Method and apparatus for measuring optical signal parameters and non-volatile storage medium

Abstract

The present invention relates to a method and device (43) for measuring at least one optical signal parameter (30), as well as a non-volatile computer-readable storage medium (111). Known methods have the drawback that they become unusable as soon as a signal of a single photon (13), i.e., a fluorescence photon event (15), is superimposed on it. To improve on known methods, the method according to the present invention comprises the following steps: illuminating a sample (49) for a predetermined measurement period (23) to excite an optical signal, in particular fluorescence, in the sample (49); detecting the optical signal emitted from the sample (49) and providing an electrical signal (3) representative of the change in the optical signal over time; counting single photon events (15) based on the electrical signal (3) over the measurement period (23) and obtaining a counter value (Σ ) representative of the number (15a) of photon events (15). E integrating the electrical signal (3) over a measurement period (23) and providing an integrated signal (Σ P ), and at least providing a counter value (Σ E ) and the integrated signal (Σ P ) and determining at least one optical signal parameter (30) based on the integrated signal (Σ P ) and counter value (Σ E The optical signal parameter (30) is determined based on the signal amplitude (V) of the optical signal.

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus, and a non-volatile computer-readable storage medium, for measuring at least one optical signal parameter. [Background technology]

[0002] Prior art methods use a light source to excite an optical signal, such as a reflectance signal, a phosphorescence signal, a second harmonic signal or fluorescence. Purely by way of example, in the case of fluorescence, the time between switching on the light source or the excitation pulse and the emitted fluorescence photon is measured. Here, for example, a TDC (time-to-digital converter) can be used. This method is known as "time-correlated single photon counting" (TCSPC). The drawback of this method is that it is limited to a photon rate of essentially one photon generated per laser pulse. More complex and expensive equipment, including parallel evaluation electronics, allows for a photon rate of around 40 Mcts (megacounts; 10 6 Measurements of the photon rate (event) can be obtained.

[0003] Other prior art methods scan the light source and the generated photon trigger signal very rapidly, i.e., at frequencies of 10 GHz or greater, so that the optical signal parameters can be determined from the data stream.

[0004] Both methods have in common that they are of limited use as soon as single photon signals, i.e. fluorescence photon events, overlap. Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is therefore to realize a method and device for measuring optical signal parameters that is structurally simple, inexpensive, and allows measuring optical signal parameters at high photon rates (>40 Mcts / s). [Means for solving the problem]

[0006] The method according to the invention solves this problem by comprising the following steps: - illuminating the sample for a predetermined measurement period for excitation of an optical signal, in particular fluorescence, in the sample; detecting an optical signal emitted by the sample and providing an electrical signal representative of changes in the optical signal over time; Counting single photon events based on the electrical signal over a measurement period and providing a counter value representing the number of photon events. integrating the electrical signal over a measurement period to provide an integrated signal; and determining at least one optical signal parameter based at least on the counter value and the integrated signal;

[0007] The device according to the invention, which may in particular be a microscope, solves the above-mentioned problem by including a detector, an integration module, a counting module, and a logic unit: the detector generates an electrical signal representative of a sequence of incident photons and outputs it at a detector output, the integration module is configured to integrate the electrical signal over a measurement period and output a resulting integral value (also called integrated value), the counting module counts the number of photon events detected during the measurement period based on the electrical signal over the measurement period and outputs a counter value representative of this number, and the logic unit determines at least one optical signal parameter in relation to the integration value and the counter value.

[0008] Furthermore, a non-volatile computer readable storage medium according to the present invention contains a program with instructions that, when executed by a computer, cause the computer to perform a method according to the present invention.

[0009] The method and device according to the present invention therefore have the advantage that complex time measurements between the switching on of the light source, i.e., between the excitation laser pulse and the detected photon event, are not required. The method according to the present invention utilizes the temporal superposition of two or more photon events that are no longer distinguishable to measure the optical signal parameters. This is based on the fact that the superposition of multiple photon events results in a counter value that is smaller than the integral value. Rather, the accuracy of the detected fluorescence lifetime increases with the photon rate. In particular, the average photon rate, i.e., the average number of photons generated per unit time, is observed.

[0010] The method according to the invention and the device according to the invention can be further improved by other features which are advantageous in themselves. The technical features of the individual features can be combined with one another in any way or omitted, provided that the technical effect achieved by the omitted technical features is not significant.

[0011] The method can in particular be carried out using a microscope, and therefore the corresponding device can in particular be a microscope.

[0012] The counting of single photon events can be preferably carried out using a digitizer, more preferably using a comparator, which may therefore be provided in a suitable device.

[0013] The illumination of the sample over the measurement period may be modulated over time, in particular by using periodic light pulses. Pulsed, short-pulse or ultrashort-pulse lasers are particularly suitable light sources here. Pulsed diode lasers are particularly preferred because they are smaller and easier to control (e.g., compared to solid-state lasers). Pulsed fiber lasers can also be used.

[0014] The illumination unit containing such a pulsed light source may be provided internally or externally. Any duration can be selected as the measurement period, preferably including multiple periods of the light pulse. This has the advantage that the pulsed light source can be easily triggered.

[0015] Preferably, a detector that can detect a single incident photon and generates a photon event for each incident photon in the detector's output electrical signal is used as a detector or photon detector.Hereinafter, only reference is made to a detector, in particular to a fluorescence photon that generates a fluorescence photon event in the detector's output electrical signal.Although this description is based on fluorescence photons and corresponding fluorescence parameters, the present invention is not limited thereto.These descriptions can also be applied in other ways to photons and corresponding optical signal parameters that are generated by, for example, reflection, phosphorescence or two-photon processes.

[0016] Specialized electron tubes, also called photomultiplier tubes (PMTs), can be used. These can be pure semiconductor detectors, PMT hybrid detectors or SiPMs (silicon photomultiplier tubes).

[0017] The detector detects light, e.g., fluorescent light, emitted from the sample and generates an electrical signal representative of the sequence or variation of incident photons, e.g., fluorescent photons, which is output at the detector output and may include multiple photon events, e.g., fluorescent photon events, which may be separated in time from one another or may be superimposed on one another.

[0018] An integration module is then used to determine from the signal all photons, e.g., fluorescence photons, detected by the detector during the measurement period and provide a signal proportional to this number, which can represent a relative photon value.

[0019] The electrical signal can be separated into a photon counting path, where the electrical signal is integrated, and an event counting path, where single photon events in the electrical signal are counted. In the integration module, a signal proportional to all detected photons is determined, whereas in the counting module, the number of photon events detected during a measurement period is determined and provided in the form of a counter value representative of this number.

[0020] Furthermore, in the logic unit, an optical signal parameter, preferably a fluorescence parameter, particularly preferably a fluorescence lifetime, can be determined based on the integrated signal and the counter value. The logic unit can be configured in the form of a single logic module or chip or as a so-called field programmable gate array (FPGA). An FPGA is an integrated circuit (IC) into which logic circuits can be loaded and executed (in the field, at the customer's location).

[0021] A non-volatile computer-readable storage medium according to the present invention includes, for example, a program with instructions for carrying out the method according to the present invention on a computer, which may be read from the medium and loaded into the memory of the above-mentioned FPGA or computer.

[0022] The method according to the present invention may further comprise counting single photon events, e.g., single fluorescence photon events, of the electrical signal over a measurement period and providing a counter value representative of the number of photon events, e.g., single fluorescence photon events. To this end, an incremental encoder may be provided in the device for outputting a counter value, which may be in digital or analog form and represents the number of photon events, e.g., single fluorescence photon events. In particular, the counter value may be reset at the end of the measurement period, i.e., at the start of the next measurement period.

[0023] The method according to the present invention includes integrating the electrical signal over a measurement period to provide an integrated signal representing the photon count of all detected fluorescence photons accumulated during the measurement period.

[0024] Many photon detectors generate an electrical pulse for each detected photon, the level of which is not related to the photon's energy. Photons of different wavelengths cannot be distinguished based on the signal, but pulses of simultaneously incident photons add up in the signal, so that the integrated signal of two simultaneously incident photons is twice that of a single photon.

[0025] The integrated signal can be obtained using an integrator or integration module that no longer distinguishes single photon events in the electrical signal, e.g., single fluorescence photon events, with a sufficiently large time constant and integrates them over the measurement period.

[0026] In other words, in the counting module, the electrical signal is compared with a threshold, and if it exceeds the threshold and then subsequently falls below the threshold, the counter reading is incremented by 1. Thus, simultaneously incident photons, e.g., fluorescence photons, give rise to temporally overlapping photon events, e.g., temporally overlapping fluorescence photon events that exceed and fall below the threshold once, and increment the counter of photon events, in particular fluorescence photon events, by 1.

[0027] In contrast, the integral path evaluates a quantitative variable, such as charge, so two fluorescence photons incident simultaneously will generate a signal in the integral path that is twice as high as two fluorescence photons incident separately.

[0028] Therefore, the integral value (ie, the integrated signal) determined within the measurement period represents the accumulated photon count of all detected photons, particularly detected fluorescence photons.

[0029] In an advantageous embodiment of the method according to the invention, integration and counting can be carried out in parallel, and preferably additionally, integration and counting are carried out simultaneously.

[0030] Furthermore, the method may include calculating a ratio value from the number of single fluorescence photon events, i.e., from the counter value and the integrated signal. In all configurations of the disclosed method or apparatus, three variables, i.e., the counter value, the integrated signal, and the ratio value, can be used in any combination, for example, two pairs, to determine an optical signal parameter, preferably a fluorescence parameter, particularly preferably a fluorescence lifetime. Thus, (1) the counter value and the integrated signal, (2) the counter value and the ratio value, or (3) the integrated signal and the ratio value can lead to an optical signal parameter. Therefore, any description in the present disclosure relating to one of the combinations (1), (2), or (3), or any other combination of variables to determine an optical signal parameter, is applicable to any other combination of these three variables and is not limited to the listed combinations, unless expressly excluded herein.

[0031] Calibration data for determining optical signal parameters, preferably fluorescence parameters such as fluorescence lifetimes, may be stored in a table (LUT: look-up table) or formula. For example, for each value pair of integrated signal and counted signal (counter value), the associated fluorescence lifetime may be stored, so that, purely by way of example, the fluorescence lifetime may be directly indicated when two measured values ​​are present (calibration LUT). A two-dimensional LUT may contain fewer grid points than all possible combinations of value pairs. In this case, purely by way of example, an optical signal parameter such as fluorescence lifetime may be interpolated from the existing grid points.

[0032] Furthermore, purely by way of example, there may be signal pairs for which it is not possible to determine optical signal parameters such as fluorescence lifetime. This may be the case, for example, when the signals are extremely small.

[0033] In another configuration, at least one other optical signal parameter may be determined using the method and device according to the present invention. For example, if a fluorescence parameter is determined, further signals may be generated in addition to the fluorescence lifetime. For example, an improved intensity signal may be generated from the combination of two signals (integrated signal / counted signal). The structure and data path are immutable; only the calculation rules / calibration LUT can be adapted to calculate the intensity signal. Such a configuration has the advantage that redundancy is provided for determining the intensity of the electrical signal, and the integrated signal, e.g., determined by integration, can be checked for accuracy and any deviations or errors in the determination can be identified.

[0034] Thus, an apparatus for carrying out this configuration of the method may include an integration module for integrating the electrical signal over a measurement period and outputting an integrated value, a counting module for counting photon events and outputting a counter value, and optionally a division module for calculating a ratio value from the counter value of the counting module and the integrated signal of the integration module and outputting the ratio value, in which case the logic unit may be configured to determine an optical signal parameter, such as the fluorescence lifetime, in relation to the integrated signal and the counter value, or also to the integrated signal (or alternatively the counter value) and the ratio value.

[0035] Therefore, this method can be further improved by including determining an optical signal parameter, preferably a fluorescence parameter, such as a fluorescence lifetime, based on the integrated electrical signal and the counter value. Optionally, a ratio value may be used to determine the optical signal parameter in combination with the counter value or the integrated signal. In other words, the optical signal parameter, preferably a fluorescence parameter, such as a fluorescence lifetime, may be determined from two variables, respectively, in different configurations. These variables may be, for example, the integrated signal and the counter value, the counter value and the ratio value, or the integrated signal and the ratio value.

[0036] The determination of the optical signal parameter by means of the counter value and the integrated signal represents a preferred configuration of the method according to the invention or the device according to the invention, but does not exclude other combinations of variables for determining the optical signal parameter.

[0037] In another configuration of the method according to the invention, this comprises calculating an optical signal parameter, preferably a fluorescence parameter, for example a fluorescence lifetime, using the integrated signal and the counter value.

[0038] Therefore, an apparatus according to the invention for carrying out this configuration of the method may comprise a calculation unit for calculating an optical signal parameter, preferably a fluorescence parameter, e.g. a fluorescence lifetime, using the integrated signal and the counter value.

[0039] Similarly, in a special configuration, the calculation unit can use the counter value (or alternatively the integrated signal) and the ratio value (ratio of the integrated signal to the counter value) to calculate an optical signal parameter, preferably a fluorescence parameter, e.g., a fluorescence lifetime, using pre-stored analytical calculation rules (analysis curves).

[0040] The calculation unit itself may contain a division module, so that all necessary calculation steps can be combined into one unit.

[0041] Alternatively or additionally, in another embodiment of the method according to the invention, it may further be provided that a light signal parameter, in particular a fluorescence parameter, such as a fluorescence lifetime, is determined in relation to the integrated signal and the counter value using pre-stored data.

[0042] Therefore, an apparatus according to the invention for carrying out this configuration of the method may comprise at least one memory module for storing data sets of mutually assigned reference values ​​of the integrated signal, the counter values ​​and the optical signal parameters, in particular the fluorescence parameters, e.g. the fluorescence lifetime.

[0043] The pre-stored data may be in the form of a two-dimensional or three-dimensional data set, which data set or this data matrix may comprise integrated electrical signals (i.e. integral values) and counter values ​​(optionally counter values ​​and ratio values, or alternatively integrated signals and ratio values), to which specific combinations of values ​​can be assigned optical signal parameters, preferably fluorescence parameters, such as fluorescence lifetimes.

[0044] In other words, in the method according to the invention, a family of measurement curves can be assumed which show the integrated signal or ratio value in relation to the counter value (or also the ratio value in the case of an integrated signal). In this case, the family of parameters are optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime. By knowing or calculating the counter value and the integrated signal (or the ratio value), a representative of the family of curves can be determined and the optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime, belonging to this representative of the family of curves can be read off.

[0045] In another embodiment of the present invention, the method may include interpolating pre-stored data, in which case the optical signal parameters, in particular the fluorescence parameters, such as the fluorescence lifetime, are determined based on the interpolated data. This has the advantage that the pre-stored data, such as the integrated signal and counter values, are stored in fixed increments. The optical signal parameters, in particular the fluorescence parameters, such as the fluorescence lifetime, can still be determined if the detected integrated signal and / or the detected counter value is between two pre-stored integrated signals and / or counter values ​​in terms of its counter value.

[0046] Thus, an apparatus for performing this aspect of the method may include an interpolation module for interpolating the pre-stored data.

[0047] As mentioned above, analytical curves, which can be the basis for the series of measurement curves provided, can be used to directly calculate optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime, from the counter values ​​and the integrated signal (or ratio values).

[0048] Here, a further configuration of the method according to the invention may provide for determining and / or calibrating the pulse shape and / or pulse duration of single photon events, for example single fluorescence photon events.

[0049] When counting photon events, for example fluorescence photon events, due to the stochastic nature of photons, preferably fluorescence photons in the case of fluorescence, there is inevitably a certain probability that single photon events, for example single fluorescence photon events, may overlap, resulting in an event that is not counted. The probability of an event that is not counted is related to: 1. Commonly known excitation illumination pulse frequency 2. Average photon rate can be calculated using the integrated signal and pulse frequency 3. Pulse shape and pulse duration of single photon events, e.g., fluorescence photon events 4. Optical signal parameters, in particular fluorescence parameters, such as the fluorescence lifetime of an excited fluorophore, and 5. Detector Gating

[0050] In particular, item 3 of the above list can be determined in this configuration of the method according to the invention, or (if fluorescence is observed) can be calibrated by comparative measurements of dyes with known fluorescence lifetimes, which makes it possible to obtain the optical signal parameters, in particular the fluorescence parameters, e.g., the fluorescence lifetime, of the excited dye directly from the ratio of the counting path to the integration path, i.e., the ratio of the count value to the integrated signal.

[0051] 5. above means that the sensitivity of the detector can be adjusted with respect to the excitation pulse over time. In other words, gating allows masking a specific time range of the counting path (i.e., during integration) to, for example, suppress the reflected excitation light that precedes the optical signal, e.g., the fluorescence pulse.

[0052] The method according to the invention can be improved by determining the optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime, taking into account the pulse shape and / or pulse duration of single photon events, in particular single fluorescence photon events.

[0053] In particular, the method according to the invention may further comprise sequential scanning or scanning of the sample and generating images of optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime, of mutually spatially spaced regions of the sample.

[0054] This particular case of the configuration of the method according to the invention therefore represents an easy-to-implement approach to examining samples by means of fluorescence lifetime imaging microscopy (FLIM). A corresponding device may be a fluorescence lifetime microscope (FLIM).

[0055] In this configuration, a scanning device or scanning arrangement may be provided that moves the excitation light across the sample in a scanning movement or scanning motion. It is also possible to move the sample relative to the illumination and detection. Thus, optical signal parameters, in particular fluorescence parameters, such as fluorescence lifetime, of each illuminated area of ​​the sample can be determined point by point, i.e., pixel by pixel, and represented as image information.

[0056] In particular, configurations of such a method can be used in scanning microscopes, especially confocal scanning microscopes.

[0057] The method according to the invention may be based in particular on a saturation characteristic occurring in the counting module (which determines the counter value). This is caused by the superposition of the counted photon events, e.g., the counted fluorescence photon events. Since the average photon rate varies significantly over the course of a pulse, this effect of superposition may be particularly relevant for optical signal parameters, in particular fluorescence parameters, e.g., fluorescence lifetime. This effect may be particularly pronounced if the width of the counted photon events, e.g., the counted fluorescence photon events, is of the same order of magnitude as the optical signal parameter, in particular the fluorescence parameter, e.g., fluorescence lifetime, of the dye being observed.

[0058] For the detector types mentioned above, the pulse duration (duration or width of a photon event, e.g., a fluorescence photon event to be counted) is typically in the range of 1 ns to 2 ns. Typical fluorescence lifetimes of dyes are generally between 1 ns and 5 ns. In another configuration of the method according to the invention, the electron pulse duration can be adapted by appropriate filtering or by threshold matching. This allows particularly advantageous fluorescence lifetime contrasts to be obtained.

[0059] A non-volatile computer-readable storage medium according to the invention contains in particular a program with instructions which, when executed by a computer, cause the computer to carry out a method according to one of the above-mentioned configurations. Any type of optical, magnetic or flash memory-based data carrier is to be understood as a storage medium.

[0060] The method according to the invention and the device according to the invention are explained in detail below on the basis of exemplary, non-limiting figures, in which the individual technical features can be combined with one another in any way and / or omitted in accordance with the dependent claims. For the sake of clarity, the same technical features and technical features with the same function are provided with the same reference signs. [Brief explanation of the drawings]

[0061] [Figure 1a]FIG. 1 is a schematic diagram of the electrical signal generated by the detector and the calculation of the counter value. [Figure 1b] Simulation of pulsed excitation with four pulses under an assumed fluorescence lifetime of 1.5 ns. [Figure 1c] 1b is a simulation of FIG. 1b with an assumed fluorescence lifetime of 5 ns. [Figure 1d] FIG. 1 is a simplified diagram of the average photon rate over time. [Figure 2a] FIG. 10 is a diagram showing a lookup table for determining a fluorescence lifetime. [Figure 2b] FIG. 10 shows a schematic look-up table for determining light intensity. [Figure 2c] FIG. 1 is a schematic diagram of pre-stored data for determining fluorescence lifetimes. [Figure 3a] 1 is a schematic diagram of the configuration of an apparatus according to the invention for measuring fluorescence lifetimes; [Figure 3b] 2 is a schematic diagram of another configuration of an apparatus according to the invention for measuring fluorescence lifetimes; FIG. [Figure 4] 1 is a schematic diagram of the scope of application of the method of the invention or the device of the invention; [Figure 5] 3 is a schematic diagram of another configuration of the method according to the invention; [Figure 6] FIG. 1 is a schematic diagram of gating.

[0062] The following figures illustrate, purely by way of example, the calculation of fluorescence lifetimes. This description is purely exemplary and can be transferred to the calculation of fluorescence parameters, or more generally, to the calculation of optical signal parameters. The following description is exemplary and does not limit the scope of protection. The optical signal may be generated, for example, by reflection, phosphorescence, fluorescence or two-photon processes.

[0063] Figure 1a shows a schematic diagram of an electrical signal 3 generated by a detector 1 (see the diagram at the top right of the graph) that is used to determine the counter value (see Figure 3). The detector may be configured, for example, as a PMT hybrid detector 1a or a silicon photomultiplier tube (SiPM for short) 1b.

[0064] The electrical signal 3 can represent, for example, the course of a voltage 5 or a current 7 with respect to time 9, and in the ideal case the measured dark current 11 is negligible.

[0065] When a fluorescence photon 13 with photon energy E=hv is incident on the detector 1, the detector 1 generates a fluorescence photon event 15. This is only possible if the assumed quantum efficiency is 1. In reality, the generation of a fluorescence photon event 15 occurs with some probability depending on the quantum efficiency. For clarity, only a few temporally separated events 15c and temporally overlapping events 15b are shown in Figure 1a.

[0066] While the time-separated events 15c do not affect each other, the time-overlapping events 15b shown in FIG. 1a give rise to voltage 5 or current 7 values ​​that are significantly larger (almost twice as large) than the voltage or current 7 values ​​of the time-separated events 15c.

[0067] Each fluorescence photon event 15 has a pulse shape 17 that includes a steep rising edge 17a and an exponentially falling edge 17b, and a pulse width or pulse duration 19. Note that the pulse shape 17 and pulse duration 19 correspond to the pulse response function 21 of the detector 1. Different detectors 1 have different pulse response functions 21. In other words, the fluorescence photon event 15 represents the change in voltage 5 or current 7 after a fluorescence photon 13 is incident on the detector 1.

[0068] In practical applications, other parameters should also be taken into account, such as the quantum efficiency or fill factor of the detector 1. For simplicity, we assume here that each incident fluorescence photon 13 always generates exactly one fluorescence photon event 15 in the detector 1.

[0069] In the area under the curve of the electrical signal 3, the 15 photons 13 incident on the detector 1 are shown by dashed lines in relation to time 9 within a given measurement period 23. These are the integrated signals Σ P The integrated signal Σ P is shown diagrammatically as being stored in memory unit 25.

[0070] 1a also shows diagrammatically two successive light pulses 27 that define a measurement period 23 in the illustrated embodiment. This clearly shows that most of the methods known from the prior art for measuring fluorescence lifetimes are inapplicable in this case, since more than one fluorescence photon 13 is detected between the two light pulses 27. Furthermore, in the prior art, a limitation to one photon per measurement period (TCSPC) is often imposed. The sequence 26 of light pulses 27 can be particularly periodic.

[0071] The threshold 29 used to count the fluorescence photon events 15 is shown in FIG. 1 a. As soon as the voltage 5 or current 7 exceeds this threshold 29 once and falls below this threshold 29 once, the counter value Σ E Counter value Σ increases by 1. E is also shown diagrammatically as being stored in memory unit 25. The counter value Σ E represents the number of fluorescence photon events 15a occurring in the electronic signal.

[0072] However, especially in the case of sufficiently large overlap, events 15b that overlap in time may occur such that multiple such events 15b exceed the counter value Σ EA sufficiently large overlap means that after the voltage 5 or current 7 exceeds the threshold 29, it has not yet fallen below the threshold 29 again before another fluorescence photon 13 is incident.

[0073] In the case of three temporally overlapping events 15b shown, the voltage 5 or current 7 falls below the threshold 29 only after the third temporally overlapping event 15b, and although there are three incident fluorescence photons 13, the counter value Σ E In the measurement period 23 shown, the counter value Σ E becomes 11.

[0074] Figures 1b and 1c each show the electrical signal 3 generated by the detector 1, where the pulses 27 used for excitation are simply indicated by the dotted lines. A sequence 26 of four light pulses 27 is shown in both figures.

[0075] In the illustrated simulation, an average photon rate 41 of 650 MHz is assumed. Thus, the measurement period 23 shown in Figures 1b and 1c includes a period of pulsed excitation of four periods 115. The duration 9 of the illustrated measurement period 23 is approximately 50 ns (thus, the laser system used for excitation has a pulse repetition frequency 113 of approximately 75 MHz).

[0076] In Figures 1b and 1c, a threshold 29 is plotted at a voltage 5 or current 7 (given in arbitrary units) value of 0.5. The threshold 29 is plotted with a dashed line.

[0077] Figures 1b and 1c differ only in the underlying fluorescence lifetime 33, which is 1.5 ns for Figure 1b and 5 ns for Figure 1c, representing a fluorescence parameter 30a or optical signal parameter 30.

[0078] As a result, in the case of short fluorescence lifetimes 33 (FIG. 1b), fluorescence photon events 15 (marked with arrows pointing to them as a whole, rather than individually) occur with high probability immediately after each light pulse 27. In contrast, in the case of fluorescence lifetimes 33 that lie within approximately the period 115 of the sequence 26 of light pulses 27 (FIG. 1c), the fluorescence photon events 15 are distributed over the period 115.

[0079] In both cases, temporally overlapping events 15b are observed, with greater overlap occurring in the case of FIG. 1b.

[0080] The effect of the fluorescence lifetime 33 will be explained in more detail based on FIGS. 2a to 2c.

[0081] FIG. 2 c shows a schematic diagram of pre-stored data 31 for determining fluorescence lifetime 33 .

[0082] Purely by way of example, five curves 35a to 35e of a family of curves 37 are shown in FIG. 2c, the curves 35 shown being used merely to illustrate the method, and in actual evaluations typically only pre-stored data sets are used.

[0083] Curves 35a-35e are the simulation results for SiPM 1b with a pulse duration 19 (electron pulse duration) of 2 ns. The integrated signal Σ for various fluorescence lifetimes 33 from 1 ns to 5 ns is P and the counter value Σ E Ratio values ​​39 have been calculated from and plotted against the average photon rate 41.

[0084] The average photon rate 41 is the integrated signal Σ P is divided by the measurement period 23, and a ratio value of 39 is simulated up to approximately 650 Mcts / s.

[0085] If the method according to the invention provides, for example, a measured average photon rate 41a of 500 Mcts / s and a measured ratio value 39a of 4 (both values ​​39a and 41a are shown in dashed lines), then a fluorescence lifetime 33 of 2 ns is determined by the method according to the invention or the device according to the invention 43 (see Figure 3).

[0086] Figure 1d shows a simplified diagram in which the photon rate 41, or more precisely the average photon rate 41, is shown over time 9. The photon rate 41 decays exponentially and is averaged over multiple fluorescence photons 13.

[0087] In the example shown in Figure 1d, several fluorescence photons 13 overlap each other, so the counter value (not shown) is now four, but based on the integrated signal (also not shown) six fluorescence photons 13 would be determined. The exponential decay of the photon rate 41 is inversely proportional to the fluorescence lifetime 33. That is, a short fluorescence lifetime 33 results in a steeper exponential decay of the average photon rate 41.

[0088] 2a and 2b each show a look-up table 117, or more precisely, data 31 that are previously stored in the look-up table 117 for determining a fluorescence lifetime 33. FIG.

[0089] Figure 2a shows five curves 35a-35e of the family of curves 37. These curves 35a-35e are only used to illustrate the method and are not shown in the method according to the invention, where a pre-stored data set is used.

[0090] Curves 35a-35e are the simulation results for SiPM 1b with a pulse duration 19 of 2 ns. In FIG. 2a, the counter value Σ E is the integrated signal Σ P Therefore, the counter value Σ E and the integrated signal Σ PUsing the set or pair of values ​​determined in the method according to the present invention for , the fluorescence lifetime 33 can be determined. Similar to the description for determining the fluorescence lifetime 33 in Fig. 2a, the fluorescence lifetime 33 can be determined from the diagram shown in Fig. 2c.

[0091] In Figure 2b, a look-up table 117 is shown, which will be referred to hereafter for short as LUT 117. The LUT 117 in Figure 2b is an intensity LUT 119. In Figure 2b, the corrected intensity 121 is shown over an average photon rate of 41.

[0092] The average photon rate 41 is expressed as the integrated signal Σ P and the measurement period 23.

[0093] Thus, the intensity LUT 119 can be used to verify and, if necessary, correct the intensity obtained by integration (i.e., the number of detected fluorescence photons).

[0094] The relationship between fluorescence lifetime 33 and ratio value 39 shown in Figures 1b and 1c is also shown in Figures 2a-2c. When fluorescence lifetime 33 is short, as in Figure 1b, the likelihood of single fluorescence photon events 15 superimposing increases, resulting in a smaller counter value Σ than when fluorescence lifetime 33 is relatively long, as in Figure 1c. E Therefore, for a relatively short fluorescence lifetime 33 with a constant average photon rate 41, a relatively small dividend and therefore a relatively large ratio value 39 can be obtained.

[0095] It is emphasized once again here that the curves 35a-35e shown are shown purely as examples for the sake of clarity, and that in the method according to the invention the data 31 may be assumed / simulated and stored with a finer gradation or with a smaller step size of the fluorescence lifetime 33. In particular, the determined fluorescence lifetime 33 (or more generally at least one optical signal parameter 30) may be interpolated from the pre-stored data 31.

[0096] In other words, the data 31 is the integrated signal Σ P , counter value Σ E and a reference value 32 of the fluorescence lifetime 33, which are preferably assigned to one another. In particular, the data 31 may be present as an at least two-dimensional data set 31a.

[0097] In addition to the measured values ​​"integrated signal" and "counted signal" (counter value), other signals can be generated that allow further refinement of the result signal. For example, the (average) measured pulse width, i.e., the time difference between the rising and falling edges, can be used in the comparator. In the case of a single pulse, this corresponds to the pulse width or pulse duration; in the case of overlapping pulses, as in Figure 1b, the measured pulse duration will be correspondingly longer. In such a case, the calibration data set would be three-dimensional: a result signal (e.g., fluorescence lifetime) would be assigned to the combination of the three input variables ("integrated signal" / "counted signal" / "average pulse duration").

[0098] Furthermore, Figures 2a to 2c show that the method according to the invention or the device according to the invention 43 ensures a higher distinguishability of the simulated curves 35, especially when the average photon rate 41 is relatively high, and can therefore preferably be used in this region.

[0099] In FIG. 3 a a schematic diagram of an apparatus 43 according to the invention for measuring fluorescence lifetime 33 is shown.

[0100] A light source 45, in particular a pulsed laser light source 45a, emits excitation light 47 in the form of a sequence 26 of light pulses 27 that are incident on a sample 49. Here, fluorescence photons 13 (only one is shown) are generated that are incident on a detector 1. Other optical elements suitable for collecting the fluorescence photons 13 are not shown, but may generally be used in other configurations of the device 43.

[0101] An electrical signal 3 is output at the detector output 51 and is fed to a preamplifier 53 where it is amplified.

[0102] An amplified electrical signal 3a is applied to the amplifier output 55, which is split and fed in the form of two signal replicas 3b to a counting path 57 and an integrating path 59. In other configurations, particularly those involving SiPMs 1b, this splitting may already take place on the detector chip.

[0103] The counting path 57 outputs the counter value Σ E The counter includes a counting module 61 that outputs the counter value Σ E represents the number of fluorescence photon events15.

[0104] The integration path 59 includes an integration module 71 that determines the number 13 a of all fluorescence photons 13 incident on the detector 1 during the measurement period 23 .

[0105] The integrated signal Σ P is output at the integrator output 75 and fed to a logic unit 77. Based on the integration in the integration module 71, the integrated signal Σ P represents the integral value 79, which represents the accumulated number of photons ΣN. Furthermore, the counter value Σ E represents the number 15a of all fluorescence photon events 15 detected in the electrical signal 3 during the measurement period 23, i.e., all time-separated events 15c and all time-overlapping events 15b.

[0106] In the configuration of the device 43 shown in FIG. 3a, the integrated signal Σ P and the counter value Σ E is transferred to the logic unit 77. Figure 3a shows a preferred configuration of the device according to the invention.

[0107] The logic unit 77 in the configuration shown in Figure 3a includes three memory modules 85 shown schematically connected to each other to form a look-up table module 87. The integrated signal ΣP , counter value Σ E Reference values ​​32 of and fluorescence lifetime 33 are stored in these memory modules 85 and are marked with the subscript "sim" to distinguish between measured values ​​and these simulated values.

[0108] In the method according to the invention in the configuration shown in FIG. 3a, the logic unit 77 in turn calculates the measured integrated signal Σ P and the measured counter value Σ E , the simulated integrated signal Σ P,sim and the simulated counter value Σ E,sim , compared with the reference value 32 stored in the look-up table module 87, resulting in a simulated fluorescence lifetime 33 sim to provide.

[0109] The logic unit 77 further includes an interpolation module 89 that allows for interpolation between a limited number of stored reference values ​​32 .

[0110] The logic unit 77 outputs the intensity result 95 provided by the intensity module 93 at the intensity output 91. The fluorescence lifetime 33 determined in this way det is output via the life output side 97.

[0111] FIG. 3a also shows a second configuration of logic units 77a, which can be used to calculate, for example, the integrated signal Σ P and the counter value Σ E Fluorescence lifetime 33 det and additionally includes a calculation unit 107 capable of determining the intensity result 95.

[0112] The configuration of the device according to the invention shown in FIG. 3b is such that the counter value Σ E 3a in that is supplied to the divider input 67 of the division module 69.

[0113] The integrated signal provided at the integrator output 75 is fed via the dividend input 65 to the division module 69 and, as in the configuration of FIG. 3a, to a logic unit 77.

[0114] In the configuration of device 43 shown in FIG. 3 b , division module 69 calculates a ratio value 39 which is transferred from division module 69 to logic unit 77 via ratio value output 83 .

[0115] The logic unit 77 of the device of Figure 3b includes three memory modules 85 shown schematically connected to each other, which form a look-up table module 87. The integrated signal Σ P , ratio values ​​39 and fluorescence lifetime 33 reference values ​​32 are stored in these memory modules 85 and are marked with the subscript "sim" to distinguish between measured values ​​and these simulated values.

[0116] The logic unit 77 of the device according to the invention of FIG. 3b now converts the measured integrated signal Σ P and the integrated signal Σ P and the counter value Σ E The ratio value 39 calculated from the simulated integrated signal Σ P,sim and simulated ratio values ​​39 sim The resulting simulated fluorescence lifetime 33 is compared with the reference value 32 stored in the look-up table module 87. sim to provide.

[0117] The configuration shown in Fig. 3b may also include an interpolation module 89, an intensity module 93 and a calculation unit 107. The counter value Σ as in Fig. 3a E Instead, in FIG. 3 b the ratio value 39 is input to the calculation unit 107 .

[0118] The device 43 of Figures 3a and 3b may be arranged, as shown diagrammatically, in a microscope 99, in particular a (confocal) scanning microscope 99a, particularly preferably a fluorescence lifetime microscope 99b (FLIM), in which the sample 49 is scanned or scanned and an image 101 of the sample 49 is generated. Here, the determined fluorescence lifetimes 33 of regions 103a, 103b spatially spaced from one another are measured. det is shown with the appropriate color or brightness distribution.

[0119] The microscope 99 may further be connected to a computer 109 that can read a non-volatile computer-readable storage medium 111, on which a program for implementing the method according to the invention may be stored. The storage medium 111 may be an optical, magnetic or flash memory based storage medium 111.

[0120] FIG. 4 shows a schematic diagram of the application area 105 of the method according to the invention or of the device 43 according to the invention and of a representative method of the prior art, the so-called time-correlated single photon counting (TCSPC).

[0121] TCSPC is usable up to an average photon rate of about 40 Mcts / s, but at higher photon rates it no longer provides reliable results for the fluorescence lifetime.

[0122] In contrast, the application area 105 of the method according to the invention and the device according to the invention 43 lies in much higher average photon rates 41, preferably in the order of magnitude of 100 to more than 1000 Mcts / s or more.

[0123] Figure 5 shows a flow chart 123 of an alternative configuration of an apparatus 43 according to the present invention, which can be used instead of the schematic structure shown in Figures 3a and 3b to determine the fluorescence lifetime 33.

[0124] 5 also includes a detector 1, a preamplifier 53 to which the electrical signal 3 is fed. The two signal replicas 3b are fed to an integrating module 71 or a counting module 61. However, in the configuration shown in FIG. 5, the counter value Σ E and the integrated signal Σ P are fed to two different look-up table modules 87. The lifetime module 125 includes a schematically illustrated lifetime LUT 127, the data of which is shown in Fig. 2a purely by way of example. Furthermore, an intensity module 129 is provided in which the intensity LUT 119 is present. The lifetime module 125 determines the fluorescence lifetime 33 by the method according to the invention, and in return the intensity module 129 outputs an intensity result 95. In other configurations not shown, at least one further optical signal parameter may be determined.

[0125] Optionally, in the structure according to FIG. 3a or 3b, a gating module 131 may further be provided, which is configured to receive the electrical signal 3, the signal replica 3b, as well as the counter value Σ E and the integrated signal Σ P are also considered only during certain time intervals.

[0126] The functioning of the gating module 131 is shown diagrammatically in Figure 6, where the counter value Σ E is shown over time 9. In the method of the present invention, configured with gating module 131, only events occurring in gating period 134 between gating start 133 and gating end 135 are observed and considered. Thus, unwanted reflections that may occur, for example, before gating start 133, can remain ignored and do not falsify the measurement results. [Explanation of symbols]

[0127] 1. Detector 1a PMT hybrid detector 1b Silicon photomultiplier (SiPM) 3 Electrical Signals 3b Signal replica 5. Voltage 7 Current 9 hours 11 Dark current 13 Fluorescence Photons 13a Number of fluorescence photons incident on the detector 15 Fluorescence Photon Events 15a Number of fluorescence photon events occurring in the electrical signal 15b Temporally overlapping events 15c Temporally separated events 17 Pulse Shape 17a Rising Edge 17b Falling Edge 19 Pulse Duration 21 Pulse response function 23 Measurement Period 25 Memory Unit 26 Sequence 27 Light Pulse 29 Threshold 30 Optical Signal Parameters 30a Fluorescence parameters 31 Data 31a Dataset 32 Reference Value 33 Fluorescence Lifetime 33 det Calculated fluorescence lifetime 33 sim Simulated fluorescence lifetime 35 curve 35a~35e 1st curve~5th curve 39 Ratio Value 39a Measured ratio value 39 sim Simulated Ratio Values 41 Average photon rate 41a Measured average photon rate 43 Equipment 45 Light source 45a Pulsed laser light source 47 Excitation Light 49 samples 51 Detector output side 53 Preamplifier 55 Amplifier output side 57 Counting Route 59 Integral Path 61 Counting Module 63 Counter output side 65 Dividend input side 67 Divider input side 69 Division Module 71 Integral Module 75 Integrator output side 77 logical units 77a Second configuration of logical units 79 integral value 83 Ratio value output side 85 memory modules 87 Lookup Table Module 89 Interpolation Module 91 Intensity output side 93 Strength Module 95 Strength Results 97 Life output side 99 Microscope 99a Scanning Microscope 99b Fluorescence Lifetime Microscopy (FLIM) 101 images 103a, 103b Areas spatially spaced apart from each other 105 Application area 107 Computational Units 109 Computer 111 Storage medium 113 Pulse Repetition Frequency 115 cycles 117 Lookup Table / LUT 119 Intensity LUT 121 corrected intensity 123 Flowchart 125 Life Module 127 Life LUT 129 Strength Module 131 Gating Module 133 Gating Start 134 Gating Period 135 Gating End E photon energy ΣN accumulated photon number Σ E Counter Value Σ P Integrated Signal Σ E,sim Simulated Counter Values Σ P,sim Simulated integrated signal

Claims

1. A method for measuring at least one optical signal parameter (30) using a device (43) disposed on or connected to a microscope (99), said method comprising: - illuminating the sample (49) for a predetermined measurement period (23) for excitation of an optical signal in said sample (49); - detecting the optical signal emitted from the sample (49) and providing an electrical signal (3) representative of the change in the optical signal over time; counting single photon events (15) based on the electrical signal (3) over the measurement period (23) and determining a counter value (Σ) representing the number (15a) of photon events (15); Ｅ ) providing a Integrating said electrical signal (3) over said measurement period (23) and obtaining an integrated signal (Σ Ｐ ) providing a At least the counter value (Σ Ｅ ) and the integrated signal (Σ Ｐ determining at least one optical signal parameter (30) based on the A method including:

2. illuminating the sample (49) over the measurement period (23) using a sequence of light pulses (27) modulated over time; The method of claim 1.

3. the optical signal parameter (30) is the fluorescence lifetime (33) or intensity (34) of the optical signal; 3. The method according to claim 1 or 2.

4. The integration and the counting are performed in parallel.

4. The method according to any one of claims 1 to 3.

5. The method further comprises: Ｅ ) and the integrated signal (Σ Ｐ ) calculating the optical signal parameters (30); 5. The method according to any one of claims 1 to 4.

6. The method uses pre-stored data (31) to calculate the counter value (Σ Ｅ ) and the integrated signal (Σ Ｐ ) determining the optical signal parameters (30) in relation to the 6. The method according to any one of claims 1 to 5.

7. The method further comprises determining and / or calibrating the pulse shape (17) and / or pulse duration (19) of the single photon event (15).

7. The method according to any one of claims 1 to 6.

8. determining the optical signal parameters (30) taking into account the pulse shape (17) and / or the pulse duration (19) of the single photon event (15); The method of claim 7.

9. The method further includes sequentially scanning or scanning the sample (49) and generating images (101) of optical signal parameters (30) of spatially spaced regions (103 a, 103 b) of the sample (49).

9. The method according to any one of claims 1 to 8.

10. A device (43) arranged on or connected to a microscope (99) for measuring an optical signal parameter (30), comprising: The device (43) includes a detector (1), an integration module (71), a counting module (61), and a logic unit (77); The detector (1) generates an electrical signal (3) representative of the sequence (26) of incident photons (13) and outputs it at the detector output (51); The integration module (71) integrates the electrical signal (3) over a measurement period (23), and the integration module (71) outputs a resulting integrated signal (Σ Ｐ ) and The counting module (61) counts the number (15a) of photon events (15) detected during the measurement period (23) based on the electrical signal (3) over the measurement period (23), and generates a counter value (Σ Ｅ ) and The logic unit (77) calculates the integrated signal (Σ Ｐ ) and the counter value (Σ Ｅ determining at least one optical signal parameter (30) in relation to the Device (43).

11. The device (43) calculates the integrated signal (Σ Ｐ ) and the counter value (Σ Ｅ a calculation unit (107) for calculating the optical signal parameters (30) using the 11. The device (43) of claim 10.

12. The device (43) calculates the integrated signal (Σ Ｐ ), the counter value (Σ Ｅ ) and at least one memory module (85) for storing a data set (31 a) of mutually assigned reference values ​​(32) of said optical signal parameters (30), 12. Apparatus (43) according to claim 10 or 11.

13. A non-volatile computer readable storage medium (111) containing a program with instructions which, when executed by a computer, causes the computer to perform the method of any one of claims 1 to 9.

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