METHOD, DEVICE AND LIGHT MICROSCOPE FOR TIME-RESOLVED SINGLE PHOTON MEASUREMENT

Abstract

The invention relates to a method for time-resolved single-photon measurement, wherein light pulses (L) are generated by a light source (3), wherein photons (P) emitted from a sample (2) on the basis of the light pulses (L) are detected by a detector (4) and registered by an evaluation device (5) coupled to the detector (4), wherein the evaluation device (5) determines detection times (t P ) of the photons (P) are determined, wherein the evaluation device (5) periodically determines pulse times (t) by evaluating a signal from the light source (3) or a sensor (11). L ) of a portion of the light pulses (L) are determined, whereby the pulse times (t L ) are determined at a lower rate than a repetition rate of the light pulses (L), based on the determined pulse times (t L ) Pulse times (t L ') further light pulses (L) are estimated, and where the pulse times (t L , t L') as reference times for the recording times (t P ) of the photons (P) as well as a device (1) for single photon measurement, a light microscope (10) with the device (1) and a computer program.

Description

Technical field of the invention

[0001] The invention relates to a method for time-resolved single-photon measurement as well as a device and a light microscope for carrying out the method. State of the art

[0002] Single photon counting is advantageous for various applications, for example in high-resolution light microscopy and optical spectroscopy.

[0003] For example, many confocal fluorescence light microscopes use detectors with a counting mode in which they accumulate the emission signal by counting individual photons.

[0004] When using detectors that not only count photons but also monitor and record the arrival times of individual photons (e.g., using a method called time-correlated single-photon counting (TCSPC)), emitter lifetimes can also be determined. For example, this additional information can be used in lifetime imaging to generate a color-coded image of the sample, with the color code reflecting the lifetime values, which depend on the emitters' local environment within the sample. Furthermore, multiple fluorophores in a microscopic image can be distinguished based on their lifetimes, even if their absorption and / or emission spectra overlap.

[0005] Specialized detector modules for time-correlated single-photon counting (TCSPC) are known from the prior art. These contain photon detectors and electronic circuits that are coupled to the photon detector and can be used to count the arriving photons and to determine the arrival times of the individual photons at the detector.

[0006] An overview of TCSPC and detector technology is given, for example, in the document "The bh TCSPC Handbook", 8 th Edition, September 2019, by Wolfgang Becker (https: / / www.beckerhickl.com / literature / documents / flim / the-bh-tcspc-handbook / ).

[0007] When measuring emission lifetime, the result from the counting electronics is usually a histogram of the photon arrival times, the shape of which resembles a temporal progression of emission decay with a corresponding time constant that yields the emission lifetime of interest.

[0008] An example of a fluorescence lifetime imaging (FLIM) technique applied in a high-resolution light microscope and based on single-photon counting is described in the publication by Marco Castello et al.: “A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM”, Nature Methods, Brief Communication, https: / / doi.org / 10.1038 / s41592-018-0291-9, published online on January 14, 2019. In the technique described in this publication, excitation light capable of exciting fluorophores is focused onto a region of the sample, which is then imaged onto an array of single-photon avalanche photodiodes (SPADs) arranged in a confocal plane. Based on the signal from the photodiodes, the arrival times of the individual photons at the detector are determined by a single-photon counting electronics system, and the fluorescence lifetime of the fluorophores is determined from histograms of the arrival times.

[0009] In DE 10 2024 112 112 A1 a method for detecting individual photons from a sample (in particular using time-correlated single-photon counting) is described, in which a dead time of a detection system is set as a function of a repetition rate of the light pulses, an expected emission lifetime of emitters in the sample and in particular a number of photons incident on the detector per light pulse.

[0010] From EP 3 431 967 A1, a method for estimating and correcting pile-up effects in TCSPC applications is known, wherein a decay histogram is obtained from a TCSPC measurement, wherein a probability is estimated that electrical pulses of the detector indicating photon detection have a distance greater than the smallest resolvable time interval of each channel of the decay histogram, and wherein a function fit of the decay histogram to a model function adapted on the basis of the estimated probability is performed.

[0011] In time-correlated single-photon counting, synchronization pulses and photon pulses are frequently recorded by the evaluation electronics. The synchronization pulses indicate the clock of the pulsed laser source, while the photon pulses originate from a single-photon detector and indicate the detection of individual photons. In many single-photon counting systems, a time interval is determined for each counted photon relative to one or more synchronization pulses. This time interval is often transmitted to downstream electronics along with the photon pulse. Calculating and / or transmitting this time interval can contribute to the dead time of the detection system.

[0012] As an alternative to directly transmitting a time interval, the detection of a photon can trigger the transmission of the time of a preceding synchronization pulse or several preceding synchronization pulses to downstream electronics.

[0013] On the one hand, determining the time interval between a photon detection and the synchronization pulses was essential from a historical point of view for early TCSPC systems, since determining absolute times with the required precision below 100 picoseconds was not yet technically feasible at that time.

[0014] On the other hand, the time interval of a photon to the preceding laser pulse, assuming that this laser pulse caused the emission of the photon, represents an important measurement from which, for example, fluorescence lifetimes can be determined in FLIM experiments.

[0015] To reliably determine such a time interval, the evaluation electronics must determine the timing of the laser pulses with the same accuracy as the timing of photon detection. The evaluation electronics have only a limited number of channels for signal timing and are therefore limited in their bandwidth.

[0016] In many measurements, the number of laser pulses per unit of time is greater than the average number of photons detected in the same time. The so-called count rate (detected photons per unit of time) is therefore often lower than the repetition rate (laser pulses per unit of time). Consequently, the bandwidth of the detection system is limited by the registration and timing of the laser pulses – the detection system thus reaches saturation at a lower count rate than if only the detected photons were registered.

[0017] Another source of error in determining the time intervals between photons and laser pulses is the so-called "jitter," i.e., the temporal variation of the laser pulses in the sample and / or the synchronization pulses in the electronics. The actual jitter of the laser pulses is sometimes not accurately represented by the synchronization pulses, as, for example, no assignment or only an erroneous assignment between individual laser pulses and their respective synchronization pulses is possible. Furthermore, the magnitude of the electronic jitter can exceed the pulse-to-pulse jitter. Both effects can contribute to measurement errors when determining the time interval between a photon and its corresponding laser pulse. Object of the invention

[0018] This leads to the task of providing a method for time-resolved single-photon measurement and a corresponding device that allows the determination of time intervals between detected photons and respective light pulses with improved bandwidth of the detection system and / or reduced measurement error. Solution

[0019] This problem is solved by the subject matter of the independent claims. Advantageous further developments arise from the dependent claims and are described below. Description of the invention

[0020] A first aspect of the invention relates to a method for time-resolved single-photon measurement, wherein light pulses are generated by a light source, wherein photons emitted from a sample due to the light pulses are detected by a detector and registered by an evaluation device coupled to the detector, wherein the evaluation device determines the detection times of the photons, wherein the evaluation device periodically determines pulse times of a portion of the light pulses by evaluating a signal from the light source or a sensor, wherein the pulse times are determined at a lower rate than a repetition rate of the light pulses, wherein pulse times of further light pulses are estimated on the basis of the determined pulse times, and wherein the estimated pulse times are used as reference times for the detection times of the photons.

[0021] Since, according to the invention, pulse times are measured for only a portion of the light pulses and the pulse times of other light pulses are estimated, the bandwidth of the system usable for the registration and arrival time determination of the photons is increased.

[0022] Furthermore, the measurement error in determining the time intervals between photon detection and the corresponding light pulse is reduced, at least by the lesser influence of measuring the pulse times. An even greater reduction in the measurement error occurs, for example, if the pulse-to-pulse jitter of the light pulses is not exactly represented by corresponding electronic synchronization pulses, or if the electronic jitter of such synchronization pulses is greater than the pulse-to-pulse jitter of the light pulses.

[0023] The light source can be, in particular, a pulsed laser. The laser pulses can be generated in a variety of ways, for example, by combining a CW laser with a pulse picker or acousto-optic modulator. The repetition rate of the light pulses, i.e., the number of light pulses per unit of time, can be in the MHz range for typical applications, but can also be orders of magnitude lower.

[0024] The detector can have a single light-sensitive element, such as a single-photon avalanche photodiode (SPAD). Alternatively, the detector can have several detector elements arranged in a detection plane, which can be individually read out. This is the case, for example, with so-called SPAD arrays.

[0025] The evaluation device can be, for example, TCSPC electronics or comprise such electronics. It can be a unit with the detector (e.g., on a common circuit board and / or enclosed in a common housing) or it can be separate from the detector and electrically connected to it.

[0026] The pulse timing of the light pulses can be determined, for example, based on electrical synchronization pulses, which are typically generated by the light source or a unit coupled to the light source. Alternatively, such synchronization pulses can also be generated based on signals from a sensor in the beam path of the device according to the invention.

[0027] The photons emitted by the light pulses can, for example, arise from the excitation of fluorophores in the sample to fluorescence by the light pulses. However, the emitted photons can also be reflected or scattered by the sample or objects within the sample. The crucial factor here is that a causal and temporal relationship exists between the light pulses striking the sample and the photons emitted by the sample.

[0028] Using the pulse times as reference times for the detection times of the detected photons can mean, in particular, that for each detected photon, at least one time interval is determined relative to a pulse time (especially the light pulse immediately preceding the photon). "Immediately preceding" means that no other light pulses occur on the time axis between the photon and the associated light pulse. Optionally, time intervals relative to multiple light pulses can also be determined.

[0029] According to one embodiment, only the estimated pulse times are used as reference times for the photon detection times. The measured pulse times, i.e., those determined by the evaluation device through the analysis of a signal from the light source or a sensor, are not used as reference times in this embodiment. This has the advantage that the error in determining the time intervals between detected photons and their corresponding light pulses does not depend on the error in measuring the pulse times. Rather, for example, when estimating the pulse times used as reference times from a large number of measured pulse times that themselves do not serve as reference times, this measurement error (especially any pulse-to-pulse jitter) can average out. The error in determining the time intervals then depends essentially only on the error in measuring the photon detection times.As mentioned above, the method results in a further improvement in measurement accuracy with regard to jitter (pulse-to-pulse jitter of the light pulses in the sample as well as, if applicable, electronic jitter of the synchronization pulses), for example, if the light pulses are not perfectly mapped by the synchronization pulses or if the electronic jitter is greater than the pulse-to-pulse jitter of the light pulses in the sample.

[0030] According to a further embodiment, for each detection time of a photon, at least one preceding pulse time and at least one subsequent pulse time are estimated. Based on these estimated pulse times, the period and phase of the respective detection time can be easily determined. The period indicates, in particular, between which successive light pulses the photon occurred, and the phase indicates, in particular, the temporal position of the photon between the light pulses. Estimating only certain pulse times, namely, for example, those pulse times necessary to determine a time interval, has the advantage of requiring less computational effort compared to estimating all pulse times.

[0031] According to one embodiment, the evaluation device comprises data channels and a switching device, wherein the switching device distributes output signals from the detector and the light source or sensor to the data channels, and the evaluation device uses the data channels to determine the detection times of photons and the pulse times of the light pulses. In particular, the switching device allows the detector and the light source or sensor to be alternately connected to the same data channel, so that the evaluation device uses the data channel to alternately determine the detection times of photons and the pulse times of the light pulses. In this way, the bandwidth of the detection system can be optimally utilized.

[0032] According to another embodiment, the evaluation device creates a time series of estimated pulse times based on the determined pulse times. From the known repetition rate of the light source and the known time intervals at which the pulse times were measured, a periodic time series can be synthesized, for example, by determining the least squares of the deviation from the measured pulse times. Pulse times measured at intervals larger than the pulse-to-pulse interval can be used, for example, as reference points for the time series. Due to the measurement error of the pulse times, the measured pulse times then generally no longer correspond exactly to the corresponding pulse times in the synthesized time series.In particular, the time series is periodic and contains pulse times for each light pulse; the adjacent synthesized pulse times in the time series are therefore spaced apart by an interval corresponding to the repetition rate of the light source. Synthesizing the entire time series has the advantage that the period and phase of a photon's detection time can be determined particularly quickly, since it is not necessary to estimate adjacent pulse times for each photon, but rather the entire time series is already known.

[0033] According to another embodiment, the time series is continuously adjusted based on further measured pulse times (determined by evaluating a signal from the light source or a sensor). In this process, all available measured pulse times or only a subset of them can be used for the estimation. When using only a subset of the measured pulse times, for example, the most recent pulse times can be used, particularly in the form of a continuous window function. This has the advantage that, for example, temporal fluctuations such as a drift in the repetition rate can be better accounted for, and the time series thus better reflects the current conditions in the sample.

[0034] According to another embodiment, the pulse times are used as reference times for the photon detection times by determining the respective periods and phases of the time series for the photon detection times.

[0035] According to a further embodiment, the rate of pulse timing determination is 20% of the repetition rate or less, in particular 10% of the repetition rate or less, and further, in particular 5% of the repetition rate or less. At a value of 10%, for example, only the pulse timing of every tenth light pulse is measured. Depending on the photon count rate, this significantly increases the usable bandwidth of the detection system. The use of only every Nth light pulse can be implemented electronically, for example, by a so-called divider in the signal line between the input of the synchronization pulses and the downstream evaluation electronics.

[0036] According to a further embodiment, first and second light pulses of different colors are generated by the light source or by the light source and a further light source, wherein the first and second light pulses are temporally offset from each other, wherein photons emitted from the sample due to the first light pulses and photons emitted from the sample due to the second light pulses are detected by the detector or by the detector and a further detector, wherein the evaluation device determines at least the pulse times of a portion of the first light pulses and / or a portion of the second light pulses by evaluating the signal. This type of alternating sample illumination and detection is also referred to in the prior art as the pulse-interleaved method.

[0037] Using the pulse times as reference points can, in particular, involve assigning the detected photons to a first or second light pulse. For example, the period of each photon in the time series of the first and second light pulses can be determined, especially when the repetition rates of the light source(s) and the time delay between the first and second light pulses are chosen such that, after a first light pulse, there is a high probability that no photon caused by the preceding second light pulse will be detected, and vice versa.

[0038] According to another embodiment, the pulse times of the second light pulses are estimated based on certain pulse times of the first light pulses, or vice versa. This can be the case, for example, when two different laser sources are used, one of which has a lower pulse-to-pulse jitter of the laser pulses. In this case, if the delay between the first and second light pulses is known, it is advantageous to estimate the pulse times of the second light pulses from the measured pulse times of the first light pulses in order to reduce the measurement error, at least if the temporal change in the delay between the first and second light pulses is sufficiently small.

[0039] According to another embodiment, the sample contains emitters, in particular fluorescent emitters, wherein the emitters are excited by the light pulses to emit photons, and wherein the lifetime of the emitters is determined by the evaluation device on the basis of the detected photons and the reference times.

[0040] The 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.

[0041] The term "emitter" as used here refers to a molecule, a molecular complex, or a particle that emits electromagnetic radiation, particularly in the visible, infrared, or ultraviolet range, when excited by external light. The emitter can be, for example, a fluorophore or 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 can be, for example, 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 can result from light scattering. Another example is photoactivation, which can consist of exciting the at least one emitter from a first, e.g.,to convert a non-fluorescent or dark state into a second, light-emitting state, e.g., by having the at least one emitter emit fluorescence in response to excitation light in the second state. In this case, the excitation light that excites the at least one emitter in the second state can originate from the light pulses that also activate the at least one emitter, or the excitation light can originate from another source, in particular, the excitation light being able to have a different wavelength than the activation light.

[0042] A second aspect of the invention relates to a device for time-resolved single-photon measurement, in particular according to a method according to the first aspect, wherein the device comprises a detector (e.g. a single-photon avalance photodiode, SPAD, or a SPAD array) configured to detect photons emitted from a sample due to light pulses, and an evaluation device coupled to the detector (e.g. a TCSPC electronics) configured to determine the detection times of the photons, wherein the evaluation device is configured to determine, by evaluating a signal from a light source (e.g.to periodically determine pulse times of a portion of the light pulses (of a laser) or a sensor at a lower rate than the repetition rate of the light pulses, to estimate pulse times of further light pulses based on the determined pulse times, and to use the pulse times as reference times for the detection times of the photons.

[0043] A third aspect of the invention relates to a light microscope comprising a light source (e.g. a laser) designed to generate light pulses, and a device for time-resolved single-photon measurement according to the second aspect.

[0044] A fourth aspect of the invention relates to a computer program comprising program code configured to cause the device for time-resolved single-photon measurement according to the second aspect or the light microscope according to the third aspect to carry out the method according to the first aspect.

[0045] Further features and advantages of the device according to the second aspect, the light microscope according to the third aspect, and the computer program according to the fourth aspect result analogously from the explanations of the method according to the first aspect.

[0046] 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.

[0047] 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.

[0048] 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 merely serve the purpose of making the patent claims easier to understand.

[0049] 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 shows an exemplary time series of light pulses and photons to illustrate an embodiment of the invention; Fig. Figure 2 shows a device for single-photon measurement according to an embodiment of the invention; Fig. Figure 3 shows a light microscope according to an embodiment of the invention; Fig. Figure 4 shows a light microscope according to a further embodiment of the invention. Description of the characters

[0050] Fig. Figure 1 schematically shows an exemplary time series of light pulses L, generated by a light source, e.g., a laser, and illuminating a sample, and photons P detected by a detector, which are emitted by the sample, e.g., by emitters within the sample, as a consequence of the light pulses L. The photons P could, for example, be fluorescence photons emitted by fluorophores in the sample, with the light pulses exciting the fluorophores to fluoresce. The detection times t are defined for each photon P. p plotted on the timeline.

[0051] In Fig. 1 only shows those light pulses L whose pulse times t L were determined by evaluating a signal from light source 3 or a sensor. Further pulse times t L ' are based on the pulse times t recorded by measurement according to the invention. L estimated (dashed lines in Fig. 1) According to the Fig. In example 1, only the pulse time t is shown. L L of every fifth light pulse is determined by measurement, the remaining pulse times t L ' are estimated, e.g. by fitting a temporal function (using the known repetition rate of the light source) to the measured pulse times t L .

[0052] In particular, the entire time series of light pulses L is derived from the pulse times t recorded by measurement. L reconstructed by estimation, i.e., even for those light pulses L whose pulse times t L Pulse times were measured t L 'estimated.

[0053] The estimated pulse times t L ' are used as reference times for the recording times t p The photons are used. In particular, time intervals between the detection times t are used. p and the pulse times t L ' certainly.

[0054] Fig. Figure 2 shows an embodiment of a device 1 according to the invention for single-photon measurement. The device 1 has a light source 3, e.g., a laser, which generates light pulses L. The light pulses L strike a sample 2 and excite emitters E in the sample 2, in particular to fluorescence, so that these photons P emit. The device 1 further has a detector 4, which is configured to detect the photons P. A signal output 40 of the detector 4 is coupled to a first signal input 53 of an evaluation device 5 of the device 1, so that the evaluation device 5 can receive signals or data from the detector 4. The evaluation device 5 has a processing unit 51, which is configured to determine detection times t. pto determine the value of individual photons P detected by the detector 4. The signal runs from the first signal input 53 via a switching device 50 and via one of several data channels 52 to the processing unit 51.

[0055] The light source 3 can also be provided as a separate component, i.e., it does not have to be part of the device 1.

[0056] The light source 3 sends synchronization pulses S via a signal output 30, which is connected via a divider 6 to a second signal input 54 of the evaluation device 5, which pulse times t L The light pulses L generated by the light source 3 are displayed. The divider 6 is designed to transmit only a portion of the synchronization pulses S, e.g., every fifth or every tenth synchronization pulse S, to the second signal input 54 of the evaluation device 5.

[0057] The switching unit 50 of the evaluation device 5 distributes incoming signals from the detector 4 via the first signal input 53 and incoming synchronization pulses S from the light source 30 via the second signal input 54 to data channels 52, via which they reach the computing unit 51.

[0058] The computing unit 51 is designed to calculate pulse times t based on the synchronization pulses S. L to determine and based on the pulse times t L further pulse times t L ' to estimate. Furthermore, the computing unit 51 is designed to estimate the pulse times t L ' as reference times for the recording times t p to use the photons, i.e., in particular, time intervals between the detection times t p and associated pulse times t L' to determine, for example, the period and phase of a photon detection with respect to the time series of light pulses L. Based on this information, emission lifetimes of a fluorophore can then be determined by the processing unit 51 or a separate processing unit.

[0059] In Fig. Figure 3 schematically illustrates an embodiment of a light microscope 10 according to the invention. In this (non-limiting) embodiment, the light microscope 10 is configured as a confocal laser scanning microscope and comprises a light source 3, in particular a laser, for generating light pulses L. The light from the light pulses L passes through a beam splitter 12, a scanning device 9 with a movable scan mirror 90, and an objective 7 into a sample 2, wherein the light is focused into the sample 2, in particular by the objective 7, and the scanning device 9 is configured to scan the focus of the light by moving (in particular rotating) the scan mirror 90 over or through the sample 2. Emitters can be arranged in the sample 2, which are excited by the light pulses L and subsequently emit photons P.The emitted photons P are collected by the lens 7, scanned by the scanning device 9, and reflected by the beam splitter 12, in particular a dichroic mirror, into a detection beam path. The detection beam path contains a pinhole aperture 8 and a detector 4 for capturing the photons P. The detector 4 is connected to an evaluation device 5. The detector 4 and the evaluation device 5 form a device 1 according to the invention for single-photon measurement, which is, for example, as shown in [reference]. Fig. 2 shown and described above, can be configured. Unlike the one in Fig. However, in the arrangement shown in Figure 2, the light source 3 is not the source; instead, a sensor 11 located in the beam path between the scanning device 9 and the lens 7 generates synchronization pulses S, which are transmitted to the evaluation device 5. The evaluation device 5 evaluates the synchronization pulses S to determine pulse times t. Lto determine the light pulses L. Based on the pulse times t L Then further pulse times will be determined. L ' estimated by the evaluation device 5. The sensor 11 can, of course, also be arranged at another suitable location in the beam path of the light microscope 10. Furthermore, the light microscope 10 according to the invention can, of course, also be equipped with a device 1 according to Fig. 2 included, i.e. the synchronization pulses S can also originate from the light source 3.

[0060] Fig. Figure 4 shows a further embodiment of the light microscope 10 according to the invention. It is the same as the embodiment shown in Figure 4. Fig. 3 also includes a confocal laser scanning microscope. Identical components are used in the Fig. 3 and Fig. 4 are provided with identical reference numerals. In this regard, please refer to the description at Fig. 3 referred.

[0061] The in Fig. The light microscope 10 shown in Figure 4 has a first light source 3a for generating first light pulses L1 and a second light source 3b for generating second light pulses L2, wherein the first light pulses L1 and the second light pulses L2 have different wavelength components. The first light pulses L1 and the second light pulses L2 can be used, for example, to excite first and second emitters of different types in the sample 2, so that the first emitters emit first photons P1 and the second emitters emit second photons P2 (in particular, of different wavelengths). The first light pulses L1 and the second light pulses L2 are coupled together into the main beam path of the light microscope 10 at a beam combiner 13.The first light pulses L1 and the second light pulses L2 are emitted at different times by the respective light sources 3a,3b (so-called Pulse-Intermittent-Excitation, PIE), whereby a time delay between the first light pulses L1 and the second light pulses L2 can be set using a control unit 15 connected to the first light source 3a and the second light source 3b.

[0062] The first photons P1 and the second photons P2 are collected by the objective 7, scanned by the scanning device 9, and reflected by the beam splitter 12 into the detection beam path of the light microscope 10. In the detection beam path, the first photons P1 and the second photons P2 pass through a pinhole 8 to a detector 4, which detects both the first photons P1 and the second photons P2. Alternatively, the first photons P1 and the second photons P2 can also be split based on their wavelengths using an additional beam splitter in the detection beam path and detected by separate detectors (not shown).

[0063] A signal output of the detector 4 is connected to the evaluation device 5. The evaluation device 5 determines the respective detection times t for the photons P1 and P2. PThe first light source 3a generates first synchronization pulses S1, and the second light source 3b generates second synchronization pulses S2. These pulses are transmitted to the respective inputs of the evaluation device 5 via an optional first divider 6a and an optional second divider 6b, respectively. Alternatively, the first and second synchronization pulses S1 and S2 can also be transmitted to the same input of the evaluation device 5, along with corresponding identifiers that encode information about the origin of the synchronization pulses S1 and S2 from the first light source 3a and the second light source 3b, respectively. The evaluation device 5 then determines pulse times t based on the first and second synchronization pulses S1 and S2. L the first light pulses L1 and the second light pulses L2. Subsequently, based on the measured pulse times t L further pulse times t L ' can be estimated. Based on the recording times t Pand the pulse times t L Based on the first light pulses L1 and the second light pulses L2, it is possible to determine, in particular, which of the light pulses L1,L2 triggered the corresponding photon P1,P2, i.e., from which emitter species it originates. The repetition rates of the light sources 3a,3b and the pulse delay are specifically chosen such that a first photon P1 caused by the first light pulse L1 is highly likely to occur before the subsequent second light pulse L2, so that the photons can be uniquely assigned to a first light pulse L1 or a second light pulse L2 based on the period of their detection in the time series of light pulses L1,L2, i.e., identified as first photons P1 or second photons P2.

[0064] As an alternative to the one in Fig.In the configuration shown in Figure 4, the evaluation device 5 can be connected to either the first light source 3a or the second light source 3b and receive either only first synchronization pulses S1 or only second synchronization pulses S2. Based on the first synchronization pulses S1, the evaluation device 5 can determine pulse times t L ' for the first light pulses L1 and the second light pulses L2. This is particularly useful if there is no significant fluctuation in the delay between the first light pulses L1 and the second light pulses L2. Reference symbol list 1 Device for single-photon measurement 2 Sample 3 light source 3a First light source 3b Second light source 4 Detector 5 Evaluation device 6-piece set 6a First divisor 6b Part Two 7 Lens 8-hole aperture 9 Scanning device 10 Light microscope 11 Sensor 12 beam splitters 13 beam combinations 15 Control unit 30 Signal output 40 Signal output 50 switching device 51 computing unit 52 data channels 53 First signal input 54 Second signal input 90 scan mirrors L light pulse L1 First light pulse L2 Second light pulse P Photon P1 First photon P2 Second photon S synchronization pulse S1 First synchronization pulse S2 Second synchronization pulse t L Specific pulse time t L 'Estimated pulse time t P Time of recording

Claims

[1] Method for time-resolved single-photon measurement, wherein light pulses (L) are generated by a light source (3), wherein photons (P) emitted from a sample (2) as a result of the light pulses (L) are detected by a detector (4) and recorded by an evaluation device (5) coupled to the detector (4), wherein the evaluation device (5) determines detection times (t P ) of the photons (P) are determined, characterized by , that the evaluation device (5) periodically determines pulse times (t) by evaluating a signal from the light source (3) or a sensor (11). L ) of a portion of the light pulses (L) are determined, whereby the pulse times (t L ) are determined at a lower rate than a repetition rate of the light pulses (L), based on the determined pulse times (t L ) Pulse times (t L ') further light pulses (L) are estimated, and where the pulse times (t L , t L') as reference times for the recording times (t P ) of the photons (P) are used. [2] Method according to claim 1, characterized by , that only the estimated pulse times (t L ') as reference times for the recording times (t P ) of the photons (P) are used. [3] Method according to claim 2, characterized by , that for each recording time (t P ) of a photon (P) at least one previous pulse time (t L ') and at least one subsequent pulse time (t L ') is estimated. [4] Method according to any one of the preceding claims, characterized by, that the evaluation device (5) has data channels (52) and a switching device (50), wherein the switching device (50) distributes output signals from the detector (4) and the light source (3) or the sensor (11) to the data channels (52), wherein the evaluation device (5) uses the data channels (52) to determine acquisition times (t P ) of photons (P) and pulse times (t L ) of the light pulses (L) are determined. [5] Method according to any one of the preceding claims, characterized by , that the evaluation device (5) determines the pulse times (t L ) a time series of the estimated pulse times (t L ') is created. [6] Method according to claim 5, characterized by , that the pulse times (t L , t L ') as reference times for the recording times (t P ) of the photons (P) are used by specifying the detection times (t P) the respective periods and phases of the time series of the photons (P) are determined. [7] Method according to any one of the preceding claims, characterized by , that the rate of determining the pulse times (t L ) 20% of the repetition rate or less, in particular 10% of the repetition rate or less, and further in particular 5% of the repetition rate or less. [8] Method according to any one of the preceding claims, characterized by, that first light pulses (L1) and second light pulses (L2) of different colors are generated by the light source (3) or by the light source (3) and another light source, wherein the first light pulses (L1) and the second light pulses (L2) are temporally offset from each other, wherein photons (P1) emitted by the sample (2) due to the first light pulses (L1) and photons (P2) emitted by the sample (2) due to the second light pulses (L2) are detected by the detector (4) or by the detector (4) and another detector, wherein at least pulse times (t) are determined by the evaluation device (5) by evaluating the signal L ) of a part of the first light pulses (L1) and / or a part of the second light pulses (L2) are determined. [9] Method according to claim 8, characterized by , that pulse times (t L ) the second light pulses (L2) based on specific pulse times (t L ) of the first light pulses (L1) can be estimated. [10] Method according to any one of the preceding claims, characterized by , that the sample contains emitters (E), in particular fluorescent emitters, wherein the emitters (E) are excited by the light pulses (L) to emit photons (P), wherein the lifetime of the emitters (E) is determined using the evaluation device (5) on the basis of the detected photons (P) and the reference times. [11] Device (1) comprising time-resolved single-photon measurement - a detector (4) designed to detect photons (P) emitted from a sample (2) on the basis of light pulses (L), - an evaluation device (5) coupled to the detector (4), which is designed to record detection times (t P ) of the photons (P), characterized by , that the evaluation device (5) is designed to periodically determine pulse times (t) by evaluating a signal from a light source (3) or a sensor (11). L) to determine a portion of the light pulses (L) with a lower rate than a repetition rate of the light pulses (L), based on the determined pulse times (t L ) Pulse times (t L ') to estimate further light pulses (L), and the pulse times (t L ) as reference times for the recording times (t P ) of the photons (P). [12] Light microscope (10) comprising a light source (3) configured to generate light pulses (L) and a device (1) for time-resolved single-photon measurement according to claim 11. [13] Computer program comprising program code configured to cause the time-resolved single-photon measurement device (1) according to claim 11 or the light microscope (10) according to claim 12 to perform the method according to any one of claims 1 to 10.

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