METHOD AND DEVICE FOR HIGH-RESOLUTION OPTICAL SCANNING OF A SAMPLE

Description

[0001] The invention relates to a method for high-resolution optical scanning of a sample, preferably using a laser scanning fluorescence microscope, wherein the sample comprises a substance that can be translocated into different energy states – first state Z1 and second state Z2 – wherein the first state Z1 and the second state Z2 differ from each other in at least one optical property, wherein the sample is illuminated with focused light of a wavelength of the excitation spectrum of the substance to generate the first state Z1 of the substance in a region, wherein the sample is illuminated in a focus edge region of the excitation with light of a suitable de-excitation wavelength to generate the second state Z2 of the substance, and wherein emission light emanating from the sample, resulting from a decay of remaining first states Z1, is detected by means of a detection device.wherein the sample comprises at least one further substance that can be converted into a first state Z1' and into a second state Z2'.

[0002] Furthermore, the invention relates to a device for high-resolution optical scanning of a sample, in particular for carrying out a method according to the invention, comprising a substance that can be converted into different energy states – first state Z1 and second state Z2 – wherein the first state Z1 and the second state Z2 differ from each other in at least one optical property, with at least one light source configured for illuminating the sample with focused light of a wavelength of the excitation spectrum of the substance for the region-wise generation of the first state Z1 of the substance, as well as for illuminating the sample with light of a suitable de-excitation wavelength for the generation of the second state Z2 of the substance in a focus edge region of the excitation, and with a detection device for detecting the emission light emanating from the sample, which results from a decay of remaining first states Z1.is trained.

[0003] Methods and devices of the type under discussion here have been known in practice for some time and are used, for example, in STED microscopy. With the imaging optical methods and devices in question, spatial resolutions beyond the theoretical limit determined by Abbe's law, which is dependent on the wavelength of the light used, can be achieved.

[0004] In STED microscopy, a substance is placed in the sample under investigation that can be excited by light and then rapidly de-excited from this excited state. Fluorescent dyes are predominantly used as such substances in STED microscopy. Generally, the substance is first excited using short-wavelength light, for example, a green laser pulse. Then, the substance is selectively de-excited at the edge of the excitation focus using a long-wavelength (e.g., red) laser pulse. To achieve de-excitation of the substance only at the edge of the focus, the de-excitation point function is specially shaped. Phase filters are generally used for this purpose. These filters are positioned in the beam path of the long-wavelength laser beam and modify the wavefront of the de-excitation light beam depending on its location.Crucially, the transition from the excited to the de-excited state induced by the de-excitation light beam is saturated, i.e., complete, at the edge of the sample, so that the substance remains in the excited state only in a (in principle arbitrarily) small central region. In this way, the de-excitation light pulse prevents the emission of fluorescence from the edge of the diffraction-limited excitation spot. The detected fluorescence therefore originates from a narrowly defined sample region, the diameter of which, due to the de-excitation saturation, can be significantly smaller than allowed by Abbe's law.

[0005] STED microscopy, like all other known methods that achieve increased resolution by saturating an de-excited state, is operated in monochromatic mode. This means that only one substance, for example, a single fluorescent dye, is present in the sample. In contrast, confocal microscopy typically involves presenting several different fluorescent dyes in the sample and acquiring multicolor images. This allows for the simultaneous investigation of different processes within the sample. Consequently, multicolor images obtained using conventional microscopy can provide significantly more information (albeit at a lower resolution) than is currently possible with known high-resolution imaging techniques.

[0006] Stefan W. Hell: "Toward fluorescence nanoscopy" NATURE BIOTECHNOLOGY, Vol. 21, No. 11, October 31, 2003 (2003-10-31), pages 1347-1355, XP002458686 discloses a method for high-resolution optical scanning of a sample using a laser scanning fluorescence microscope. The sample comprises a substance that can be shifted into different energy states.

[0007] T. LACOSTE, X. MICHALET, F. PINAUD, D. CHEMLA, P. ALIVISATOS, S. WEISS: "ultrahigh-resolution multicolor colocalization of single fluorescent probes" PNAS, Vol. 97, No. 17, August 15, 2000 (2000-08-15), pages 9461-9466, XP002458687 discloses a non-STED-based method for multicolor analysis.

[0008] WO 2005 / 040771 A discloses a method for optically scanning a sample, wherein the sample has at least two fluorescent substances.

[0009] DE 10 2006 011 556 A1 discloses a method for high-resolution optical scanning of a sample. The sample contains two substances that can be converted into an excited and an de-excited state.

[0010] Katrin I Willig et al, "Nanoscale resolution in GFP-based microscopy", Nature Methods, Vol. 3, No. 9, Sep. 2006, pages 721 to 723, discloses the use of a GFP-based dye in STED microscopy.

[0011] The present invention is based on the objective of providing a method and a device of the type mentioned at the outset, according to which a high spatial resolution beyond the diffraction limit is achieved with simultaneously high information yield using structurally simple and cost-effective means.

[0012] The problem is solved by a method characterized in that the substances differ from one another in at least one property, namely with respect to their excitation spectra, and that the substances at least partially agree in at least one other property, namely with respect to their emission spectra, wherein the substances are excited with light of different excitation wavelengths but are de-excited with light of the same de-excitation wavelength, and in that the steps of excitation and / or de-excitation and / or detection are carried out separately for each substance when scanning the sample, and the detection device comprises several detectors, wherein one or more detectors of the detection device are assigned to each substance.

[0013] Furthermore, the problem is solved by a device of the type mentioned above, characterized in that the device is configured to carry out the steps of excitation and / or de-excitation and / or detection for at least one further substance encompassed by the sample and which can be converted into a first state Z1' and a second state Z2', wherein the substances differ from each other in at least one property, namely with respect to their excitation spectra, and at least partially agree in at least one other property, namely with respect to their emission spectra, and in that the device is configured to excite the substances with light of different excitation wavelengths.but stimulated to excite with light of the same de-excitation wavelength, and that when scanning the sample, the steps of excitation and / or de-excitation and / or detection can be carried out separately in time for the individual substances, and that the detection device comprises several detectors and that at least one of the detectors can be assigned to each substance.

[0014] According to the invention, it has first been recognized that the information obtained from high-resolution optical scanning of a sample can be increased by producing a multi-colored image of the sample. According to the invention, at least one further substance is provided in the sample for this purpose, which can be converted into a first state and a second state, wherein the substances differ from each other in at least one property and at least partially correspond in at least one other property. Properties of the substances include, for example, their excitation wavelengths or excitation spectra, their emission spectra, de-excitation wavelengths, or lifetimes. The selection of substances according to the invention, whereby they differ from each other in at least one property, is used to separate the two substances from one another.The further at least partial agreement in another property according to the invention enables a particularly simple construction, namely, it makes it possible to use the same components or assemblies, such as illumination light sources or detector devices, for several substances together.

[0015] Furthermore, it has been recognized that simultaneous excitation and de-excitation of several substances can lead to the de-excitation of one substance being excited by another. Due to the high power required to induce a saturated transition to the de-excitation state, a substance excited by a de-excitation wavelength is bleached very quickly and is therefore no longer available for detection. To circumvent this problem, the excitation and / or saturated de-excitation and / or detection steps for each individual substance are performed separately during sample scanning according to the invention.

[0016] It should be emphasized at this point that, within the scope of the present invention, the terms "excitation spectrum" or "excitation wavelength" and "de-excitation spectrum" or "de-excitation wavelength" are not to be understood narrowly, and the invention is by no means limited to stimulated emission. Rather, the invention relates to all methods in which an increase in resolution is generated by saturating a reversible molecular transition. This includes, for example, the use of photoswitchable or photoconvertible proteins, which can be switched between fluorescent and non-fluorescent states by irradiation with light of specific wavelengths. The switching on and off of fluorescence is preferably effected by different wavelength bands, so that an increase in resolution can be achieved by saturating the switched-off state.In general, these types of high-resolution optical imaging techniques are referred to as RESOLFT techniques. Besides its use in STED microscopy, the invention can also be used in STED-4Pi microscopy, up-conversion microscopy, and ground-state depletion (GSD).

[0017] The substances are selected such that their de-excitation spectra overlap at least partially, allowing them to be converted to the second state using the same wavelength. From a design perspective, it proves advantageous to use only one common illumination source for converting the substances to the second state. Since illumination sources used for stimulated de-excitation generally require high light output and are therefore expensive, significant cost savings can be achieved in this way. The excitation spectra could differ for the substances in this case, allowing them to be excited separately with different wavelengths.

[0018] According to a further preferred embodiment, the substances may be selected such that their excitation spectra at least partially overlap. In this case, excitation can be achieved using a single illumination light source. One or more illumination light sources may be provided for transitioning the substances to the second state.

[0019] The substances are selected such that their emission spectra overlap at least partially. This design proves advantageous insofar as the detection of the emission light is particularly easy in this case.

[0020] In one specific embodiment, the temporal sequence of the excitation, de-excitation, and / or detection steps is determined according to the magnitude of the respective de-excitation wavelengths of the substances. Advantageously, each scanning step begins with the substance exhibiting the longest de-excitation wavelength. This approach has the advantage that the emission light emitted by the substance can be detected without interference from another substance. Consequently, excitation of the substance by de-excitation light from another substance prior to the detection of its emission light is avoided.

[0021] Advantageously, the first energy state is a fluorescent state. Fluorescence light is very easy to handle for detection, and a wide variety of substances are available with which the sample can be labeled.

[0022] Due to the low quantum yield of fluorescence, it may be advantageous to perform several excitation, de-excitation and / or detection cycles, i.e., that the steps of excitation and / or (saturated) de-excitation and / or detection of emission light are carried out several times in succession for one substance before moving on to the next substance.

[0023] Sequential sampling of individual substances can be performed point by point, row by row, frame by frame, or even in batches. In point-by-point sampling, for example, all substances present in the sample are initially examined in the manner described before the scanning process continues at the next point. In frame-by-frame sampling, a sample area to be examined is first completely scanned, with only one substance being examined in the manner described. In a subsequent complete scan of the sample area, only a different substance is examined, and so on.

[0024] In a specific embodiment, the detection device for detecting the emission light emanating from the sample may include a detector with a wavelength-dependent element. The wavelength-dependent element may, for example, be configured as a filter. According to the invention, the detection device comprises several detectors, wherein one or more detectors of the detection device can be assigned to each substance.

[0025] Advantageously, the detectors of the detection device can be switched off individually and / or in groups. This prevents the detection of emission light from one substance that results from excitation of that substance with light of a de-excitation wavelength from another substance. The switch-off can be provided for the duration of the irradiation with the de-excitation wavelength. Alternatively or additionally, detection can be prevented by using blocking filters and / or apertures.

[0026] To achieve particularly high efficiency, the light used to generate the de-excited state of one substance can be used simultaneously to generate the excited state of another substance. Preferably, light pulses with a pulse width of 100 ps or longer are used to generate the de-excited state. This minimizes the phototoxicity of the de-excitation pulse.

[0027] With a view to a particularly comprehensive avoidance of mutual interference between the individual substances, the substances and the de-excitation wavelengths can be selected in a particularly advantageous way such that the de-excitation wavelengths of the substances are each outside the excitation spectra of the other substances.

[0028] With a view to high flexibility and user-friendliness, it can be provided that the excitation and de-excitation wavelengths for each substance can be freely selected. This enables optimal excitation and stimulated de-excitation of the substances. Preferably, a software interface is provided through which a user can select the excitation and de-excitation wavelengths. The wavelengths can, for example, be entered or freely set by the user. Alternatively or additionally, it is possible to provide a table to the user from which excitation and / or de-excitation wavelengths can be selected.

[0029] There are now various ways to advantageously elaborate and further develop the teaching of the present invention. For this purpose, reference should be made, on the one hand, to the claims subordinate to the independent claims and, on the other hand, to the following explanation of preferred embodiments of the invention with reference to the drawings. In conjunction with the explanation of the preferred embodiments of the invention with reference to the drawings, generally preferred embodiments and further developments of the teaching are also explained. The drawings show schematic representations. Fig. 1 a spectral distribution of an excitation and an emission spectrum of a single fluorescent dye, Fig. 2 the spectra of two fluorescent dyes as used according to a first embodiment of the method according to the invention, Fig. 3 the spectra of two fluorescent dyes as used according to an example not belonging to this invention, Fig. 4 an embodiment of a device according to the invention, which is particularly useful in connection with the methods described in Fig. 2 Fig. 5 shows another example of a device not belonging to this invention, which is particularly useful in connection with the spectral conditions shown in Fig. 3 The spectral conditions shown are applicable, and Fig. 6 is an embodiment of a device according to the invention with separate excitation and separate de-excitation of two fluorescent dyes.

[0030] Fig. 1 Figure 1 shows the spectral distribution of an excitation spectrum 1 and an emission spectrum 2 of a substance S1, specifically a fluorescent dye. The use of fluorescent dyes with the shown spectral distribution is typically employed in monochromatic STED microscopy, according to the known state of the art. In this process, a sample under investigation is illuminated with a short-wavelength laser pulse of excitation wavelength 3, preferably from a pulsed laser diode. Subsequently, the fluorescent molecules at the excitation focus edge are de-excited by applying a long-wavelength laser pulse of de-excitation wavelength 4.To achieve de-excitation of the fluorescence molecules exclusively at the focal edge, the de-excitation point image function is specially shaped by inserting phase filters in the beam path of the long-wavelength laser beam. These filters alter the wavefront of the de-excitation beam depending on its position. The de-excitation of the fluorescence molecules is carried out to saturation, so that only in a very small central region do fluorescence molecules remain in the excited state. The emission light resulting from the spontaneous decay of these remaining excited states is then detected with high spatial resolution.

[0031] Fig. 2 Figure 1 schematically shows the excitation spectra 1, 1' and the emission spectra 2, 2' of two substances S1 and S2, as they can be used according to a first embodiment of the method according to the invention. In the illustrated embodiment, two fluorescent dyes are used as substances S1 and S2, which are excited with different excitation wavelengths but de-excited with the same de-excitation wavelength.

[0032] To separate the two dyes, they are excited sequentially. For example, one dye is first excited by a laser pulse of wavelength λ1, then de-excited by a laser pulse of wavelength λ3. This sequence is repeated several times until sufficient fluorescence light has been collected from this dye. Then the second dye is excited by a laser pulse of wavelength λ2 and de-excited by a laser pulse of wavelength λ3. This second sequence is also repeated several times until sufficient fluorescence light has been detected.

[0033] Alternatively, a pulse sequence can be used with the following order: pulse of wavelength λ₁, pulse of wavelength λ₃, pulse of wavelength λ₂, pulse of wavelength λ₃, ... Such a pulse sequence proves particularly advantageous, as it allows the use of laser light sources where the laser light source with wavelength λ₃ has twice the pulse repetition frequency of the laser light sources with wavelengths λ₁ and λ₂. Mixtures of the pulse sequences described above can also be used.

[0034] Sequential acquisition of the different color channels can be performed, for example, point by point, line by line, frame by frame, or stack by stack. To separate the dyes, the pulse sequence is advantageously synchronized with the detection so that the light detected during the first sequence can be assigned to one dye, and the light detected during the second sequence can be assigned to the second dye. The laser pulses are also synchronized with each other. In particular, the excitation and de-excitation pulses belonging to each dye are synchronized. The pulse intervals are generally different in the first and second sequences. If the emission spectra of the two dyes are sufficiently different, the fluorescence light can also be separated by a wavelength-sensitive element (filter). This then allows, for example,The use of the pulse sequence wavelength λ1, wavelength λ2, wavelength λ3, etc. The laser light source used to generate the de-excitation pulses with wavelength λ3 must deliver high laser power to achieve de-excitation of the excited states to saturation and is therefore relatively expensive. In this respect, the case described here of a common de-excitation wavelength for both substances S1 and S2 is particularly preferable, since only one de-excitation laser is required. Fig. 4 shows a possible setup of a scanning microscope, which is adapted to the situation described here in terms of its design.

[0035] In the context of an example not belonging to this invention, which is described in Fig. 3 As shown, fluorescent dyes S1 and S2 are used as substances such that their excitation spectra 1 and 1' allow excitation at the same wavelength λ4. For stimulated de-excitation, however, different wavelengths λ5 and λ6 are used. If the fluorescence spectra 2 and 2' of the two dyes differ sufficiently, their fluorescence can be separated by a wavelength-sensitive element (filter), and each dye's fluorescence can be recorded with its own detector.

[0036] Since the lifetimes of the excited states of the two dyes are generally different, it is particularly advantageous to select a different pulse interval between the excitation pulse and the first de-excitation pulse, as well as between the excitation pulse and the second de-excitation pulse. The pulses are temporally synchronized. In particular, the excitation and de-excitation pulses belonging to a single dye are synchronized. Additionally, the pulse sequences can be synchronized with the detection. This is especially useful when the fluorescence spectra 2, 2' of the two dyes differ only slightly. The pulse sequence excitation pulse (λ4), first de-excitation pulse (λ5), second de-excitation pulse (λ6), etc., is preferably chosen. This sequence is repeated several times until sufficient fluorescence light has been collected. Fig. 5 shows a possible setup of a scanning microscope, which is adapted to the situation described here in terms of its design.

[0037] Fig. 4 Figure 1 shows – in a schematic representation – an embodiment of a device according to the invention for high-resolution optical scanning of a sample 10 labeled with two (not shown) dyes S1 and S2. The device, designed as a scanning microscope, is suitable for use with dyes with a spectral characteristic such as that found in Fig. 2 The scanning microscope is adapted as shown. It comprises a light source 49 designed as a pulsed laser 53, which emits a first excitation light beam 51. The excitation light beam 51, with a wavelength λ1, excites the first dye S1 and, after passing through the excitation aperture 55, strikes the lens arrangement 20 and is collimated by the lens arrangement 20. The excitation light beam 51 then strikes the beam splitters 26 and 73 and the main beam splitter 57, which directs the excitation light beam 51 to the beam deflection device 59, which includes a gimbal-mounted scanning mirror 61.

[0038] The scanning microscope further comprises a second light source 21, which emits a second excitation light beam 22 with a wavelength λ 2 to excite the second dye S2. The second light source 21 is also designed as a pulsed laser 23. After passing through the excitation aperture 24, the excitation light beam 22 strikes the optics 25 and is collimated by the optics 25 and combined with the first excitation light beam 51 by means of the optical element 26.

[0039] The scanning microscope includes a third light source 63, configured as a pulsed laser 65, to which the first light source 49 and the second light source 21 are synchronized. The third light source 63 emits a light beam 19, hereinafter referred to as the stimulation beam 67. The stimulation beam 67 comprises light of wavelength λ3, with which both dye S1 and dye S2 are stimulated and de-excited. The stimulation beam 67 is collimated by an optical element 5 and strikes a phase filter 69. The stimulation beam 67 emerging from the phase filter 69 strikes the dichroic beam splitter 73. The phase filter 69 is imaged into the pupil of the objective lens by the optical element 9. The dichroic beam splitter 73 combines the two already combined excitation beams 51 and 22 and the stimulation beam 67 into a common beam path.The stimulation light beam 67 is also directed by the main beam splitter 57 to the beam deflection device 59. The beam deflection device 59 guides the excitation light beams 51 and 22, as well as the stimulation light beam 67, together through the scanning optics 75, the tube optics 77, and the focusing optics 79, namely the microscope objective 81, over or through the sample 10.

[0040] The focus of the excitation light beams 51 and 22 optically excites the sample 10 at a point on the sample, while the focus of the stimulation light beam 67 excites and de-excites an outer area of ​​the excited sample region. The focus of the stimulation light beam 67 is designed as a hollow focus for this purpose. In this variant, the phase filter 69 is arranged in a plane conjugate to the focal plane of the microscope objective 81 (Fourier plane).

[0041] The detection light 85 emanating from sample 10 passes through the microscope objective 81, the tube optics 77, the scan optics 75 to the beam deflection device 59, from there to the main beam splitter 57, passes through this and the subsequent detection aperture 87, and finally reaches the detection device 89, which is designed as a multiband detector 91. The detector 91 generates electrical signals proportional to the light intensity of the detection light 85, which, together with the position signals of the beam deflection device 59, are transmitted to a processing unit (not shown) for image display.

[0042] Fig. 5 shows another example of a scanning microscope according to the invention, not belonging to this invention, for two dyes S1 and S2, which is based on a spectral characteristic of the dyes as described in Fig. 3 is shown, is adapted. The in Fig. 5 The scanning microscope shown is largely similar to the one in Fig. 5 The scanning microscope shown. Identical components are marked with the same reference symbols. In contrast to the scanning microscope according to Fig. 5 The scanning microscope shown here has only one light source 49 for generating an excitation light beam 51. The excitation light beam 51 comprises light with wavelength λ 4, which serves to excite both the first dye S1 and the second dye S2.

[0043] As the scanning microscope according Fig. 4 This also includes the scanning microscope according to Fig. 5 A light source 63, configured as a pulsed laser 65, is synchronized to the first light source 49. The second light source 63 emits a light beam 19, hereinafter referred to as the stimulation light beam 67. In this case, the stimulation light beam 67 comprises light of wavelength λ5, with which the first dye S1 is stimulated and de-excited. The stimulation light beam 67 is collimated by an optic 5 and strikes a phase filter 69.

[0044] The scanning microscope also includes a third light source 30, configured as a pulsed laser 31, to which the light source 49 is synchronized. The third light source 30 emits a light beam 35, hereinafter referred to as the stimulation beam 32. The stimulation beam 32 comprises light with a wavelength of λ6, which is used to stimulate and de-excite the second dye S2. The stimulation beam 32 is collimated by an optic 33 and strikes a phase filter 34. The stimulation beams 67 and 32, originating from the phase filters 69 and 34, are deflected by the dichroic beam splitters 73 and 37. The phase filters 69 and 34 are imaged into the pupil of the objective by the optics 9 and 36. The beams are directed by the main beam splitter 57 to the beam deflection device 59.The beam deflection device 59 guides the excitation light beam 51 and the stimulation light beams 67 and 32 together through the scan optics 75, the tube optics 77 and the focusing optics 79, namely the microscope objective 81, over or through the sample 10.

[0045] Regarding the foci of the excitation light beam 51 and the stimulation light beams 67 and 32, as well as regarding the detection of the detection light 85, the same applies as in connection with Fig. 4 As already explained above.

[0046] By adjusting the optics 20, 25, 5 and 33 in the axial direction, the chromatic longitudinal aberration of the lens 81 can be compensated and the foci of excitation and de-excitation can be superimposed.

[0047] Fig. 6 Finally, an embodiment of a scanning microscope according to the invention is shown, the structure of which is largely the same as the structure of the one described in the Fig. 4 and 5The scanning microscopes shown are shown. The only difference is that two separate light sources 49 and 23 are provided for the excitation of the two substances S1 and S2, as well as two separate light sources 63 and 30 for the stimulated de-excitation.

[0048] Regarding further advantageous embodiments of the device according to the invention, reference is made to the general part of the description and to the attached patent claims in order to avoid repetition.

[0049] Finally, it should be expressly pointed out that the exemplary embodiments of the device according to the invention described above serve only to discuss the claimed teaching, but do not limit it to these exemplary embodiments.

Claims

1. A method for high-resolution optical scanning of a sample, preferably using a laser scanning fluorescence microscope, wherein the sample (10) comprises a substance (S1) that can be set to different energy states - first state Z1 and second state Z2 - wherein the first state Z1 and the second state Z2 differ from one another in at least one optical property, wherein the sample (10) is illuminated with focused light of a wavelength of the excitation spectrum (1) of the substance (S1) in order to generate the first state Z1 of the substance (S1) in specific regions, wherein the sample (10) is illuminated with light of a suitable de-excitation wavelength in a focus edge region of the excitation in order to generate the second state Z2 of the substance (S1), and wherein emission light (85), which emanates from the sample (10) and which results from a decay of remaining first states Z1, is detected by means of a detection device (89), wherein the sample (10) comprises at least one further substance (S2) that can be transferred to a first state Z1' and to a second state Z2', wherein the substances (S1, S2) differ from one another in at least one property, namely in terms of their excitation spectra, characterised in that the substances (S1, S2) coincide at least partially in at least one other property, namely in terms of their emission spectra, wherein the substances (S1, S2) are excited with light of different excitation wavelengths but are de-excited with light of the same de-excitation wavelength in a stimulated manner, and when scanning the sample (10), the steps of excitation and / or de-excitation and / or detection for the individual substances (S1, S2) are carried out separately from one another in terms of time, and the detection device (89) comprises a plurality of detectors, wherein one or a plurality of detectors of the detection device (89) are each assigned to a substance (S1, S2).

2. The method according to claim 1, characterised in that the substances (S1, S2) are selected in such a manner that the excitation spectra (1, 1') of the substances (S1, S2) partially overlap one another.

3. The method according to claim 1 or 2, characterised in that a sequential recording of the individual substances (S1, S2) is carried out point by point, line by line, image by image or stack by stack.

4. The method according to any one of claims 1 to 3, characterised in that a separate detector is used for each substance (S1, S2).

5. The method according to any one of claims 1 to 4, characterised in that during the illumination of the sample (10) with the de-excitation wavelength, the detection of emission light (85) of another substance (S2), the excitation spectrum of which (1') comprises the irradiated de-excitation wavelength, is suppressed.

6. The method according to claim 5, characterised in that the detection of the emission light (85) of another substance (S2), the excitation spectrum of which (1') comprises the irradiated de-excitation wavelength, is suppressed by means of a wavelength-dependent element, for example a filter, and / or by switching off the corresponding detector and / or by connecting a blocking filter or an aperture upstream.

7. The method according to any one of claims 1 to 4, characterised in that the substances (S1, S2) are selected in such a manner that the de-excitation wavelength in each case lies outside the excitation spectra (1, 1') of the other substances (S1, S2).

8. A device, in particular for carrying out a method according to any one of claims 1 to 7, for high-resolution optical scanning of a sample (10) comprising a substance (S1) that can be set to different energy states - first state Z1 and second state Z2 - wherein the first state Z1 and the second state Z2 differ from one another in at least one optical property, with at least one light source (49, 23, 63, 30) which is designed to illuminate the sample (10) with focused light of a wavelength of the excitation spectrum of the substance (S1) in order to generate the first state Z1 of the substance (S1) in specific regions as well as to illuminate the sample (10) with light of a suitable de-excitation wavelength in order to generate the second state Z2 of the substance (S1) in a focus edge region of the excitation, and with a detection device (89) which is designed to detect the emission light (85), which emanates from the sample (10) and which results from a decay of remaining first states Z1, characterised in that the device is designed to carry out the steps of excitation and de-excitation and detection for at least one further substance (S2) which is comprised of the sample (10) and which can be transferred to a first state Z1' and to a second state Z2', wherein the substances (S1, S2) differ from one another in at least one property, namely in terms of their excitation spectra, and coincide at least partially in at least one other property, namely in terms of their emission spectra, and in that the device is designed to excite the substances (S1, S2) with light of different excitation wavelengths, but to de-excite with light of the same de-excitation wavelength, and in that when scanning the sample (10), the steps of excitation and / or de-excitation and / or detection for the individual substances (S1, S2) can be carried out separately from one another in terms of time, and the detection device (89) comprises a plurality of detectors and at least one of the detectors can be assigned to each substance (S1, S2).

9. The device according to claim 8, characterised in that a separate detector can be used for each substance (S1, S2).

10. The device according to claim 8 or 9, characterised in that the device is a laser scanning fluorescence microscope.