JP2022541036A - Method and apparatus for measuring optical signal parameters and non-volatile storage media - Google Patents

Abstract

The present invention relates to a method and apparatus (43) for measuring at least one optical signal parameter (30) and a non-volatile computer readable storage medium (111). The known method has the disadvantage that as soon as the single photon (13) signal, ie the fluorescence photon event (15) is superimposed, the method becomes unusable. In order to improve the known method, the method according to the invention comprises the following steps: For excitation of the light signal, in particular fluorescence, in the sample (49), the sample ( 49); detecting the optical signal emitted from the sample (49) and providing an electrical signal (3) representing the change in the optical signal over time; counting single photon events (15) based on the signal (3) to provide a counter value (ΣE) representing the number (15a) of photon events (15); the electrical signal ( 3) to provide an integrated signal (ΣP) and determining at least one optical signal parameter (30) based at least on the counter value (ΣE) and the integrated signal (ΣP) step to do. The device (43) according to the invention solves the above-mentioned problems at least by The solution is to include a logic unit (77) that determines one optical signal parameter (30).

Description

The present invention relates to methods and apparatus and non-volatile computer-readable storage media for measuring at least one optical signal parameter.

Prior art methods use light sources to excite optical signals such as reflected, phosphorescent, second harmonic or fluorescent signals. Purely by way of example, in the case of fluorescence, the time between the switch-on of the light source or 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). A drawback of this method is that it is limited to a photon rate at which substantially one photon is generated per laser pulse. Measurements up to photon rates of about 40 Mcts (megacounts; 10 ⁶ events) per second can be obtained with more complex and expensive instruments that include parallel evaluation electronics.

Other prior art methods scan the light source and the generated photon trigger signal very rapidly, ie at frequencies above 10 GHz, so that optical signal parameters can be determined from the data stream.

Both methods have in common that they can only be used to a limited extent as soon as single-photon signals, ie fluorescence photon events, overlap.

The object of the present invention is therefore to provide a method for measuring optical signal parameters that is structurally simple, inexpensive and allows optical signal parameters to be measured at high photon rates (>40 Mcts/s). It is to implement a method and apparatus.

The method according to the invention solves this problem by comprising the following steps.
Illuminating the sample over a predetermined measurement period for the excitation of a light signal, in particular fluorescence, in the sample Detecting the light signal emitted by the sample and electrical Counting single photon events based on the electrical signal over a step-measurement period that provides a signal; Integrating the electrical signal over a step-measurement period that provides a counter value representing the number of photon events; and determining at least one optical signal parameter based on at least the counter value and the integrated signal

The device according to the invention may in particular be a microscope, which solves the above-mentioned problem by comprising a detector, an integrating module, a counting module and a logic unit. The detector generates an electrical signal representing the sequence of incident photons and outputs at the detector output, and the integration module integrates the electrical signal over the measurement period, where the integration module results therefrom. The counting module is configured to output an integrated value (also referred to as an integrated value), the counting module counting 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 integral value and the counter value.

Furthermore, a non-volatile computer-readable storage medium according to the invention contains a program comprising instructions which, when executed by a computer, cause the computer to perform the method according to the invention.

The method according to the invention and the device according to the invention thus have the advantage that switching on of the light source, ie for example a complex time measurement between the excitation laser pulse and the detected photon event, is not required. The method according to the invention utilizes the temporal superposition of two or more photon events that are no longer distinguishable to measure optical signal parameters. That is, it is based on the fact that the counter value is less than the integral value due to the superposition of multiple photon events. Rather, the accuracy of the detected fluorescence lifetime improves with photon rate. In particular, the average photon rate, ie the average photons generated per unit time, is observed.

The method according to the invention and the device according to the invention can be further improved by other configurations which are advantageous in themselves. Technical features of individual configurations may be arbitrarily combined with each other or omitted, provided that the technical effect obtained by the omitted technical features is not significant.

This method can in particular be carried out using a microscope. A corresponding device can therefore be, in particular, a microscope.

Counting of single photon events can be performed preferably using a digitizer, more preferably using a comparator. It can therefore be provided in a corresponding device.

The illumination of the sample over the measurement period may be modulated over time, in particular using periodic light pulses. Pulsed lasers, short-pulsed lasers or ultra-short-pulsed lasers are particularly suitable light sources here. Pulsed diode lasers may be particularly preferably used because they are smaller (eg compared to solid-state lasers) and can be easily controlled. Pulsed fiber lasers are also available.

A lighting unit containing such a pulsed light source may be provided within the device or provided externally. Any duration can be selected as the measurement period, preferably a measurement period comprising multiple periods of the light pulse. This has the advantage that the trigger can be easily tapped from the pulsed light source.

Preferably, a detector capable of detecting a single incident photon and generating a photon event for each incident photon in the output electrical signal of the detector is used as the detector or photon detector. be done. In the following, reference will only be made to fluorescence photons that generate fluorescence photon events in the detector, in particular the output electrical signal of the detector. Although the description is based on fluorescence photons and corresponding fluorescence parameters, the invention is not limited thereto. These descriptions can also be transferred in other ways, for example to photons generated by reflection, phosphorescence or two-photon processes and corresponding optical signal parameters.

Special electron tubes, also called photomultiplier tubes or photomultiplier tubes (in English photomultiplier tubes, PMTs) can be used. These may be pure semiconductor detectors, PMT hybrid detectors or SiPMs (silicon photomultiplier tubes).

The detector detects light, eg, fluorescent light, emitted from the sample and produces an electrical signal representative of the sequence or variation of incident photons, eg, fluorescent photons. The electrical signal is output at the detector output and may include multiple photon events, eg fluorescence photon events, which may be temporally separated from one another or may be superimposed on one another.

An integration module is then used to determine from the signal all photons, for example fluorescence photons, detected at the detector during the measurement period and to provide a signal proportional to this number. This signal can represent relative photon values.

An electrical signal can be divided into a photon-counting path, where the electrical signal is integrated, and an event-counting path, where single photon events of 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 the measurement period is determined and in the form of a counter value representing this number. provided in

Furthermore, in the logic unit a light signal parameter, preferably a fluorescence parameter, particularly preferably a fluorescence lifetime, can be determined on the basis of the integrated signal and the counter value. The logic units may be configured in the form of single logic modules or chips or as so-called Field Programmable Gate Arrays (FPGA). The FGPA is an integrated circuit (IC) on which logic circuits can be loaded (in the field, at the customer) and executed on it.

A non-volatile computer readable storage medium according to the invention contains, for example, a program comprising instructions for executing the method according to the invention on a computer, which is read from the medium and loaded into the above mentioned FPGA or computer memory. good.

A method according to the invention comprises counting single photon events, e.g. single fluorescence photon events of the electrical signal over a measurement period and providing a counter value representing the number of photon events, e.g. single fluorescence photon events. may further include To this end, an incremental encoder may be provided in the device that outputs a counter value, which may be present 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, ie at the start of the next measurement period.

A method according to the invention includes integrating an electrical signal over a measurement period to provide an integrated signal representative of the photon count of all detected fluorescence photons accumulated during the measurement period.

Many photon detectors produce an electrical pulse for each photon detected, the level of which is not related to the photon's energy. Photons of different wavelengths cannot be distinguished based on the signal, but because pulses of photons incident at the same time add in the signal, for example, the integrated signal of two photons incident at the same time is the integral of a single photon twice the signal applied.

An integrated signal can be obtained using an integrator or integration module. The integrator or integration module no longer distinguishes single photon events of the electrical signal, eg single fluorescence photon events, with a sufficiently large time constant and integrates them over the measurement period.

In other words, in the counting module the electrical signal is compared with a threshold value and the reading of the counter is incremented by one if the threshold value is exceeded and subsequently falls below the threshold value. Thus, simultaneously incident photons, e.g. fluorescence photons, give rise to temporally overlapping photon events, e.g. Increment the counter by one.

In contrast, the integration path evaluates quantitative variables such as charge. Thus, two fluorescence photons incident at the same time will produce a signal twice as high in the integration path as fluorescence photons incident individually.

Therefore, the integrated value (ie the integrated signal) determined within the measurement period represents the accumulated photon count of all detected photons, especially detected fluorescence photons.

In an advantageous configuration of the method according to the invention, integration and counting can take place in parallel. Furthermore, integration and counting are preferably additionally performed simultaneously.

Additionally, the method may include calculating a ratio value from the number of single fluorescence photon events, ie from the counter value and the integrated signal. In all configurations of the method or apparatus of the present disclosure, the three variables, namely the counter value, the integrated signal and the ratio value, are combined with any , for example in two pairs. Thus, (1) counter value and integrated signal, (2) counter value and ratio value, or (3) integrated signal and ratio value can lead to optical signal parameters. Accordingly, descriptions of this disclosure relating to one of combinations (1), (2) or (3), or other combinations of variables for determining optical signal parameters, unless expressly excluded in the text: Any other combination of these three variables is transferable and is not limited to each combination listed.

Calibration data for determining optical signal parameters, preferably fluorescence parameters such as fluorescence lifetimes, may be stored in tables (LUTs: look-up tables) or formulas. For example, for each value pair of integrated signal and counted signal (counter value) the attributed fluorescence lifetime may be stored, thus purely by way of example if there are two measurements may directly represent the fluorescence lifetime (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, optical signal parameters such as fluorescence lifetimes can be interpolated from the existing grid points.

Furthermore, purely by way of example, there may be signal pairs for which optical signal parameters such as fluorescence lifetime cannot be determined. This is the case, for example, if the signal is very small.

At least one further optical signal parameter may be determined in another configuration using the method according to the invention and the device according to the invention. For example, when fluorescence parameters are determined, further signals may be generated in addition to fluorescence lifetime. As an example, an improved intensity signal can be generated from a combination of two signals (integrated signal/counted signal). The structure and data path cannot be changed, only the calculation rule/calibration LUT can be adapted to the calculation of the intensity signal. Such an arrangement provides redundancy for determining the strength of the electrical signal, e.g. the integrated signal determined by integration can be checked for its correctness and the It has the advantage that deviations or errors are identifiable.

Accordingly, the apparatus for carrying out this aspect of the method comprises an integration module that integrates the electrical signal during a measurement period and outputs an integrated value, a counting module that counts photon events and outputs a counter value, and optionally may include a division module; A division module is provided for calculating a ratio value from the counter value of the counting module and the integrated signal of the integration module and for outputting the ratio value, in which case the logic unit is for example the fluorescence lifetime. It may be configured to determine the optical signal parameter in relation to the integrated signal and the counter value, or the integrated signal (or alternatively the counter value) and also the ratio value.

Therefore, the method can be further improved in that it includes determining an optical signal parameter, preferably a fluorescence parameter, such as fluorescence lifetime, based on the integrated electrical signal and the counter value. Optionally, ratio values may be used in combination with counter values or integrated signals to determine optical signal parameters. In other words, an optical signal parameter, preferably a fluorescence parameter, eg fluorescence lifetime, may be determined from two variables respectively in different configurations. These variables may be, for example, an integrated signal and a counter value, a counter value and a ratio value, or an integrated signal and a ratio value.

Determination of the optical signal parameter by 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, excluding other combinations of variables for determining the optical signal parameter. is not.

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

A device according to the invention for carrying out this aspect of the method may therefore comprise a calculation unit for calculating an optical signal parameter, preferably a fluorescence parameter, eg a fluorescence lifetime, using the integrated signal and the counter value. .

Similarly, in a special configuration, the computation unit uses pre-stored analytical computation rules (analytic curves) to compute optical signal parameters, preferably fluorescence parameters, e.g. fluorescence lifetimes, counter values (or Alternatively, an integrated signal) and a ratio value (the ratio of the integrated signal and the counter value) can be used.

Since the calculation unit itself can contain a division module, all necessary calculation steps can be combined in one unit.

Alternatively or additionally, in another configuration of the method according to the invention, further, using pre-stored data, optical signal parameters, in particular Determining a fluorescence parameter, such as fluorescence lifetime, may be provided.

Therefore, the device according to the invention for carrying out this aspect of the method stores data sets of mutually assigned reference values of integrated signals, counter values and optical signal parameters, in particular fluorescence parameters, e.g. fluorescence lifetimes. may include at least one memory module for

The pre-stored data may exist in the form of two-dimensional or three-dimensional data sets. This data set or this data matrix contains integrated electrical signals (i.e. integral values) and counter values (optionally counter values and ratio values, or alternatively integrated signals and ratio values). Well, a particular combination of these values can be assigned an optical signal parameter, preferably a fluorescence parameter, eg a fluorescence lifetime.

In other words, the method according to the invention makes it possible to assume a set of measurement curves representing the integrated signal or the ratio value in relation to the counter value (or also the ratio value in the case of an integrated signal). . In this case, the group of parameters are optical signal parameters, in particular fluorescence parameters, eg fluorescence lifetime. By knowing or calculating the counter value and the integrated signal (or ratio value), a family of curve representatives can be determined and the light signal parameters, in particular fluorescence parameters, e.g. Life can be read.

In another configuration of the invention, the method may comprise interpolation of pre-stored data, in which case, based on this interpolated data, optical signal parameters, in particular fluorescence parameters, e.g. Fluorescence lifetime is determined. This has the advantage that pre-stored data, such as integrated signals and counter values, are stored in fixed stepping. Even if the detected integrated signal and/or detected counter value lies between two pre-stored integrated signals and/or counter values with respect to its own counter value, the optical signal Determination of parameters, particularly fluorescence parameters, such as fluorescence lifetime, can be performed.

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

Analytical curves that can form the basis of a series of provided measurement curves for directly calculating optical signal parameters, in particular fluorescence parameters, e.g. fluorescence lifetimes, from counter values and integrated signals (or ratio values), as described above. can be used.

Here, another configuration of the method according to the invention may provide for determining and/or calibrating the pulse shape and/or pulse duration of a single photon event, for example a single fluorescence photon event. .

Due to the stochastic nature of photons, preferably fluorescence photons in the case of fluorescence, in counting photon events, e.g. Fluorescence photon events may overlap, resulting in uncounted events. The probability of uncounted events is related to:
1. Pulse frequency of commonly known excitation illumination2. average photon rate, which can be calculated using the integrated signal and pulse frequency;3. 4. Pulse shape and pulse duration for single photon events, eg fluorescence photon events. 5. optical signal parameters, in particular fluorescence parameters, eg fluorescence lifetimes of excited fluorophores; Detector gating

In particular, 3. from the list above. can be determined with 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. This makes it possible to obtain the optical signal parameters, in particular the fluorescence parameters, e.g. Become.

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 certain time range of the counting path (i.e. during integration) in order to suppress the reflected excitation light that precedes the optical signal, e.g. the fluorescence pulse. do.

The method according to the invention is improved by determining an optical signal parameter, in particular a fluorescence parameter, e.g. a fluorescence lifetime, taking into account the pulse shape and/or pulse duration of a single photon event, in particular a single fluorescence photon event. can be

In particular, the method according to the invention may further comprise the sequential scanning or scanning of the sample and the generation of images of optical signal parameters, in particular fluorescence parameters, e.g. fluorescence lifetimes, of mutually spatially spaced regions of the sample. .

A special case of this configuration of the method according to the invention thus represents an easy-to-implement technique for examining samples using fluorescence lifetime imaging microscopy (FLIM in English). A corresponding device may be a fluorescence lifetime microscope (FLIM).

In this arrangement, a scanning device or scanning device may be provided for moving the excitation light across the sample in a scanning motion or scanning motion. It is also possible to move the sample relative to illumination and detection. Thus, point by point, ie pixel by pixel, a light signal parameter, in particular a fluorescence parameter, eg a fluorescence lifetime, of each illuminated area of the sample can be determined and represented as image information.

In particular, such a method configuration can be used in scanning microscopes, in particular confocal scanning microscopes.

The method according to the invention may in particular be based on the saturation behavior that occurs in the counting module (which determines the counter value). This is caused by the superposition of counted photon events, eg counted fluorescence photon events. This effect of superposition can be related in particular to optical signal parameters, in particular fluorescence parameters, eg fluorescence lifetime, since the average photon rate varies greatly in the course of the pulse. This effect becomes particularly pronounced when the width of the counted photon event, e.g. the counted fluorescence photon event, is of the same order of magnitude as the optical signal parameter, especially the fluorescence parameter, e.g. the fluorescence lifetime, of the observed dye. obtain.

For the detector types mentioned above, the pulse duration (the duration or width of a photon event, eg a fluorescence photon event counted) is typically in the range of 1 ns to 2 ns. Typical fluorescence lifetimes of dyes are generally 1 ns to 5 ns. In another configuration of the method according to the invention, the electronic pulse duration can be adapted by suitable filtering or by threshold matching. This makes it possible to obtain a particularly advantageous fluorescence lifetime contrast.

A non-volatile computer-readable storage medium according to the invention in particular contains a program comprising instructions which, when executed by a computer, cause the computer to perform a method according to one of the arrangements described above. A data carrier based on any type of optical memory, magnetic memory or flash memory is to be understood as storage medium.

In the following, the method according to the invention and the device according to the invention are explained in detail on the basis of exemplary, non-limiting figures. Here, individual technical features can be combined with one another and/or left out arbitrarily according to 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 symbols.

Fig. 2 is a schematic diagram of the electrical signal produced by the detector and the calculation of the counter value; FIG. 13A is a simulation of pulsed excitation with four pulses under an assumed fluorescence lifetime of 1.5 ns. FIG. 1b is a simulation of FIG. 1b with an assumed fluorescence lifetime of 5 ns; FIG. 4 is a simplified diagram of the average photon rate over time; FIG. 4 is a diagram showing a lookup table for obtaining fluorescence lifetime; FIG. 11 shows a schematic lookup table for determining light intensity; Schematic representation of pre-stored data for determining fluorescence lifetime. 1 is a schematic diagram of the set-up of an apparatus according to the invention for measuring fluorescence lifetime; FIG. Fig. 3 is a schematic diagram of another configuration of an apparatus according to the invention for measuring fluorescence lifetime; 1 is a schematic illustration of the scope of application of the method of the invention or of the device of the invention; FIG. Fig. 4 is a schematic diagram of another configuration of the method according to the invention; FIG. 3 is a schematic diagram of gating;

The figures described below show the calculation of the fluorescence lifetime purely by way of example. 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 not intended to limit the scope of protection. Optical signals may be generated by, for example, reflection, phosphorescence, fluorescence or two-photon processes.

FIG. 1a shows a schematic diagram of the electrical signal 3 generated by the detector 1 (see diagram on the top right of the graph) used to determine the counter value (see FIG. 3). The detector can be configured, for example, as a PMT hybrid detector 1a or as a silicon photomultiplier tube 1b (in English Silicon photomultiplier, abbreviated SiPM).

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

Detector 1 generates a fluorescence photon event 15 when a fluorescence photon 13 with photon energy E=hv is incident on detector 1 . This is only for the hypothetical quantum efficiency of unity. In practice, the generation of fluorescence photon events 15 occurs with some probability depending on the quantum efficiency. For clarity, only some temporally separated events 15c and temporally overlapping events 15b are shown in FIG. 1a.

While the temporally separated events 15c do not affect each other, the temporally overlapping events 15b shown in FIG. This produces a large (almost doubled) voltage 5 or current 7 value.

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

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

In the lower region of the curve of the electrical signal 3, 15 photons 13 incident on the detector 1 are indicated by dashed lines in relation to the times 9 within the predetermined measurement period 23. FIG. These may be summed into an integrated signal Σ _P representing the number of photons 13 . The integrated signal Σ _P is shown schematically as being stored in memory unit 25 .

Furthermore, FIG. 1a schematically shows two consecutive light pulses 27 which define the measurement period 23 in the illustrated embodiment. This is because more than one fluorescence photon 13 is detected between two light pulses 27, so most of the methods known from the prior art for measuring fluorescence lifetime apply in this case. It clearly shows that it cannot be done. Furthermore, in the prior art, a limitation to one photon per measurement period is often used (TCSPC). The sequence 26 of light pulses 27 can be particularly periodic.

A threshold 29 used for counting fluorescence photon events 15 is shown in FIG. 1a. As soon as the voltage 5 or the current 7 exceeds this threshold 29 once and falls below this threshold 29 once, the counter value _ΣE is incremented by one. The counter value Σ _E is also shown schematically as being stored in memory unit 25 . The counter value _ΣE represents the number 15a of fluorescence photon events occurring in the electronic signal.

However, in particular for sufficiently large overlaps, events 15b that overlap in time cause a plurality of such events 15b to increment the counter value _ΣE by one. A sufficiently large superposition means that after the voltage 5 or current 7 has exceeded the threshold 29 and before another fluorescence photon 13 is incident, the threshold 29 has not yet fallen below the threshold 29 again.

In the shown case of three temporally overlapping events 15b, the voltage 5 or the current 7 falls below the threshold 29 only after the third temporally overlapping event 15b and the incident fluorescence photons 13 are 3, but increments the counter value _ΣE by one. During the measurement period 23 shown, the counter value _ΣE is 11.

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 respective dashed lines. A sequence 26 of a total of four light pulses 27 is shown in both figures.

The simulations shown assume an average photon rate 41 of 650 MHz. Thus, the measurement period 23 shown in FIGS. 1b and 1c comprises four periods 115 of pulsed excitation. Time 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).

In FIGS. 1b and 1c, the threshold value 29 is plotted under a value of voltage 5 or current 7 (shown in arbitrary units) of 0.5. The threshold value 29 is entered with a dash-dotted line.

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. The fluorescence lifetime represents the fluorescence parameter 30a or the optical signal parameter 30. FIG.

As a result, in the case of short fluorescence lifetime 33 (Fig. 1b), fluorescence photon events 15 (they are not marked individually, but marked with arrows pointing to them as a whole) have a high probability of , occurs immediately after each light pulse 27 . In contrast, in the case of fluorescence lifetimes 33 ( FIG. 1 c ) lying approximately within period 115 of sequence 26 of light pulses 27 , fluorescence photon events 15 are distributed over period 115 .

In both cases temporally overlapping events 15b are observed, with a larger overlap occurring in the case of FIG. 1b.

The effect of fluorescence lifetime 33 is explained in more detail on the basis of FIGS. 2a-2c.

FIG. 2 c shows a schematic representation of pre-stored data 31 for determining fluorescence lifetime 33 .

Purely by way of example, five curves 35a-35e of a family of curves 37 are shown in FIG. In practice, generally only pre-stored datasets are used.

Curves 35a-35e are simulation results for SiPM1b with a pulse duration 19 (electron pulse duration) of 2 ns. Ratio values 39 have been calculated from the integrated signal Σ _P and the counter value Σ _E for various fluorescence lifetimes 33 from 1 ns to 5 ns and plotted against the mean photon rate 41 .

The average photon rate 41 is the result of dividing the integrated signal Σ _P by the measurement period 23 and the ratio value 39 is simulated up to about 650 Mcts/s.

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), the or a device 43 according to the invention (see FIG. 3) a fluorescence lifetime 33 of 2 ns is determined.

FIG. 1d shows a simplified diagram in which the photon rate 41, or more precisely the average photon rate 41, is shown over time 9. FIG. The photon rate 41 falls off exponentially and is averaged over multiple fluorescence photons 13 .

In the example shown in FIG. 1d, several fluorescence photons 13 are superimposed on each other, so that the counter value (not shown) is now four, but the integrated signal (also shown (not shown) would have required six fluorescence photons 13 . The exponential decay of photon rate 41 is inversely proportional to fluorescence lifetime 33 . That is, a short fluorescence lifetime 33 results in a steep exponential drop in mean photon rate 41 .

FIGS. 2 a and 2 b each show the lookup table 117 , more precisely the data 31 previously stored in the lookup table 117 for determining the fluorescence lifetime 33 .

FIG. 2a shows five curves 35a-35e of the family of curves 37. FIG. These curves 35a-35e are only used to explain the method and are not shown in the method according to the invention, where pre-stored data sets are used.

Curves 35a-35e are simulation results for SiPM1b having a pulse duration 19 of 2 ns. In FIG. 2a the counter value _ΣE is shown over the integrated signal _ΣP . The set or pair of values determined in the method according to the invention for the counter value Σ _E and the integrated signal Σ _P can therefore be used to determine the fluorescence lifetime 33 . Analogously 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.

The lookup table 117 is shown in FIG. 2b. Henceforth, this is called LUT117 for short. LUT 117 in FIG. 2b is intensity LUT 119 . The corrected intensity 121 is shown over the average photon rate 41 in FIG. 2b.

An average photon rate 41 can be calculated from the integrated signal Σ _P and the measurement period 23 .

Therefore, the intensity LUT 119 can be used to verify the intensity (ie, the number of fluorescence photons detected) resulting from the integration and correct if necessary.

The relationship between fluorescence lifetime 33 and ratio value 39, shown in FIGS. 1b and 1c, is also shown in FIGS. 2a-2c. When the fluorescence lifetime 33 is short, as in FIG. 1b, the single fluorescence photon events 15 are more likely to overlap than when the fluorescence lifetime 33 is relatively long, as in FIG. 1c. The resulting counter value _ΣE will be lower. Thus, for a relatively short fluorescence lifetime 33 with a constant mean photon rate 41, a relatively small dividend and thus a relatively large ratio value 39 can be obtained.

It should be noted here that the curves 35a-35e shown are shown purely by way of example for the sake of clarity and that in the method according to the invention the data 31 can be obtained with finer grading or with the fluorescence lifetime 33 It is emphasized again that smaller step sizes may have been assumed/simulated and stored. In particular, the determined fluorescence lifetime 33 (or more generally at least one optical signal parameter 30 ) can be interpolated from pre-stored data 31 .

In other words, the data 31 may be understood as the integrated signal Σ _P , the counter value Σ _E and the reference value 32 of the fluorescence lifetime 33, which are preferably assigned to each other. In particular, the data 31 may exist as an at least two-dimensional data set 31a.

In addition to the measured values "integrated signal" and "counted signal" (counter value), further signals can be generated that allow further refinement of the resulting signal. For example, the (average) measured pulse width, ie the time difference between the rising edge and the falling edge, can be used in the comparator. In the case of a single pulse, this corresponds to the pulse width or pulse duration 19, and if the pulses overlap, as in FIG. 1b, the measured pulse duration is correspondingly longer. It will be. In such cases, the calibration data set will be three dimensional. That is, a combination of three input variables (“integrated signal”/“counted signal”/“average pulse duration”) will be assigned a resulting signal (eg fluorescence lifetime).

Furthermore, FIGS. 2a to 2c show that the method according to the invention or the device 43 according to the invention ensures a higher distinguishability of the simulated curve 35, especially when the average photon rate 41 is relatively high, Therefore, it indicates that it can preferably be used in this region.

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

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

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

An amplified electrical signal 3a is applied to the amplifier output 55, which is split and fed to a counting path 57 and an integrating path 59 in the form of two signal replicas 3b. In other configurations, particularly including SiPM1b, this splitting may already be done on the detector chip.

The counting path 57 includes a counting module 61 which outputs a counter value _ΣE at a counter output 63 . The counter value Σ _E represents the number of fluorescence photon events 15 .

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

The integrated signal Σ _P is output at integrator output 75 and supplied to logic unit 77 . Based on the integration in the integration module 71, the integrated signal ΣP represents the integrated value 79, which represents the accumulated photon number _ΣN . Furthermore, the counter value Σ _E is superimposed in time with all the number 15a of fluorescence photon events 15 detected in the electrical signal 3 in the measurement period 23, i.e. all temporally separated events 15c. event 15b.

In the configuration of device 43 shown in FIG. 3a, integrated signal Σ _P and counter value Σ _E are forwarded to logic unit 77 . Figure 3a shows a preferred configuration of the device according to the invention.

Logic unit 77 of the configuration shown in FIG. The integrated signal Σ _P , the counter value Σ _E and the reference values 32 of the fluorescence lifetime 33 are stored in these memory modules 85, which contain the measured values and their simulated values. A subscript “sim” is added for differentiation.

In the method according to the invention of the configuration shown in FIG. 3a, the logic unit 77 in turn _converts the measured integrated signal ΣP and the measured counter value _ΣE into the simulated integrated signal Σ _P,sim and the simulated counter value Σ _E,sim are compared with reference values 32 stored in the lookup table module 87 to provide the resulting simulated fluorescence lifetime 33 _sim .

Logic unit 77 further includes an interpolation module 89 that allows interpolating a limited number of stored reference values 32 .

Logic unit 77 outputs at intensity output 91 the intensity result 95 provided by intensity module 93 . The fluorescence lifetime 33 _det determined in this way is output via lifetime output 97 .

Figure 3a also shows a second configuration 77a of the logic unit, which can, for example, determine the fluorescence lifetime 33 _det from the integrated signal _ΣP and the counter value _ΣE , and additionally the intensity result It includes a calculation unit 107 capable of outputting 95.

The configuration of the device according to the invention shown in _FIG . 3b differs from the device of FIG. different.

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

In the configuration of device 43 shown in FIG.

The logic unit 77 of the device of FIG. The integrated signal Σ _P , the ratio value 39 and the reference value 32 of the fluorescence lifetime 33 are stored in these memory modules 85, which distinguish between the measured values and their simulated values. The subscript "sim" is attached to this purpose.

_Logic unit ₇₇ of the device according to the invention of _FIG . 39 and compared to the reference values 32 stored in the lookup table module 87 of the simulated integrated signal Σ _P,sim and the simulated ratio values 39 _sim , resulting in the simulated fluorescence Provides a lifetime of 33 _sim .

The arrangement shown in FIG. 3b may also include an interpolation module 89, an intensity module 93 and a calculation unit 107. FIG. Instead of the counter value _.SIGMA.E as in FIG. 3a, the ratio value 39 is input to the calculation unit 107 in FIG. 3b.

The device 43 of FIGS. 3a and 3b may be arranged in a microscope 99, in particular a (confocal) scanning microscope 99a, particularly preferably a fluorescence lifetime microscope 99b (FLIM), as shown schematically. At FLIM 99b, sample 49 is scanned or scanned to generate image 101 of sample 49. FIG. Here, the determined fluorescence lifetimes 33 _det of the spatially spaced regions 103a, 103b are shown in a suitable color or brightness distribution.

The microscope 99 may furthermore be connected to a computer 109 capable of reading a non-volatile computer-readable storage medium 111, on which the program for implementing the method according to the invention is stored. you can The storage medium 111 may be optical memory, magnetic memory, or flash memory based storage medium 111 .

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). ing.

TCSPC is usable up to an average photon rate 41 of about 40 Mcts/s, but for higher photon rates 41 it no longer provides reliable results of fluorescence lifetime 33 .

In contrast, the application area 105 of the method according to the invention and of the device 43 according to the invention lies at significantly higher mean photon rates 41, preferably ranging from 100 to over 1000 Mcts/s on the order of magnitude or higher.

FIG. 5 shows a flowchart 123 of another configuration of device 43 according to the invention. This can be used instead of the schematic structure shown in FIGS. 3a and 3b to determine the fluorescence lifetime 33. FIG.

The schematic structure of FIG. 5 also includes a detector 1, a preamplifier 53 to which the electrical signal 3 is supplied. The two signal replicas 3b are fed to the integrating module 71 or the counting module 61. FIG. However, in the configuration shown in FIG. 5, the counter value _.SIGMA.E and the integrated signal _.SIGMA.P are fed to two different lookup table modules 87. The lifespan module 125 contains a schematically illustrated lifespan LUT 127, the data of which is shown purely by way of example in FIG. 2a. Additionally, an intensity module 129 is provided in which the intensity LUT 119 resides. The lifetime module 125 determines the fluorescence lifetime 33 by the method according to the invention, whereas the intensity module 129 outputs the intensity result 95 . In other configurations not shown, at least one additional optical signal parameter may be determined.

Optionally, in the structure according to FIG. 3a or 3b, a gating module 131 may also be provided, which may also be provided with the electrical signal 3, the signal replica 3b, the counter value _ΣE and the integrated signal _ΣP are also considered only at certain time intervals.

In FIG. 6 the mode of functioning of the gating module 131 is shown schematically. Here the counter value Σ _E is shown over time 9 . In the method of the present invention configured with gating module 131, only events that occur during gating period 134 between gating start 133 and gating end 135 are observed and considered. Thus, unwanted reflections, which may occur, for example, before the gating start 133, can remain ignored and do not falsify the measurement results.

1 detector 1a PMT hybrid detector 1b silicon photomultiplier (SiPM)
3 electrical signal 3b signal replica 5 voltage 7 current 9 time 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 light signal parameter 30a fluorescence parameter 31 data 31a dataset 32 reference value 33 fluorescence lifetime 33 _det determined fluorescence lifetime 33 _sim simulated fluorescence lifetime 35 curves 35a-35e first curve to fifth curve 39 ratio value 39a measured ratio value 39 _sim simulated ratio value 41 average photon rate 41a measured average photon rate 43 device 45 light source 45a pulsed laser light source 47 excitation light 49 sample 51 detector output 53 preamplifier 55 amplifier output 57 counting path 59 integration path 61 counting module 63 counter output 65 dividend input 67 divider input 69 division module 71 integration module 75 integrator output 77 logic unit 77a second configuration of logic unit 79 integral value 83 ratio value output 85 memory module 87 lookup table module 89 interpolation module 91 intensity output 93 intensity module 95 intensity result 97 lifetime output 99 microscope 99a scanning microscope 99b fluorescence lifetime microscope (FLIM)
101 image 103a, 103b spatially spaced regions 105 application region 107 computation unit 109 computer 111 storage medium 113 pulse repetition frequency 115 period 117 lookup table/LUT
119 Intensity LUT
121 Corrected Intensity 123 Flowchart 125 Lifetime Module 127 Lifetime LUT
129 intensity module 131 gating module 133 gating start 134 gating period 135 gating end E photon energy ΣN accumulated photon count Σ _E counter value Σ _P integrated signal Σ _E,sim simulated counter value Σ _{P , sim} Simulated integrated signal

Claims (13)

A method for measuring at least one optical signal parameter (30), said method comprising:
- illuminating said sample (49) for a predetermined measurement period (23) for excitation of a light signal, in particular fluorescence, in said sample (49);
- detecting the optical signal emitted from the sample (49) and providing an electrical signal (3) representative of the variation of the optical signal over time;
- counting single photon events (15) based on said electrical signal (3) over said measurement period (23) to provide a counter value (Σ _E ) representing the number (15a) of photon events (15); and
- integrating said electrical signal (3) over said measurement period (23) to provide an integrated signal (Σ _P );
- determining at least one optical signal parameter (30) based at least on said counter value (Σ _E ) and said integrated signal (Σ _P );
method containing. illuminating said sample (49) over said measurement period (23), in particular using a sequence of light pulses (27) modulated over time,
The method of claim 1. said optical signal parameter (30) is the fluorescence lifetime (33) or intensity (34) of said optical signal;
3. A method according to claim 1 or 2. performing said integration and said counting in parallel;
A method according to any one of claims 1 to 3. said method further comprising calculating said optical signal parameter (30) using said counter value (Σ _E ) and said integrated signal (Σ _P );
A method according to any one of claims 1 to 4. The method uses pre-stored data (31) to determine the optical signal parameter (30) in relation to the counter value (Σ _E ) and the integrated signal (Σ _P ). further comprising the steps of
A method according to any one of claims 1 to 5. The method further comprises determining and/or calibrating a pulse shape (17) and/or pulse duration (19) of a single photon event (15).
A method according to any one of claims 1 to 6. determining said optical signal parameters (30) taking into account said pulse shape (17) and/or said pulse duration (19) of said single photon event (15);
8. The method of claim 7. The method comprises sequential scanning or scanning of the sample (49) and an image (101) of optical signal parameters (30) of spatially spaced regions (103a, 103b) of the sample (49). further includes generation,
A method according to any one of claims 1 to 8. An apparatus (43) for measuring optical signal parameters (30), comprising:
Said device (43) comprises a detector (1), an integrating module (71), a counting module (61) and a logic unit (77),
said detector (1) produces an electrical signal (3) representing a sequence (26) of incident photons (13) and outputs at a detector output (51),
Said integration module (71) integrates said electrical signal (3) over a measurement period (23) so that said integration module (71) outputs therefrom a resulting integrated signal (Σ _P ). is composed of
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 outputting a counter value (Σ _E ) representing said number (15a),
said logic unit (77) determines at least one optical signal parameter (30) in relation to said integrated signal (Σ _P ) and said counter value (Σ _E );
device (43). said apparatus (43) further comprising a calculation unit (107) for calculating said optical signal parameter (30) using said integrated signal (Σ _P ) and said counter value (Σ _E );
Device (43) according to claim 10. Said device (43) comprises a data set (31a) of said integrated signal (Σ _P ), said counter value (Σ _E ) and mutually assigned reference values (32) of said optical signal parameter (30) further comprising at least one memory module (85) for storing
Device (43) according to claim 10 or 11. A non-volatile computer readable storage medium (111) containing a program comprising instructions which, when executed by a computer, cause said computer to perform the method of any one of claims 1 to 9.

Images