WO2021008811A1 - Procédé et dispositif pour mesurer un paramètre de signal lumineux et support d'enregistrement non volatile - Google Patents

Procédé et dispositif pour mesurer un paramètre de signal lumineux et support d'enregistrement non volatile Download PDF

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Publication number
WO2021008811A1
WO2021008811A1 PCT/EP2020/067090 EP2020067090W WO2021008811A1 WO 2021008811 A1 WO2021008811 A1 WO 2021008811A1 EP 2020067090 W EP2020067090 W EP 2020067090W WO 2021008811 A1 WO2021008811 A1 WO 2021008811A1
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Prior art keywords
light signal
fluorescence
signal
counter value
photon
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PCT/EP2020/067090
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German (de)
English (en)
Inventor
Holger Birk
Arnold Giske
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Leica Microsystems Cms Gmbh
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Priority to JP2022502805A priority Critical patent/JP2022541036A/ja
Publication of WO2021008811A1 publication Critical patent/WO2021008811A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J1/44—Electric circuits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2803—Investigating the spectrum using photoelectric array detector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J1/44—Electric circuits
    • G01J2001/4413—Type
    • G01J2001/442—Single-photon detection or photon counting

Definitions

  • the invention relates to a method and a device for measuring at least one light signal parameter, as well as a non-volatile computer-readable storage medium.
  • the methods from the prior art use a light source to excite a light signal, such as a reflection signal, a phosphorescence signal, the second harmonic or fluorescence.
  • a light signal such as a reflection signal, a phosphorescence signal, the second harmonic or fluorescence.
  • the time between switching on the light source or an excitation pulse and the emitted fluorescence photons is measured, for example a TDC (time-to-digital converter) can be used.
  • TDC time-to-digital converter
  • the process is known as “time-correlated single photon counting” (TCSPC).
  • TCSPC time-correlated single photon counting
  • Both methods have in common that they can only be used to a limited extent as soon as the signals of the individual photons, i.e. the fluorescence photon events, overlap.
  • the object of the present invention is therefore to create a method and a device for measuring a light signal parameter which are simple and inexpensive in terms of apparatus, as well as a measurement of the light signal parameter at high photon rates (> 40 Mcts / s).
  • the method according to the invention solves this problem in that it comprises the following procedural steps:
  • Light signal especially from fluorescence in the sample
  • the device according to the invention can in particular be a microscope and solves the above problem in that it has a detector for generating and outputting an electrical signal representing a sequence of incoming photons at a detector output, an integration module for integrating the electrical signal over a measurement period, where the integration module is designed to output a resulting integration value (also: integrated value), a counting module for counting a number of photon events detected in the measuring period on the basis of the electrical signal over the measuring period and for outputting a counter value representing this number, as well as a logic unit for determination comprises at least one light signal parameter depending on the integration value and the counter value.
  • non-volatile computer-readable storage medium comprises a program with instructions which, when executed by a computer, cause the computer to carry out the method according to the invention.
  • the method according to the invention and the device according to the invention thus have the advantage that no complex time measurement is necessary between switching on the light source, ie, for example, the exciting laser pulse and a detected photon event.
  • the inventive method uses a temporal overlap of two or more photon events, which can no longer be distinguished, to measure the light signal parameter, ie it is based on the fact that the counter value is smaller than the integral value due to the overlap of several photon events. Rather, the accuracy of the detected fluorescence lifetime increases with the photon rates. In particular, the mean photon rates are considered, ie the photons occurring on average per unit of time.
  • the method according to the invention and the device according to the invention can be further improved by further refinements that are advantageous per se.
  • the technical features of the individual configurations can be combined with one another or omitted as desired, provided that the technical effect achieved with the omitted technical feature is not important.
  • the method can in particular be carried out by means of a microscope.
  • a corresponding device can thus in particular be a microscope.
  • the counting of individual photon events can preferably be carried out by means of a digitizer, more preferably by means of a comparator. This can thus be provided in the corresponding device.
  • the illumination of the sample over the measurement period can be modulated over time, in particular with periodic light pulses.
  • Pulse lasers, short pulse lasers or ultrashort pulse lasers are particularly suitable light sources.
  • Pulsed diode lasers can particularly preferably be used, since they are smaller (compared to solid-state lasers, for example) and are easily controllable.
  • Pulsed fiber lasers can also be used.
  • a lighting unit which comprises such a pulsed light source can be provided in the device or can be provided externally. Any time period can be selected as the measurement period, with a measurement period preferably being able to be selected which comprises several period lengths of the light pulses. This has the advantage that a trigger can be tapped off in a simple manner from the pulsed light source.
  • Detectors are preferably used as detectors or photon detectors which can detect individual incoming photons and generate a photon event for each incoming photon in an electrical signal output by the detector.
  • fluorescence photons are spoken of, which generate a fluorescence photon event in the detector, in particular in the electrical signal output by the detector.
  • the explanations can also be transferred to photons and corresponding light signal parameters that have been generated in other ways, for example by reflection, phosphorescence or two photon processes.
  • Special electron tubes can be used, also known as photomultiplier or photo electron multiplier (English photomultiplier tube, PMT). These can also be pure semiconductor detectors or PMT hybrid detectors or SiPM (silicon photomultiplier).
  • the detector detects the light emitted by the sample, for example fluorescent light, and generates an electrical signal which represents a sequence or variation of the incoming photons, for example fluorescent photons.
  • the electrical signal is output at a detector output and can comprise a multiplicity of photon events, for example fluorescence photon events, which can be temporally separated from one another or can be overlapped with one another.
  • all photons detected with the detector during the measurement period for example fluorescence photons, can be determined from the signal by means of the integration module and a signal proportional to this number can be made available. This signal can represent a total photon value.
  • the electrical signal can be split into a photon counting path, in which the electrical signal is integrated, and an event counting path, in which the individual photon events of the electrical signal are counted.
  • the integration module the signal proportional to all detected photons is determined, whereas in the counting module the number of photon events detected in the measurement period is determined and provided in the form of the counter value representing this number.
  • the light signal parameter preferably a fluorescence parameter and particularly preferably the fluorescence lifetime, can also be determined in the logic unit on the basis of the integrated signal and the counter value.
  • the logic unit can be designed in the form of a single logic module or chip or as a so-called field programmable gate array (FPGA).
  • An FGPA is an integrated circuit (IC) in which (on site, at the customer's) a logic circuit can be loaded and executed on it.
  • the non-volatile computer-readable storage medium according to the invention comprises a program with instructions for carrying out the method according to the invention, for example on a computer, which can be read from the medium and loaded into an above-mentioned FPGA or into the memory of a computer.
  • the method according to the invention can also count individual photon events, for example individual fluorescence photon events of the electrical signal via the Measuring period and providing a counter value representing the number of photon events, for example individual fluorescence photon events.
  • an incremental encoder can be provided in the device which outputs the counter value, the counter value being available in digital or analog form and representing the number of photon events, for example individual fluorescence photon events.
  • the counter value can be reset at the end of the measuring period, that is to say at the beginning of the subsequent measuring period.
  • the method according to the invention comprises integrating the electrical signal over the measuring period and providing an integrated signal which represents a photon number of all detected fluorescence photons accumulated in the measuring period.
  • photon detectors generate an electrical pulse for each photon detected, the height of which is independent of the energy of the photon.
  • Photons of different wavelengths cannot be differentiated on the basis of the signal, but the pulses of simultaneously hitting photons add up in the signal, so that, for example, the integrated signal of two simultaneously hitting photons is twice as large as the integrated signal of a single photon.
  • the integrated signal can be obtained by means of an integrator or integration module which no longer differentiates the individual photon events, for example individual fluorescence photon events of the electrical signal, with a sufficiently large time constant, but integrates them over the measurement period.
  • the electrical signal is compared with a threshold value in the counter module, the counter reading being increased by one when the threshold value is exceeded and subsequently fallen below.
  • Simultaneously incident photons for example fluorescence photons
  • temporally overlapping photon events for example temporally overlapping fluorescence photon events that only exceed and fall below the threshold value once and the counter of the photon events, in particular the fluorescence photon events, only by one increase.
  • a quantitative variable such as a charge
  • Two fluorescence photons arriving at the same time thus generate a signal in the integrating path that is twice as high as a single incoming fluorescence photon.
  • An integration value ie the integrated signal re determined within the measurement period thus presents the accumulated number of photons of all detected photons, in particular the detected fluorescence photons.
  • the integration and the counting can take place in parallel. Furthermore, the integration and the counting also preferably take place at the same time.
  • the method can comprise calculating a ratio value from the number of individual fluorescence photon events, i.e. from the counter value and the integrated signal.
  • the three variables counter value, integrated signal and ratio value can be used in any desired, e.g. pairing of two, in order to determine the light signal parameter, preferably the fluorescence parameter and particularly preferably the fluorescence lifetime.
  • (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 the light signal parameter. Descriptions in this disclosure that relate to one of the combinations (1), (2) or (3), or to combinations of other variables for determining the light signal parameter, are therefore - unless explicitly excluded in the text - to any other combination these three sizes are transferable and not limited to the combination mentioned.
  • the calibration data for determining the light signal parameter preferably a fluorescence parameter, such as the fluorescence lifetime
  • a fluorescence parameter such as the fluorescence lifetime
  • an associated fluorescence lifetime can be stored for each value pair of the integrated and counted signal (counter value), so that, purely by way of example, the fluorescence lifetime can be indicated directly when the two measured values are available (calibration LUT).
  • the two-dimensional LUT can also contain fewer support points than possible combinations of all value pairs. Then the light signal parameter, such as, purely by way of example, the fluorescence lifetime, can be interpolated from the existing support points.
  • At least one further light signal parameter can be determined in a further embodiment.
  • other signals can be generated in addition to the fluorescence life.
  • an improved intensity signal can be generated from the combination of both signals (integrated / counted).
  • the structures and the data paths can be unchanged, only the calculation rule / calibration LUT can be adapted accordingly to the calculation of the intensity signal.
  • Such a refinement has the advantage that redundancy is provided for determining the intensity of the electrical signal, the integrated signal determined, for example, by integration being checked for correctness and deviations or errors in the determination being able to be recognized.
  • a device for carrying out this embodiment of the method can thus include the integration module for integrating the electrical signal in the measurement period and for outputting an integration value and the counting module for counting the photon events and outputting the 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 for outputting the ratio value, whereby the logic unit can be configured to provide the light signal parameters, such as the fluorescence lifetime as a function of the integrated signal and the counter value or also the integrated signal (or age native from the counter value) and from the ratio value.
  • the method can consequently be further improved in that it comprises the determination of the light signal parameter, preferably the fluorescence parameter, such as, for example, the fluorescence lifetime using the integrated electrical signal and the counter value.
  • the ratio value can optionally be used in combination with the counter value or the integrated signal to determine the light signal parameter.
  • the light signal parameter, preferably the fluorescence parameter, such as, for example, the fluorescence lifetime can be determined in different configurations from two variables each. These variables can be, for example, the integrated signal and the counter value, the counter value and the ratio value or also the integrated signal and the ratio value.
  • the determination of the light signal parameter by means of the counter value and the integrated signal represents the preferred embodiment of the method according to the invention or the device according to the invention, but does not exclude the combination of other variables for determining the light signal parameter.
  • this includes calculating the light signal parameter, preferably the fluorescence parameter such as the fluorescence lifetime with the aid of the integrated signal and the counter value.
  • a device for carrying out this embodiment of the method can thus include a computing unit for calculating the light signal parameter, preferably the fluorescence parameter such as the fluorescence lifetime with the aid of the integrated signal and the counter value.
  • the computing unit can use the counter value (or, alternatively, the integrated signal) and the ratio value (the ratio of the integrated signal to the counter value) in order to use a previously stored, analytical calculation rule (analytical curve) to determine the light signal parameter, preferably the To calculate fluorescence parameters, for example the fluorescence lifetime.
  • the computing unit itself can include a division module and thus combine all the necessary computing steps in one unit.
  • the determination of the light signal parameter, in particular the fluorescence parameter such as the fluorescence lifetime using previously stored data can be provided as a function of the integrated signal and the counter value.
  • the device according to the invention for carrying out this embodiment of the method can thus comprise at least one memory module for storing a data set of mutually assigned reference values of the integrated signal, the counter value and the light signal parameter, in particular the fluorescence parameter such as the fluorescence lifetime.
  • the previously stored data can be in the form of a two-dimensional or three-dimensional data record.
  • This data set or this data matrix can include the integrated electrical signal (i.e. the integration value) and the counter value (optionally counter value and ratio value or alternatively integrated signal and ratio value) and certain combinations of these values include a light signal parameter, preferably a fluorescence parameter, such as a fluorescence lifetime assign.
  • a family of measurement curves can be posed which show the integrated signal or the ratio value as a function of the counter value (or in the case of the integrated signal also the ratio value).
  • the array parameter is the light signal parameter, in particular the fluorescence parameter such as the fluorescence lifetime.
  • a representative of the family of curves can be determined and the light signal parameters associated with this representative of the family of curves, in particular the fluorescence parameters, such as the fluorescence lifetime can be read.
  • the method can include interpolating the previously stored data, with the light signal parameters, in particular the fluorescence parameters, such as the fluorescence lifetime, being determined on the basis of the interpolated data.
  • the previously stored data for example the integrated signal and the counter value
  • the light signal parameter, in particular the fluorescence parameter, such as the fluorescence lifetime can be determined.
  • a device for carrying out this refinement of the method can consequently comprise an interpolation module for interpolating the previously stored data.
  • the analytical curve which can be the basis of the set of measurement curves provided, can be used to determine the light signal parameter, in particular the fluorescence parameter, such as the fluorescence lifetime, directly from the counter value and the integrated signal (or the Ratio value).
  • the determination and / or calibration of a pulse shape and / or pulse duration of a single photon event can be provided.
  • the photon events such as, for example, the fluorescence photon events
  • the fluorescence photons in fluorescence due to the stochastic behavior of the photons, preferably the fluorescence photons in fluorescence, it inevitably occurs with a certain probability also for superimposing individual photon events, such as individual fluorescence photon events and thus for non-counted events.
  • the probability of non-counted events depends on:
  • the light signal parameter in particular the fluorescence parameter, such as the fluorescence lifetime of the excited fluorophore.
  • point 3 of the above list can be determined with this embodiment of the method according to the invention or (if fluorescence is considered) can be calibrated by means of comparison measurements of dyes with known fluorescence lifetimes.
  • the light signal parameter in particular the fluorescence parameter, such as the fluorescence parameter, to be determined directly from the ratio of counting and integrating path, i.e. from the ratio of counter value and integrated signal. B. to get the fluorescence lifetime of the excited dye.
  • Point 5 above means that the sensitivity of the detector can be adjusted over time with respect to the excitation pulse.
  • gating allows certain time ranges of the counting path to be masked out (i.e. during the integration) in order, for example, to suppress reflected excitation light leading to the light signal, such as the fluorescence pulse.
  • the method according to the invention can be improved by determining the light signal parameter, in particular the fluorescence parameter, such as the fluorescence lifetime, taking into account the pulse shape and / or the pulse duration of the individual photon events, in particular the individual fluorescence photon events.
  • the fluorescence parameter such as the fluorescence lifetime
  • the method according to the invention can furthermore comprise the sequential rasterization or scanning of a sample and generating an image of light signal parameters, in particular of fluorescence parameters, such as fluorescence lifetimes of spatially spaced apart regions of the sample.
  • fluorescence parameters such as fluorescence lifetimes of spatially spaced apart regions of the sample.
  • FLIM fluorescence lifetime imaging microscopy
  • the corre sponding device can be a fluorescence lifetime microscope (FLIM).
  • a raster or scanning device can be provided which moves the excitation light over the sample in a scanning or raster movement. It is also possible for the sample to be moved relative to the illumination and the detection.
  • the light signal parameters in particular the fluorescence parameters, such as the fluorescence lifetime of the respective illuminated area of the sample, can be determined and displayed as image information.
  • the configuration of such a method can be used in a scanning microscope, in particular a confocal scanning microscope.
  • the method according to the invention can in particular be based on the saturation behavior that occurs in the counting module (for determining the counter value). This is caused by the superimposition of the photon events to be counted, such as the fluorescence photon events to be counted.
  • This superimposition effect can depend in particular on the light signal parameter, in particular on the fluorescence parameter, such as the fluorescence lifetime, since the average photon rate varies greatly in the course of a pulse.
  • the effect can be particularly pronounced if the width of the photon event to be counted, such as the fluorescence photon event to be counted, is of the same order of magnitude as the light signal parameter, in particular the fluorescence parameter, such as the fluorescence lifetime of the observed dye.
  • the pulse durations are typically in the range between one and two nanoseconds.
  • Typical fluorescence lifetimes of the dyes are generally one to five nanoseconds.
  • the electronic pulse durations can be adapted by suitable filtering or by adapting the threshold value. This can achieve a particularly advantageous fluorescence lifetime contrast.
  • the non-volatile computer-readable storage medium comprises in particular a program with commands which, when executed by a computer, cause the computer to carry out the method according to one of the aforementioned embodiments.
  • Storage medium is any type of optical, magnetic or flash memory based data carrier.
  • 1 a shows a schematic representation of the electrical generated by a detector
  • 1 b shows the representation of a simulation for a pulsed excitation with four pulses with an assumed fluorescence lifetime of 1.5 ns;
  • 1 c shows the simulation of FIG. 1 b with an assumed fluorescence life of 5 ns; 1d shows a simplified representation of the mean photon rate over time;
  • 2a shows a look-up table for determining a fluorescence lifetime
  • 2b shows a schematic look-up table for determining a light intensity
  • 2c is a schematic representation of previously stored data for determining the
  • Fluorescence lifetime 3a shows a schematic representation of an embodiment of the device according to the invention for measuring the fluorescence lifetime
  • 3b shows a schematic representation of a further embodiment of the device according to the invention for measuring the fluorescence lifetime
  • 5 shows a schematic representation of a further embodiment of the method according to the invention.
  • 6 shows a schematic representation of the gating.
  • the figures described below show, purely by way of example, the determination of the fluorescence lifetime. This description is purely exemplary and can be applied to the determination of a fluorescence parameter or, more generally, to the determination of a light signal parameter. Since the following descriptions are exemplary, they do not limit the scope of protection.
  • the light signal can be generated for example by reflection, phosphorescence, fluorescence or two photon processes.
  • FIG. 1 a shows a schematic representation of the electrical signal 3 generated by a detector 1 (see representation in the graph at the top right), which is used to determine a counter value (see FIG. 3).
  • the detector can be configured, for example, as a PMT hybrid detector 1a or as a silicon photomultiplier 1b (English: silicon photomultiplier, SiPM for short).
  • the electrical signal 3 can, for example, represent the course of a voltage 5 or an electrical current 7 as a function of time 9, with a negligible dark current 11 being measured in the ideally assumed case.
  • FIG. 1a shows only a few temporally isolated events 15c and temporally overlapping events 15b.
  • the temporally isolated events 15c do not mutually influence one another, whereas the temporally overlapping events 15b, which are shown in FIG. 1a, lead to a value of the voltage 5 or the electrical current 7 that is significantly greater (almost a factor of 2 ) of the value of the voltage or the electrical current 7 of the temporally isolated events 15c.
  • Each fluorescence photon event 15 has a pulse shape 17, comprising a steep rising edge 17a and an exponentially falling edge 17b, and a pulse width or pulse duration 19. It should be noted that the pulse shape 17 and the pulse duration 19 correspond to an impulse response function 21 of a detector 1. Different detectors 1 have different impulse response functions 21. In other words, the fluorescence photon event 15 represents the change in the voltage 5 or the electrical current 7 after which a fluorescence photon 13 has hit the detector 1.
  • the five ten photons 13 impinging on the detector 1 as a function of the time 9 within a predetermined measurement period 23 are indicated by dashed lines. These can be added together in an integrated signal Sr, which represents the number of photons 13.
  • the integrated signal Sr is shown schematically as being stored in a memory unit 25.
  • FIG. 1 a schematically shows two successive light pulses 27 which define the measurement period 23 in the exemplary embodiment shown.
  • TCSPC photon per measurement period
  • a sequence 26 of the light pulses 27 can in particular be periodic.
  • a threshold value 29 is shown in FIG. 1 a, which is used to count the fluorescence photon events 15. As soon as the voltage 5 or the electrical current 7 exceeds this threshold value 29 once and falls below it once, a counter value S E is increased by one.
  • the counter value S E is also shown schematically as being stored in a memory unit 25.
  • the counter value S E represents the number of fluorescence photon events 15a occurring in the electronic signal.
  • the temporally overlapping events 15b given a sufficiently large overlap, result in several such events 15b increasing the counter value S E by only one.
  • a sufficiently large overlap means that the voltage 5 or the electrical current 7 has not yet fallen below the threshold value 29 again after the threshold value 29 has been exceeded and before a further fluorescence photon 13 arrives.
  • the voltage 5 or the electric current 7 only falls below the threshold value 29 after the third temporally overlapping event 15b and increases the counter value S E by only one despite three incoming fluorescence photons 13.
  • the counter value S E is eleven in the measurement period 23 shown.
  • FIGS. 1b and 1c each show an electrical signal 3 generated by a detector 1, the pulses 27 used for excitation being merely provided by a dotted line. The sequence 26 of a total of four light pulses 27 is shown in both figures.
  • the measurement period 23 shown in FIGS. 1 b and 1c consequently comprises a period of pulsed excitation of four period lengths 115.
  • the time 9 of the measurement periods 23 shown is approx. 50 ns (the laser system used for excitation consequently has a pulse repetition frequency 113 of approx. 75 MHz).
  • the threshold value 29 is shown at a value of the voltage 5 or the electrical current 7 (shown in arbitrary units) of 0.5.
  • the threshold value 29 is shown with a dash-dotted line.
  • the fluorescence lifetime represents a fluorescence parameter 30a or a light signal parameter 30.
  • FIG. 2 c shows a schematic representation of previously stored data 31 for determining the fluorescence lifetime 33.
  • five curves 35a to 35e of a group of curves 37 are drawn in FIG. 2c, the drawn curves 35 only serving to illustrate the method and generally only referring to the previously stored data set when an evaluation is actually carried out.
  • the curves 35a to 35e are the results of a simulation for a SiPM 16 with a pulse duration 19 (electronic pulse duration) of 2 ns.
  • a ratio 39 was calculated from the integrated signal S R and the counter value S E and plotted against an average photon rate 41.
  • the mean photon rate 41 results from the integrated signal S R divided by the measurement period 23 and the ratio value 39 is simulated up to approximately six hundred and fifty Mcts / s.
  • the method according to the invention delivers, for example, a measured average photon rate 41a of five hundred Mcts / s and a measured ratio value 39a of four (both values 39a and 41a are shown by dashed lines), then with the method according to the invention or the device 43 according to the invention ( see FIG. 3) a fluorescence lifetime 33 of two nanoseconds is determined.
  • FIG. 1d shows a simplified illustration in which the photon rate 41, more precisely the averaged photon rate 41, is shown over time 9.
  • the photon rate 41 drops exponentially and is averaged over a number of fluorescence photons 13.
  • FIG. 1d some fluorescence photons 13 overlap with one another, so that the counter value (not shown) would result in four here, but six fluorescence photons 13 would be determined on the basis of the integrated signal (likewise not shown).
  • the exponential decrease in the photon rate 41 is inversely proportional to the fluorescence lifetime 33, i.e. a short fluorescence lifetime 33 leads to a rapid exponential decrease in the mean photon rate 41.
  • FIGS. 2a and 2b each show a look-up table 117, more precisely data 31 previously stored in the look-up table 117 for determining the fluorescence lifetime 33.
  • FIG. 2a shows five curves 35a to 35e of a family of curves 37. These curves 35a to 35e serve only to illustrate the method and are not shown in the method according to the invention, but rather the previously stored data set is used.
  • the curves 35a to 35e are the results of a simulation for a SiPM 16 with a pulse duration 19 of 2 ns.
  • the counter value S E is shown over the integrated signal S R.
  • a set or pair of values determined in the method according to the invention for the counter value S E and the integrated signal S R can thus be used to determine a fluorescence lifetime 33.
  • the fluorescence lifetime 33 can be determined from the illustration shown in FIG. 2c.
  • a look-up table 117 is shown in FIG. 2b. This will be referred to as LUT 117 for short in the following.
  • the LUT 117 of FIG. 2b is an intensity LUT 119.
  • a corrected intensity 121 over the mean photon rate 41 is shown.
  • the mean photon rate 41 can be calculated from the integrated signal S R and the measurement period 23.
  • the intensity obtained by means of integration i.e. the number of detected fluorescence photons
  • the intensity obtained by means of integration i.e. the number of detected fluorescence photons
  • the relationship between the fluorescence lifetime 33 and the ratio 39 shown in FIGS. 1b and 1c is also shown in FIGS. 2a to 2c. If, with short fluorescence lifetimes 33, as is the case in FIG. 1b, there is a greater probability of overlapping individual fluorescence photon events 15, then a lower counter value S E is obtained than is the case with longer fluorescence lifetimes 33, as in FIG. 1c is. Accordingly, for shorter fluorescence lifetimes 33, with the average photon rate 41 remaining the same, a smaller dividend and consequently a larger ratio value 39 can be obtained.
  • the curves 35a to 35e shown are shown purely by way of example for clarification and in the method according to the invention the data 31 can be postulated / simulated and stored in a finer gradation or with a smaller step size of the fluorescence lifetime 33.
  • the determined fluorescence lifetimes 33 (or more generally at least one light signal parameter 30) can be interpolated from the previously stored data 31.
  • the data 31 can be understood as reference values 32 of the integrated signal SR, the counter value S E and the fluorescence lifetime 33 which are mutually exclusive are assigned preferentially.
  • the data 31 can be present as at least two-dimensional data record 31a.
  • the (average) measured pulse duration i.e. the time difference between the rising and falling edge on the comparator
  • the calibration data set would be three-dimensional, i.e. a combination of the 3 input variables ("integrated signal7" counted signal "/" average pulse duration ") would be assigned a result signal (e.g. fluorescence lifetime).
  • FIGS. 2a to 2c show that the method according to the invention and the device 43 according to the invention, in particular at higher average photon rates 41, ensure greater distinguishability between the simulated curves 35 and can thus be used preferably in this area.
  • FIG. 3a A schematic representation of the device 43 according to the invention for measuring the fluorescence lifetime 33 is shown in FIG. 3a.
  • a light source 45 in particular a pulsed laser light source 45a, emits excitation light 47 in the form of the sequence 26 of light pulses 27 which strikes a sample 49.
  • excitation light 47 in the form of the sequence 26 of light pulses 27 which strikes a sample 49.
  • fluorescence photons 13 (only one is shown) are generated, which fall on the detector 1 De.
  • Further optical elements that are suitable for collecting the fluorescence photons 13 are not shown, but can generally be used in other configurations of the device 43.
  • the electrical signal 3 is output at a detector output 51 and fed into a preamplifier 53 and amplified.
  • the amplified electrical signal 3a is present at an amplifier output 55, which is split up and fed into a counting path 57 and an integrating path 59 in the form of two signal replicas 3b. In other configurations, which in particular include a SiPM 1b, the division can already take place on the detector chip.
  • the counting path 57 includes a counting module 61 which outputs the counter value S E at a counting output 63.
  • the counter value S E represents a number of fluorescence photon events 15.
  • the integrating path 59 comprises an integration module 71 which determines the number 13a of all fluorescence photons 13 impinging on the detector 1 in the measurement period 23.
  • the integrated signal S R is output at an integrator output 75 and fed into a logic unit 77. Due to the integration in the integration module 71, the integrated signal S R represents an integration value 79, this representing an accumulated number of photons SN. Furthermore, the counter value S E describes a number 15a of all fluorescence photon events 15 detected in the electrical signal 3 in the measurement period 23, that is to say all time-isolated events 15e and all time-overlapping events 15b.
  • FIG. 3 a shows the preferred configuration of the device according to the invention.
  • the logic unit 77 of the configuration shown in FIG. 3 a comprises three memory modules 85 shown schematically and connected to one another, which form a look-up table module 87.
  • the reference values 32 of the integrated signal SR, the counter value S E and the fluorescence lifetime 33 are stored in these memory modules 85 and are identified by the subscript “sim” in order to distinguish the measured values from these simulated values.
  • the logic unit 77 now compares the measured integrated signal S R and the measured counter value S E with the reference values 32 of the simulated integrated signal Zp , Sim stored in the look-up table module 87 and the simulated counter value Z E, sim and provides the simulated fluorescence lifetime 33 Sim as a result.
  • the logic unit 77 further comprises an interpolation module 89, which makes it possible to interpolate a limited number of stored reference values 32.
  • the logic unit 77 outputs an intensity result 95 provided by an intensity module 93 at an intensity output 91.
  • a fluorescence lifetime 33 det determined in the method is output via lifetime output 97.
  • 3a also shows a second embodiment of the logic unit 77a, which includes a computing unit 107 which, for example, can determine the fluorescence lifetime 33 det from the integrated signal S R and the counter value S E and can also output the intensity result 95.
  • the embodiment of the device according to the invention shown in FIG. 3b differs from the device in FIG. 3a in that the counter value Se ⁇ h output at counter output 63 is fed into a divider input 67 of a division module 69.
  • the integrated signal output in the integrator output 75 is fed into the division module 69 via a divide input 65 and, as in the embodiment of FIG. 3a, into the logic unit 77.
  • the division module 69 calculates a ratio value 39 which is transferred from the division module 69 to the logic unit 77 via a ratio value output 83.
  • the logic unit 77 of the device of FIG. 3 b comprises three memory modules 85, shown schematically connected to one another, which form a look-up table module 87.
  • the reference values 32 of the integrated signal SR, the ratio 39 and the fluorescence lifetime 33 are stored in these memory modules 85, which are identified by the subscript “sim” to distinguish the measured values from these simulated values.
  • the logic unit 77 in the inventive device of FIG. 3b now compares the measured integrated signal S R and the ratio value 39 calculated from the integrated signal S R and counter value S E with those stored in the look-up table module 87 in the method according to the invention Reference values 32 of the simulated integrated signal Zp , Sim and the simulated ratio value 39 Sim , and provides the simulated fluorescence lifetime 33 Sim as a result.
  • the embodiment shown in FIG. 3 b can also include an interpolation module 89, an intensity module 93 and a computing unit 107.
  • the ratio value 39 is entered into the arithmetic unit 107 in FIG. 3b.
  • the devices 43 of FIGS. 3a and 3b can, as is shown schematically, be arranged in a microscope 99, in particular a (confocal) scanning microscope 99a, and particularly preferably in a fluorescence lifetime microscope 99b (FLIM).
  • the sample 49 is scanned or rasterized in the FLIM 99b and an image 101 of the sample 49 is generated, in which the determined fluorescence lifetimes 33 det of spatially spaced areas 103a, 103b are shown with a suitable color or brightness distribution.
  • the microscope 99 can also be connected to a computer 109, which can contain a non-volatile computer-readable storage medium 111, wherein a program for carrying out the method according to the invention can be stored on the storage medium 111.
  • the storage medium 111 can be an optical, magnetic or flash memory-based storage medium 111.
  • TCSPC time-correlated single photon counting
  • the TCSPC can be used up to average photon rates 41 of approx. 40 Mcts / s, but no longer provides reliable results for the fluorescence lifetime 33 for higher photon rates 41.
  • the area of application 105 of the method according to the invention and of the device 43 according to the invention is at significantly higher mean photon rates 41 and preferably extends over more than an order of magnitude of one hundred to over one thousand Mcts / s.
  • FIG. 5 a flow chart 123 of a further embodiment of the device 43 according to the invention is shown. This can be used instead of the schematic structures shown in FIGS. 3a and 3b for determining the fluorescence lifetime 33.
  • the schematic structure of FIG. 5 also includes a detector 1, a preamplifier 53, into which the electrical signal 3 is fed. Two signal replicas 3b are fed into the integration module 71 and into the counting module 61, respectively. However, in the embodiment shown in FIG. 5, the counter value S E and the integrated signal S R are fed into two different look-up table modules 87.
  • a life module 125 includes one schematically illustrated lifetime LUT 127, the data of which is shown purely by way of example in FIG. 2a.
  • An intensity module 129 is also provided, in which the intensity LUT 119 is located. The service life module 125 determines a fluorescence service life 33 using the method according to the invention, whereas the intensity module 129 outputs the intensity result 95. In further refinements not shown, at least one other light signal parameter can be determined.
  • a gating module 131 can also be provided in the structure according to FIG. 3a or according to FIG. 3b, which only takes into account the electrical signal 3, the signal replica 3b as well as the counter value S E and the integrated signal S R only in a certain time interval - done.
  • the mode of operation of the gating module 131 is shown schematically in FIG. 6.
  • the counter value S E is shown here over time 9.
  • only events are considered and taken into account which occur in a gating time segment 134 between a gating start 133 and a gating end 135.
  • unwanted reflections that can occur before the gating start 133 can be ignored and do not falsify the measurement result.
  • SiPM silicon photomultiplier

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Abstract

L'invention concerne un procédé et un dispositif (43) pour mesurer au moins un paramètre de signal lumineux (30), ainsi qu'un support d'enregistrement (111) non volatile lisible par ordinateur. Des procédés connus présentent l'inconvénient de ne plus pouvoir être utilisés dès que les signaux des différents photons (13), c'est-à-dire les événements (15) de photons de fluorescence, se superposent. Le procédé selon l'invention comprend les étapes suivantes pour l'amélioration des procédé connus : éclairage d'un échantillon (49) pendant une période de mesure (23) prédéfinie afin d'exciter un signal lumineux, notamment la fluorescence, dans l'échantillon (49) ; détection du signal lumineux émis par l'échantillon (49), et fourniture d'un signal électrique (3) représentant la variation temporelle du signal lumineux ; comptage de différents événements (15) de photons sur la base du signal électrique (3) pendant la période de mesure (23), et fourniture d'une valeur de comptage (ΣE) représentant le nombre (15a) d'événements (15) de photons ; intégration du signal électrique (3) pendant la période de mesure (23), fourniture du signal intégré (ΣP) et détermination d'au moins un paramètre de signal lumineux (30) au moins à l'aide de la valeur de comptage (ΣE) et du signal intégré (ΣP). Le dispositif (43) selon l'invention permet d'atteindre l'objectif ci-dessus du fait qu'il comprend un détecteur (1), un module d'intégration (71), un module de comptage (61) ainsi qu'une unité logique (77) permettant de déterminer au moins un paramètre de signal lumineux (30) en fonction du signal intégré (ΣP) et de la valeur de comptage (ΣE).
PCT/EP2020/067090 2019-07-15 2020-06-19 Procédé et dispositif pour mesurer un paramètre de signal lumineux et support d'enregistrement non volatile WO2021008811A1 (fr)

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