DE102012019471A1 - Method for determining e.g. fluorescence lifetime of paper to identify banknote, involves recording phase difference values of sample light by runtime camera, and determining material characteristic based on difference values - Google Patents

Abstract

The method involves illuminating a sample (2) with light pulses (L) of a pulse rate. Phase difference values of a sample light (P) are recorded by a runtime camera (4) with a photon mixing device (PMD) sensor (4.1), where the sample light is spectrally splitted on a dispersive element (9) i.e. holographic grating. The camera is synchronized with the light pulses. The sample is illuminated with another pulse rate that is different from the former pulse rate. Other phase difference values of the sample light are recorded. A material characteristic is determined based on the difference values. Independent claims are also included for the following: (1) a device for determining a material characteristic of a sample (2) an object (3) a control unit or control program comprising a set of instructions for performing a method for determining a material characteristic of a sample.

Description

The invention relates to a method for determining a (or more) substance property (s), in particular a fluorescence lifetime, of a sample by illuminating a sample with light pulses and thus synchronized recording of spectrally split sample light by means of a runtime camera and a device for determining one (or more) Substance property (s), in particular a fluorescence lifetime, of a sample comprising a modulatable light source for emitting light pulses, a spatially resolving runtime camera, a pulse generator with adjustable pulse rate and a control unit, wherein the light source and the runtime camera are connected to the pulse generator and in the beam path of the runtime camera for the spectrally resolved recording of sample light a dispersive element is arranged. The spatial resolution of the camera preferably runs along the dispersion direction of the dispersive element. It preferably serves for the simultaneous recording of light from different locations on the sample into different detector elements, so that a one-dimensional or two-dimensional image can be determined.

For the purposes of the invention, the term light comprises any electromagnetic radiation which can be manipulated by optical means, that is to say in particular ultraviolet, visible and infrared radiation. For the purposes of the invention, sample light is light which is emitted by fluorescence from the sample, excited by the illuminating light pulses. It may also contain components which are formed by reflection and / or scattering of the illuminating light pulses on the sample. The substance property to be determined can be represented by any measurable variable, in particular it can be a physical, biological or chemical property of a single ingredient of the sample or, for example in the case of a composition, the sample as a whole.

In the context of the invention, a runtime camera is any optoelectronic detector which detects a time difference between a reference signal indicating the emission of an illuminating light pulse and the detection of light (correlated with the light pulse with respect to time), in particular the fluorescence response of the sample to the illuminating light pulse. in the form of a phase shift, time duration, path length, or other equivalent quantity (analog or digital) (hereinafter referred to as the phase difference value). The runtime camera is synchronized by means of the reference signal with the light source. The runtime camera can be arranged to the light source either so that it receives backscattered / reflected sample light (remission), or so that it receives forward scattered / transmitted sample light (transmission).

In the prior art, such methods and devices are made DE 199 51 154 A1 and from US 2011/007311 A1 known. This makes it possible to differentiate materials with very similar optical properties, in particular spectrally similar fluorescent substances, by the additional evaluation of the transit time of the light emitted by the sample from each other. For this purpose, in particular fluorescence lifetimes can be measured on the basis of the transit time. In DE 199 51 154 A1 For example, as methods for measuring different fluorescence lifetimes in the same sample, phase fluorometry, fluorescence correlation spectroscopy (FCS), and time-correlated single photon counting (TCSPC) are described. The runtime camera used here is a photonic mixing device (PMD), which may be part of an optical spectrometer. However, the accuracy of the measurement is limited by the methods described, in particular in the presence of different types of fluorophore with significantly different fluorescence lifetimes in the sample, for example in mixtures.

The invention has for its object to improve a device and a method of each type mentioned, so that a greater measurement accuracy is possible.

The object is achieved by a method having the features specified in claim 1, and by a device having the features specified in claim 7.

Advantageous embodiments of the invention are specified in the subclaims.

• a) Beleuchten der Probe mit Lichtpulsen einer ersten Pulsfrequenz (mittels einer Lichtquelle),
• b) Aufnehmen von ersten Phasendifferenzwerten von an einem dispersiven Element spektral aufgespaltetem Probenlicht mittels einer mit den Lichtpulsen synchronisierten Laufzeitkamera mit einem (längs der Dispersionsrichtung) ortsauflösenden Detektor,
• c) Beleuchten der Probe mit Lichtpulsen einer zweiten, von der ersten verschiedenen Pulsfrequenz,
• d) Aufnehmen von zweiten Phasendifferenzwerten von an dem dispersiven Element spektral aufgespaltetem Probenlicht mittels der mit den Lichtpulsen synchronisierten Laufzeitkamera und
• e) Ermitteln mindestens einer Stoffeigenschaft anhand (aufgenommener) erster und zweiter Phasendifferenzwerte.

According to the invention, the following steps are carried out for the time-resolved, spatially resolved and spectrally resolved measurement of a substance property of a sample:

• a) illuminating the sample with light pulses of a first pulse frequency (by means of a light source),
• b) recording first phase difference values of sample light spectrally split at a dispersive element by means of a time-of-flight camera synchronized with the light pulses with a detector (spatially resolving along the dispersion direction),
• c) illuminating the sample with light pulses of a second, different from the first pulse rate,
• d) spectrally recording second phase difference values of at the dispersive element split sample light by means of synchronized with the light pulses runtime camera and
• e) determining at least one substance property based on (recorded) first and second phase difference values.

Before determining the substance property in step e), it is preferably identified in an intermediate step that the recorded phase difference values are significant. Non-significant phase difference values are preferably not taken into account in the determination of the substance property in step e) (in the intermediate step then non-significant phase difference values are discarded). The significance is preferably identified on the basis of at least one predetermined (upper and / or lower) limit value for the phase difference values, in particular by a comparison of the recorded phase difference values with the predetermined limit value (s). These may be determined, for example, by the user through a visual assessment of the amplitude spectrum (or intensity spectrum). Alternatively, they can be automatically determined based on predetermined limits for energy values and a minimum distance between excitation wavelengths and fluorescence wavelengths within the method according to the invention, for example by a control unit.

• a) Einstellen des Pulsgebers zur Abgabe von Pulsen einer ersten Pulsfrequenz,
• b) Ermitteln von ersten Phasendifferenzwerten von (an dem dispersiven Element spektral aufgespaltetem) Probenlicht mittels der Laufzeitkamera,
• c) Einstellen des Pulsgebers zur Abgabe von Pulsen einer zweiten, von der ersten verschiedenen Pulsfrequenz,
• d) Ermitteln von zweiten Phasendifferenzwerten von (spektral aufgespaltetem) Probenlicht mittels der Laufzeitkamera,
• e) Ermitteln mindestens einer Stoffeigenschaft anhand (aufgenommener) erster und zweiter Phasendifferenzwerte.

Accordingly, it is provided for a device according to the invention that the control unit is set up to carry out the following steps:

• a) setting the pulser to deliver pulses of a first pulse rate,
• b) determining first phase difference values of sample light (spectrally split at the dispersive element) by means of the time-of-flight camera,
• c) setting the pulser for delivery of pulses of a second, different from the first pulse rate,
• d) determining second phase difference values of (spectrally split) sample light by means of the time-of-flight camera,
• e) determining at least one substance property based on (recorded) first and second phase difference values.

Subsequently, the determined substance property can be output by the control unit via an interface. Before determining the fluorescence lifetime in step e), it is preferably identified in an intermediate step that the recorded phase difference values are significant. Non-significant phase difference values are preferably not taken into account in determining the fluorescence lifetime in step e).

Each phase difference value indicates the time difference between the emission of a respective illuminating light pulse and the arrival of the associated fluorescence response in the time-of-flight camera. Alternatively, as a phase difference value, any variable equivalent to the time difference may be used, for example the corresponding angular difference with respect to the pulse frequency or the corresponding traveled path length. In addition to the phase difference values, spectral intensity values of the sample light can also be determined. In advantageous embodiments, the determination of the material property can additionally be carried out on the basis of the spectral intensity values.

Expediently, separate subphase difference values are determined for each phase difference value for a plurality of different (disjoint) wavelength ranges. Each phase difference value may then be a scalar formed from a mean value of its (wavelength dependent) subvalues (weighted, for example, based on the errors of the subvalues). Alternatively, the phase difference value may be a vector whose components are the subvalues.

By the sequential modulation with at least two (preferably three or more) different pulse frequencies and thereby separate determination of the phase difference values at different pulse frequencies, one or more mutually different material properties can be determined with significantly higher accuracy than before. This is especially true for strongly different fluorescence lifetimes and also when the respective fluorophores have a spectrally overlapping or even similar emission spectrum. The determination of the substance property succeeds in particular on the basis of a matrix of the ascertained phase difference values which in a first dimension are represented by the respective wavelength (more precisely: by the relevant wavelength range) of the sample light (or an equivalent thereof) and in a second dimension by the pulse frequency (or Equivalent of the same) is parametrized. Expediently, such a matrix is therefore created before the determination of the substance property on the basis of the determined phase difference values. The more nodes this matrix has, ie by measuring at more pulse frequencies or at more wavelength ranges, the higher the accuracy in determining the fluorescence lifetime.

Since it is possible to determine the property of the substance and hence the evaluation of a single sample in a short time, preferably all products of a production process can be investigated in this way instead of (possibly first rejected) samples.

It may be advantageous to discard phase difference values that correspond to light propagation times below a predetermined minimum run time and above a predefined maximum run time (not to be taken into account when determining the fluorescence lifetime). As a result, it can be avoided that light from outside of the sample or sample light originating from a packaging impairs the accuracy of the determination of the property of the substance. Even higher accuracy can be achieved by using a sample with fluorescence-free packaging.

In particular embodiments of the method, a fluorescence quencher may advantageously be introduced into the sample prior to illuminating and picking up sample light. As a result, one or more fluorescence lifetimes can be changed and thus the measurement accuracy can be increased. This is achieved in particular by carrying out a first measuring run (with at least two different pulse frequencies) without a fluorescence quencher and after adding the fluorescence quencher a second measuring pass (with at least two different pulse frequencies, preferably the same as in the first pass). On the basis of the fluorescence lifetime in the presence of the extinguisher, for example, a chemometric model can be created and used in a later determination of the substance property of a regular (extinguisher-free) sample. In addition to the fluorescence lifetime, it is then also possible, for example, to determine the concentration of a fluorescent substance involved.

Advantageous are process forms in which a sample is used, which is surrounded by a closed shell, in particular a liquid sample. For the purposes of the invention, the shell is part of the sample, so that a material property can also be determined by it. For example, the fluorescence lifetime can be determined noninvasively by the sheath. The envelope may in particular be a package. As a result, already packaged products can be evaluated. Preferably, a fluorescence lifetime of an empty shell or a shell filled with a nonfluorescent substance, which can be determined, for example, in a calibration measurement, can be used to determine the material property of the sample. Such a method is particularly advantageous for the examination of a beverage bottled in a bottle.

For example, an upper or lower limit value or an interval of permissible values can be specified for the determined substance property. If an upper limit value is exceeded, a lower limit value is exceeded or an interval is left out, the examined sample can be treated differently from other samples. For example, it can be singled out. Another possibility of the treatment is the classification of examined samples in different quality levels, in particular with identification of the samples with the relevant quality level.

In a particular embodiment of the method according to the invention, a production process for the sample can be controlled on the basis of the determined material property. This can significantly reduce the impact of errors in the manufacturing process.

A problem with a spectroscopic transreflection measurement, in which the illumination light in particular provides a spectroscopic response signal from the upper region of the sample, is the undesired measurement of gloss, ie a directed reflection of excitation light on the sample surface. Therefore, embodiments in which phase difference values that are smaller than a predefined phase difference limit value are not used in determining the property of the substance are advantageous. In this way, for example, a higher accuracy of the determined material property can be achieved by scattered or reflected illumination light (which occurs exclusively during the illuminating light pulse) is not taken into account, because the gloss information reaches the sensor before the actual measurement information in the sample light, which from the inside of the Sample comes. By means of time-resolved spectroscopy, the gloss can be calculated out of the measurement or filtered out by discarding an individual measurement response, ie the measured phase difference value, before a specific time, preferably the pulse duration of the illuminating light pulses. In this way, the effort required in the prior art in the optical design to avoid the effects of gloss on the determined substance property (s) can be reduced or even completely avoided.

It can also be advantageous to use the runtime camera (repeatedly, in particular quasi-permanently) to determine a spatial distance of the sample from the runtime camera. This can be dispensed with an additional distance sensor. With the additional information from which distance the spectroscopically decomposed measurement response comes, correction methods can be used to increase the measurement accuracy and to estimate the plausibility of the measurement. Such a method with distance measurement can be used, for example, in process measurement technology if samples are to be measured spectroscopically at different distances from the measuring device (for example, on a conveyor belt with a different high filling profile). The distance to the sample and thus the optical path, which pass twice will vary. Thus, the influence of the atmosphere in the optical path varies, for example due to moisture. By measuring the exact sample distance, corrections of the absorption of given wavelengths, for example caused by atmospheric moisture, can be included in the spectroscopic measurement in order to compensate for a measurement error resulting from the absorption. It may therefore be expedient to use the determined distance when determining the fluorescence lifetime or for determining a (different) material property of the sample, for example as a correction factor in a calculation rule. Alternatively or additionally, the determined distance can be used in a time-parallel measurement and in the evaluation of such a measurement.

The runtime camera preferably has a one-dimensional or two-dimensional field ("array") of photonic mixer detectors. As a result, spectrally resolved sample light can be recorded with little effort. In the case of a two-dimensional field, not only an approximately punctiform sample spot but also a line-shaped sample region can be measured in a spatially resolved manner. In this way, the measuring accuracy can either be improved (for example by combining the measuring locations by weighted averaging of the relevant phase difference values) or the measurement can be accelerated (for example by separate treatment of the measuring locations).

Advantageously, such embodiments in which the beam path of the runtime camera comprises at least one optical waveguide, wherein the dispersive element between the optical waveguide and the runtime camera is arranged. As a result, the device can be made compact.

Conveniently, the dispersive element is a planitter or an imaging grating or a prism.

The light source preferably comprises one or more semiconductor light sources, for example LED, OLED or laser. Alternatively, gas discharge flash lamps can be used. If the light source itself emits continuously, the illuminating light pulses can be generated from the continuous luminous flux by means of a modulator, which in the sense of the invention is part of the light source. For example, acoustooptic or electrooptical modulators (AOM, EOM) or polarization optical modulators such as Pockels cells or liquid crystal switches can be used. Conveniently, the light source can be cooled to achieve a reproducible excitation wavelength and intensity. This is particularly advantageous because of the different power losses at different pulse frequencies.

The use of multiple (spectrally non-contiguous) excitation wavelengths (excitation wavelength ranges) allows the more accurate determination of time-resolved fluorescence events and phase angles, especially if different fluorophores are present in the sample. Different excitation wavelengths can be generated by means of different light sources or by means of a variable spectral filter in front of a light source emitting in several spectral bands. It is advantageous if the light source (s) emit a spectrally spaced excitation wavelength for each (expected) spectral range of the fluorescence response.

Embodiments are advantageous in which a second dispersive element is arranged in the beam path of the time-of-flight camera whose dispersion direction is arranged at an angle other than zero to the dispersion direction of the first dispersive element. This can improve the spectral resolution.

By designing the device such that a part of each illuminating light pulse of the light source as reference light (without interaction with the sample) arrives at the transit time camera (two-beam setup), the measurement accuracy can be significantly improved. Alternatively, the device may comprise an additional optoelectronic detector, wherein a portion of each illuminating light pulse of the light source passes as a reference light to the detector. Thus, an accurate reference time stamp of the optical excitation is available for each measurement. The runtime camera may, for example, have two optical inputs or a combined optical input for the sample light and the reference light for this purpose. The combined optical input may be, for example, a segmented cross-section converter. With two inputs, the reference light can be imaged into an area of the sensor of the runtime camera, which is not used for the spectroscopic evaluation. Alternatively, the two inputs (including the separate inputs of a combined input) may be provided with optical shutters to allow either sample light or reference light to pass to the sensor.

In all design variants, the reference light can be used as a time reference, for example for a distance measurement, and / or as a spectral intensity reference. The separation of the reference light succeeds in all design variants, for example by means of a beam splitter and / or an optical waveguide. Alternatively, a free-beam optics is conceivable, so that the spectrometer receives light from the light source directly or via mirrors.

The determination of the substance property (s) preferably takes place by means of a compensation calculation, in particular by minimizing a deviation function, in particular deviation squares. This allows a high measurement accuracy with low cost.

Embodiments in which an adaptation of a predetermined or predefinable (two-dimensional or multidimensional) mathematical model, which depends on illumination pulse frequencies and fluorescence wavelengths, to the determined phase difference values are advantageous in the compensation calculation. This allows a high measurement accuracy despite low computational effort, which can optionally be further increased by the inclusion of the intensity and / or amplitude values.

Embodiments in which the determination of the substance property (s) does not take place on the basis of intensity values and / or amplitude values of the runtime camera are particularly preferred. As a result, the substance property (s) can be measured with high precision using the phase difference values despite the low computational complexity.

The determination of the substance property (s) on the basis of a predetermined or predeterminable emission ratio of at least two different types of fluorophore is particularly fast and accurate since the solution space of the mathematical model can be limited by the emission ratio. The emission ratio is preferably assumed and specified as known information about the sample.

Preferably, before the compensation calculation, the fluorescence lifetime of the dominant type of fluorophore can first be determined on the basis of the determined phase difference values or on the basis of phase difference values derived from an empty measurement of a packaging. This allows a particularly high accuracy of the measurement.

The determination of one or more substance properties in the compensation calculation is particularly accurate in that a prescribable or predefined or determined fluorescence lifetime of a dominant type of fluorophore is left constant.

In particular, a first fluorescence lifetime of zero (for a fictitious type of fluorophore representing the excitation light) can advantageously be used in the compensation calculation, and a second fluorescence lifetime can be determined as the substance property. As a result, in particular in cases where the light source emits both in a wavelength range for exciting fluorescence in the sample and in a wavelength range of the fluorescence, the fluorescence of reflected / scattered excitation light in the sample light can be separated. This is preferably used to subsequently determine a reflectance of the sample with compensation of the interfering fluorescence on the basis of the second fluorescence lifetime. Advantageously, further fluorescence lifetimes can be determined in the compensation calculation, in particular based on a model with a corresponding number of fluorophore species (plus the fictitious fluorophore variety with the first fluorescence lifetime of zero).

Preferably, the first and second pulse frequencies are between 1 MHz and 1000 MHz. The same applies to a possible third and further pulse frequencies in further (also synchronized with each other) lighting and recording steps. These frequencies are advantageous because in the majority of known fluorescents, the fluorescence lifetimes are in the range of 0.01 ns to 100 ns.

In an advantageous embodiment, the illuminating light pulses have a sinusoidal or cosinusoidal intensity profile. As a result, the measurement accuracy can be significantly improved. The device expediently comprises means for generating light pulses with sinusoidal or cosinusoidal intensity profile. It may be, for example, a digital signal processor (DSP). Another possibility is to switch behind the output of the (rectangular) pulser a signal delay unit, which causes on average temporal mean sinusoidal-wave delay of the electrical pulses, the delayed pulses are output to the light source and / or to the runtime camera. As an alternative to a signal delay, the harmonic error rejection method can also be used. The generation of the illuminating light pulses preferably takes place in such a way that harmonics of the basic excitation are largely avoided.

It may be advantageous to use a broadband emitting light source and to evaluate multilayer samples using the knowledge of absorption processes of the layer structure, a time-resolved spectral response of the back of the sample and / or the respective penetrated layer structure. In this case, the excitation light passes through each layer of the sample as an optical path twice (transflection). In each layer, part of the excitation light is absorbed by fluorophores and the excitation light for the next layer is spectrally changed in amplitude. The control unit expediently simulates this behavior in a corresponding multilayer model. A broadband emitting light source in the context of the invention is a light source which is in a significant spectral Portion (over a width of at least 400 nm) of the range of 300 nm to 3000 nm emitted. It may be, for example, a thermal radiator, a halogen lamp, or - with a limited wavelength range - a xenon or deuterium lamp. Semiconductor light sources, such as white LEDs, can also emit broadband in the VIS range, for example from 350 nm to 780 nm. It is important that the excitation wavelength range of the fluorophores expected in the sample is included.

• – ein Softwaremodul zum Einstellen des Pulsgebers zur Abgabe von Pulsen einer ersten Pulsfrequenz,
• – ein Softwaremodul zum Ermitteln von ersten Phasendifferenzwerten von Probenlicht mittels der Laufzeitkamera,
• – ein Softwaremodul zum Einstellen des Pulsgebers zur Abgabe von Pulsen einer zweiten, von der ersten verschiedenen Pulsfrequenz,
• – ein Softwaremodul zum Ermitteln von zweiten Phasendifferenzwerten von Probenlicht mittels der Laufzeitkamera,
• – ein Softwaremodul zum Ermitteln mindestens einer Stoffeigenschaft anhand (aufgenommener) erster und zweiter Phasendifferenzwerte.

The invention also includes a control unit and a computer program, configured for carrying out the method according to the invention. The control unit or the computer program may comprise the following components for this purpose:

• A software module for setting the pulser for delivering pulses of a first pulse frequency,
• A software module for determining first phase difference values of sample light by means of the runtime camera,
• A software module for setting the pulser for delivering pulses of a second, different from the first pulse rate,
• A software module for determining second phase difference values of sample light by means of the runtime camera,
• A software module for determining at least one substance property based on (recorded) first and second phase difference values.

These may be different software modules, but it may also be the same software module in the case of several or even all of the named software modules.

Particularly advantageous is the use of a device according to the invention for identifying an object, in particular a banknote, in particular with identification of a fluorescent constituent of the object on the basis of a fluorescence wavelength and / or a fluorescence lifetime. This use thus allows a new security assessment of banknotes, in which not only on the basis of the fluorescence of one or more ingredients in the banknotes the existence of these / these ingredients (s) detected, but also their fluorescence lifetime can be compared with a predetermined value. Expediently, the error intervals of the predefined value on the one hand and the fluorescence lifetime determined on the other hand are compared with one another and the banknote in question is identified as genuine only if the error intervals overlap. The fluorescence lifetime (s) of the ingredient (s) can thus be used as an additional security feature.

• a) Einstellen des Pulsgebers zur Abgabe von Pulsen,
• b) Aufnehmen von Intensitätswerten und zugehörigen Phasendifferenzwerten von an einem dispersiven Element spektral aufgespaltetem Probenlicht mittels der Laufzeitkamera,
• c) Identifizieren einer Teilmenge der Intensitätswerte, deren zugehörige Phasendifferenzwerte kleiner als ein vorgegebener Schwellwert sind,
• e) Ermitteln mindestens einer Stoffeigenschaft anhand der Teilmenge der Intensitätswerte.

A further aspect of the invention relates to the task of improving the accuracy of the measurement of a substance property of a fluorescent sample by means of optical spectroscopy. For this purpose, a device for determining a material property, in particular one of a reflectance, a sample, in particular paper, comprising a modulatable light source for emitting light pulses, a spatially resolving runtime camera, a pulser and a control unit, wherein the light source and the runtime camera with the Pulse generator are connected and arranged in the beam path of the runtime camera for spectrally resolved recording of sample light, a dispersive element provided, the control unit is adapted to perform the following steps:

• a) setting the pulser for delivery of pulses,
• b) recording intensity values and associated phase difference values of spectrally split sample light on a dispersive element by means of the time-of-flight camera,
• c) identifying a subset of the intensity values whose associated phase difference values are less than a predetermined threshold,
• e) determining at least one substance property based on the subset of the intensity values.

• a) Beleuchten der Probe mit Lichtpulsen mittels einer Lichtquelle,
• b) Aufnehmen von Intensitätswerten und zugehörigen Phasendifferenzwerten von an einem dispersiven Element spektral aufgespaltetem Probenlicht mittels einer mit den Lichtpulsen synchronisierten Laufzeitkamera mit einem ortsauflösenden Detektor,
• c) Identifizieren einer Teilmenge der Intensitätswerte, deren zugehörige Phasendifferenzwerte kleiner als ein vorgegebener Schwellwert sind,
• e) Ermitteln der Stoffeigenschaft anhand der Teilmenge der Intensitätswerte.

The control unit preferably carries out the following method according to the invention for determining a substance property, in particular a reflectance, of a sample, in particular of paper, wherein the following steps are carried out:

• a) illuminating the sample with light pulses by means of a light source,
• b) recording intensity values and associated phase difference values of sample light spectrally split at a dispersive element by means of a time-of-flight camera synchronized with the light pulses with a spatially resolving detector,
• c) identifying a subset of the intensity values whose associated phase difference values are less than a predetermined threshold,
• e) Determining the substance property based on the subset of the intensity values.

This further aspect of the invention makes it possible to determine the reflectance both in a wavelength-dependent form and integrated over all the measured wavelengths without the error arising from the fluorescence emission of the sample. This form of determination is particularly advantageous when the light source emits both in a wavelength range for excitation of fluorescence in the sample and in a wavelength range of the fluorescence.

• a) Softwaremodul zum Einstellen des Pulsgebers zur Abgabe von Pulsen,
• b) Softwaremodul zum Aufnehmen von Intensitätswerten und zugehörigen Phasendifferenzwerten von an einem dispersiven Element spektral aufgespaltetem Probenlicht mittels der Laufzeitkamera,
• c) Softwaremodul zum Identifizieren einer Teilmenge der Intensitätswerte, deren zugehörige Phasendifferenzwerte kleiner als ein vorgegebener Schwellwert sind,
• e) Softwaremodul zum Ermitteln mindestens einer Stoffeigenschaft anhand der Teilmenge der Intensitätswerte.

This second aspect of the invention also includes a control unit and a computer program configured to carry out the method. The control unit or the computer program may comprise the following components for this purpose:

• a) software module for setting the pulser for delivery of pulses,
• b) software module for recording intensity values and associated phase difference values of spectrally split sample light on a dispersive element by means of the time-of-flight camera,
• c) software module for identifying a subset of the intensity values whose associated phase difference values are smaller than a predetermined threshold value,
• e) software module for determining at least one substance property based on the subset of the intensity values.

These may be different software modules, but it may also be the same software module in the case of several or even all of the named software modules.

The invention enables the use of fluorescence, phosphorescence and luminescence processes in substances in a non-invasive, technically simple, cost-effective and rapid manner for the determination of sample properties and / or for the differentiation of samples and / or for the quantification / concentration determination of sample components of quiescent or moving samples , Plausibility checks are also possible. Due to the high measuring speed and measuring accuracy, the described methods and devices can be used to determine the ingredients used in the sample and / or their packaging in process measuring technology, bioanalytics, food monitoring and materials technology. For this purpose, the device can be mounted on a crossbeam over a transport device on which samples can be moved past. It is possible to analyze multiple and multi-fluorescence processes and - if necessary - to process the determined data in such a way that they can be used for immediate use in process engineering, especially in ongoing production processes ("in-line", "on-line"). or "at-line"). Also, the optical quality control of the absence of certain unwanted portions of the sample, such as impurities, is possible in this way (indirect measurement method).

The invention will be explained in more detail by means of exemplary embodiments.

In the drawings show:

1 a measuring device in a schematic representation,

2 a detailed section of a measuring device,

3 a measured amplitude and phase spectrum,

4 a measured phase difference matrix,

5 a determined fluorescence lifetime as a function of the wavelength and

6 an explanation of the distinction of reflected light and fluorescence emission in the measurement of reflectivity.

In all drawings, like parts bear like reference numerals.

1 shows a device 1 for time-resolved spectroscopy, in particular for measuring one or more fluorescence lifetimes of in a sample 2 contained fluorophore, in a schematic representation. The sample 2 may be homogeneous or a mixture of substances and each packaged or unpackaged. The device 1 comprises a modulatable, preferably spectrally broadband light source 3 for the emission of light pulses L with adjustable pulse frequency and adjustable pulse shape, for example a light-emitting diode 3.1 (LED) with a driver circuit 3.2 , A higher intensity of the illumination light L allows shorter integration times or higher measurement frequencies, thus a faster analysis of the time-resolved fluorescence and thus a higher measurement accuracy.

The device 1 further includes a spatially resolving runtime camera 4 for example, a PMD sensor 4.1 , in particular in matrix form such as the type "PMD [vision] 3k-S" with a resolution of 64 × 48 pixels, as well as a pulse generator 5 with adjustable pulse rate and a control unit 6 , contains. The pulser 5 here is part of the runtime camera 4 , wherein it has an input for the specification of the pulse frequency to be generated. In alternative embodiments (not shown), the pulser 5 Part of the light source 3 or part of the control unit 6 be. The input for the pulse rate is, for example, with the control unit 6 connected, so that they can specify the pulse rate. The light source 3 and the sensor 4.1 the runtime camera 4 are with the pulser 5 connected and thereby synchronized with each other.

The so-called SBI correction ("Suppression of Background Illumination") of the sensor 4.1 for the suppression of the ambient light / extraneous light by Scaling is preferably switched off so as not to falsify the measurement results. The intensity course of the on the sample 2 The incident illumination light is preferably sinusoidal or cosinusoidal. This can, for example, by a corresponding form of the pulser 5 output electric pulses can be achieved.

From the sample 2 Sample light P emitted in transmission and / or reflection is transmitted via optical elements, here for example an optical waveguide 7 and an entrance slit 8th , on the arranged in the image plane sensor of the runtime camera 4 displayed. In this detection beam path is a dispersive element 9 , For example, a holographic grating, arranged, which causes a spatial-spectral splitting of the incident sample light P. The runtime camera 4 forms with the optical elements 7 . 8th and 9 a spectrometer 10 , with which the sample light P can be detected spectrally and temporally resolved. In 2 is the spectrometer 10 shown in more detail in cross section. In alternative embodiments (not shown), the spectrometer may be coupled to the sample only in free-beam optics.

The intensity and light transit time response of the sample 2 is detected via the excitation phase position of the illuminating light pulses L, wherein the pulse frequency can be varied. This is preferably done independently by the runtime camera 4 , for example by means of an internal algorithm in their sensor 4.1 or in a special (camera-internal or camera-external) control and evaluation unit (not shown). In this case, the time-resolved fluorescence response of the sample is determined from the grouped intensity and light transit time responses of the sample light P and optionally using measurement results from separate dark and / or reference measurements 2 , in amplitude and phase position, determined. The in the sensor 4.1 detected matrix images then simultaneously contain the spectral intensity information of the sample light P and their time resolution relative to the pulses of the pulser 5 in the form of phase difference values (or other physical equivalents of the light transit time).

With a one-dimensional, spatially resolving runtime camera 4 For example, if the dispersion direction of the dispersive element is parallel to the direction of the spatial resolution (pixel line), exactly one time-resolved spectrum of exactly one sample area can be recorded for a sample light pulse. The measurement then yields a number of interpolation points corresponding to the number of pixels for the phase difference and the intensity of the respectively detected sample light, the number of spectral interpolation points being able to be smaller. In 3 is the phase difference Δφ and the intensity I applied as a detector counting rate from such a measurement emission wavelength dependent. Repeating the measurement at other pulse frequencies, one can represent the phase shift both over the emission wavelength and the pulse frequency (see 4 ). With a two-dimensionally spatially resolving runtime camera 4 For example, from different sample areas simultaneously a respective spectrum in each pixel line of the runtime camera 4 when the dispersion direction of the dispersive element 9 is parallel to the lines. Each line then represents a separate detection channel, each having the same number of nodes. Such a multi-channel arrangement can simultaneously measure the spectrally resolved phase difference values and intensities of the sample light from different locations of the sample.

The control unit 6 For this purpose, in a first step, the pulser 5 to a first pulse rate f _{pulse, 1} and turns on the light source 3 one, so the sample 2 illuminated with light pulses and contained fluorophores are excited to fluoresce. By the controlled acquisition of matrix image groups with a specific phase shift of the charge carrier swing of each pixel of the PMD sensor 4.1 of the runtime camera 4 At least two different pulse frequencies of the illumination light L produce raw data of spectral intensities I and the associated phase information, from which the phase difference values Δφ are determined and sent to the control unit 6 be issued.

While the light source 3 the sample 2 Illuminated with fluorescence-exciting light pulses L, the control unit takes 6 over a given integration time by means of the runtime camera 4 a matrix of intensity values and first phase difference values of the spectrally split sample light P on. For example, a coordinate of the matrix indicates the detection channel to which the relevant column of the matrix belongs. It describes the location of the sample 2 from which the sample light P integrated in a matrix element originates. A second coordinate of the matrix indicates, for example, the spectral support point to which a row of the matrix belongs. It therefore describes the spectral interval from which the sample light P integrated in a matrix element originates. Each matrix element contains on the one hand an intensity value and on the other hand a phase difference value.

The control unit 6 then sets the pulser in a second step 5 to a second pulse rate f _{pulse, 2} , which is different from the first pulse rate, and decreases during illumination of the sample 2 by means of the runtime camera 4 another matrix of intensity values and second phase difference values. Accordingly, in a third step, at a third (different from the first and the second) pulse frequency, a third Matrix of intensity values and third phase difference values and generally in the n-th step at an n-th pulse frequency f _{pulse, n} an n-th matrix of intensity values and n-th phase difference values recorded.

The control unit 6 evaluates the phase difference values to reflect one or more fluorescence lifetimes of one or more ingredients of the sample 2 to investigate. There is no analytical way to determine the solution, but mathematical algorithms and regressions are used, which evaluate the possible solutions of the multifluorescence of the fluorophore mixture on the calculation of a best-fit calculation and of several possible solutions / determine the plausible (s).

Preferably, the control unit forms 6 for the compensation calculation from the previously recorded n matrices, a matrix Δφ (f _pulse , λ) of phase difference values whose one coordinate indicates the pulse frequency at which the relevant matrix column was recorded and whose second coordinate indicates the spectral support point from which the relevant matrix row comes. Thus, a separate phase difference matrix or a phase difference matrix averaged from a plurality of (or all) detection channels can be formed for each detection channel. 4 shows such a phase difference matrix. For example, 24 pulse frequencies were measured.

For further evaluation, the control unit 6 For example, a mathematical model of the determined fluorescence lifetime (τ) τ _(i) . The control unit 6 then optimizes the model against the measured phase position difference matrix via a compensation calculation by minimizing an error function towards a (global) minimum. This can include errors such as aliasing. An initial value to be optimized can be specified. 5 shows a (wavelength-dependent) result of an evaluation model of a compensation calculation with minimized deviation χ _φ . In the case of multiple and multi-fluorescence processes, the predetermined optical emission ratio, ie a quantity equivalent to the concentration ratio of the respective fluorophore species, is additionally taken into account in the evaluation and included in the optimization of the model of the fluorescence lifetime τ. As solutions arise one or more fluorescence lifetime (τ) τ _(i) and their emission spectra, which reflect the properties of the sample 2 , therefore, if necessary, of packaging and contents. For example, to determine the fluorescence lifetime at the relevant emission wavelength, a quasi-stable state of the optimization in which the error function is minimal is identified and the fluorescence lifetime is determined from this range. The further course of the curves can serve for the plausibility assessment of the model used, preferably during the calibration of the control unit 6 ,

The compensation calculation becomes more accurate if the (plausible) potential solution space is restricted by knowledge of individual fluorescence processes in the sample, for example from materials engineering or by targeted blank measurements of packaging. To minimize measurement errors, measured values from reference measurements, such as light and dark measurements, can also be used for correction. For example, one or more fluorescence lifetime (s) and / or phase difference value (s) may be derived from one or more dark measurements and / or from one or more measurements of known portions of the sample 2 , For example, a fluorescence response of the empty packaging, are given. This is particularly advantageous in Mehrfachbeziehungsweise Multifluoreszenzvorgängen in the sample 2 , In this case, the determined fluorescence lifetime of the empty packaging and / or of the dominant fluorophore is preferably fixed and (further) fluorescence lifetime (s) released as a variable (s) in the evaluation algorithm. When determining the fluorescence lifetime (s), a plausibility check is performed on the error deviation function.

In addition, the amplitude spectrum (spectral intensity) of the sample light P from the control unit 6 be evaluated. Appropriately, the emission wavelength of the dominant fluorophore is first determined. On the basis of the ripple of the amplitude spectrum, for example, a number of (possible) contained fluorescence transitions can be determined. For each transition, the emission wavelength and the associated fluorescence lifetime are then determined by the method described above. From these values, a chemometric model can be created that can be used in process measurement, for example, for process control or regulation.

Preferably, the sample light P is picked up by regular samples 2 with identical integration time and identical pulse frequencies as in a dark measurement and / or a packaging blank measurement. If cyclic measurement is carried out, for example to reduce measurement errors, the averaging of the measured values of the sample light P from regular samples is preferably carried out 2 , For example, the phase difference values, identical to a dark measurement and / or a packaging blank.

With the described methods and devices, it is possible to spectrally resolve fluorescence processes and at the same time to differentiate substances via the time resolution and to determine solution approaches for unknown fluorophore mixtures via an evaluation of the phase response. By creating a chemometric model and (empirical) calibration of the model for a particular application, it is possible to determine the concentration of one or more fluorescent substances in mixtures. With the reference structure shown, an absolute measurement accuracy for fluorescence lifetimes in the ns range of 0.1 ns and a standard deviation of less than 50 ps is achieved, which can still be significantly increased. The chemometric models can be created with even higher accuracy by additional calibrations after adding "quenchers" to the fluorescers. With the once created chemometric models, the use in the process measuring technology for quantification / concentration monitoring and material plausibility control, even of packaged samples, cost and quickly possible.

A first proof of the measuring accuracy of the described method and the devices described was carried out by analyzing fluorescent security features in bank notes using the example of € 20 and € 50 notes. The individual fluorescence lifetimes of areal green and fibrous blue security features could be detected in spatially close areas.

A second proof of the measurement accuracy of the described method and the described devices was made by the detection of the fluorescence properties in bottles filled with tonic beverage. The quinine content in the tonic beverage was analyzed through the likewise fluorescent packaging (polyethylene with unknown admixtures). The bottles represent a closed envelope around the measured sample and are part of the sample. The (relevant) fluorescence lifetimes and emission spectra on the one hand of the quinine ingredient and on the other hand of the packaging (multifluorescent response) could be determined from the superimposed emission spectrum. By measuring the empty packaging as a reference measurement, it could be demonstrated that the analysis of the multifluorescence processes in the sample leads to the correct solution for the fluorescence lifetime of quinine, in which case prior knowledge of the material properties can support the plausibility of the solution found.

By adding quenchers, in particular chlorides, for example NaCl - in this application, for example, as a quencher for chemically functional groups of quinine -, a change in the fluorescence lifetime could be effected and detected. The invention makes it possible to create a respective chemometrics model for a specific application, with which the concentration of quencher substances in samples can be determined by time-resolved spectroscopy, and thus makes possible an indirect measurement method, for example for quality features of a sample.

The method described and the device described 1 represent a new, cost effective and fast form of optical quality control suitable for direct and indirect measurements.

A particularly advantageous aspect of the invention relates to the determination of the reflectivity of fluorescent samples as a property of matter, for example in the form of a reflectance. For example, principles for determining the (wavelength-dependent) reflectivity of fluorescent samples in the testing of paper and paperboard are known DIN 53145-2: March 2000 , The standard describes a measurement with high equipment complexity, which requires a comparison measurement and also makes special demands on the lighting. For example, excitation light below 400 nm must be excluded by filters. The problem is that the spectrum recorded during the measurement also includes a time-delayed remitted fluorescent light and thus falsifies the resulting reflectance. 6 illustrates the problem that occurs especially in spectral broadband illumination, for example, between 350 nm and 800 nm, which is particularly necessary for white samples such as paper. During the emission of the illumination light L, the sample responds with sample light P, which is stronger by fluorescence remission E than the sample light T (hypothetical) based exclusively on reflection and scattering.

By the use according to the invention of the time-resolved fluorescence measurement, the disadvantages of the measurement can be demonstrated DIN 53145-2 be overcome and a quick, inexpensive measurement possible: When determining a reflectance of a sample 2 , in particular paper, from by means of a runtime camera 4 measured intensity and phase difference values, it may be advantageous to intensity values, their associated phase difference values of a pure reflection in the sample 2 discard (not to be used in the determination of the reflectance). The disturbing fluorescence emission E can be compensated in this way. Phase difference values which correspond to a pure reflection / scattering in the sample can, for example, in a calibration measurement on a (preferably almost ideal) reflector (not imaged) can be determined as a calibration sample. The maximum phase difference value determined in this case for discarding the relevant intensity value can be specified as a comparison condition in a regular measurement: If the phase difference value belonging to an intensity value is greater than the predetermined maximum phase difference value, the relevant intensity value (and the associated phase difference value) is discarded (not included) the determination of the substance property used). Such a measurement of the reflection by means of time-resolved spectroscopy allows reflection measurements without error component by fluorescence emission E or at least with exactly known error component. Thus, with time-resolved spectroscopy, a new, qualitatively better measuring method is available.

As an alternative to discarding readings as a function of sample light response time, a measured reflectance of a sample, particularly paper, can be corrected by fitting a model of a sample with (at least) two fluorophore species, with a first fictitious fluorescence lifetime of zero for one species is given. It represents the excitation light reflected / scattered by the sample. If the sample contains several real fluorophore species, they can be simulated in the model approximately as a single (with a common second fluorescence lifetime). For example, the real / simulated fluorophore species in the sample has a lifetime of 2 ns, which is to be determined to correct for the measured reflectance. For this purpose, the sample is illuminated as described above with at least two different pulse frequencies and recorded spectrally resolved phase difference values. Based on the phase difference values, the model with the two different types of fluorophore is adapted to the measured phase difference values (and optionally to the measured intensity values). By separating into two fluorophore species, the reflection T and the fluorescence E are separated, so that the reflectance can be corrected. Since both a broadband emitting light source and the fluorophores have complex properties and can be described inaccurately by a single fluorescence lifetime, the second fluorescence lifetime is preferably determined as a function of wavelength from the model and the reflectivity (reflectance) is corrected according to wavelength dependent.

LIST OF REFERENCE NUMBERS

1

measuring device

2

sample

3

light source

4

Runtime Camera

5

pulser

6

control unit

7

optical fiber

8th

entrance slit

9

Dispersive element

10

spectrometer

e

fluorescence emission

L

Illuminating light pulses

P

sample light

T

Sample light from reflection

λ

wavelength

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 19951154 A1 [0004, 0004]
• US 2011/007311 A1 [0004]

Cited non-patent literature

• DIN 53145-2: March 2000 [0079]
• DIN 53145-2 [0080]

Claims (21)

Method for determining a substance property, in particular a fluorescence lifetime, of a sample ( 2 ), wherein the following steps are carried out: a) illuminating the sample ( 2 ) with light pulses (L) of a first pulse frequency, b) recording first phase difference values of at a dispersive element ( 9 ) Spectrally split sample light (P) by means of a synchronized with the light pulses (L) runtime camera ( 4 ) with a spatially resolving detector ( 4.1 ), c) illuminating the sample ( 2 ) with light pulses (L) of a second, different from the first pulse frequency, d) recording of second phase difference values of at the dispersive element ( 9 ) spectrally split sample light (P) by means of synchronized with the light pulses (L) runtime camera ( 4 ) and e) determining at least one material property based on first and second phase difference values. Method according to the preceding claim, wherein a fluorescence quencher is introduced into the sample prior to illumination and recording. Method according to one of the preceding claims, wherein a sample is used, which is surrounded by a closed shell, in particular a liquid sample, in particular using a fluorescence lifetime of an empty or filled with a non-fluorescent material envelope for determining the material property of the sample. Method according to one of the preceding claims, wherein a manufacturing process for the sample is controlled on the basis of the determined material property. Method according to one of the preceding claims, wherein by means of the runtime camera, a spatial distance of the sample from the runtime camera is determined, in particular with use of the determined distance in the determination of a feature of the sample. Method for determining a substance property, in particular a reflectance, of a sample, in particular of paper, wherein the following steps are carried out: a) illuminating the sample with light pulses by means of a light source, b) recording intensity values and associated phase difference values of sample light spectrally split at a dispersive element by means of a time-of-flight camera synchronized with the light pulses with a spatially resolving detector, c) identifying a subset of the intensity values whose associated phase difference values are less than a predetermined threshold, and d) Determining the substance property based on the subset of the intensity values. Contraption ( 1 ) for determining a substance property, in particular a fluorescence lifetime, of a sample ( 2 ) comprising a modulatable light source ( 3 ) for the emission of light pulses (L), a spatially resolving runtime camera ( 4 ), a pulser ( 5 ) with adjustable pulse rate and a control unit ( 6 ), the light source ( 3 ) and the runtime camera ( 4 ) with the pulser ( 5 ) and in the beam path of the runtime camera ( 4 ) for the spectrally resolved recording of sample light (P) a dispersive element ( 9 ), characterized in that the control unit ( 6 ) is set up to carry out the following steps: a) setting the pulser ( 5 ) for emitting pulses of a first pulse frequency, b) determining first phase difference values of sample light (P) by means of the time-of-flight camera ( 4 ), c) setting the pulser ( 5 ) for the delivery of pulses of a second, different from the first pulse rate, d) determination of second phase difference values of sample light (P) by means of the runtime camera ( 4 ) and e) determining at least one material property based on first and second phase difference values. Contraption ( 1 ) for determining a substance property, in particular one of a reflectance, of a sample ( 2 ), in particular paper, comprising a modulatable light source ( 3 ) for the emission of light pulses (L), a spatially resolving runtime camera ( 4 ), a pulser ( 5 ) and a control unit ( 6 ), the light source ( 3 ) and the runtime camera ( 4 ) with the pulser ( 5 ) and in the beam path of the runtime camera ( 4 ) for the spectrally resolved recording of sample light a dispersive element ( 9 ), characterized in that the control unit ( 6 ) is set up to carry out the following steps: a) setting the pulser ( 5 b) picking up intensity values and associated phase difference values from at the dispersive element ( 9 ) spectrally split sample light (P) by means of the runtime camera ( 4 ), c) identifying a subset of the intensity values whose associated phase difference values are smaller than a predetermined threshold value, and d) determining at least one substance property based on the subset of the intensity values. Apparatus according to any one of the preceding apparatus claims, wherein the runtime camera comprises a one-dimensional or two-dimensional array of photonic mixer detectors. Device according to one of the preceding device claims, wherein the beam path of the runtime camera comprises at least one optical waveguide, wherein the dispersive element between the optical waveguide and the runtime camera is arranged. Device according to one of the preceding device claims, wherein in the beam path of the runtime camera, a second dispersive element is arranged, the dispersion direction is arranged at a non-zero angle to the dispersion direction of the first dispersive element. Device according to one of the preceding device claims, wherein a portion of each illuminating light pulse of the light source passes as a reference to the runtime camera, in particular by means of a beam splitter and / or an optical waveguide. Device according to one of the preceding device claims, comprising an additional optoelectronic detector, wherein a portion of each illuminating light pulse of the light source reaches the detector, in particular by means of a beam splitter and / or an optical waveguide. Article according to one of the preceding claims, wherein the determination of the material property is carried out by means of a compensation calculation, in particular while minimizing a deviation function, in particular deviation squares. An object according to the preceding claim, wherein an adaptation of a predetermined or predefinable mathematical model, which depends on illumination pulse frequencies and fluorescence wavelengths, to the determined phase difference values takes place in the compensation calculation. An article according to the preceding claim, wherein before the compensation calculation, the fluorescence lifetime of the dominant type of fluorophore is first determined on the basis of the determined phase difference values or on the basis of phase difference values derived from an empty measurement of a packaging. The article according to any one of claims 13 to 15, wherein a first fluorescence lifetime of zero is used in the compensation calculation and a second fluorescence lifetime is determined as the substance property, in particular with subsequent determination of a reflectance based on the second fluorescence lifetime. Article according to one of the preceding claims, wherein the illuminating light pulses have a sinusoidal or cosinusoidal intensity profile. The article of any one of the preceding claims, wherein the light source emits both in a wavelength range for exciting fluorescence in the sample and in a wavelength range of the fluorescence. Control unit or computer program, configured to carry out a method according to one of the method claims. Use of a device according to one of the device claims for identifying an object, in particular a banknote, in particular with identification of a fluorescent constituent of the object on the basis of a fluorescence wavelength and / or a fluorescence lifetime.

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