first with the second variant, Fig. 7 a representation similar to the Fig. 2 for a third variant for wavelength-selective and spatially high-resolution fluorescence microscopy and Fig. 8 a state-of-the-art fluorescence microscope.

[0033] Fig. Figure 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 source(s) 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 needs not be repeated. The camera represents a detector.

[0034] 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 sample 2 is transmitted via a diffractive element 11.

[0035] The PALM procedure described above, consisting of isolation and localization, is performed using a microscope. The relevant literature cited above is fully incorporated into this description.

[0036] The diffractive element ensures that fluorescent fluorescence emitters in the sample are imaged at different locations on the camera depending on their color, i.e., wavelength. The following variants are possible: In variant 1, the diffractive element 11 is designed as a blazed grating. It has a blazed wavelength that is efficiently retroreflected. This grating is tilted about its non-diffracting axis (so-called off-axis Littrow configuration), so that incident radiation with the blazed wavelength is imaged onto the camera K1 unmodified, but in the first diffraction order. To ensure this occurs efficiently, both the spectrally selective element 11 and the camera K are arranged in an intermediate image plane Z of the imaging beam path 10. Optics 14 to 16 are provided to generate the intermediate image planes Z, and deflecting mirrors 12 and 13 are provided for folding to keep the beam path compact. The number of intermediate image planes and the folding are optional. Furthermore, the imaging beam path 10 has a tube lens 17, as is known for fluorescence microscopes.

[0037] In order to enable the assignment of color to the detected locations of a fluorescence emitter in PALM microscopy, each fluorescence emitter is designed as an emitter pair, ie a first emitter, whose spectral behavior at a location in the sample P is unknown, is coupled to a second emitter, which has a known spectral behavior.

[0038] Sample 2 is labeled with fluorescent emitters consisting of coupled emitter pairs. The first emitters in each pair can vary, but each emitter pair has the same second emitter. In other words, for all fluorescent emitters, the second emitter is identical in type. The first emitter comes from an emitter group. Therefore, the color of a first emitter in a fluorescent emitter pair is unknown, but the set of possible colors is known. Therefore, the chromaticity of a first emitter must always be determined.

[0039] The diffractive element 11 in the form of a grating causes the camera K to record images in the first diffraction order for each isolated fluorescence emitter, i.e., images from both the same (second) emitters and the differently colored first emitters. This is shown schematically in Fig. 2. In Fig. 2 shows various fluorescence emitters, each consisting of a second emitter 18 and a first emitter 19 to 21, with the first emitters differing in their chromaticity. In the diffraction pattern on camera K, a diffraction pattern 22 of the second emitter 18 appears for the first emitter pair, and a diffraction pattern 23 shifted by a distance D3 appears for the first emitter 19. The distance is given by half the spectral difference between the fluorescence radiation from the second emitter 18 and the first emitter 19.

[0040] Below the emitter pair 18, 19, a pair of emitters 18, 20 is shown, the first emitter 20 of which fluoresces at a different wavelength than the first emitter 19 of the pair drawn above. Accordingly, a diffraction pattern 24 of the first emitter 20 results, which is at a different distance D2 from the diffraction pattern 22 of the second emitter 18.

[0041] Yet another distance, namely D1, is present for a diffraction pattern 25 of a first emitter 21 of a third chromaticity.

[0042] Since the first emitters 19, 20, 21 are known with regard to their color properties, but one does not know which chromaticity the first emitter in an emitter pair has, one receives an image on the camera K after PALM-compliant localization, as shown exemplarily in Fig. 3. Continuous sample structures 26 are recognizable therein based on the images of the second emitters 18. The images of the first emitters 19 to 21 are located at a distance that depends on the color of the first emitter. Since only known states (color types), in this case the three distances D1, D2, and D3, need to be distinguished (the number of color types of the first emitters can, of course, be varied), it is easy to determine at what distance the corresponding diffraction pattern of the first emitter lies for each diffraction pattern 22. The distance encodes the color type and thus the wavelength, so that after this processing in Fig. 4 can assign the colours 23f, 24f and 25f to the structures, whereby the reference numerals indicate the corresponding colour types of the first emitters 23 to 25.

[0043] Fig. Figure 5 shows a second variant of the evaluation of the images taken with microscope 1 of the Fig. 5 when using the described fluorescence emitter pairs. Here, the distance between the diffraction patterns is not evaluated, but rather the diffraction-induced distortion of the diffraction patterns 23 to 25 of the second fluorophores 19 to 21 indicates their chromaticity and thus their color.

[0044] Fig. Figure 6 shows a situation in which the number of possible different fluorescent labels has been further increased by using triple pairs in addition to pairs of two, i.e. emitter pairs consisting of the second emitter 18 and two different first emitters 19, 20; 20, 21; 19, 21. Accordingly, for each diffraction pattern, in addition to the diffraction pattern 22 of the second emitter 18, a combination of two diffraction patterns is obtained, namely the diffraction patterns 23 and 24, 24 and 25, or 23 and 25. Combining these triple pairs with the conventional pairs according to Fig. 5, six different fluorescent labels are obtained using three different fluorescent emitters, where only one is common in all pairs.

[0045] Of course, it is possible to combine the first and second variants, i.e., to evaluate the distance, i.e., the spectral difference between diffraction pattern 22 and the diffraction pattern of the first emitters 19 to 21, as well as the spectral rotation asymmetry, i.e., the blurring of the diffraction patterns of the first fluorescence emitters. This increases the discrimination accuracy in sample sections where the sections do not allow for clear resolution because the luminous fluorescence emitters are too close together.

[0046] A modification of the microscope of the Fig.1 consists in using a conventional grating instead of a blazed grating or a DOE as the diffractive element 11, and only individual markings (i.e., no emitter pairs) are made when marking the sample with fluorescence emitters. In this case, the imaging in the imaging beam path 10 is designed such that the zeroth and first diffraction orders of the grating are imaged simultaneously onto the camera K. This results in a respective first diffraction image 23 to 25 for each fluorescence emitter 19 to 21, as well as an additional non-diffracted image (zeroth diffraction order) 26. The first diffraction order, in turn, carries the color information, either through its distance from the image 26 of the zeroth diffraction order, or through its smearing. The evaluation is therefore analogous to the first variant.

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 of 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 for a subset of the fluorescence emitters, their images are isolated in each individual image (16), and wherein the sample is imaged onto a detector with the fluorescent fluorescence emitters, b) in the individual images (16) generated, the positions of the isolated fluorescent fluorescence emitters in the sample are localized with a spatial accuracy exceeding optical resolution and a high-resolution overall image is generated therefrom, c) the imaging beam path (10) of the microscope (1) has a diffractive element (11) whose diffraction properties are known and which, during imaging, diffracts the image of the sample with the isolated fluorescent fluorescence emitters into a first diffraction order, so that each individual image contains the images of the isolated fluorescent fluorescence emitters in the first diffraction order, d) in the individual images (16), a parameter of the images of the isolated fluorescent fluorescence emitters is evaluated in the first diffraction order and an indication of the wavelength of the isolated fluorescent fluorescence emitters is derived therefrom, characterized bythat fluorescence emitters are used which each consist of emitter pairs which are formed by one of a plurality of first emitters and a second emitter, the second emitter being present in each emitter pair, whereby in the individual images the image of each isolated fluorescent fluorescence emitter consists of a diffraction image of the first emitter and a separate diffraction image of the second emitter, and that in step b) the positions of the isolated fluorescent fluorescence emitters in the sample are determined from the positions of the diffraction images of the second emitters, taking into account the known diffraction properties of the spectrally selective element (11). [2] Method according to claim 1 characterized by that in step d) -- the distance of the diffraction pattern of each second emitter from the diffraction pattern of the corresponding first emitter is evaluated as a parameter and / or -- the diffraction causes a non-rotationally symmetric smearing of the images of the isolated fluorescent fluorescence emitters and an amount of the non-rotationally symmetric smearing of the first emitters is evaluated as a parameter. [3] Method according to claim 1 or 2, characterized by that for each image of an isolated fluorescent fluorescence emitter, a location information is corrected by a diffraction-related offset on the basis of the determined information on the wavelength of the isolated fluorescent fluorescence emitter and taking into account the known diffraction properties of the spectrally selective element (11). [4] Method according to one of the above claims, characterized by that a component is used as the diffractive element (11) which diffracts the predominant part of incident radiation into the first diffraction order, wherein in particular the component is a blazed grating or a diffractive optical element (DOE). [5] Method according to one of the above claims, characterized by that the image of each isolated fluorescent fluorescence emitter is also imaged onto the detector in a zeroth diffraction order and a distance between the image in the zeroth diffraction order and the image of this fluorescence emitter in the first diffraction order is evaluated as a parameter. [6] Method according to one of the above claims, characterized by that the image of each isolated fluorescent fluorescence emitter is also imaged onto the detector in a zeroth diffraction order and in step b) the position of each fluorescence emitter is determined from the image of the zeroth diffraction order. [7] 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 and a detector, which is designed to image the sample onto the detector with the optical resolution and thus generate individual images (16), - 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), wherein - 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, - the imaging beam path (10) of the microscope has a diffractive element (11) whose diffraction properties are known and which, during imaging, diffracts the image of the sample with the isolated fluorescent fluorescence emitters into a first diffraction order, so that each individual image contains the images of the isolated fluorescent fluorescence emitters in the first diffraction order, - the control device is designed to evaluate the images of the isolated fluorescence emitters in the first diffraction order in the individual images (16) and to derive therefrom an indication of the wavelength of the isolated fluorescent fluorescence emitters, characterized by , that - the control device is designed for the use of fluorescence emitters, each consisting of emitter pairs formed by one of a plurality of first emitters and a second emitter, the second emitter being present in each emitter pair, whereby in the individual images the image of each isolated fluorescent fluorescence emitter consists of a diffraction image of the first emitter and a separate diffraction image of the second emitter, and the control device is designed to determine the position of the isolated fluorescent fluorescence emitters in the sample from the position of the diffraction images of the second emitters, taking into account the known diffraction properties of the spectrally selective element (11). [8] Fluorescence microscope according to claim 7, characterized by that the control device is designed to carry out one of the methods according to one of claims 1 to 6.

Images