Light microscopy method, light microscopy apparatus and computer program

Abstract

The invention relates to a light microscopy method, wherein a sample (2) is illuminated with focused pulsed excitation light (A) to excite at least two types of emitters (E) with different emission lifetimes to emit light, wherein the sample (2) is additionally illuminated with an intensity distribution of suppression light (V) with a local minimum to de-excite emitters (E) outside the local minimum, wherein photons emitted from the sample (2) are detected by a detector (4), wherein a phasor analysis is performed on the basis of the detected photons (P) to separate photons of the different types of emitters (E) based on their emission lifetime, wherein photon gating is performed, wherein for excitation pulses (p A) of the excitation light (A) respective gating time intervals (10) are provided, and wherein in the phasor analysis only those photons (P) are taken into account which are detected in the respective gating time interval (10), as well as a light microscopic device (1) and a computer program for carrying out the procedure.

Description

Technical field of the invention

[0001] The invention relates to a light microscopy method, in particular using obscuring light to increase resolution and lifetime analysis to separate the signals of different emitters. Further aspects of the invention include a device and a computer program for carrying out the method. State of the art

[0002] In light microscopy, lifetime analysis, particularly fluorescence lifetime analysis, can be used to separate the signals of different types of emitters, especially fluorophores or sample structures labeled with fluorophores. This method also allows the separation of signals from emitters with strongly overlapping or even identical emission spectra, whose emitted light cannot be separated using wavelength-dependent optical filters, for example, as long as the emitters have different lifetimes. Here, lifetime is understood as the average time after excitation by excitation light after which a photon is emitted. For fluorescent dyes commonly used in fluorescence microscopy, lifetimes typically range from fractions of nanoseconds to several nanoseconds.

[0003] Lifetimes can be determined experimentally, for example, by time-correlated single-photon counting (TCSPC). In this method, the sample is periodically illuminated with excitation laser pulses, and the detection light from the sample (especially fluorescence light) is captured by a detector capable of detecting individual photons and determining their arrival time at the detector with picosecond accuracy, such as an avalanche photodiode coupled with appropriate evaluation electronics. From the time difference of a large number of photon signals to the respective preceding laser pulse, a temporal distribution of arrival times can be determined, which can be represented, for example, as a histogram. From this histogram, the lifetime can be determined, for example, by functional fitting or phasor plot analysis (su).

[0004] As an alternative to time-correlated single-photon counting, lifetime analysis can also be performed based on frequency data by illuminating a sample in a wide-field manner with time modulation and recording the emission signals with a gating-capable camera. In this case, the lifetime of emitters can be determined from the phase shift between the periodic excitation and the modulated emission signal, as well as from the modulation depth.

[0005] A lifetime analysis based on time data, i.e., using time-correlated single-photon counting, can be performed, for example, with a confocal laser scanning microscope. In this method, the laser pulses are focused into the sample, and the laser focus is scanned through the sample, for example, with a galvo scanner. Pixel intensities of a raster image are then calculated from the intensities or photon counts of the detection light captured at the various sample positions. In addition to the total intensity, a lifetime analysis can be performed for each pixel. This typically involves analyzing the arrival times of a large number of photons for each sample position.

[0006] Lifetimes can be determined, for example, by fitting the arrival time distribution to a monoexponential or multiexponential function. If the signal originates from a single emitter species whose arrival times are monoexponentially distributed (this is the case for many fluorescent dyes), the corresponding signal can be assigned to that emitter species in this way.

[0007] In the case of a mixed signal originating from several emitter types with different lifetimes, the contribution of the different emitter types to the signal can, in principle, be determined by fitting the arrival time data to a multi-exponential function. This allows the signals to be separated, and, for example, two raster images of different lifetime channels can be calculated and displayed.

[0008] However, the separation of emission signals using function fitting is particularly challenging and error-prone for weak signals, i.e., relatively few detected photons, especially since the underlying model of the function fit is not known in advance, and there are no clearly defined criteria for when a match with the model exists.

[0009] As an alternative to functional fit, phasor plot analysis allows a graphical evaluation of lifetime data and is fundamentally able to separate the signals from emitters with different characteristic lifetimes.

[0010] In phasor plot analysis based on arrival time data (i.e., especially TCSPC-based), coordinates of a phasor, namely a real part, are assigned to each scan pixel. g(ω)=∫t0∞I(t)cos(ωt)∫t0∞I(t) and an imaginary part s(ω)=∫t0∞I(t)sin(ωt)∫t0∞I(t) determined where ω corresponds to the repetition rate of the pulsed light source multiplied by 2π, and where I(t) is the decay function or the temporal

[0011] The distribution of the detected photon numbers is given. The real part g(ω) corresponds to the cosine transform of the arrival time data, and the imaginary part s(ω) corresponds to the sine transform of the arrival time data.

[0012] The phasors can be graphically represented in a g / s coordinate system, where each pixel corresponds to a data point (so-called phasor plot).

[0013] Pixels containing pure signals from an emitter with a single characteristic lifetime are arranged in the phasor plot on a semicircle centered at (0,5;0) with a radius of 0.5. The longer the lifetime, the closer the corresponding phasor is to the point (0,0), and the shorter the lifetime, the closer the phasor is to the point (1,0). The phasor (0,0) represents the limit for lifetime ∞, and the phasor (1,0) represents the limit for lifetime 0.

[0014] Mixtures of two signals, e.g., pixels containing emitters of several different species, lie under ideal conditions on a connecting line between the two corresponding pure lifetime components (i.e., on a secant of the semicircle), with the position on the line depending on the mixing ratio of the signals. For example, a phasor of a pixel whose signal is composed of 50% each of two lifetime components lies at the midpoint of a connecting line between the phasors of the corresponding pure lifetime components.

[0015] In this way, the pixel intensities can be assigned to different detection channels (emitter species) according to their position on the secant; a raster image with a mixed signal can therefore be separated into two raster images of the corresponding detection channels (so-called lifetime unmixing).

[0016] Due to measurement errors and noise, phasor data typically scatter around the ideal positions on the phasor plot described above.

[0017] STED microscopy allows light microscopic imaging with super-resolution (so-called super-resolution microscopy), a better spatial resolution than the Abbé diffraction limit of classical light microscopy.

[0018] According to the state of the art, STED microscopes are based on confocal laser scanning microscopes, where the excitation focus is superimposed with a light distribution of STED (stimulated emission depletion) light with a central intensity zero. The STED light excites fluorophores in the region around the intensity zero back from the excited state to the ground state without emitting fluorescence. In this way, the effective detection point spread function can be narrowed to values ​​far below the diffraction limit; that is, the fluorescence light detected for a pixel originates predominantly from a region near the intensity zero.

[0019] A combination of STED microscopy with a TCSPC-based lifetime analysis is generally known from the prior art (E. Auksorius et al., “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging” (2008), Optics Letters 33 (2), 113-115).

[0020] Additional STED light has a spatially and temporally complex influence on the lifetime of the emitters in the sample. In addition to the spontaneous decay of the excited fluorophores, fluorophores illuminated with STED light can also transition to the ground state without light emission via the STED effect, at a rate that differs from the decay rate by fluorescence and depends on the local STED light intensity, i.e., the spatial distribution of the fluorophores within the illuminated area.

[0021] STED microscopy can be performed with both pulsed STED light (p-STED) and continuous-wave STED light illumination (CW-STED). In p-STED, the STED pulse is often delayed relative to the excitation pulse to increase the effectiveness of the stimulated emission.

[0022] In the CW-STED method with pulsed excitation light and continuously applied STED light, the spatial resolution can be increased by so-called time gating. In this technique, photons detected during a time interval after the excitation pulse are filtered out or not used for image processing (G. Vicidomini et al., “Sharper low-power STED nanoscopy by time gating” (2011), Nature Methods 8(7), 571-573). This filters out photons with shorter lifetimes, which are more likely to have been spontaneously emitted before the STED light could excite the corresponding emitter to the ground state. A disadvantage of CW-STED with gating is that short-life photons are also filtered out from the central region of the effective PSF, resulting in a loss of usable signal.

[0023] p-STED with time gating is also known from the prior art (G. Vicidomini et al., “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects (2013), PLoS ONE 8(1): e54421. doi:10.1371 / journal.pone.0054421), however, gating only results in a minimal increase in spatial resolution.

[0024] From the publication “Encoding and decoding spatio-temporal information for superresolution microscopy” (2015) by L. Lanzano et al., Nat Commun 6, 6701 (2015). https: / / doi.org / 10.1038 / ncomms7701, a method is known in which the resolution of CW-STED images is increased by phasor plot analysis. Faster and slower lifetime components of the fluorescence signal are separated from each other by solving a system of linear equations.

[0025] A similar method for increasing resolution based on phasor analysis is described for pulsed STED microscopy in the publication “Photon-separation to enhance the spatial resolution of pulsed STED microscopy” (2019) by G. Tortarolo et al., Nanoscale 11, 1754-1761. Due to the even more complex spatiotemporal distribution of lifetime components, a simulation of the emitter distribution is used here instead of solving a linear system of equations.

[0026] In “A novel pulsed STED microscopy method using FastFLIM and the phasor plots” (2017) by Y. Sun et al., Proc. of SPIE Vol. 10069 100691C-1, a method is described that combines STED microscopy with TCSPC analysis and phasor plots. In the phasor plot analysis, a binary mask is applied based on prior knowledge of the position of phasors of the pure lifetime component without STED light and at different STED light intensities to filter out the unwanted low-resolution components.

[0027] In the application note “TauSTED: pushing STED beyond its limits with lifetime” by LAJ Alvarez (2021) in Nature Methods, a method called “TauBackgroundSuppression” is described. In this method, a phasor plot is generated from time-resolved STED microscopy data, and phasors on a “STED trajectory” are separated from uncorrelated background signals in other regions of the phasor plot by applying a binary mask. In a separate method called “TauStrength,” phasors are assigned different weights according to their position on the “STED trajectory,” which are then used to weight the corresponding photons during image generation.

[0028] The publication “Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation” (2022) by MG Pisfil et al., Nature 12:14017, describes the separation of signals from different fluorescent dyes using phasor analysis in the presence of STED light. A comparison of the phasor plots with and without STED light reveals a shift of the phasor distribution to the right (towards shorter lifetimes) and a blurring of the phasor distribution due to the STED light. In some samples, the phasor plot analysis in the presence of STED light did not provide satisfactory separation of the lifetime components. The resulting images are therefore not shown in the publication. Object of the invention

[0029] The disadvantages of the state of the art discussed above give rise to the task of providing a super-resolution light microscopy method that is able to reliably and flexibly separate photons from different emitters based on their lifetime, with reduced optimization effort, for different dyes and samples. Solution

[0030] This problem is solved by the subject matter of the independent claims. Advantageous embodiments of the invention are specified in the dependent claims and are described below. Description of the invention

[0031] A first aspect of the invention relates to a light microscopy method, wherein a sample is illuminated with focused pulsed excitation light to excite at least two types of emitters with different emission lifetimes to emit light, wherein the sample is additionally illuminated with an intensity distribution of suppression light with a local minimum to de-excite emitters outside the local minimum, wherein photons emitted from the sample are detected by a detector, in particular with time resolution, wherein a phasor analysis is carried out on the basis of the detected photons in order to separate photons of the different types of emitters based on their emission lifetime, wherein a gating of the photons is carried out, wherein respective gating time intervals are provided for excitation pulses of the excitation light, and wherein in the phasor analysis only those photons are taken into account,which are recorded within the respective gating time interval.

[0032] According to the invention, time gating is combined with phasor plot analysis to improve the separation of different emitter signals (in particular, different dyes in the sample). In contrast, gating in the prior art is used either as an alternative to phasor analysis or to increase the resolution of a STED image by filtering out the early photons, or methods of phasor plot analysis are known to separate early and late photons and thus improve the resolution. An increase in resolution can, of course, be an additional advantageous side effect of the method according to the invention in certain cases.

[0033] By omitting the early photons in the phasor plot analysis according to the invention, the blurring of the phasor distribution caused by the STED effect is reduced or completely eliminated.

[0034] This allows the phasor populations assigned to the different emitters to be better separated in the phasor plot, and lifetime unmixing is easier and more reliable, especially for dye combinations and samples that cannot be separated when analyzing all photons in the phasor plot.

[0035] The excitation light can be, for example, fluorescence excitation light, which excites fluorophores in the sample to fluorescence. However, other excitation mechanisms are also conceivable, as long as the excited emitters emit photons when they return to the ground state within timeframes measurable by the evaluation electronics.

[0036] The excitation light is pulsed, meaning it consists of a non-continuous electromagnetic wave or wave packet with a pulse width. The pulse width and repetition rate are arbitrarily selectable; the term "pulse" is therefore not limited to specific pulse widths or repetition rates. Typically, the pulse duration can range from femtoseconds to picoseconds. Typical repetition rate values ​​are in the MHz range.

[0037] The blocking light can be, for example, STED light, which converts excited emitters to the ground state without emission. Alternatively, the blocking light can also be, for example, deactivation light, which converts emitters to a dark state, as in RESOLFT microscopy.

[0038] Regarding the effect of the excitation light and the suppression light on the at least two types of emitters, different constellations are possible.

[0039] For example, one type of excitation light can be used to excite several types of emitters, or several types of excitation light with different wavelengths or spectra can be used, each exciting a different type of emitter. In the latter case, the corresponding excitation pulses can be alternately directed into the sample, as is known from the prior art for pulse-interleaved excitation microscopy (PIE).

[0040] The blocking light (especially STED light) can also act on one or more types of emitters and de-excite them. If blocking light acts on only one type of emitter in the sample, it is possible to introduce two or more different types of blocking light with different wavelengths or spectra, e.g., as alternately introduced blocking pulses, which can, for example, be time-delayed relative to the corresponding excitation pulse. However, the invention is also practical if only one type of blocking light is used and a first type of emitter in the sample is de-excited by the blocking light, while at least a second type of emitter in the sample is not affected by the blocking light at all. Even then, the blocking light can, for example,The phasors assigned to the first type of emitters become so blurred that they overlap in the phasor plot with the phasors assigned to the second type of emitters. This effect can then be reduced by the time gating according to the invention, thus enabling better signal separation.

[0041] The gating time intervals can have the same length or different lengths for different excitation pulses.

[0042] The term "emitter" as used here refers to a molecule, molecular complex, or particle that emits electromagnetic radiation, particularly in the visible, infrared, or ultraviolet range, when excited by external light. The emitter may, for example, be a fluorophore or be coupled to a fluorophore (i.e., covalently or non-covalently bonded) that emits fluorescent light when excited by a light pulse of suitable wavelengths. Alternatively, the emitter may, for example, be a quantum dot. Besides fluorescence, other mechanisms are conceivable within the scope of the present invention for exciting the at least one emitter to emit a photon. For example, emitted photons may result from light scattering. Another example is photoactivation, which may consist of exciting the at least one emitter from a first, e.g.,to convert a non-fluorescent state or dark state into a second, light-emitting state, e.g. by having at least one emitter emit fluorescence in response to excitation light in the second state.

[0043] The emission lifetime is defined here as a parameter that specifies the typical (or average) time after which a photon is emitted following a light pulse. In the case of fluorescence emission by emitters in response to excitation light, the emission lifetime is the fluorescence lifetime. If the light emission can be described, for example, as a monoexponential process, then the emission lifetime is specifically defined as the reciprocal of the time constant of a monoexponential decay. For an ensemble of emitters, this lifetime describes the time after which the light intensity of the emission has decreased to a fraction of 1 / e of the initial intensity value. In the case of a single emitter, the emission of a photon after triggering by the light pulse follows a probability distribution, which, for example,can be described by a monoexponential decay, and the emission lifetime is the time after which the probability that a particular emitter has emitted a photon is equal to 1-1 / e.

[0044] The intensity distribution can, for example, be a donut shape, where the local minimum in the focal plane is surrounded laterally (but not along the optical axis, i.e., axially) by areas of increasing intensity, or a so-called bottle beam (sometimes also referred to as a 3D donut), where the local minimum is surrounded both laterally and axially by areas of increasing intensity. A donut shape can be created, for example, by phase modulation of the illumination light in or near a pupil plane conjugate to the back aperture of the microscope objective, using a vortex phase pattern (also called a phase clock). A bottle beam can be generated by phase modulation of the illumination pattern with an annular phase pattern in or near the pupil plane.Finally, intensity distributions with an area-like local minimum (sometimes also called a crescent-shaped intensity distribution) can be achieved by phase modulation with a simply segmented phase pattern. The local minimum can be a zero point of intensity. Under real-world conditions, however, the intensity at the local minimum is often not zero, for example, due to optical aberrations. The local minimum can be centrally located, i.e., at the geometric focus. In this case, the intensity distribution can be arranged symmetrically around the local minimum.

[0045] The term "phasor analysis" here refers to a pixel-by-pixel evaluation of the arrival time distribution of photons, in which at least one Fourier coefficient (in particular, a real part and / or an imaginary part of a phasor) is calculated for each pixel. A phasor plot can be displayed to the user during phasor analysis, but this is not strictly necessary, as the phasor analysis and separation of emitter signals can also be performed automatically in the background.

[0046] The term "gating" here refers to the exclusion or disregard of certain photons depending on their arrival time during phasor analysis. This can be achieved through "physical gating," meaning that photons are not detected within a specific time interval, for example, because the detection beam path is obstructed, the detection light is deflected or blocked so that it does not reach the detector, or because the detector is inactivated during the relevant period. Alternatively, "electronic gating" can be used. In this case, all photons are detected and recorded, and the gating takes place at the level of a downstream electronic circuit or is performed through data analysis.

[0047] According to one embodiment, the zero point of a time scale underlying the phasor analysis is set to an initial time point of the gating time interval. In other words, the phasors are normalized to the beginning of the gating time interval. Specifically, for example, when determining the Fourier coefficients or the real and imaginary parts of the phasors, the lower limit of the summation or integration can be set to the initial time point of the gating time interval.

[0048] According to another embodiment, the blocking light is directed onto the sample in blocking pulses, with a time interval between an excitation pulse and the start time of the respective gating time interval overlapping with a respective blocking pulse. This allows at least some of the photons emitted during the effect of the respective blocking pulse on the emitters in the sample to be excluded from the phasor analysis. Depending on the type of blocking pulse, and in particular on how quickly the effect of the blocking pulse on the emitters occurs, it can be advantageous for the gating time interval to have a certain time interval from the respective blocking pulse.

[0049] According to another embodiment, the time interval between an excitation pulse and the start time of the respective gating time interval corresponds to at least one pulse length of the respective suppression pulse. Advantageously, all photons emitted during a suppression pulse are excluded from the phasor plot analysis. Depending on the speed of action of the suppression pulses, it can also be advantageous if the time interval is longer than the pulse duration of the suppression pulse and the gating time interval only begins after the suppression pulse has ended, with a certain time interval.

[0050] According to a further embodiment, the suppression pulses are applied to the sample with a pulse delay relative to each preceding excitation pulse, wherein the time interval between an excitation pulse and the start time of the respective gating time interval is at least equal to the sum of the pulse length of the suppression pulse and the pulse delay. This pulse delay results in particularly high efficiency in suppressing the emitters in regions outside the minimum of the intensity distribution. In this case, the aforementioned time interval is advantageously longer than the suppression pulse so that the gating time interval begins only after the suppression pulse has finished.

[0051] According to another embodiment, the photons emitted by the sample are only detected by the detector or registered by an evaluation device coupled to the detector only during the gating time interval. This embodiment includes, in particular, so-called physical gating, in which, for example, the detection light can be shaded or deflected, or the detector or electronics coupled to the detector can be switched off.

[0052] According to another embodiment, the photons emitted by the sample between an excitation pulse and the start time of the respective gating time interval are indeed detected and recorded, but excluded from a data set used in phasor analysis. In this case, the gating is therefore performed electronically, at the data processing level.

[0053] According to a further embodiment, the sample is scanned with a focus of the excitation light, with which the intensity distribution of the obstruction light is superimposed, whereby photons detected at the respective positions of the focus in the sample are assigned to the respective image pixels. The focus can be moved, in particular, by confocal laser scanning of the excitation light and the obstruction light over or through the sample. In this process, especially during phasor analysis, respective phasors are calculated for the respective image pixels.

[0054] According to a further embodiment, the detector is coupled to an evaluation device, wherein the evaluation device calculates, on the basis of a signal pulse sequence of photon signals transmitted from the detector to the evaluation device, in particular without storing individual photon signals, the real parts and imaginary parts of the respective phasors, on the basis of which the phasor analysis is carried out.

[0055] In this embodiment, the phasors required for separating the different types of emitters are quickly obtained without the need to subsequently know the captured photons and their individual arrival times for data analysis. This advantageously reduces the data streams and the demands on the processors used, especially in experiments with many data points (pixels).

[0056] The evaluation device can comprise one unit or several interconnected units. For example, the evaluation device can include TCSPC electronics configured to determine the arrival times of individual photons detected by the detector. Alternatively, or additionally, the evaluation device can also include, for example, a microprocessor, an FPGA (field programmable gate array), or similar components, for instance, to quickly calculate phasors from a data stream during the measurement, as described above, without the need to store the individual photon arrival times.

[0057] According to another embodiment, the evaluation device calculates a real part and an imaginary part for each image pixel.

[0058] According to another embodiment, the blocking light, in particular the blocking pulses, acts at least on first emitters in the sample by de-exciting the first emitters outside the local minimum of the blocking light, wherein the first emitters have a longer emission lifetime than second emitters in the sample. In this configuration, a particularly strong deterioration of the separability of the two emitters can occur, since the blocking light blurs the phasor of the longer-lived first emitters into an ensemble of shorter-lived species, whose phasors in the phasor plot can overlap with the population of phasors of the shorter-lived second emitters.

[0059] A second aspect of the invention relates to a light microscopy apparatus, in particular for carrying out a method according to the first aspect, wherein the apparatus comprises at least the following components: an illumination device configured to illuminate a sample with focused pulsed excitation light in order to excite at least two types of emitters with different emission lifetimes to emit light, and additionally to illuminate the sample with an intensity distribution of suppression light with a local minimum in order to de-excite emitters outside the local minimum; a detector configured to detect photons emitted by the sample, in particular with time resolution; and a computing unit configured to perform a phasor analysis on the basis of the detected photons in order to separate photons of the different types of emitters based on their emission lifetimes.wherein the light microscopic device, in particular an evaluation device or the computing unit of the light microscopic device, is configured to perform photon gating by providing respective gating time intervals for excitation pulses of the excitation light, wherein the computing unit is configured to consider only those photons in the respective gating time interval during phasor analysis.

[0060] A third aspect of the invention relates to a computer program comprising commands that cause the light microscopic device according to the second aspect to perform the method according to the first aspect.

[0061] Further advantages and embodiments of the light microscopic device according to the second aspect and of the computer program according to the third aspect result analogously from the description of the method according to the first aspect.

[0062] Advantageous embodiments of the invention are described in the claims, the description, the drawings, and the accompanying explanations. The described advantages of features and / or combinations of features of the invention are merely examples and can have an effect alternatively or cumulatively.

[0063] Regarding the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be seen in the drawings – in particular the depicted relative arrangements and functional connections. The combination of features from different embodiments of the invention or from features of different claims is also possible, deviating from the chosen cross-references in the claims, and is hereby encouraged. This also applies to features that are illustrated in separate drawings or mentioned in their description. These features can also be combined with features from different claims. Likewise, features listed in the claims can be omitted for further embodiments of the invention, but this does not apply to the independent claims of the granted patent.

[0064] The reference numerals contained in the patent claims do not constitute a limitation of the scope of the subject matter protected by the patent claims. They serve only the purpose of making the patent claims easier to understand.

[0065] Exemplary embodiments of the invention are described below with reference to figures. These do not limit the subject matter of this disclosure or the scope of protection. Brief description of the characters Fig. Figure 1 schematically shows an example of an arrival time distribution of detected photons with time-gating and a phasor plot derived from it; Fig. 2 shows a light microscopy device according to an embodiment; Fig. Figure 3 shows a light microscopic device according to a further embodiment. Description of the characters

[0066] Fig. Figure 1 schematically illustrates an embodiment of the method according to the invention, in which a sample 2 is equipped with at least two different types of emitters E with focused excitation light A in the form of excitation pulses p A and with an intensity distribution of blocking light V (especially STED light) superimposed on the focus of the excitation light A, with a local minimum (especially a central zero) in the form of blocking pulses p VThe sample 2 is illuminated and individual photons P emitted from the sample 2 are detected with time resolution, wherein the blocking light V transitions at least one type of emitter E in the sample 2 from a state excited by the excitation light A to a ground state in a region outside the local minimum. In particular, the focus of the excitation light A and the intensity distribution of the blocking light V are scanned across the sample 2 using a scanning device 36, and the number of detected photons P and the arrival times of the individual photons P are determined pixel by pixel.

[0067] The left diagram in Fig. Figure 1 shows an excitation pulse p on a time axis t. A , relative to the excitation pulse p A delayed prevention pulse p V as well as a frequency distribution represented as a histogram (frequency n) p ) of arrival times due to the excitation light A from a sample 2 (see Fig. 2 and Fig. 3) emitted photons P, which are detected by a detector 4 (see Fig. 2 and Fig. 3) were recorded. The right-hand diagram of the Fig. Figure 1 shows a phasor plot 12 with exemplary phasors P1, P2, P3, which were calculated on the basis of the photons P detected in the gating time interval 10 (see left diagram).

[0068] The photons P detected before the gating time interval 10 were not considered in the calculation of the phasors P1, P2, P3. The gating time interval 10 begins at time t0'. The time interval between the excitation pulse p A and the starting time t0' of the gating time interval 10 corresponds in this example to the duration of the prevention pulse p VBy omitting the photons P emitted by the emitters E before the gating time interval 10, the phasor analysis can be largely free from the effects of the blocking light V (for example, a smearing of the phasor distribution due to the reduction of the emission lifetime caused by the blocking pulse p). V ) are carried out. This improves the separability of different phasor populations, which are assigned to the different types of emitters E in sample 2.

[0069] When calculating the phasors, for example, the real parts of the phasors can be determined by calculating the cosine transformation. g(ω)=∫t0∞,I(t)cos(ωt)∫t0∞,I(t) and imaginary parts of the phasors by calculating the sine transform s(ω)=∫t0∞,I(t)sin(ωt)∫t0∞,I(t) to be determined, where ω is the repetition rate of the excitation pulses p A, I(t) is the time intensity profile of the signal and t0' is the starting time of the gating time interval 10.

[0070] The phasors P1 and P2, labeled as examples in phasor plot 12, are located on the semicircle with center (0.5;0) and radius 0.5 and correspond to signals with a single emission lifetime, as would be expected at sample positions where only one type of emitter E with monoexponential decay behavior is present. Phasor P3 is approximately located on a line between phasor P1 and phasor P2 and is thus an example of a phasor representing a mixed signal of the emitters E with the corresponding emission lifetimes. For the pixel to which phasor P3 is assigned, the contributions of the two emitter types can be determined by identifying the position of phasor P3 on the line between phasors P1 and P2, and the corresponding photon numbers can be assigned to different detection channels for this pixel.This separation of signals works better through the gating according to the invention, since the effects of the obstructing light V are at least reduced in the phasor analysis.

[0071] Fig. Figure 2 shows a first embodiment of a light microscopy device 1 according to the invention, comprising an illumination device 3 for illuminating a sample 2 with excitation light A and suppression light V, a detector 4 for time-resolved detection of photons P emitted from the sample 2, and a computing unit 5 for performing a phasor analysis based on the photons P detected by the detector 4. The illumination device 3 includes a first light source 31, in particular a laser, for generating the excitation light A in the form of excitation light pulses p. A , a second light source 32, in particular another laser, for generating obstruction light V (in particular STED light) in the form of obstruction pulses p V, a light modulator 33 (e.g., a so-called spatial light modulator, SLM) for influencing a spatial phase and / or amplitude distribution of the blocking light V, a beam combiner 34 (e.g., a dichroic mirror) for combining the excitation light A with the blocking light V, and a lens 38 for jointly focusing the excitation light A and the blocking light V into a sample 2 with at least two different types of emitters E (e.g., fluorophores with different emission lifetimes). The light modulator 33 and the lens 38 interact such that an intensity distribution of the blocking light V with a central zero intensity (which is superimposed on the intensity maximum of the excitation light A) is formed at the common focus of the excitation light A and the blocking light V in the sample 2. The intensity distribution can be, for example,This involves a donut, a bottle beam, or a so-called crescent light distribution. Photons P emanating from sample 2 are detected by a detector 4 (e.g., a single-photon avalanche photodiode, SPAD) and time-resolved by an evaluation device 41 coupled to the detector 4 (e.g., comprising TCSPC electronics). The evaluation device 41 is coupled to the processing unit 5.

[0072] The evaluation device 41 can transmit the arrival times of individual photons P to the processing unit 5. The processing unit 5 can then, based on the arrival times of the photons in the gating time interval 10 (see Fig. 1) Perform a phasor analysis of the detected photons P to separate the signals of the different types of emitters E in the sample 2.

[0073] Gating can be performed, for example, as electronic gating by the evaluation device 41 or by the computing unit 5.

[0074] According to an alternative approach, the evaluation device 41, which in this case can, for example, comprise an FPGA connected to the TCSP electronics, determines the real and imaginary parts of the phasors from a data stream coming from the TCSPC electronics and transmits these values ​​to the processing unit 5. This has the advantage that an arrival time does not need to be stored for each individual photon P.

[0075] Fig. Figure 3 shows a second embodiment of the light microscopy device 1 according to the invention, which here is configured as a confocal laser scanning microscope and / or STED microscope. Regarding the embodiment of the Fig. Two identical or analogous components are designated with the same reference numerals. For further information, please refer to the description above. Fig. 2 referred. In the case of the light microscope apparatus 1 according to Fig.3. The focused excitation light A and the intensity distribution of the blocking light V are scanned across the sample 2. The objective 38 also collects the photons P emitted by the sample 2, and these photons P are detected confocally. For this purpose, a beam splitter 35 (in particular, another dichroic mirror) and a scanning device 36 (e.g., a galvanometer scanner) with at least one controllable, movable scan mirror 37, in particular at least one scan mirror 37 for each spatial direction of the scan (not shown), are located in the beam path between the beam combiner 34 and the objective 38. The scanning device 36 scans the common focus of the excitation light A and the blocking light V across the sample 2.

[0076] The photons P emitted from sample 2 are focused by the lens 38, scanned by the scanning device 36, and reflected by the beam splitter 35 in a wavelength-dependent manner. The reflected light passes through a confocal aperture 6 to the detector 4, which detects the photons P, which are then registered pixel by pixel with time resolution by the evaluation device 41. Reference symbol list 1 Light microscope apparatus 2 Sample 3 Lighting device 4 Detector 5 computing unit 6-hole aperture 10 Gating time interval 12 Phase plot 31 First light source 32 Second light source 33 Light modulator 34 beam combinations 35 beam splitters 36 Scanning device 37 scan mirrors 38 lens 41 Evaluation device A excitation light E Emitter P photons V Preventive light p A Stimulation pulse p V Prevention pulse t0' Start time of the gating time interval

Claims

[1] Light microscopy method, wherein a sample (2) is illuminated with focused pulsed excitation light (A) to excite at least two types of emitters (E) with different emission lifetimes to emit light, wherein the sample (2) is additionally illuminated with an intensity distribution of blocking light (V) with a local minimum to de-excite emitters (E) outside the local minimum, wherein photons (P) emitted from the sample (2) are detected by a detector (4), wherein a phasor analysis is performed on the basis of the detected photons (P) to separate photons (P) of the different types of emitters (E) based on their emission lifetime, characterized by , that a gating of the photons (P) is performed, wherein for excitation pulses (p A) of the excitation light (A) respective gating time intervals (10) are provided, and in the phasor analysis only those photons (P) are taken into account which are detected in the respective gating time interval (10). [2] Method according to claim 1, characterized by , that a zero point of a time scale underlying the phasor analysis is set to an initial time point (t0') of the gating time interval (10). [3] Method according to claim 1 or 2, characterized by , that the prevention light (V) in prevention pulses (p V ) is irradiated onto the sample (2), with a time interval between an excitation pulse (p A ) and a starting time (t0') of the respective gating time interval (10) with a respective prevention pulse (p V ) overlap in time. [4] Method according to claim 3, characterized by , that the time interval between an excitation pulse (p A) and the start time (t0') of the respective gating time interval (10) of at least one pulse length of the respective prevention pulse (p V ) corresponds. [5] Method according to claim 3 or 4, characterized by , that the prevention pulses (p V ) with a pulse delay relative to each preceding excitation pulse (p A ) are irradiated onto the sample (2), whereby the time interval between an excitation pulse (p A ) and the start time (t0') of the respective gating time interval (10) at least the sum of the pulse length of the prevention pulse (p V ) and corresponds to the pulse delay. [6] Method according to any one of claims 1 to 5, characterized by, that the photons (P) emitted by the sample (2) are only detected by the detector (4) during the gating time interval (10) or are only registered by an evaluation device (41) coupled to the detector (4) during the gating time interval (10). [7] Method according to any one of claims 1 to 5, characterized by , that the photons (P) emanating from the sample (2) between an excitation pulse (p A ) and the start time (t0') of the respective gating time interval (10) are indeed captured and recorded, but are excluded from a data set used in the phasor analysis. [8] Method according to any one of the preceding claims, characterized by , that the sample (2) is scanned with a focus of the excitation light (A) with which the intensity distribution of the prevention light (V) is superimposed, whereby photons (P) detected for respective positions of the focus in the sample (2) are assigned to respective image pixels. [9] Method according to any one of the preceding claims, characterized by , that the detector (4) is coupled to an evaluation device (41), wherein the evaluation device (41) calculates real parts (G) and imaginary parts (S) of the respective phasors (P1,P2,P3) on the basis of a signal pulse sequence of photon signals transmitted from the detector (4) to the evaluation device (41) without storing individual photon signals, on the basis of which the phasor analysis is carried out. [10] Method according to claim 9, insofar as it relates back to claim 8, characterized by , that the evaluation device (41) calculates a real part (G) and an imaginary part (S) of a phasor (P1,P2,P3) for each image pixel. [11] Method according to any one of the preceding claims, characterized by, that the obstruction light (V) acts at least on first emitters (E) in the sample (2) by de-exciting the first emitters (E) outside the local minimum of the obstruction light (V), wherein the first emitters (E) have a longer emission lifetime than second emitters (E) in the sample (2). [12] Light microscopy apparatus (1), in particular for carrying out a method according to any one of claims 1 to 11, wherein the apparatus (1) comprises at least the following components: - a lighting device (3) configured to illuminate a sample (2) with focused pulsed excitation light (A) to excite at least two types of emitters (E) with different emission lifetimes to emit light, and to additionally illuminate the sample (2) with an intensity distribution of suppression light (V) with a local minimum to de-excite emitters (E) outside the local minimum, - a detector (4) designed to detect photons (P) emanating from the sample (2), and - a computing unit (5) which is trained to perform a phasor analysis on the basis of the detected photons (P) in order to separate photons (P) of the different types of emitters (E) on the basis of their emission lifetimes, characterized by , that the light microscopic device (1), in particular an evaluation device (41) or the computing unit (5) of the light microscopic device (1), is configured to perform gating of the photons (P) by providing for excitation pulses (p A ) of the excitation light (A) respective gating time intervals (10) are provided, wherein the computing unit (5) is designed to only consider those photons (P) that are detected in the respective gating time interval (10) during the phasor analysis. [13] Computer program comprising commands that cause the light microscopic device (1) according to claim 12 to perform the method according to any one of claims 1 to 11.

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