Method for flexible and super-resolved adjustment of the axial resolution of a fluorescence microscope

Abstract

The invention relates to a method for the flexible and super-resolved adjustment of the axial resolution of a fluorescence microscope. The object of the present invention, to provide a method for the flexible and super-resolved adjustment of the axial resolution of a confocal fluorescence microscope, is achieved by carrying out the method using a fluorescence microscope, at least two lasers which are pulsed, intensity-modulated or continuous, a radiolucent material and a measuring device for determining the difference between the fluorescence lifetimes of fluorescent molecules, wherein an evanescent wave formed by supercritical angle illumination of a laser in the fluorescence microscope is used, with a wavelength which causes stimulated emission in the fluorescent molecules of a sample, and light with a wavelength which excites the fluorescence emission of the fluorescent molecules by at least one further laser.A fluorescence excitation of the fluorescent molecules is caused by a stimulated emission of these fluorescent molecules, whereby the strength of the stimulating emission, through superposition with the evanescent illumination, leads to a readout parameter in the form of the fluorescence lifetime for an axial nanometer-accurate spatial resolution, and with the measuring device, by measuring the difference between the fluorescence lifetimes of the fluorophores in the presence and absence of the evanescent wave, the information about the axial position is obtained according to the equation τ'(z) / τ(z) = (1 + σl0τ(z)e, -z / d ) is won.

Description

[0001] The invention relates to a method for flexible and super-resolved adjustment of the axial resolution of a fluorescence microscope of the type claimed.

[0002] Optical fluorescence microscopy is an extremely important tool for investigating and uncovering molecular processes in cells, e.g. for the development of improved drugs.

[0003] Since the beginning of the 2000s, super-resolution fluorescence microscopy has revolutionized basic research in the life sciences, as several approaches such as STED (STimulated Emission Depletion), (d)STORM [(direct)STochastic Reconstruction Microscopy] or (f)P ALM - ((fluorescence) PhotoActivation Localization Microscopy), SIM (Structured Illumination Microscopy) or MINFLUX microscopy have been developed and commercialized, which allow the observation of cellular structures with higher resolution, which was awarded the Nobel Prize in Chemistry in 2014.

[0004] The axial (i.e., in the direction of light propagation) resolution of fluorescence microscopy is known to be far inferior to the lateral (i.e., in the plane of the sample) resolution. The lateral resolution is defined by the formula: rlateral=λem / 2NA where λ emis the emission wavelength of a fluorescent sample, and NA is the numerical aperture of the objective. In this case, we assumed an inverted microscope geometry, where the condenser and objective are the same object, hence the factor 2 in the denominator.

[0005] However, for the same type of system, the axial resolution is: raxial=2λem / NA2 that is, much worse than the lateral resolution (e.g. r lateral = 200 mm and raxial = 600 nm for λ em 500 nm and NA = 1.25).

[0006] Super-resolution fluorescence microscopy techniques were initially approaches to overcome the lateral resolution limit, but later solutions for improved axial resolution were also developed and implemented in commercial devices, such as the Spectrophotometer. • STED (via a 3-dimensional donut), • (d)STORM / (f)PALM (via astigmatism, 2-plane observation or double-helix detection), • SIM (via 3-dimensional structured lighting) and • MINFLUX (via a 3-dimensional donut).

[0007] A detailed summary of these technical approaches to overcome the lateral resolution limit can be found, for example, in the publications C. Eggeling et al. 2015 Q Rev Biophys. 48:178-243 or SJ Sahl, et al. 2017 Nat Rev Mol Cell Biol. 18:685-701.

[0008] One technical approach to improving axial resolution in fluorescence microscopy is MIET (metal-induced electron transfer) microscopy, which allows the distance of a fluorescently labeled molecule from the microscope coverslip to be measured with nanometer precision. In this technique, the coverslips are coated with a very thin and transparent coating of gold or graphene, which leads to a distance-dependent energy transfer between the fluorescent marker and the coating. This allows the axial distance to be determined with nanometer precision based on the fluorescence lifetime of the fluorescent marker and calibration measurements.

[0009] This technical approach has already been applied to investigate various biological questions at the cellular level (e.g., in the detection of axial membrane organizations).

[0010] A detailed description of this technical approach can be found, for example, in the publication Karedla, N., AI Chizhik, I. Gregor, AM Chizhik, O. Schulz, and J. Enderlein. 2014. Single-molecule metal-induced energy transfer (smMIET): resolving nanometer distances at the single-molecule level. Chemphyschem. 15:705-711.

[0011] Other known methods are based on the following two approaches, although these also have limitations: • Beam tuning in super-resolution microscopy

[0012] First of all, some of the most successful approaches require some form of beam shaping.

[0013] In super-resolution STED microscopy, for example, it is possible to extend the resolution enhancement to the third dimension by using active optical elements, such as spatial light modulators, to shape the depletion beam to have two lobes above and below the focal plane.

[0014] This approach enables an axial resolution of about 50 nm.

[0015] In SIM microscopy, an improvement in axial resolution is achieved in a similar way. A three-dimensional illumination pattern is generated, which is then shifted to produce a series of images, which are combined by reconstruction algorithms to generate an image with an axial resolution on the order of 250 nm.

[0016] These two approaches are very effective, but they require expensive optical components and increase the complexity of the setup so significantly that upgrading existing microscopes is extremely costly. Setups based on these principles are expensive and unnecessary for many microscopy applications.

[0017] Finally, the reconstruction algorithms required for this method are highly complex and contained in software packages that are often expensive or, if available as open source, unreliably maintained. • Metal-induced and graphene energy transfer

[0018] A simple approach that can be implemented on most laser scanning microscopes with less demanding technical requirements is the exploitation of the phenomena of metal-induced energy transfer and graphene energy transfer (MIET-GET) (I. Gregor et al., 2019 and Ghosh et al., 2021). This is achieved by using a MIET-GET on a metal (e.g., gold) or graphene-coated glass slide with an existing dependence of the fluorescence lifetime on the distance between the slide and the fluorophore.

[0019] Due to the coupling of the emission dipole of a fluorophore to the substrate, the energy is transferred to the substrate more efficiently the closer the emitting molecule is to the substrate. This leads to a shortening of the fluorescence lifetime according to the following formula: τf(θ,z0) / τ0=S0 / ϕS0+(1−ϕ)S0 where θ and z0 are the spherical polar coordinates of the emitting dipole, ϕ is the quantum yield of the fluorophore, S0 is the emission rate of the fluorescent molecule in the absence of the substrate, and S(θ, z0) is the emission rate of the fluorescent probe altered by interaction with the metal substrate. The fluorescence lifetime increases as a function of z, the axial distance of the fluorophore from the metal or graphene substrate. This approach has been used with great effectiveness in conjunction with laser scanning microscopy and pulsed MINFLUX to achieve axial resolution on the order of nanometers (Ghosh et al., 2017).

[0020] A disadvantage of this approach is that the substrate introduces strong scattering and backreflection, which adversely affects the final image quality. Furthermore, the effect is not tunable, meaning the range of energy transfer cannot be changed or controlled.

[0021] Finally, the analysis of the data is highly empirical and heuristic, meaning it requires extensive calibration before it can be used effectively.

[0022] In summary, the disadvantages of these previously known technical solutions in the 3-dimensional extensions to the known super-resolution microscopy techniques can be stated that they are: • are technically very complex and therefore expensive, such as STED and MINFLUX, • require a high degree of additional data analysis, such as (d)STORM / (f)PALM, MINFLUX, SIM, MIET, • are often still resolution-limited due to an insufficiently high detected signal level and a too low signal-to-background ratio, such as 3D-SIM and partly (d)STORM / (f)PALM, or • are not optimally designed for live cell imaging with very low acquisition speed and are therefore not suitable for dynamic measurements, such as (d)STORM / (f)PALM, MINFLUX, partly 3D-STED • complex sample preparation and calibration measurements with specific infrastructure, such as MIET, • increased background signals, such as MIET, and • have limited flexibility in setting the axial resolution range, such as with MIET.

[0023] In particular, MIET requires intensive and very specific sample preparation through the use of coated surfaces and precise calibration for each new measurement condition, and many laboratories do not have the appropriate infrastructure to produce such coatings.

[0024] In addition, the MIET is very limited in the length range, which is determined by the type of coating and the dye marker used, whereby this length range lies between 1 nm and 15 nm and is hardly changeable.

[0025] A disadvantage of optical fluorescence microscopy is the spatial resolution of conventional non-invasive (and lens-based) fluorescence microscopes, which is limited to approximately 200 nm in the lateral direction and approximately 600-800 nm in the axial direction. Due to the focusing of the light with lenses, the axial resolution is much more limited than the lateral resolution.

[0026] WO 2020212563 A1 discloses a fluorescence scanning microscope and a method for imaging a sample.

[0027] The fluorescence scanning microscope includes: • an excitation light source designed to generate an excitation light distribution that excites fluorophores present in the sample to spontaneously emit fluorescence photons, • a de-excitation light source designed to generate a de-excitation light distribution that de-excites the fluorophores in the sample excited by the excitation light distribution to generate stimulated emission of fluorescence photons, • an illumination unit designed to combine the excitation light distribution and the de-excitation light distribution to form a light distribution that scans across multiple illumination targets of the sample, such that the intensity maximum of the excitation light distribution and the intensity minimum of the de-excitation light distribution spatially overlap in the respective illumination target, • a detector designed to detect the fluorescence photons emitted by each illumination target based on the arrival time of the photons, • and a processor.

[0028] The processor is designed to analyze the fluorescence photons detected in each illumination target with respect to the arrival time of the photons, generate a first and a second pixel representing each illumination target based on this analysis, combine the first pixels to form a first sample image and the second pixels to form a second sample image, and determine a spatial offset between the intensity maximum of the excitation light distribution and the intensity minimum of the de-excitation light distribution based on the two sample images.

[0029] The procedure for imaging a sample using this fluorescence scanning microscope includes the following steps: • Generating an excitation light distribution that excites fluorophores present in the sample to spontaneously emit fluorescence photons, • Generating a de-excitation light distribution that de-excites the fluorophores in the sample excited by the excitation light distribution through stimulated emission of fluorescence photons, combining the excitation light distribution and the de-excitation light distribution to form a light distribution that is scanned over several illumination target points of the sample in such a way that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed in the respective illumination target points, • Detection of the fluorescence photons emitted by the respective illumination target point depending on their arrival times, • Evaluation of the fluorescence photons recorded at the respective illumination target point with regard to their arrival times, • Generating a first pixel and a second pixel representing the respective illumination target point on the basis of this evaluation, combining the first pixels to form a first sample image and the second pixels to form a second sample image, and • Determine a spatial offset between the intensity maximum of the excitation light distribution and the intensity minimum of the de-excitation light distribution using the two sample images.

[0030] Thus, according to the technical teaching of WO 2020212563 A1, the resolution improvement is based on a difference in the arrival time of the photons.

[0031] In order to improve the resolution of the system, it is necessary that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed at the respective illumination target point.

[0032] In addition, this technical teaching requires that the excitation and de-excitation light must be combined before imaging.

[0033] The object of the present invention is therefore to improve a method for the flexible and super-resolved adjustment of the axial resolution of a lens-based fluorescence microscope in order to avoid the aforementioned disadvantages of the prior art.

[0034] In particular, the axial spatial resolution is to be improved compared to the currently known technical solutions, especially MIET microscopy, so that, for example, the possibility is opened up to observe living cells and their dynamics with sufficient temporal resolution (sub-seconds) and high information content (e.g. environmental properties such as fluidity, pH value, etc.).

[0035] The use of this method is intended to enable axial super-resolution fluorescence microscopy images using a simple, cost-effective and flexible instrument, requiring no special infrastructure and sample preparation, and providing nanometer resolution in a tunable length range.

[0036] In addition, images with high temporal resolution and under live cell conditions should be possible and the imaging should not require intensive data analysis.

[0037] According to the invention, this object is achieved by the features of the first patent claim. Further advantageous embodiments of the invention are specified in the subordinate patent claims.

[0038] The method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection is carried out using at least two lasers, a radiation-transmissive carrier, e.g. a glass carrier in the form of a microscope cover glass, and a measuring device for determining the difference between the fluorescence lifetimes of fluorescent molecules, whereby • the radiation-permeable carrier (e.g. the microscope cover glass) carries a sample in which fluorescent molecules are located, and the sample is irradiated by the radiation of both lasers, • the first laser, which is pulsed, intensity-modulated or continuous and has a wavelength or wavelength range that excites the fluorescence emission of the fluorescent molecules, causes fluorescence excitation of the fluorescent molecules, • with the second laser, which is superimposed on the first laser in the sample, which is pulsed, intensity-modulated or continuous and has a wavelength or a wavelength range such that this laser leads to stimulated emission in the excited fluorescent molecules, a supercritical angle illumination is generated at an interface near the sample, which is achieved by illumination with a supercritical angle at the interface between the carrier and the sample in order to generate an evanescent wave with a wavelength that causes stimulated emission in the fluorescent molecules, • the evanescent wave of the second laser produces a variable intensity throughout the sample due to the exponential decay of the illumination intensity, • due to the phenomenon of stimulated emission, the fluorescence lifetime of fluorescent molecules exposed to a higher intensity of the evanescent wave is reduced more than that of fluorophores exposed to a lower intensity, and thus the strength of stimulated emission leads to a flexible and dynamic readout parameter in the form of the fluorescence lifetime for an axial nanometer-precise spatial resolution, • by measuring the difference between the fluorescence lifetimes of the fluorescent molecules in the presence and absence of the evanescent wave of the second laser, the information about the axial position according to equation τ'(z) / τ(z)=(1+σI0τ(z)e−z / d) is obtained, where τ'(z) is the fluorescence lifetime of the fluorescent molecules at the axial position z above the radiation-transmissive support (e.g., the microscopy cover glass) in the presence of the evanescent wave of the second laser, τ(z) is the fluorescence lifetime of the fluorescent molecules at the axial position z above the radiation-transmissive support (e.g., the microscopy cover glass) in the absence of the evanescent wave of the second laser, σ is the cross section for the stimulated emission of the fluorescent molecules by the second laser, l0 is the incident intensity of the second laser at the radiation-transmissive support (e.g., the microscopy cover glass) at z = 0, and d is the strength of the exponential decrease of the intensity of the second laser in the evanescent wave, • by means of the total power l0 of the second laser and its angle of incidence at the interface between the radiation-permeable carrier and the sample, which determines the order of magnitude of the parameter d and thus the penetration depth of the stimulating laser, the axial length range of the sample area intended for increased axial resolution can be adjusted very flexibly between approx. 10 nm to approx. 1 µm. See Fig. 1 to 3.)

[0039] The fluorescence lifetime is measured using time-correlated single photon counting.

[0040] Supercritical angle illumination at the interface between the carrier and the sample is achieved by an objective lens with a large numerical aperture, a prism above or below the sample, or a waveguide device.

[0041] By illuminating the first laser at different angles of incidence at the interface between the carrier and the sample, an evanescent wave with different penetration depths is also flexibly generated for the first laser.

[0042] More than two lasers can be used for fluorescence excitation and stimulated emission, whereby the sample is illuminated by the lasers in pulsed, intensity-modulated or continuous form, whereby the lasers used are solid-state lasers, diode lasers, fiber lasers or gas lasers with continuous laser radiation or with time-modulated laser power.

[0043] The lasers used are pulsed solid-state lasers, diode lasers, fiber lasers or gas lasers with pulse repetition rates in the range of 250 kilohertz to 100 megahertz and pulse widths in the range of 1 picosecond to 100 nanoseconds, whereby several lasers with different wavelengths excite different fluorescent molecules to emit fluorescence and the fluorescent light of the different fluorescent molecules is detected separately.

[0044] In this case, all or only some of the different fluorescent molecules, but at least one type of fluorescent molecule, are stimulated to emit and are used accordingly to improve the axial resolution.

[0045] The fluorescence detectors used are cameras or point detectors.

[0046] The microscope used is a wide-field microscope with wide-field illumination or a confocal microscope with wide-field illumination.

[0047] The evanescent wave is generated using an axicon, whereby the fluorescence detection is extended by detection of light transmitted through the sample and the lateral resolution is improved by well-known super-resolution methods such as STED, (d)STORM, (f)PALM, SIM, Airy-Scan, ISM or MINFLUX microscopy.

[0048] Particularly advantageously, a radiation-transparent support in the form of a microscope cover glass is used, whereby the axial decay of the power of the second laser is caused by any illumination technique other than evanescent illumination and the equation for determining the axial position is adapted accordingly.

[0049] It is also within the scope of the invention that in the method for flexible and super-resolved adjustment of the axial resolution for optical microscopy, the axially dependent fluorescence emission reduction can also be caused by fluorescence parameters other than the fluorescence lifetime for determining the axial position by determining the fluorescence parameter via adapted polarization settings of the laser and polarization-sensitive detection of the emitted signal.

[0050] It is also within the scope of the invention that the axially dependent fluorescence emission reduction by the second laser is caused by any process other than stimulated emission, in that the fluorescence emission reduction occurs by light-induced switching on and off.

[0051] The proposed technical solution in the form of the method consists in using a LITED method (LITED = Lifetime Tuning by Evanescent wave depletion) using supercritical angle illumination with evanescent illumination to induce stimulated emission of fluorescent dye markers, whereby the strength of the stimulated emission leads to a flexible and dynamic readout parameter (= in the form of the fluorescence lifetime) for an axial nanometer-precise spatial resolution in a fluorescence microscope by superimposing additional fluorescence illumination.

[0052] For this purpose, a normal epi- or evanescent TIRF (= Total Internal Reflection Fluorescence) fluorescence illumination (which generates a fluorescence emission in the illumination field) is superimposed with a second laser (a fluorescence depletion laser in an evanescent illumination mode on a radiation-transmissive support (e.g. a glass support in the form of a microscope coverslip on or on which a sample bearing a fluorescent dye marker is located), whereby this second laser leads to a fluorescence excitation-stimulated emission in the fluorescent dye markers.

[0053] The latter is not detected, but it leads to the quenching of the actual fluorescence signal and, above all, to a shortening of the fluorescence lifetime of the dye marker. This quenching, and thus the reduction in fluorescence lifetime, is proportional to the power of the second laser and, due to its evanescent illumination mode, depends on the distance to the radiation-transmissive support (e.g., a glass support, which can be designed as a coverslip).

[0054] Thus, the fluorescence lifetime acts as a readout parameter for the distance from the radiation-permeable support (e.g. glass support, which can be designed as a cover glass).

[0055] A preferred mode for fluorescence illumination is confocal microscopy.

[0056] In contrast to the previously known MIET method, the method for adjusting the axial resolution of a fluorescence microscope using this supercritical angle illumination requires no coating or complex calibration. Furthermore, the overall power and the angle of incidence (and thus the penetration depth) of the stimulating laser allow for a very flexible adjustment of the wavelength range between approximately 10 nm and approximately 1 µm, making it easy to handle.

[0057] The resolution improvement according to the proposed new technical method can be derived from lifetime tuning and is based on the principle of photon separation by lifetime tuning, where the resolution improvement in conventional STED microscopy beyond the diffraction limit results from the fact that the stimulated emission process effectively reduces the fluorescence lifetime of excited fluorophores.

[0058] This is due to the fact that the depletion beam creates a steep gradient in the sample, so that excited fluorophores in the periphery are more effectively depleted than those in the center. This results in a reduced fluorescence lifetime for molecules receiving a higher dose of depletion photons, meaning that resolution improvement can be achieved through lifetime tuning.

[0059] The key feature of the proposed new LITED (Lifetime Tuning by Evanescent wave depletion) technique is to utilize the evanescent wave of the depletion laser, which is generated by supercritical angle illumination in a fluorescence microscope.

[0060] The intensity of the evanescent light propagating through the interface decreases rapidly beyond the incident region, with an exponential behavior being observed: I(z)=I0e−z / d with d=λ / 4π(sin2(θc)−sin2(θ)) where I0 is the light intensity at the interface, z is the axial distance coordinate to the radiation-permeable support (e.g. a microscopy coverslip), d is the distance of the fluorescent sample to the radiation-permeable support (e.g. the microscopy coverslip), λ is the wavelength of the incident wave, and θc is the critical angle according to Snell's law (θc = arcsn(n1 / n2)). (See Fig. 1 to 3.)

[0061] The realization of the supercritical angle has become easier thanks to the introduction of high NA lenses (numerical aperture of the lens 1.4 and higher), with illumination angles up to 73° possible.

[0062] To illustrate the proposed method, a sample with a series of excited fluorophores is considered below.

[0063] A pulsed beam with a wavelength that excites stimulated emission is used, and this pulsed beam is emitted at a time interval that corresponds to the fluorescence emission kinetics (10 -9 seconds), so that the lifetime of the fluorophores is increased by an equal amount: τ' / τ=(1+σIτ)−1 is reduced, where τ and τ' are the original and the shortened fluorescence emission lifetimes (due to stimulated emission), σ is the stimulated emission cross section, and I is the emission intensity. This equation can also be derived for the case where the intensity decays exponentially after the interface, i.e., it has a dependence on the axial z-direction: τ'(z) / τ=(1+σI(z)τ)−1

[0064] Inserting equation 4 into this expression, we get: τ'(z) / τ=(1+σI0τe−z / d)−1

[0065] This relationship opens the door to tunable axial resolution, as the dependence of d on the wavelength ensures the angle of the incident light. The dependence on the light intensity I0 may seem like a disadvantage at first glance, but potential photodamage can be minimized by using, for example, pulsed light.

[0066] The LITED microscopy technique described here, based on fluorescence detection, represents a new way to utilize the stimulated emission phenomenon in fluorescence microscopy to obtain spatial information beyond the xy plane. By combining this stimulated emission phenomenon with the evanescent wave phenomenon, it is now possible to generate a favorable anisotropy in the fluorescence emission of a sample. The advantage of this technique is that appropriate tuning of the stimulated emission is easily achieved using standard equipment.

[0067] Equipping conventional optical fluorescence microscopy techniques with this method is easy to accomplish, as a user can adjust the lifetime of a fluorescent molecule depending on its position in the axial direction (z) in a completely predictable manner.

[0068] Considering the low equipment complexity required, this solution represents a technically very feasible way to achieve high resolution in the z-direction (which is known to be the most critical of the three directions in space) without having to resort to additional complex tools and expensive equipment to measure the fluorescence lifetime using, for example, time-correlated single-photon counting in the optical system based on fluorescence detection.

[0069] For this, the fluorescent molecules must be present in a solid sample, as is common in fluorescence microscopy. Then, an evanescent wave with a wavelength capable of eliciting stimulated emission from the fluorescent target molecules must be generated in the sample. This is achieved by supercritical angle reflection at an interface near the sample. This can be achieved, for example, by supercritical angle illumination at the interface between the support and the sample through a large numerical aperture objective lens, a prism above or below the sample, or a waveguide device.

[0070] By measuring the difference between the fluorescence lifetimes of the fluorophores in the presence and absence of the evanescent wave, the information about the axial position can be obtained according to Equation 7.

[0071] Due to the total power and the angle of incidence (and thus the penetration depth) of the stimulating laser, the length range can be adjusted very flexibly between approx. 10 nm and approx. 1 µm and is easy to handle.

[0072] The advantage of this technical solution is that the setup is inexpensive and of low complexity and does not require any additional sample preparation or infrastructure.

[0073] In addition, this technical solution allows for easy tuning of the length range to be examined as well as the investigation of living cells with their fast dynamics, all with simple routine data analysis.

[0074] All features presented in the description, the embodiments and the following claims can be essential to the invention both individually and in any combination with one another.

Claims

[1] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection, using at least two lasers, a radiation-transparent carrier and a measuring device for determining the difference between the fluorescence lifetimes of fluorescent molecules, characterized by , that • the carrier carries a sample containing fluorescent molecules and the sample is irradiated by the radiation of both lasers, • the first laser, which is pulsed, intensity-modulated or continuous and has a wavelength or wavelength range that excites the fluorescence emission of the fluorescent molecules, causes fluorescence excitation of the fluorescent molecules, • with the second laser, which is superimposed on the first laser in the sample, is pulsed, intensity-modulated or continuous and has a wavelength or a wavelength range such that this laser causes stimulated emission from the excited fluorescent molecules, a supercritical angle illumination is generated at an interface near the sample, which is achieved by supercritical angle illumination at the interface between the carrier and the sample in order to generate an evanescent wave with a wavelength that causes stimulated emission from the fluorescent molecules, • the evanescent wave of the second laser produces a variable intensity throughout the sample due to the exponential decay of the illumination intensity, • due to the phenomenon of stimulated emission, the fluorescence lifetime of fluorescent molecules exposed to a higher intensity of the evanescent wave is reduced more than that of fluorophores exposed to a lower intensity, and thus the strength of stimulated emission leads to a flexible and dynamic readout parameter in the form of the fluorescence lifetime for an axial nanometer-precise spatial resolution, • by measuring the difference between the fluorescence lifetimes of the fluorescent molecules in the presence and absence of the evanescent wave of the second laser, the information about the axial position according to equation τ'(z) / τ(z)=(1+σI0τ(z)e−z / d) where τ'(z) is the fluorescence lifetime of the fluorescent molecules at the axial position z above the support in the presence of the evanescent wave of the second laser, τ(z) is the fluorescence lifetime of the fluorescent molecules at the axial position z above the support in the absence of the evanescent wave of the second laser, σ is the cross section for the stimulated emission of the fluorescent molecules by the second laser, l0 is the incident intensity of the second laser at the support at z = 0 and d is the strength of the exponential decrease of the intensity of the second laser in the evanescent wave, • by means of the total power l0 of the second laser and its angle of incidence at the interface between carrier and sample, which determines the order of magnitude of the parameter d and thus the penetration depth of the stimulating laser, the axial length range of the sample area intended for increased axial resolution can be adjusted very flexibly between approx. 10 nm to approx. 1 µm. [2] Method according to claim 1, characterized by that the fluorescence lifetime is measured using time-correlated single photon counting. [3] Method according to claim 1, characterized by that supercritical angle illumination at the interface between the support and the sample is achieved by a large numerical aperture objective lens, a prism above or below the sample, or a waveguide device. [4] Method according to claim 1, characterized bythat the illumination for the first laser also occurs at different angles of incidence at the interface between carrier and sample in order to flexibly achieve an evanescent wave with different penetration depths for the first laser as well. [5] Method according to claim 1, characterized by that more than two lasers are used for fluorescence excitation and stimulated emission. [6] Method according to claim 4 or 5, characterized by that the sample is illuminated by the laser in pulsed, intensity-modulated or continuous form. [7] Method according to claim 1, characterized by that the lasers used are solid-state lasers, diode lasers, fiber lasers or gas lasers with continuous laser radiation. [8] Method according to claim 1, characterized by that the lasers used are solid-state lasers, diode lasers, fiber lasers or gas lasers with time-modulated laser power. [9] Method according to claim 1, characterized by that the lasers used are pulsed solid-state lasers, diode lasers, fiber lasers or gas lasers with pulse repetition rates in the range of 250 kilohertz to 100 megahertz and pulse widths in the range of 1 picosecond to 100 nanoseconds. [10] Method according to claim 5, characterized by that different fluorescent molecules are excited to emit fluorescence by several lasers with different wavelengths and the fluorescent light of the different fluorescent molecules is detected separately. [11] Method according to claim 10, characterized by that all or only some of the different fluorescent molecules, but at least one fluorescent molecule type, is used for stimulated emission for axial nanometer-precise spatial resolution. [12] Method according to claims 1, 10 and 11, characterized bythat the fluorescence detectors used are cameras or point detectors. [13] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized by that the microscope used is a wide-field microscope with wide-field illumination. [14] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized by that the microscope used is a confocal microscope with wide-field illumination. [15] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 14, characterized by that the evanescent wave is generated by means of an axicon. [16] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized by that fluorescence detection is extended by detection of light transmitted through the sample. [17] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized by that the lateral resolution is also improved by well-known super-resolution techniques such as STED, (d)STORM, (f)PALM, SIM, Airy-Scan, ISM or MINFLUX microscopy. [18] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized bythat the axially dependent fluorescence emission reduction is caused by a fluorescence parameter other than the fluorescence lifetime for determining the axial position, by providing the fluorescence parameter via adapted polarization settings of the lasers and via polarization-sensitive detections of the emitted signal. [19] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to claim 1, characterized by that the axially dependent fluorescence emission reduction by the second laser is caused by a different process than stimulated emission, in that the fluorescence emission reduction occurs by light-induced switching on and off. [20] Method for flexible and super-resolved adjustment of the axial resolution for optical microscopy based on fluorescence detection according to one or more of claims 1 to 19, characterized by that a microscope cover glass is used as a radiolucent support.

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