Optical microscopic method, optical microscopic device, and computer program

Abstract

The present application relates to an optical microscopic method, an optical microscopic device and a computer program. The invention relates to an optical microscopic method, wherein a sample (2) is illuminated with a focused pulsed excitation light (A) to excite at least two types of emitters (E) with different emission lifetimes to emit light, wherein the sample is also illuminated with an intensity profile of a quenching light (V) having local minima to de-excite emitters outside the local minima, wherein photons emitted from the sample are detected by a detector (4), wherein a phasor analysis is performed based on the detected photons (P) to separate photons of different types of emitters based on the emission lifetime of the emitters, wherein a gating is performed on the photons, wherein a respective gating time interval (10) is set for an excitation pulse (p A ) of the excitation light, and wherein only photons detected in the respective gating time interval are considered in the phasor analysis. The invention also relates to an optical microscopic device (1) and a computer program for performing the method.

Description

Technical Field

[0001] This invention relates to optical microscopy methods, particularly optical microscopy methods for using suppressed light to improve resolution and lifetime analysis to separate signals from different emitters. Other aspects of the invention include apparatus and computer programs for performing these methods. Existing technology

[0002] In optical microscopy, lifetime analysis, particularly fluorescence lifetime analysis, can be applied to separate signals from different types of emitters, especially fluorophores or fluorophore-labeled sample structures. In this way, signals from emitters with highly overlapping or even identical emission spectra (i.e., the light emitted by these emitters cannot be separated, for example, by wavelength-dependent optical filters) can also be separated, provided that the emitters have different lifetimes. Lifetime here is specifically understood as the average time from excitation by light to the emission of a photon. For fluorescent dyes commonly used in fluorescence microscopy, lifetimes are typically in the range of fractions of a nanosecond to several nanoseconds.

[0003] Lifetime can be determined experimentally, for example, by time-correlated single-photon counting (TCSPC). Here, the sample is periodically illuminated with excitation laser pulses, and the detection light (particularly fluorescence) from the sample is detected by a detector (e.g., an avalanche photodiode coupled to corresponding evaluation electronics). This detector is capable of detecting individual photons and determining their arrival times with picosecond accuracy. The temporal distribution of arrival times can be determined by the time differences of multiple photon signals relative to their respective preceding laser pulses, which can be displayed, for example, as a histogram. The lifetime can be determined from this histogram, for example, by function fitting or by phasor diagram analysis (see below).

[0004] As an alternative to time-correlated single-photon counting, lifetime analysis can also be performed based on frequency data by irradiating the sample with wide-field time modulation and recording the emission signal with a gated camera. In this case, the lifetime of the emitter can be determined based on the phase shift between the periodically excited and modulated emission signals, as well as the modulation depth.

[0005] Lifetime analysis based on time data (i.e., by means of time-correlated single-photon counting) can be performed, for example, using a confocal laser scanning microscope, in which a laser pulse is focused onto the sample, and a galvanometer scanning device is used to scan the sample by traversing the laser focus. The pixel intensity of the grating image is calculated based on the intensity or number of photons detected at different sample locations. In addition to the total intensity, lifetime analysis can also be performed for each pixel. Here, the arrival time of multiple photons is typically analyzed for each sample location.

[0006] Here, lifetime can be determined, for example, by fitting the arrival time distribution function to a single exponential function or a multi-exponential function. If the signal comes from a single emitter species with an arrival time distribution of a single exponential (as is the case with many fluorescent dyes), the corresponding signal can be assigned to an emitter species in this way.

[0007] In the case of mixed signals originating from multiple emitter types with different lifetimes, the proportion of each emitter type in the signal can, in principle, be determined by fitting the arrival time data function to a multi-exponential function. This allows for signal separation, and, for example, the calculation and display of raster images with different lifetime channels.

[0008] However, separating the emitted signal by means of function fitting is demanding and error-prone, especially in the case of weak signals (i.e., relatively few photons are detected), particularly because the underlying model of the function fitting is unknown beforehand, and there is no well-defined standard for when it is consistent with the model.

[0009] As an alternative to function fitting, phasor diagram analysis allows for graphical evaluation of lifetime data and essentially enables the separation of signals from emitters with different characteristic lifetimes.

[0010] In phasor diagram analysis based on time-of-arrival data (i.e., particularly TCSPC-based data), the coordinates, i.e., the real part, of the phasor are determined for each scan pixel. and the virtual part ,in This corresponds to the repetition rate of the pulsed light source multiplied by 2π, and where It is the time distribution of the decay function or the number of detected photons. The real part g( ) corresponds to the cosine transform of the arrival time data, while the imaginary part s( This corresponds to the sine transformation of the arrival time data.

[0011] Phasors can be graphically represented in a g / s coordinate system, where each pixel corresponds to a data point (the so-called phasor diagram).

[0012] Pixels containing a pure signal from an emitter with a single characteristic lifetime are arranged in the phasor diagram 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). Here, the phasor (0, 0) is the limiting value of the lifetime. The phasor (1, 0) is the lifetime limit of 0.

[0013] Under ideal conditions, the mixture of two signals (e.g., a pixel containing multiple different types of emitters) lies on the line connecting the two corresponding pure lifetime components (i.e., the secant of a semicircle), where the position on the line depends on the mixing ratio of the signals. For example, the phasor of a pixel whose signal is composed of 50% of each of the two lifetime components lies at the midpoint of the line connecting the phasors of the corresponding pure lifetime components.

[0014] In this way, pixel intensity can be assigned to different detection channels (emitter types) based on the position on the secant line. Therefore, a raster image with mixed signals can be separated into two raster images for the corresponding detection channels (so-called lifetime demixing).

[0015] Due to measurement errors and noise, phasor data are typically scattered around the aforementioned ideal locations on the phasor diagram.

[0016] STED microscopy enables super-resolution optical microscopy imaging, with spatial resolution superior to the Abbe diffraction limit of classical optical microscopy.

[0017] STED microscopy, as known in existing technology, is based on confocal laser scanning microscopy, in which the excitation focal point is superimposed with the light distribution of STED (stimulated emission depletion) light, which has a central intensity zero. The STED light causes excited fluorophores in the region near the intensity zero to return from the excited state to the ground state without emitting fluorescence. In this way, the effective detection point spread function can be narrowed to a value far below the diffraction limit, meaning that the fluorescence detected at a pixel mainly originates from the region near the intensity zero.

[0018] In principle, the combination of STED microscopy with TCSPC-based lifetime analysis is known from existing technologies (E. Auksorius et al., “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging” (2008), OpticsLetters 33 (2), 113-115).

[0019] Additional STED light has a complex spatiotemporal effect on the lifetime of emitters in the sample. In addition to the spontaneous decay of excited fluorophores, fluorophores irradiated by STED light can also transition to the ground state without emitting light through the STED effect, and the rate is different from the fluorescence decay rate and depends on the local STED light intensity, i.e., the positional distribution of the fluorophore within the irradiated area.

[0020] STED microscopy can be performed using either pulsed STED light (so-called p-STED) or continuous STED light (CW-STED). In the case of p-STED, the STED pulse is typically delayed relative to the excitation pulse to improve the efficiency of stimulated emission.

[0021] In the CW-STED method using pulsed excitation light and continuously incident STED light, spatial resolution can be improved through so-called time gating. Here, photons detected within a certain time interval after the excitation pulse are either shielded or excluded from image computation (G. Vicidomini et al., “Sharper low-power STED nanoscopy by time gating” (2011), Nature Methods 8(7), 571-573). This filters out short-lived photons, which are likely to be spontaneously emitted before the STED light can induce the corresponding emitter to its ground state. A drawback of gated CW-STED is that short-lived photons from the central region of the effective PSF are also filtered out, resulting in the loss of useful signals.

[0022] The use of time-gated p-STED is also known from existing technology (G. Vicidomini et al., “STEDNanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects (2013), PLoS ONE 8(1): e54421. doi: 10.1371 / journal.pone.0054421”), but here the gating only achieves a very small improvement in spatial resolution.

[0023] A method for improving the resolution of CW-STED images through phasor diagram analysis is known from the published paper "Encoding and decoding spatio-temporalinformation for super-resolution microscopy" (2015) by L. Lanzano et al., Nat Commun 6, 6701 (2015). https: / / doi.org / 10.1038 / ncomms7701. Here, the faster and slower lifetime components of the fluorescence signal are separated from each other by solving a system of linear equations.

[0024] A similar approach to improving resolution based on phasor analysis is described in G. Tortarolo et al.’s publication, “Photon-separation to enhance the spatial resolution of pulsed STED microscopy” (2019), Nanoscale 11, 1754–1761, for pulsed STED microscopy. Because the spatiotemporal distribution of the lifetime component is more complex, this approach relies on simulations of the emitter distribution rather than solving linear equations.

[0025] In Y. Sun et al.'s paper, "A novel pulsed STED microscopy method using FastFLIM and the phasor plots" (2017), Proc. of SPIE Vol. 10069 100691C-1, a method combining STED microscopy with TCSPC analysis and phasor plots is described. In phasor plot analysis, based on prior knowledge of the phasor positions of the pure lifetime components in the absence of STED light and under different STED light intensities, a binary mask is used to filter out unwanted low-resolution components.

[0026] In LAJ Alvarez's application note "TauSTED: pushing STED beyond its limits with lifetime" (2021), Nature Methods describes a method called "TauBackgroundSuppression," which generates a phasor map of STED microscopy data with temporal resolution. Within the phasor map, a binary mask is applied to separate phasors on the "STED trajectory" from irrelevant background signals in other regions of the phasor map. On the other hand, a method called "TauStrength" assigns different weights to phasors based on their position on the "STED trajectory," and these weights are used to weight the corresponding photons during image generation.

[0027] The publication "Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetimephasor separation" (2022), Nature 12:14017, by MG Pisfil et al., describes the separation of signals from different fluorescent dyes by phasor analysis in the presence of STED light. A comparison of phasor diagrams with and without STED light shows that the phasor distribution is shifted to the right (towards lower lifetimes) and becomes diffuse and blurred due to STED light. In some samples, phasor diagram evaluation in the presence of STED light failed to provide satisfactory lifetime component separation. Therefore, the resulting images are not shown in the published document. Purpose of the invention

[0028] The objective derived from the shortcomings of the prior art discussed above is to provide a super-resolution optical microscopy method that can reliably and flexibly separate photons from different emitters based on emitter lifetime, reducing optimization work for different dyes and samples.

[0029] Solution

[0030] This objective is achieved through the subject matter of the independent claims. Advantageous improvements of the invention are given in the dependent claims and described below. Invention Description

[0031] The first aspect of the invention relates to an optical microscopy method in which a sample is irradiated with focused pulsed excitation light to excite at least two types of emitters with different emission lifetimes, wherein the sample is additionally irradiated with an intensity distribution of blocking light having local minima to de-excite emitters outside the local minima, wherein photons emitted from the sample are detected by a detector, particularly in a manner with time resolution, wherein phasor analysis is performed based on the detected photons to separate photons of different types of emitters based on the emission lifetime of the emitters, wherein gating is performed on the photons, wherein a corresponding gating time interval is set for the excitation pulse of the excitation light, and wherein only photons detected within the corresponding gating time interval are considered in the phasor analysis.

[0032] According to the present invention, time gating is combined with phasor diagram analysis to improve the separation of signals from different emitters (particularly different dyes in a sample). In contrast, prior art gating is known either as an alternative to phasor analysis, or for the purpose of improving the resolution of STED images by filtering out early photons, or for phasor diagram analysis methods used to separate early and late photons and thus improve resolution. Of course, in some cases, improved resolution can be an additional advantageous side-band effect of the method according to the present invention.

[0033] According to the present invention, by excluding early photons in phasor diagram analysis, the diffuse ambiguity of phasor distribution caused by the STED effect is reduced or completely eliminated. Consequently, phasor groups assigned to different emitters are better separated in the phasor diagram, and lifetime unmixing can be performed more easily and reliably, especially in cases where dye combinations and samples cannot be separated when analyzing all photons in the phasor diagram.

[0034] The excitation light could be, for example, fluorescence excitation light that excites a fluorophore in a sample to fluoresce. However, other excitation mechanisms are also conceivable, as long as the excited emitter emits photons during the time it takes for the electronic device to be evaluated to return to its ground state.

[0035] The excitation light is pulsed, meaning it consists of a discontinuous electromagnetic wave or wave packet with a pulse width. Here, the pulse width and repetition rate can be arbitrarily chosen; that is, the term "pulse" is not limited to a specific pulse width or repetition rate. Typically, the pulse length can range from femtoseconds to picoseconds. Typical values ​​for the repetition rate can be in the MHz range.

[0036] The suppressor light can be, for example, STED light, which causes the excited emitter to transition to the ground state in a non-emission manner. Alternatively, the suppressor light can also be, for example, inactivating light that causes the emitter to transition to a dark state, such as in the case of RESOLFT microscopy.

[0037] There can be different configuration schemes for the effects of excitation light and suppression light on at least two types of emitters.

[0038] For example, one type of excitation light can be used to excite multiple types of emitters, or multiple types of excitation light with different wavelengths or spectra can be used, each of which excites one type of emitter. In the latter case, the corresponding excitation pulses can be incident on the sample alternately, for example, as is known in the art in what is called pulse-interleaved excitation microscopy (PIE).

[0039] Suppressive light (especially STED light) can also act on one or more types of emitters and de-excite them. When a suppressive light acts on only one type of emitter in a sample, two or more different types of suppressive light with different wavelengths or different spectra can be incident, for example as alternating suppressive pulses, which may, for example, be time-delayed relative to the associated excitation pulses. However, the subject matter of the invention is also applicable when only one type of suppressive light is used and the first type of emitter in the sample is de-excited by the suppressive light, while at least the second type of emitter in the sample is not affected by the suppressive light at all. This is because even in this case, the suppressive light can, for example, cause the phasors associated with the first type of emitter to become diffusely blurred, such that they overlap in the phasor diagram with those associated with the second type of emitter. This effect can then be reduced by time gating according to the invention, thereby enabling better signal separation.

[0040] For different excitation pulses, the gating time interval can have the same length or different lengths.

[0041] As used herein, the term "emitter" 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. An emitter can be, for example, a fluorophore or coupled to a fluorophore (i.e., covalently or non-covalently), which fluoresces when excited by a light pulse of suitable wavelength. Alternatively, an emitter can be, for example, a quantum dot. Besides fluorescence, other mechanisms for exciting at least one emitter to emit photons are conceivable within the scope of this invention. For example, the emitted photons can be a result of light scattering. Another example is photoactivation, which can refer to transitioning at least one emitter from a first state (e.g., a non-fluorescent or dark state) to a second luminescent state, for example, by the at least one emitter emitting fluorescence in response to excitation light in the second state.

[0042] The emission lifetime is defined here as a parameter indicating the typical (or average) time from the time of light pulse to the emission of a photon. In the case where the emitter fluoresces in response to excitation light, the emission lifetime is the fluorescence lifetime. For example, if light emission can be described as a single exponential process, the emission lifetime is specifically defined as the reciprocal of the time constant of the single exponential decay. For an ensemble of emitters, the lifetime describes the time it takes for the emitted light intensity to decrease to 1 / e of its initial intensity value. In the case of a single emitter, the emission of photons after being triggered by a light pulse follows a probability distribution that can be described, for example, by single exponential decay, and the emission lifetime is the time it takes for the probability of a particular emitter emitting a photon to equal 1 - 1 / e.

[0043] The intensity distribution can be, for example, a donut, where the local minimum is within the focal plane, i.e., surrounded by an intensity-increasing region in the transverse direction (but not along the optical axis, i.e., the axial direction), or it can be, for example, a so-called bottle-shaped beam (sometimes also called a 3D donut), where the local minimum is surrounded by intensity-increasing regions in both the transverse and axial directions. For example, a donut can be formed by phase modulation of the illumination light using a vortex phase mode (also called a phase clock) in or near the pupil plane conjugate to the rear aperture of the microscope objective. A bottle-shaped beam can be generated by phase modulation of the illumination mode using a donut phase mode in or near the pupil plane. Finally, an intensity distribution with planar local minimums (sometimes also called a crescent-shaped intensity distribution) can be achieved by phase modulation using a simple piecewise phase mode. The local minimum can be a point of zero intensity. However, under practical conditions, the intensity at the local minimum is usually not zero, for example, due to optical aberrations. The local minimum can be located at the center, i.e., at the geometric focal point. Here, the intensity distribution can be arranged particularly symmetrically about the local minimum.

[0044] The term "phasor analysis" is understood here as a pixel-by-pixel evaluation of the photon arrival time distribution, where at least one Fourier coefficient (specifically the real and / or imaginary parts of the phasor) is formed for each pixel. In phasor analysis, a phasor diagram can be displayed to the user, but this is not absolutely necessary, as phasor analysis and the separation of the emitter signal can also be performed automatically, particularly in the background.

[0045] The term "gating" here refers to the exclusion or disregard of photons in phasor analysis based on their arrival time. This can be described as "physical gating," where photons are not detected within a specific time interval because, for example, the detection path is blocked, the detection light is deflected or obstructed, preventing it from reaching the detector, or the detector is deactivated for the corresponding time period. Alternatively, "electronic gating" can be used. In this case, all photons are detected and recorded, and gating occurs at the level of downstream electronic circuitry or is performed through data analysis.

[0046] According to one implementation, the zero point of the time scale on which phasor analysis is based is set as the starting point of the gated time interval. In other words, the phasor is normalized based on the starting point of the gated time interval. Specifically, for example, when determining the Fourier coefficients or the real and imaginary parts of the phasor, the lower limit of the summation or integration can be set as the starting point of the gated time interval.

[0047] According to another embodiment, the suppressing light is incident on the sample in the form of a suppressing pulse, wherein the time interval between the excitation pulse and the start point of the corresponding gate time interval overlaps temporally with the corresponding suppressing pulse. Thus, at least a portion of the photons emitted during the period when the corresponding suppressing pulse acts on the emitter in the sample can be excluded from phasor analysis. Depending on the type of suppressing pulse, and particularly on the speed at which the suppressing pulse takes effect on the emitter, a certain time interval between the gate time interval and the corresponding suppressing pulse can be advantageous.

[0048] According to another embodiment, the time interval between the excitation pulse and the start point of the corresponding gating time interval corresponds to at least one pulse length of the corresponding suppressor pulse. Advantageously, all photons emitted during the suppressor pulse are excluded from phasor diagram analysis. Depending on the speed at which the suppressor pulse takes effect, it is also advantageous that this time interval is longer than the pulse duration of the suppressor pulse, and that the gating time interval begins only after a certain time interval following the end of the suppressor pulse.

[0049] According to another embodiment, the suppressor pulse is incident on the sample with a pulse delay relative to the corresponding preceding excitation pulse, wherein the time interval between the excitation pulse and the start point of the corresponding gating time interval corresponds at least to the sum of the pulse length of the suppressor pulse and the pulse delay. This pulse delay achieves particularly high efficiency in de-excitation of the emitter in regions outside the minimum intensity distribution. In this case, the time interval is advantageously longer than the suppressor pulse, so the gating time interval begins only after the suppressor pulse has ended.

[0050] According to another embodiment, the detector detects photons emitted from the sample only during the gating time interval, or the evaluation device coupled to the detector records these photons only during the gating time interval. This embodiment specifically includes so-called physical gating, where, for example, the detection light can be blocked or deflected, or the detector or the electronics coupled to the detector can be turned off.

[0051] According to another implementation, although photons emitted from the sample between the start time of the excitation pulse and the corresponding gating time interval are detected and recorded, they are excluded from the dataset used in phasor analysis. That is, in this case, gating is specifically performed electronically at the data processing level.

[0052] According to another embodiment, the sample is scanned with the focal point of an excitation light superimposed on the intensity distribution of the suppressing light, wherein photons detected at corresponding positions of the focal point within the sample are assigned to corresponding image pixels. The focal point can be moved over or traversed across the sample via confocal laser scanning of the excitation and suppressing light. Here, particularly in phasor analysis, a phasor is calculated for each image pixel.

[0053] According to another embodiment, the detector is coupled to an evaluation device, wherein the evaluation device calculates the real and imaginary parts of each phasor based on a sequence of signal pulses of photon signals transmitted from the detector to the evaluation device, particularly without storing individual photon signals, and performs phasor analysis on this basis.

[0054] Therefore, in this embodiment, the phasors required to separate different types of emitters can be quickly derived without needing to know the detected photons and their respective arrival times afterward for data analysis. This advantageously reduces the data flow and the requirements on the processor used, especially in experiments with many data points (pixels).

[0055] Evaluation equipment may include one or more units coupled to each other. For example, evaluation equipment may include TCSPC electronics designed to determine the arrival times of individual photons detected by the detector. Evaluation equipment may alternatively or additionally include, for example, a microprocessor, an FPGA (Field Programmable Gate Array), etc., to rapidly compute phasors from the data stream during measurement within the scope of the above embodiments, without having to store the arrival times of individual photons.

[0056] According to another embodiment, the evaluation device calculates the real and imaginary parts for each image pixel.

[0057] According to another embodiment, the suppressing light, particularly the suppressing pulse, acts at least on the first emitter by de-exciting the first emitter in a sample outside the local minimum of the suppressing light, wherein the first emitter in the sample has a longer emission lifetime than the second emitter. This situation can lead to a particularly severe deterioration in the separability of the two emitters, because the phasor of the longer-lived first emitter is diffused and blurred into a set of shorter-lived species due to the suppressing light, and these shorter-lived phasors may overlap with the phasor group of the shorter-lived second emitter in the phasor diagram.

[0058] The second aspect of the invention relates to an optical microscopy apparatus, particularly for performing the method according to the first aspect, wherein the apparatus comprises at least the following components: an illumination device designed to illuminate a sample with focused pulsed excitation light to excite at least two types of emitters with different emission lifetimes, and additionally to illuminate the sample with an intensity distribution of blocking light having local minima to de-excite emitters outside the local minima; a detector designed to detect photons emitted from the sample, particularly in a manner having temporal resolution; and a computing unit designed to perform phasor analysis based on the detected photons to separate photons of different types of emitters based on the emission lifetime of the emitters, wherein the computing unit of the optical microscopy apparatus, particularly an evaluation apparatus or an optical microscopy apparatus, is designed to perform gating of the photons by setting a corresponding gating time interval for the excitation pulse of the excitation light, wherein the computing unit is designed to consider only the photons detected within the corresponding gating time interval in the phasor analysis.

[0059] The third aspect of the invention relates to a computer program having instructions that cause an optical microscope apparatus according to the second aspect to perform the method according to the first aspect.

[0060] Other advantages and implementations of the optical microscope according to the second aspect and the computer program according to the third aspect are similarly derived from the description of the method according to the first aspect.

[0061] Advantageous improvements of the invention are derived from the patent claims, specification, and drawings, as well as the related descriptions of the drawings. The advantages described in the features and / or combinations of features of the invention are merely exemplary and may have alternative or cumulative effects.

[0062] Regarding the disclosures in the original application and patent (but not concerning the scope of protection), further features can be found in the accompanying drawings, particularly the relative arrangements and operational connections shown. Combinations of features from different embodiments of the invention or combinations of features from different patent claims may also deviate from the selected reference relationships in the patent claims, as hereby stated. This also applies to features shown in separate drawings or mentioned in their descriptions. These features may also be combined with features from different patent claims. Similarly, for further embodiments of the invention, features listed in the patent claims may be omitted, but this does not apply to the independent patent claims of the granted patent.

[0063] Reference marks included in patent claims do not constitute a limitation on the scope of protection of the patent claims. Reference marks are only used to make the patent claims easier to understand.

[0064] Embodiments of the present invention are described below with reference to the accompanying drawings. These embodiments do not limit the subject matter and scope of protection of this disclosure. Brief description of the attached diagram

[0065] Figure 1 An example of the arrival time distribution of photons detected using time gating and the phasor diagram derived therefrom are schematically shown. Figure 2 An optical microscopy apparatus according to an embodiment is shown; Figure 3 An optical microscopy apparatus according to another embodiment is shown. Attached Figure Description

[0066] Figure 1 An embodiment of the method according to the invention is schematically illustrated, in which an excitation pulse p is used. A The form of a focused excitation light A and a suppression pulse p superimposed on the focal point of the excitation light A. V A sample 2 having at least two different types of emitters E is illuminated by an intensity distribution of a suppressor light V (particularly STED light) (which has local minima, particularly a central zero point), and individual photons emitted from the sample 2 are detected in a time-resolution manner. The suppressor light V transitions at least one type of emitter E in the sample 2 from an excited state by excitation light A to its ground state in a region outside the local minima. Specifically, a scanning device is used to scan the focal point of excitation light A and the intensity distribution of suppressor light V over the sample 2, and the number of detected photons and the arrival time of each photon are determined pixel-by-pixel.

[0067] Figure 1 The left-hand diagram shows the excitation pulse p on the time axis t. A Relative to the excitation pulse p A Delayed suppression pulse p V And by detector 4 (see Figure 2 and Figure 3 ) detected by excitation light A from sample 2 (see Figure 2 and Figure 3 Frequency distribution of the arrival time of emitted photons (frequency n) p The frequency distribution is presented as a histogram. Figure 1 The right-hand side of the diagram shows a phasor diagram 12 with exemplary phasors P1, P2, and P3, which are calculated based on photons detected in the gated time interval 10 (see the left-hand diagram).

[0068] When calculating phasors P1, P2, and P3, photons detected before the gated time interval 10 are not considered. The gated time interval begins at time t0'. In this example, the excitation pulse p AThe time interval between the start time point t0' of the gated time interval 10 corresponds to the blocking pulse p. V The duration. By excluding photons emitted by the emitter before the gate time interval 10, it is possible to largely eliminate the influence of the suppressor light (e.g., due to the suppressor pulse p). V Phasor analysis is performed under conditions where shortened launch lifetime leads to diffuse and blurred phasor distribution. This improves the separability of different phasor groups associated with different types of emitters in the sample.

[0069] When calculating phasors, for example, this can be achieved by calculating the cosine transform. Determine the real part of the phasor and calculate the sine transform To determine the imaginary part of the phasor, where It is the excitation pulse p A The repetition rate is given by I(t), which is the temporal intensity distribution of the signal, and t0' is the starting time point of the gated time interval 10.

[0070] Phasors P1 and P2, exemplarily identified in phasor diagram 12, lie on a semicircle centered at (0.5; 0) with a radius of 0.5, and correspond to the respective signals with a single emission lifetime, as expected at the sample location where only one type of emitter E with single exponential decay behavior is present. Phasor P3 lies approximately on the line between phasors P1 and P2, and is therefore a phasor example of a mixed signal of emitters with corresponding emission lifetimes. For a pixel associated with phasor P3, the proportion of the two emitter types can be determined by determining the position of phasor P3 on the line between phasors P1 and P2, and the corresponding photon number can be assigned to different detection channels for that pixel. This signal separation works better due to the gating according to the invention, as the influence of the suppressor light V is at least reduced in phasor analysis.

[0071] Figure 2 A first embodiment of an optical microscopy device 1 according to the present invention is shown. The optical microscopy device 1 includes an illumination device 3 for illuminating a sample 2 with excitation light A and a blocking light V, a detector 4 for detecting photons P emitted from the sample 2 with time resolution, and a computing unit 5 for performing phasor analysis based on the photons P detected by the detector 4. The illumination device 3 includes a first light source 31, particularly a laser, for generating excitation light pulses p. A The form of excitation light A; the second light source 32, in particular another laser, is used to generate the suppression pulse p. VThe sample 2 consists of a suppressed light V (particularly STED light); an optical modulator 33 (e.g., a so-called spatial light modulator, SLM) for influencing the spatial phase and / or amplitude distribution of the suppressed light V; a beam combiner 34 (e.g., a dichroic mirror) for combining the excitation light A and the suppressed light V; and an objective lens 38 for co-focusing the excitation light A and the suppressed light V onto a sample 2 having at least two different types of emitters E (e.g., fluorophores with different emission lifetimes). The optical modulator 33 and the objective lens 38 work together to form an intensity distribution of the suppressed light V with a central zero intensity point (which superimposed on the maximum intensity of the excitation light A) at the common focal point of the excitation light A and the suppressed light V in the sample 2. The intensity distribution can be, for example, a ring, a bottle-shaped beam, or a so-called crescent-shaped light distribution. Photons P emitted from the sample 2 are detected by a detector 4 (e.g., a single-photon avalanche photodiode, SPAD) and recorded in a time-resolution manner by an evaluation device 41 coupled to the detector 4 (e.g., including TCSPC electronics). The evaluation device 41 is coupled to the computing unit 5.

[0072] The evaluation device 41 can send the arrival times of each photon P to the calculation unit 5. The calculation unit 5 can then base its calculations on the arrival times of the photons within the gated time interval 10 (see...). Figure 1 The arrival time of the photon P detected in sample 2 was used to perform phasor analysis in order to separate the signals of different types of emitters E in sample 2.

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

[0074] According to an alternative, the real and imaginary parts of the phasors are determined directly from the data stream from the TCSPC electronics by the evaluation device 41, and these values ​​are transmitted to the computing unit 5. In this case, the evaluation device 41 may include, for example, an FPGA connected to the TCSPC electronics. The advantage of doing so is that it is not necessary to consider the arrival time of each individual photon storage.

[0075] Figure 3 A second embodiment of the optical microscopy apparatus 1 according to the present invention is shown, which is herein designed as a confocal laser scanning microscope and / or a STED microscope. Figure 2 Components that are identical or similar to those in the embodiments are indicated by the same reference numerals. Refer to the above for further details. Figure 2 The description. Based on Figure 3In the optical microscope 1, the intensity distribution of the focused excitation light A and the suppression light V is scanned above the sample 2. The objective lens 38 also collects photons P emitted from the sample 2 and performs confocal detection on the photons P. For this purpose, a beam splitter 35 (in particular another dichroic mirror) and a scanning device 36 (e.g., a galvanometer scanner) are located in the optical path between the beam combiner 34 and the objective lens 38. The scanning device 36 has at least one drivable, movable scanning mirror 37, in particular at least one scanning mirror (not shown) in each scanning spatial direction. The scanning device 36 scans the common focal point of the excitation light A and the suppression light V above the sample 2.

[0076] Photons P emitted from sample 2 are focused by objective lens 38, descanned by scanning device 36, and reflected by beam splitter 35 according to wavelength. The reflected light passes through confocal pinhole aperture 6 to reach detector 4, where these photons P are recorded pixel-by-pixel by evaluation device 41 in a time-resolution manner. Reference tag list

[0077] 1. Optical Microscopy Equipment 2 samples 3. Lighting equipment 4 Detectors 5. Calculation Unit 6. Pinhole aperture 10 Gating Time Interval 12 Phasor Diagrams 31 First Light Source 32 Second Light Source 33 Optical modulator 34 Beam combiner 35 beam splitter 36 Scanning equipment 37 Scanning mirror 38 Objective lenses 41. Evaluation Equipment A Excitation Light E emitter P photon V. Light blocking p A Excitation pulse ps Suppression Pulse t0' is the starting time point of the gated time interval.

Claims

1. An optical microscopy method, wherein, A sample (2) is irradiated with a focused pulsed excitation light (A) to excite at least two types of emitters (E) with different emission lifetimes, wherein the sample (2) is additionally irradiated with an intensity distribution of a blocking light (V) having 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 phasor analysis is performed based on the detected photons (P) to separate photons (P) of different types of emitters (E) based on the emission lifetime of the emitters. Its features are, Gating is performed on the photon (P), where is the excitation pulse (p) of the excitation light (A). A Set a corresponding gated time interval (10), wherein only photons (P) detected in the corresponding gated time interval (10) are considered in the phasor analysis.

2. The method according to claim 1, characterized in that, The zero point of the time scale on which the phasor analysis is based is set as the starting time point (t0') of the gated time interval (10).

3. The method according to claim 1 or 2, characterized in that, The suppressing light (V) is a suppressing pulse (p) V The excitation pulse (p) is incident on the sample (2) in the form of an excitation pulse (p). A The time interval between the start time point (t0') of the corresponding gated time interval (10) and the corresponding blocking pulse (p) V They overlap in time.

4. The method according to claim 3, characterized in that, Excitation pulse (p) A The time interval between the start time point (t0') of the corresponding gated time interval (10) and the corresponding blocking pulse (p) corresponds to the corresponding blocking pulse. V At least one pulse length.

5. The method according to claim 3 or 4, characterized in that, The suppression pulse (p) V ) relative to the corresponding previous excitation pulse (p A The pulse of excitation pulse (p) is delayed and incident on the sample (2), wherein the excitation pulse (p) is delayed and incident on the sample (2). A The time interval between the start time point (t0') of the corresponding gated time interval (10) and the time interval between the start time point (t0') corresponds at least to the blocking pulse (p) V The sum of the pulse length and the pulse delay.

6. The method according to any one of claims 1 to 5, characterized in that, The detector (4) detects photons (P) emitted from the sample (2) only during the gated time interval (10), or the evaluation device (41) coupled to the detector (4) records photons (P) emitted from the sample (2) only during the gated time interval (10).

7. The method according to any one of claims 1 to 5, characterized in that, Although detected and recorded in the excitation pulse (p A The photons (P) emitted from the sample (2) between the start time (t0') of the corresponding gated time interval (10) and the corresponding gating time interval (10), but excluded from the dataset used in the phasor analysis.

8. The method according to any one of the preceding claims, characterized in that, The sample (2) is scanned with the focus of the excitation light (A) superimposed on the intensity distribution of the blocking light (V), wherein photons (P) detected at the corresponding positions in the sample (2) for the focus are assigned to the corresponding image pixels.

9. The method according to any one of the preceding claims, characterized in that, The detector (4) is coupled to the evaluation device (41), wherein the evaluation device (41) calculates the real part (G) and imaginary part (S) of each phasor (P1, P2, P3) without storing each photon signal based on the signal pulse sequence of the photon signal transmitted from the detector (4) to the evaluation device (41), and performs the phasor analysis on this basis.

10. The method according to claim 9 of claim 8, characterized in that, The evaluation device (41) calculates the real part (G) and imaginary part (S) of the phasors (P1, P2, P3) for each image pixel.

11. The method according to any one of the preceding claims, characterized in that, The suppressor light (V) acts at least on the first emitter (E) in the sample (2) outside the local minimum of the suppressor light (V), wherein the first emitter (E) in the sample has a longer emission lifetime than the second emitter (E).

12. An optical microscopy apparatus (1), said apparatus (1) being specifically designed for performing the method according to any one of claims 1 to 11, said apparatus (1) comprising at least the following components: Lighting device (3), the lighting device being designed to illuminate 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 additionally illuminating sample (2) with an intensity distribution of blocking light (V) having local minima to de-excite emitters (E) outside the local minima. Detector (4), the detector being designed to detect photons (P) emitted from the sample (2), and The computing unit (5) is designed to perform phasor analysis based on the detected photons (P) to separate photons (P) of different types of emitters based on the emitter's emission lifetime. Its features are, The optical microscopy device (1), particularly the evaluation device (41), or the computing unit (5) of the optical microscopy device (1) is designed to transmit excitation pulses (p) to the excitation light (A). A The corresponding gate time interval (10) is set to perform gating on the photon (P), wherein the computing unit (5) is designed to consider only the photons (P) detected in the corresponding gate time interval (10) in the phasor analysis.

13. A computer program having instructions that cause the optical microscope (1) according to claim 12 to perform the method according to any one of claims 1 to 11.

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