DE102018129807A1 - Method and system for classifying chemical and biological substances - Google Patents

Abstract

The invention relates to a method and a system for characterizing chemical and / or biological substances.

Description

The present invention relates to a method for the classification of chemical and biological substances, in particular of hazardous substances.

Furthermore, the present invention relates to a system for the classification of chemical and biological substances, in particular of hazardous substances.

It is known to investigate and classify chemical substances by means of time-resolved fluorescence spectroscopy. Is for example in the US 6,272,376 B1 , in the US 8,743,356 B1 , in the WO 2014/145786 A1 as well as in the US 2010/0067003 A1 described.

For the detection of hazardous substances, however, other spectroscopic methods such as Raman spectroscopy or laser-induced plasma spectroscopy can be used. This is for example in the US 8,665,433 B2 disclosed.

Disadvantages of known time-resolved fluorescence spectroscopy method is in particular that a data acquisition takes a long time. It takes about 6 minutes for a measuring cycle. The measuring time can be extended even further if spectroscopy is carried out at different excitation wavelengths. In addition, superstructures are highly complex for this purpose.

Raman spectroscopic methods give very long measurement times due to the low signal intensity typical of the method.

Laser-induced plasma spectroscopy also requires very high laser intensities for the generation of the plasma. This does not allow eye-safe operation and thereby limits in particular an application for the detection of hazardous substances, for example in person checks at airports.

It is therefore an object of the present invention to improve methods and systems for classifying chemical and biological substances.

This object is achieved according to the invention by a method for classifying chemical and biological substances, in particular hazardous substances, in which process a comprehensive or supporting sample containing at least one chemical substance is excited to fluoresce with at least one excitation pulse of electromagnetic radiation, in particular a laser pulse, of an excitation wavelength in which method an intensity of fluorescence radiation emitted by the sample on the basis of the excitation with the at least one excitation pulse is measured as a function of wavelength and in which method a decrease of the intensity of the fluorescence radiation emitted by the sample is measured as a function of time for at least one wavelength and / or one wavelength range, in which method at least one reference spectrum in the form of at least one wavelength-dependent reference fluorescence spectrum of at least one chemical and / or biological substance and in Form the at least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance, the measured wavelength-dependent intensity and the measured time-dependent intensity of the fluorescence radiation emitted by the sample being compared with the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent reference fluorescence spectrum for determining whether or not the sample contains at least one chemical and / or biological substance of a class.

The method proposed according to the invention makes it possible in a simple manner to detect and reliably classify chemical and biological substances by both recording a wavelength-dependent fluorescence spectrum of the sample and measuring a time-dependence of the fluorescence radiation of the sample. Fluorescence signatures are spectrally very broad, so that, for example, different bacteria can only be distinguished from one another under very limited conditions. As a result of the two different measuring methods, virtually two non-redundant signatures of the sample can be taken and compared with corresponding reference spectra. In particular, these measurements can take place simultaneously or virtually simultaneously, so that a measuring time can be significantly reduced in comparison with known methods. In particular, such chemical substances can be classified practically in real time. Only a few seconds of measurement time are required for this. By the wavelength-dependent measurement on the one hand and the time-dependent measurement on the other hand, samples in a shorter time can be classified much more reliable. Through the application of fluorescence spectroscopy with excitation in the In particular, eye-safe radiation sources can be used in the ultraviolet spectral range, making it possible to use the method for the real-time examination of persons for contamination with hazardous substances, for example in security controls.

It is favorable if the wavelength-dependent measurement of the intensity of the fluorescence radiation emitted by the sample and the measurement of the time-dependent decrease of the fluorescence radiation emitted by the sample are carried out simultaneously or substantially simultaneously. This approach has the particular advantage that the total measurement time for the detection and classification of chemical and / or biological substances can be significantly reduced. The wavelength-dependent spectrum and the time-dependent spectrum are therefore not recorded successively, but simultaneously or substantially simultaneously. In this way it can also be achieved that the fluorescence radiation emitted on the basis of the excitation with the at least one pulse is measured both as a function of wavelength and as a time-resolved method. In particular, this ensures that the signals actually originate from the same sample. For example, part of the fluorescence radiation can be coupled out before the wavelength-dependent measurement with a spectrometer via a beam splitter, in order to measure the time signal, that is to say the time profile of the drop in fluorescence radiation.

Advantageously, the sample is excited with at least two excitation pulses of electromagnetic radiation in a short time interval from each other to the fluorescence. In particular, a plurality of excitation pulses can be used. The measurements can thus be carried out, for example, with a high repetition rate, which makes it possible to add up and time the measurement signals. This makes it possible in particular to achieve a good signal-to-noise ratio. The at least two excitation pulses of magnetic radiation can in particular have the same wavelength.

Furthermore, it is advantageous if the sample is excited with at least two excitation pulses of electromagnetic radiation of different excitation wavelengths in a short time interval from one another to the sample. For example, two excitation pulses with different excitation wavelength spaced apart from each other in time can excite the sample to fluoresce. Both a wavelength-dependent fluorescence spectrum and a time-resolved course of the fluorescence signal are then measured for each excitation pulse. This procedure can also be carried out with a high repetition rate, so that both a time-averaged wavelength-dependent spectrum and a time-resolved spectrum can be determined for two different excitation wavelengths. In this way, samples can be characterized even better, as a total of four or more characteristic spectra of the sample can be determined. In principle, this procedure can also be carried out with three, four or more excitation pulses, each of which has a different excitation wavelength. However, the total measurement time will be extended accordingly.

In order to obtain a sufficiently strong fluorescence signal, it is advantageous if the at least one excitation pulse of electromagnetic radiation of an excitation wavelength is generated with at least one excitation laser. In particular, only a single excitation laser can be used here.

Preferably, the at least two excitation pulses are generated with a single radiation source. This significantly simplifies a construction of a system for carrying out a method for classifying chemical and / or biological substances.

Conveniently, the at least two excitation pulses of different wavelengths are generated with a monochromatic radiation source in cooperation with a nonlinear optics. It is also monochromatic radiation sources, so in particular lasers are used, which emit monochromatic electromagnetic radiation having two or more defined wavelengths.

It is advantageous if the at least two excitation pulses are generated simultaneously by the radiation source and then separated in time. In particular, this makes possible a simple system structure in that a temporal correlation of the at least two excitation pulses, in particular if they have a different wavelength, does not have to be effected by complex activation, in particular triggering, of the laser. Rather, a defined temporal separation can be achieved for example by a delay device in the form of a delay line.

Preferably, the at least two excitation pulses will be separated in time such that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample. In particular, it can be ensured that the spectra of the sample, the be measured wavelength dependent and time-dependent, are each assigned to only one of the at least two excitation pulses. This is particularly advantageous if the two excitation pulses have different wavelengths.

Conveniently, the same spatial region of the sample is excited with the at least two excitation pulses. Thus, in particular, it can be achieved that the same substances adhering to the sample are always detected. In particular, when excited with different excitation wavelengths can thus be ensured that the measured spectra can be assigned to the same substance.

It is advantageous if the fluorescence radiation emitted by the sample on the basis of the at least one excitation pulse is measured with a spectrometer depending on the wavelength. In this way, in particular a wavelength-dependent fluorescence spectrum can be determined in a simple manner. With the spectrometer, the fluorescence radiation is spatially split, for example with a dispersive optical element such as a grating or a prism. By means of this wavelength-dependent spatial splitting of the fluorescence radiation, the intensity of the fluorescence radiation for different wavelengths or wavelength ranges can then be detected in a simple manner with one or more detectors and converted into electrical or electronic measurement signals.

An evaluation, in particular an automatic evaluation, of the determined spectra can be simplified, in particular, by generating electronic measuring signals with the spectrometer depending on the wavelength.

It is favorable if a part of the wavelength-dependent generated electronic measuring signals is coupled out and an intensity profile of the same is measured as a function of time. This can be done in particular with an oscilloscope or a fast analog-to-digital converter card for a computer. With this procedure, therefore, not a part of the fluorescence radiation is coupled out and its temporal course is measured, but a part of the already generated electronic measurement signals is decoupled and their time course measured. In this way no influence on the measurement of the wavelength-dependent fluorescence spectrum is taken. In addition, it can thus be ensured in particular that the measurement of both the wavelength-dependent spectrum and the time-dependent spectrum takes place simultaneously. Furthermore, such a construction of a system for carrying out the method for classifying chemical and / or biological substances can be significantly simplified.

Advantageously, the wavelength-dependent generated electronic measurement signals are averaged over a measurement period. In this way, both wavelength-dependent and time-dependent fluorescence spectra can be measured with a good signal-to-noise ratio. As a result, chemical substances can be classified safely and with high accuracy in a short time.

The object stated at the outset is furthermore achieved by a system for classifying chemical and / or biological substances, in particular hazardous substances, which system has at least one electromagnetic radiation source, in particular in the form of a laser, for generating at least one excitation pulse for exciting at least one chemical and / or which system comprises a first spectroscopy device for wavelength-dependent measurement of an intensity of fluorescence radiation emitted by the sample due to excitation with the at least one excitation pulse and a second spectroscopy device for time-dependent measurement of an intensity of the emitted fluorescence radiation for at least one wavelength and / or a wavelength range, which system comprises a memory device for storing at least one reference spectrum in the form of at least one wel and at least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance and which system comprises comparison means for comparing the measured wavelength-dependent intensity and the measured time-dependent intensity of the fluorescence radiation emitted by the sample with the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent reference fluorescence spectrum comprises for determining whether or not the sample contains the at least one chemical and / or biological substance of a substance class.

In such a system can be determined significantly faster compared to known systems with the same accuracy or accuracy in the classification of chemical and / or biological substances, whether the sample of a particular chemical and / or biological class can be assigned. Time-dependent fluorescence spectroscopy can provide additional information on the Characterization of chemical substances that are characteristic of them. For example, a better distinction can be achieved between different substances that have very similar wavelength-dependent fluorescence spectra. Conversely, very similar time dependencies of the fluorescence of different substances can be distinguished more clearly if wavelength-dependent fluorescence spectra are recorded at the same time. Furthermore, it is particularly possible to use such a system to examine in real time samples, for example, to characterize localized aerosols or contaminants on remote surfaces, for example cars.

It is favorable if the system is designed to carry out the wavelength-dependent measurement of the intensity of the fluorescence radiation emitted by the sample simultaneously with the first spectroscopy device and to measure the time-dependent decrease of the fluorescence radiation emitted by the sample with the second spectroscopy device. In particular, the first spectroscopy device may at least partially comprise the second spectroscopy device. For example, an optical structure may be common to or shared by both spectroscopy devices. The simultaneous or substantially simultaneous performance of the measurements makes it possible, in particular, to ensure that the fluorescence radiation under investigation actually originates from the same substances of the sample. In addition, time is gained so significantly in the measurement that, in real time, within a few seconds, it can provide a reliable result as to whether the sample comprises, contains or carries a particular chemical.

It is favorable if the system is designed to excite the sample for fluorescence with at least two excitation pulses of electromagnetic radiation in a short time interval from one another. In particular, the system may be designed to carry out and repeatedly excite the sample so that the measurement signals can be added up and averaged over the total measurement time in order to optimize a signal-to-noise ratio of the measurement. The at least two excitation pulses may in particular have identical or different wavelengths.

It is advantageous if the system is designed to generate at least two excitation pulses of electromagnetic radiation of different excitation wavelengths in a short time interval from one another. Thus, with a first excitation pulse, a wavelength-dependent fluorescence spectrum and a time-dependent fluorescence spectrum can be recorded if the sample is excited at the first excitation wavelength. In a short time interval thereafter, the sample is excited with a second excitation pulse having a different excitation wavelength, and a wavelength-dependent fluorescence spectrum and a time-dependent fluorescence spectrum are also recorded for this excitation pulse. In this way, four measurements can be carried out within the defined short time interval, which provide characteristic results for the chemical and / or biological substances to be detected and classified. These double measurements can in turn be carried out several times in an identical manner with a high repetition rate, wherein it is preferably ensured that the individual measurements do not overlap in time. By carrying out the measurements several times over a few seconds, in particular the respective spectra can be added up and averaged in order to improve a signal-to-noise ratio of the examination results.

Preferably, the at least one radiation source is designed in the form of an excitation laser. With a laser, sufficiently high excitation intensities can be generated, in particular also at distances of a few to a few hundred meters, in order to obtain easily measurable fluorescence signals from the sample. In particular, two or more different radiation sources and in particular excitation lasers may also be included in the system in order to generate excitation pulses with correspondingly different excitation wavelengths.

It is advantageous if the system is designed to generate the at least two excitation pulses with a single radiation source. In this way, a total structure of the system can be significantly simplified. Preferably, excitation pulses of different wavelengths can be generated with the single radiation source. For this purpose, in particular lasers can be used which have two or more emission lines.

It is favorable if the system comprises at least one monochromatic radiation source and a nonlinear optical system cooperating therewith for producing at least two excitation pulses of different wavelengths. If, for example, a laser is used as the monochromatic radiation source, an emission line of the laser can be changed by frequency multiplication to a different wavelength with a nonlinear optics cooperating therewith. So can easily With a single radiation source electromagnetic radiation with different wavelengths are generated.

According to a further preferred embodiment of the invention, it can be provided that the system is designed to simultaneously generate the at least two excitation pulses and that the system comprises a delay device for temporally separating the simultaneously generated at least two excitation pulses. Such a delay device simplifies the structure of the system, since in particular no complicated triggering of the excitation pulses is required. If the two excitation pulses are emitted simultaneously, they can be separated in time by the delay device, for example an optical delay path, by sending one of the two pulses directly to the sample, the other pulse first passing through the delay device. Thus, defined time intervals between successive pulses can be specified with high precision, namely in particular by setting a length of a delay path of the delay device.

It is advantageous if the delay device is designed to separate the at least two excitation pulses over time in such a way that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample. In this way it can be ensured that a fluorescence of the sample has completely decayed as a result of the excitation with a first pulse when the sample is excited again to fluorescence with a second, subsequent pulse. Thus, in addition, the emissions of the sample can be completely separated from one another as a result of the excitations with the different pulses, so that defined spectra can be recorded in particular when excited with different excitation wavelengths.

In order to ensure that the fluorescence spectra which are measured as a result of excitations with different excitation wavelengths also originate from the same substances of the sample, it is favorable if the system is designed to excite the same spatial region of the sample with the at least two excitation pulses. This can be achieved, in particular, by providing an optical setup such that optical axes coincide for the successive excitation pulses.

It is favorable if the first spectroscopy device comprises a wavelength-dependent detection device. In particular, this may be an electron multiplier array. For example, the first spectroscopy device may be in the form of an optical spectrometer comprising a dispersive element, in particular an optical grating or a prism, in order to spectrally disperse the fluorescence radiation emitted by the specimen, that is to spatially split it in particular. Thus, a plurality of detectors arranged spatially next to one another can detect radiation intensities for a plurality of wavelength ranges. Or it can be used for this purpose, an electron multiplier array, with the spatially selective radiation intensities can be converted into electronic signals.

In order to evaluate the results of the measurements quickly and fully automatically, it is advantageous if the detection device is designed for the wavelength-dependent generation of electronic measurement signals. In particular, the measurement signals can be assigned to individual wavelengths or wavelength ranges.

It is advantageous if the system comprises a signal processing device for decoupling a part of the wavelength-dependent generated electronic measurement signals and for measuring a time-dependent intensity profile of the decoupled measurement signals. Such a design of the system makes it possible in particular to manage with a single optical spectrometer. In addition, no splitting of the optical fluorescence signal is required, whereby the measurement results could be falsified. As it were, a time course is measured only after conversion of the optical signal of the impinging optical radiation into electronic signals. This also ensures that both the wavelength-dependent spectrum and the temporal intensity curve originate from the same fluorescence emission of the sample. For example, part of the fluorescence radiation can be coupled out before the wavelength-dependent measurement with a spectrometer via a beam splitter, in order to measure the time signal, that is to say the time profile of the drop in fluorescence radiation.

In a simple manner, a time profile of the intensity of the decoupled measuring signals can be measured if the signal processing device comprises an oscilloscope and / or a fast analog-to-digital converter card for a computer. In particular, it can be a very fast oscilloscope with a time resolution of well below 0.1 ns.

In accordance with a further preferred embodiment of the invention, the system may further be designed such that the signal processing device comprises an integration device for averaging the wavelength-dependent generated electronic measurement signals over a measurement period. With the integration device, the individual electronic signals can in particular be added up and averaged over the measurement period. In this way, a signal-to-noise ratio of the measurement for the characterization of chemical substances can be improved.

Furthermore, the use of one of the systems described above for carrying out one of the methods described above is proposed.

In this way, in particular chemical and biological substances, such as hazardous substances, can be easily and quickly classified. Thus, the system performing any of the methods described above may be used, in particular, for real-time checking of passengers at security controls. This makes it easy to determine whether people and / or their luggage were in contact with hazardous substances, in particular explosives, or not.

1. 1. Verfahren zur Klassifizierung chemischer und/oder biologischer Stoffe, insbesondere von Gefahrstoffen, bei welchem Verfahren eine mindestens einen chemischen und/oder biologischen Stoff enthaltende, umfassende oder tragende Probe (16) mit mindestens einem Anregungspuls elektromagnetischer Strahlung, insbesondere einem Laserpuls, einer Anregungswellenlänge zur Fluoreszenz angeregt wird, bei welchem Verfahren eine Intensität von von der Probe (16) aufgrund der Anregung mit dem mindestens einen Anregungspuls emittierter Fluoreszenzstrahlung (24) wellenlängenabhängig gemessen wird und bei welchem Verfahren ein Abfall der Intensität der von der Probe (16) emittierten Fluoreszenzstrahlung (24) für mindestens eine Wellenlänge und/oder einen Wellenlängenbereich zeitabhängig gemessen wird, bei welchem Verfahren mindestens ein Referenzspektrum in Form mindestens eines wellenlängenabhängigen Referenzfluoreszenzspektrums mindestens eines chemischen und/oder biologischen Stoffs und in Form mindestens eines zeitabhängigen Referenzfluoreszenzspektrums des mindestens einen chemischen und/oder biologischen Stoffs bereitgestellt wird, wobei die gemessene wellenlängenabhängige Intensität und die gemessene zeitabhängige Intensität der von der Probe (16) emittierten Fluoreszenzstrahlung (24) mit dem mindestens einen wellenlängenabhängigen Referenzfluoreszenzspektrum und dem mindestens einen zeitabhängigen Referenzfluoreszenzspektrum verglichen werden zum Bestimmen, ob die Probe (16) den mindestens einen chemischen und/oder biologischen Stoff einer Stoffklasse enthält oder nicht.
2. 2. Verfahren nach Satz 1, dadurch gekennzeichnet, dass die wellenlängenabhängige Messung der Intensität der von der Probe (16) emittierten Fluoreszenzstrahlung (24) und die Messung des zeitabhängigen Abfalls der von der Probe emittierten Fluoreszenzstrahlung (24) gleichzeitig oder im Wesentlichen gleichzeitig durchgeführt werden.
3. 3. Verfahren nach Satz 1 oder 2, dadurch gekennzeichnet, dass die Probe (16) mit mindestens zwei Anregungspulsen elektromagnetischer Strahlung in einem kurzen Zeitabstand voneinander zur Fluoreszenz angeregt wird.
4. 4. Verfahren nach einem der voranstehenden Sätze, dadurch gekennzeichnet, dass die Probe (16) mit mindestens zwei Anregungspulsen elektromagnetischer Strahlung unterschiedlicher Anregungswellenlänge in einem kurzen Zeitabstand voneinander zur Fluoreszenz angeregt wird.
5. 5. Verfahren nach einem der voranstehenden Sätze, dadurch gekennzeichnet, dass der mindestens eine Anregungspuls elektromagnetischer Strahlung einer Anregungswellenlänge mit mindestens einem Anregungslaser (14) erzeugt wird.
6. 6. Verfahren nach einem der Sätze 3 bis 5, dadurch gekennzeichnet, dass die mindestens zwei Anregungspulse mit einer einzigen Strahlungsquelle (12) erzeugt werden.
7. 7. Verfahren nach einem der Sätze 3 bis 6, dadurch gekennzeichnet, dass die mindestens zwei Anregungspulse unterschiedlicher Wellenlänge mit einer monochromatischen Strahlungsquelle (12) im Zusammenwirken mit einer nichtlinearen Optik erzeugt werden.
8. 8. Verfahren nach Satz 6 oder 7, dadurch gekennzeichnet, dass die mindestens zwei Anregungspulse von der Strahlungsquelle (12) gleichzeitig erzeugt und anschließend zeitlich separiert werden.
9. 9. Verfahren nach einem der Sätze 3 bis 8, dadurch gekennzeichnet, dass die mindestens zwei Anregungspulse zeitlich separiert werden derart, dass ein zeitlicher Abstand zwischen zwei aufeinanderfolgenden Anregungspulsen größer ist als eine Fluoreszenzabklingzeit der Probe (16).
10. 10. Verfahren nach einem der Sätze 3 bis 9, dadurch gekennzeichnet, dass mit den mindestens zwei Anregungspulsen derselbe räumliche Bereich der Probe (16) angeregt wird.
11. 11. Verfahren nach einem der voranstehenden Sätze, dadurch gekennzeichnet, dass die aufgrund des mindestens einen Anregungspulses von der Probe (16) emittierte Fluoreszenzstrahlung (24) mit einem Spektrometer (34) wellenlängenabhängig gemessen wird.
12. 12. Verfahren nach Satz 11, dadurch gekennzeichnet, dass mit dem Spektrometer (34) wellenlängenabhängig elektronische Messsignale erzeugt werden.
13. 13. Verfahren nach Satz 12, dadurch gekennzeichnet, dass ein Teil der wellenlängenabhängig erzeugten elektronischen Messsignale ausgekoppelt und ein Intensitätsverlauf derselben zeitabhängig gemessen wird, insbesondere mit einem Oszilloskop (50) oder einer schnellen Analog-Digital-Wandler-Karte für einen Computer.
14. 14. Verfahren nach Satz 12 oder 13, dadurch gekennzeichnet, dass die wellenlängenabhängig erzeugten elektronischen Messsignale über einen Messzeitraum gemittelt werden.
15. 15. System (10) zur Klassifizierung chemischer und/oder biologischer Stoffe, insbesondere von Gefahrstoffen, welches System (10) mindestens eine elektromagnetische Strahlungsquelle (12), insbesondere in Form eines Lasers (14), zum Erzeugen mindestens eines Anregungspulses zum Anregen einer mindestens einen chemischen und/oder biologischen Stoff enthaltenden, umfassenden oder tragenden Probe (16) zur Fluoreszenz umfasst, welches System (10) eine erste Spektroskopieeinrichtung (34) zum wellenlängenabhängigen Messen einer Intensität von von der Probe (16) aufgrund der Anregung mit dem mindestens einen Anregungspuls emittierter Fluoreszenzstrahlung (24) und eine zweite Spektroskopieeinrichtung (34) zum zeitabhängigen Messen einer Intensität der von der emittierten Fluoreszenzstrahlung (24) für mindestens eine Wellenlänge und/oder einen Wellenlängenbereich umfasst, welches System (10) eine Speichereinrichtung zum Speichern mindestens eines Referenzspektrums in Form mindestens eines wellenlängenabhängigen Referenzfluoreszenzspektrums mindestens eines chemischen und/oder biologischen Stoffs und mindestens eines zeitabhängigen Referenzfluoreszenzspektrums des mindestens einen chemischen und/oder biologischen Stoffs umfasst und welches System (10) eine Vergleichseinrichtung (44) zum Vergleichen der gemessenen wellenlängenabhängigen Intensität und der gemessenen zeitabhängigen Intensität der von der Probe (16) emittierten Fluoreszenzstrahlung (24) mit dem mindestens einen wellenlängenabhängigen Referenzfluoreszenzspektrum und dem mindestens einen zeitabhängigen Referenzfluoreszenzspektrum umfasst zum Bestimmen, ob die Probe (16) den mindestens einen chemischen und/oder biologischen Stoff einer Stoffklasse enthält oder nicht.
16. 16. System nach Satz 15, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum zeitgleichen oder im Wesentlichen zeitgleichen Durchführen der wellenlängenabhängigen Messung der Intensität der von der Probe emittierten Fluoreszenzstrahlung (24) mit der ersten Spektroskopieeinrichtung (34) und der Messung des zeitabhängigen Abfalls der von der Probe emittierten Fluoreszenzstrahlung (24) mit der zweiten Spektroskopieeinrichtung (34).
17. 17. System nach Satz 15 oder 16, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum Anregen der Probe (16) zur Fluoreszenz mit mindestens zwei Anregungspulsen elektromagnetischer Strahlung in einem kurzen Zeitabstand voneinander.
18. 18. System nach einem der Sätze 15 bis 17, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum Erzeugen von mindestens zwei Anregungspulsen elektromagnetischer Strahlung unterschiedlicher Anregungswellenlänge in einem kurzen Zeitabstand voneinander.
19. 19. System nach einem der Sätze 15 bis 18, dadurch gekennzeichnet, dass die mindestens eine Strahlungsquelle (12) in Form eines Anregungslasers (14) ausgebildet ist.
20. 20. System nach einem der Sätze 17 bis 19, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum Erzeugen der mindestens zwei Anregungspulse mit einer einzigen Strahlungsquelle (12), insbesondere mit unterschiedlicher Wellenlänge.
21. 21. System nach einem der Sätze 17 bis 20, dadurch gekennzeichnet, dass das System (10) mindestens eine monochromatische Strahlungsquelle (12) und eine mit dieser zusammenwirkende nichtlineare Optik umfasst zum Erzeugen von mindestens zwei Anregungspulsen unterschiedlicher Wellenlänge.
22. 22. System nach einem der Sätze 17 bis 21, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum gleichzeitigen Erzeugen der mindestens zwei Anregungspulse und dass das System (10) eine Verzögerungseinrichtung (18) umfasst zum zeitlichen Separieren der gleichzeitig erzeugten mindestens zwei Anregungspulse.
23. 23. System nach Satz 22, dadurch gekennzeichnet, dass die Verzögerungseinrichtung (18) ausgebildet ist zum zeitlichen Separieren der mindestens zwei Anregungspulse derart, dass ein zeitlicher Abstand zwischen zwei aufeinanderfolgenden Anregungspulsen größer ist als eine Fluoreszenzabklingzeit der Probe (16).
24. 24. System nach einem der Sätze 17 bis 23, dadurch gekennzeichnet, dass das System (10) ausgebildet ist zum Anregen desselben räumlichen Bereichs der Probe (16) mit den mindestens zwei Anregungspulsen.
25. 25. System nach einem der Sätze 15 bis 24, dadurch gekennzeichnet, dass die erste Spektroskopieeinrichtung (34) eine wellenlängenabhängige Detektionseinrichtung (54) umfasst, insbesondere in Form eines Elektronenvervielfacher-Arrays (56).
26. 26. System nach Satz 25, dadurch gekennzeichnet, dass die Detektionseinrichtung (54) ausgebildet ist zum wellenlängenabhängigen Erzeugen elektronischer Messsignale.
27. 27. System nach Satz 26, dadurch gekennzeichnet, dass das System eine Signalverarbeitungseinrichtung (40) umfasst zum Auskoppeln eines Teils der wellenlängenabhängig erzeugten elektronischen Messsignale und zum Messen eines zeitabhängigen Intensitätsverlauf der ausgekoppelten Messsignale.
28. 28. Vorrichtung nach Satz 27, dadurch gekennzeichnet, dass die Signalverarbeitungseinrichtung (40) ein Oszilloskop (50) oder eine schnelle Analog-Digital-Wandler-Karte für einen Computer umfasst.
29. 29. System nach einem der Sätze 27 oder 28, dadurch gekennzeichnet, dass die Signalverarbeitungseinrichtung (40) eine Integrationseinrichtung umfasst zum Mitteln der wellenlängenabhängig erzeugten elektronischen Messsignale über einen Messzeitraum.
30. 30. Verwendung eines Systems nach einem der Sätze 15 bis 29 zur Durchführung eines Verfahrens nach einem der Sätze 1 bis 14.

The above description thus includes in particular the embodiments of methods and systems for the classification of chemical and / or biological substances which are defined below in the form of numbered sentences:

1. 1. A method for classifying chemical and / or biological substances, in particular hazardous substances, in which method a comprehensive or carrying sample containing at least one chemical and / or biological substance ( 16 ) is excited with at least one excitation pulse of electromagnetic radiation, in particular a laser pulse, an excitation wavelength to fluorescence, in which method an intensity of the sample ( 16 ) due to the excitation with the at least one excitation pulse emitted fluorescence radiation ( 24 ) is measured as a function of wavelength and in which method a drop in the intensity of the sample ( 16 ) emitted fluorescence radiation ( 24 ) is measured for at least one wavelength and / or wavelength range in a time-dependent manner, in which method at least one reference spectrum in the form of at least one wavelength-dependent reference fluorescence spectrum of at least one chemical and / or biological substance and in the form of at least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance with the measured wavelength-dependent intensity and the measured time-dependent intensity of the sample ( 16 ) emitted fluorescence radiation ( 24 ) are compared with the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent reference fluorescence spectrum for determining whether the sample ( 16 ) contains at least one chemical and / or biological substance of a class or not.
2. 2. Procedure according to sentence 1 , characterized in that the wavelength-dependent measurement of the intensity of the sample ( 16 ) emitted fluorescence radiation ( 24 ) and the measurement of the time-dependent decrease of the fluorescence radiation emitted by the sample ( 24 ) simultaneously or substantially simultaneously.
3. 3. Procedure according to sentence 1 or 2 , characterized in that the sample ( 16 ) is excited with at least two excitation pulses of electromagnetic radiation in a short time interval from each other to fluorescence.
4. 4. Method according to one of the preceding sentences, characterized in that the sample ( 16 ) is excited with at least two excitation pulses of electromagnetic radiation of different excitation wavelength in a short time interval from each other to the fluorescence.
5. 5. The method according to one of the preceding sentences, characterized in that the at least one excitation pulse of electromagnetic radiation of an excitation wavelength with at least one excitation laser ( 14 ) is produced.
6. 6. Method according to one of the sentences 3 to 5 , characterized in that the at least two excitation pulses with a single radiation source ( 12 ) be generated.
7. 7. Method according to one of the sentences 3 to 6 , characterized in that the at least two excitation pulses of different wavelengths with a monochromatic radiation source ( 12 ) are generated in cooperation with a non-linear optics.
8. 8. Procedure according to sentence 6 or 7 , characterized in that the at least two excitation pulses from the radiation source ( 12 ) are generated simultaneously and then separated in time.
9. 9. Method according to one of the sentences 3 to 8th , characterized in that the at least two excitation pulses are separated in time such that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample ( 16 ).
10. 10. Method according to one of the sentences 3 to 9 , characterized in that with the at least two excitation pulses the same spatial region of the sample ( 16 ) is stimulated.
11. 11. Method according to one of the preceding sentences, characterized in that, due to the at least one excitation pulse from the sample ( 16 ) emitted fluorescence radiation ( 24 ) with a spectrometer ( 34 ) is measured as a function of wavelength.
12. 12. Procedure according to sentence 11 , characterized in that with the spectrometer ( 34 ) Depending on the wavelength electronic measuring signals are generated.
13. 13. Procedure according to sentence 12 , characterized in that a part of the wavelength-dependent generated electronic measuring signals coupled out and an intensity profile of the same is measured time-dependent, in particular with an oscilloscope ( 50 ) or a fast analog-to-digital converter card for a computer.
14. 14. Procedure according to sentence 12 or 13 , characterized in that the wavelength-dependent generated electronic measurement signals are averaged over a measurement period.
15. 15. System ( 10 ) for the classification of chemical and / or biological substances, in particular of hazardous substances, which system ( 10 ) at least one electromagnetic radiation source ( 12 ), in particular in the form of a laser ( 14 ) for generating at least one excitation pulse for exciting a comprehensive or carrying sample containing at least one chemical and / or biological substance ( 16 ) to fluorescence, which system ( 10 ) a first spectroscopy device ( 34 ) for wavelength-dependent measurement of an intensity of the sample ( 16 ) due to the excitation with the at least one excitation pulse emitted fluorescence radiation ( 24 ) and a second spectroscopy device ( 34 ) for time-dependent measurement of an intensity of the emitted fluorescence radiation ( 24 ) for at least one wavelength and / or wavelength range, which system ( 10 ) comprises a memory device for storing at least one reference spectrum in the form of at least one wavelength-dependent reference fluorescence spectrum of at least one chemical and / or biological substance and at least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance and which system ( 10 ) a comparison device ( 44 ) for comparing the measured wavelength-dependent intensity and the measured time-dependent intensity of the sample ( 16 ) emitted fluorescence radiation ( 24 ) comprising the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent reference fluorescence spectrum for determining whether the sample ( 16 ) contains at least one chemical and / or biological substance of a class or not.
16. 16. System by sentence 15 , characterized in that the system ( 10 ) is designed for simultaneously or substantially simultaneously carrying out the wavelength-dependent measurement of the intensity of the fluorescence radiation emitted by the sample ( 24 ) with the first spectroscopy device ( 34 ) and the measurement of the time-dependent decrease of the fluorescence radiation emitted by the sample ( 24 ) with the second spectroscopy device ( 34 ).
17. 17. System by sentence 15 or 16 , characterized in that the system ( 10 ) is designed to excite the sample ( 16 ) for fluorescence with at least two excitation pulses of electromagnetic radiation in a short time interval from each other.
18. 18. System according to one of the sentences 15 to 17 , characterized in that the system ( 10 ) is configured to generate at least two excitation pulses of electromagnetic radiation of different excitation wavelengths in a short time interval from each other.
19. 19. System according to one of the sentences 15 to 18 , characterized in that the at least one radiation source ( 12 ) in the form of an excitation laser ( 14 ) is trained.
20. 20. System according to one of the sentences 17 to 19 , characterized in that the system ( 10 ) is configured to generate the at least two excitation pulses with a single radiation source ( 12 ), in particular with different wavelengths.
21. 21. System according to one of the sentences 17 to 20 , characterized in that the system ( 10 ) at least one monochromatic radiation source ( 12 ) and a non-linear optic cooperating therewith for generating at least two excitation pulses of different wavelengths.
22. 22. System according to one of the sentences 17 to 21 , characterized in that the system ( 10 ) is configured to simultaneously generate the at least two excitation pulses and that the system ( 10 ) a delay device ( 18 ) comprises for temporally separating the simultaneously generated at least two excitation pulses.
23. 23. System by sentence 22 , characterized in that the delay device ( 18 ) is formed for temporally separating the at least two excitation pulses such that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample ( 16 ).
24. 24. System according to one of the sentences 17 to 23 , characterized in that the system ( 10 ) is designed to excite the same spatial area of the sample ( 16 ) with the at least two excitation pulses.
25. 25. System according to one of the sentences 15 to 24 , characterized in that the first spectroscopy device ( 34 ) a wavelength-dependent detection device ( 54 ), in particular in the form of an electron multiplier array ( 56 ).
26. 26. System by sentence 25 , characterized in that the detection device ( 54 ) is designed for the wavelength-dependent generation of electronic measurement signals.
27. 27. System by sentence 26 , characterized in that the system comprises a signal processing device ( 40 ) comprises for decoupling a portion of the wavelength-dependent generated electronic measurement signals and for measuring a time-dependent intensity profile of the decoupled measurement signals.
28. 28. Device by sentence 27 , characterized in that the signal processing device ( 40 ) an oscilloscope ( 50 ) or a fast analog-to-digital converter card for a computer.
29. 29. System according to one of the sentences 27 or 28 , characterized in that the signal processing device ( 40 ) comprises an integration device for averaging the wavelength-dependent generated electronic measurement signals over a measurement period.
30. 30. Use of a system according to one of the sentences 15 to 29 to carry out a method according to one of the sentences 1 to 14 ,

• 1: eine schematische Darstellung eines Ausführungsbeispiels eines Systems zur Klassifizierung chemischer Stoffe;
• 2: eine schematische Darstellung eines Ausführungsbeispiels eines Teils einer Verzögerungseinrichtung;
• 3: eine schematische Darstellung gemessener Laserleistung von Laserpulsen bei einer Wellenlänge von 266 nm (erster Puls) und 355 nm (zweiter Puls) für unterschiedliche Anzahlen von Reflexionen im Bereich der Verzögerungseinrichtung;
• 4: Fluoreszenzspektren für bacillus thuringiensis (gepunktete Linie), micrococcus luteus (gestrichelte Linie) und oligella urethralis (durchgezogene Linie);
• 5: Fluoreszenzpektren mit Anregungspulsen mit einer Wellenlänge von 355 nm für bacillus thuringiensis (gepunktete Linie), micrococcus luteus (gestrichelte Linie) und oligella urethralis (durchgezogene Linie);
• 6: zeitlicher Intensitätsverlauf der Fluoreszenz bei Anregung mit Anregungspulsen mit Wellenlänge von 266 nm für bacillus thuringiensis (gepunktete Linie), micrococcus luteus (gestrichelte Linie) und oligella urethralis (durchgezogene Linie);
• 7: zeitlicher Intensitätsverlauf der Fluoreszenz bei Anregung mit Anregungspulsen mit Wellenlänge von 355 nm für bacillus thuringiensis (gepunktete Linie), micrococcus luteus (gestrichelte Linie) und oligella urethralis (durchgezogene Linie);
• 8: Tabelle mit Klassifikationsergebnissen, wobei a) wellenlängenabhängige Spektroskopiedaten berücksichtigt, b) zeitabhängige Fluoreszenzspektroskopiedaten und c) kombinierte wellenlängenabhängige und zeitabhängige Spektroskopiedaten; Spalten zeigen Vorhersagewerte und Zeilen Referenzwerte an;
• 9: eine schematische Darstellung eines weiteren Ausführungsbeispiels eines Systems zur Klassifizierung chemischer und/oder biologischer Stoffe;
• 10: eine Darstellung gemessener Laserleistung am Ausgang des Lasers für 355 nm für unterschiedliche transversale Positionen eines Metallkeils, gemessen in einem Abstand von 2,1 m vom Ausgang des Anregungslasers mit der sogenannten Knife-Edge-Methode;
• 11: eine Darstellung des Strahldurchmessers für verschiedene Distanzen des Metallkeils relativ zum Ausgang des Anregungslasers;
• 12: eine Darstellung der Abhängigkeit der zeitlichen Verzögerung der Laserpulse in Abhängigkeit von der Anzahl an Durchläufen durch die Verzögerungseinrichtung entsprechend 3;
• 13: eine schematische Darstellung des Aufbaus zur Detektion von Fluoreszenzsignalen;
• 14: eine schematische Darstellung eines Ausführungsbeispiels einer Ladungsintegrationseinheit vom Typ Vertilon Photoniq mit Steuerung;
• 15: eine schematische Darstellung des zeitlichen Verlaufs einer Ladungsintegration mit der Ladungsintegrationseinheit aus 14;
• 16: eine schematische Darstellung des Ablaufs eines Ausführungsbeispiels eines Computerprogramms zur Steuerung eines Systems zur Klassifizierung chemischer und/oder biologischer Stoffe;
• 17: eine schematische Darstellung des Ablaufs der Messprozedur für eine kontinuierliche Messung;
• 18: eine schematische Darstellung der Messprozedur für eine Einzelmessung;
• 19: eine schematische Darstellung der Messprozedur für eine spektrale und zeitliche Messung;
• 20: eine tabellarische Darstellung einer Abklingzeit verschiedener Proben bei Anregung mit unterschiedlichen Wellenlängen, nämlich 266 nm und 355 nm, mit einfacher exponentieller Anpassung;
• 21: Fluoreszenzspektren von Fluorescin in Wasser bei Anregung mit 266 nm und 355 nm;
• 22: zeitabhängige Fluoreszenzspektren von Fluorescin in Wasser von vier Messkanälen entsprechend vier Wellenlängenbereichen;
• 23: Fluoreszenzspektren von Diesel in Wasser bei Anregung mit 266 nm und 355 nm;
• 24: zeitabhängige Fluoreszenzsignale von Diesel in Wasser von vier Messkanälen entsprechend vier Wellenlängenbereichen;
• 25: Fluoreszenzspektren von Curcumin in Aceton bei Anregung mit 266 nm und 355 nm;
• 26: zeitabhängige Fluoreszenzsignale von Curcumin in Aceton von vier Messkanälen entsprechend vier Wellenlängenbereichen;
• 27: Fluoreszenzspektren von NADH und Tryptophan in Wasser bei Anregung mit 266 nm und 355 nm;
• 28: zeitabhängige Fluoreszenzsignale von NADH und Tryptophan in Wasser von vier Messkanälen entsprechend vier Wellenlängenbereichen;
• 29: Fluoreszenzspektren von bacillus thuringiensis bei Anregung mit 266 nm und 355 nm;
• 30: zeitabhängige Fluoreszenzsignale von bacillus thuringiensis von vier Messkanälen entsprechend vier Wellenlängenbereichen;
• 31: einen Beispielaufbau;
• 32: eine interne Taktung des Ladungsintegrators in Abhängigkeit zu den Laserpulsen unterschiedlicher Wellenlänge;
• 33: einen experimentellen Aufbau;
• 34: eine schematische Darstellung der optischen Verzögerungsstrecke;
• 35: eine Verzögerung von zwei aufeinanderfolgenden Pulsen mit unterschiedlichen Wellenlängen;
• 36: Tabellen a) bis c);
• 37: eine schematische Darstellung des experimentellen Aufbaus; die Laserstrahlen des 266 nm und des 355 nm Ausgang werden zur Leistungsanpassung zuerst durch eine Wellenplatte und ein Glan-Taylor Prisma geleitet; Abkürzungen: GT: Glan-Taylor Prisma;
• 38: Bild der Verzögerungsstrecke; und
• 39: eine Programmoberfläche des Experimentkontrollprogramms.

The following description of preferred embodiments of the invention is used in conjunction with the drawings for further explanation. Show it:

• 1 a schematic representation of an embodiment of a system for the classification of chemical substances;
• 2 a schematic representation of an embodiment of a part of a delay device;
• 3 : a schematic representation of measured laser power of laser pulses at a wavelength of 266 nm (first pulse) and 355 nm (second pulse) for different numbers of reflections in the region of the delay device;
• 4 : Fluorescence spectra for bacillus thuringiensis (dotted line), micrococcus luteus (dashed line) and oligella urethralis (solid line);
• 5 : Fluorescence spectra with excitation pulses with a wavelength of 355 nm for bacillus thuringiensis (dotted line), micrococcus luteus (dashed line) and oligella urethralis (solid line);
• 6 : time intensity course of the fluorescence when excited with excitation pulses with wavelength of 266 nm for bacillus thuringiensis (dotted line), micrococcus luteus (dashed line) and oligella urethralis (solid line);
• 7 : time intensity curve of the fluorescence upon excitation with excitation pulses with wavelength of 355 nm for bacillus thuringiensis (dotted line), micrococcus luteus (dashed line) and oligella urethralis (solid line);
• 8th Table with classification results, where a) takes into account wavelength-dependent spectroscopy data, b) time-dependent fluorescence spectroscopy data and c) combined wavelength-dependent and time-dependent spectroscopy data; Columns display predictive values and rows reference values;
• 9 a schematic representation of another embodiment of a system for classifying chemical and / or biological substances;
• 10 : a plot of measured laser power at the output of the laser for 355 nm for different transverse positions of a metal wedge, measured at a distance of 2.1 m from the output of the excitation laser with the so-called Knife Edge method;
• 11 a representation of the beam diameter for different distances of the metal wedge relative to the output of the excitation laser;
• 12 : A representation of the dependence of the time delay of the laser pulses as a function of the number of passes through the delay device accordingly 3 ;
• 13 a schematic representation of the structure for the detection of fluorescence signals;
• 14 a schematic representation of an embodiment of a charge integration unit of the type Vertilon Photoniq with control;
• 15 : A schematic representation of the time course of a charge integration with the charge integration unit 14 ;
• 16 a schematic representation of the sequence of an embodiment of a computer program for controlling a system for classifying chemical and / or biological substances;
• 17 : a schematic representation of the sequence of the measuring procedure for a continuous measurement;
• 18 : a schematic representation of the measurement procedure for a single measurement;
• 19 : a schematic representation of the measurement procedure for a spectral and temporal measurement;
• 20 : a tabular representation of a cooldown of different samples when excited at different wavelengths, namely 266 nm and 355 nm, with a simple exponential fit;
• 21 : Fluorescence spectra of fluorescein in water when excited at 266 nm and 355 nm;
• 22 Time-dependent fluorescence spectra of fluorescein in water from four measurement channels corresponding to four wavelength ranges;
• 23 : Fluorescence spectra of diesel in water when excited at 266 nm and 355 nm;
• 24 : time-dependent fluorescence signals from diesel in water from four measuring channels corresponding to four wavelength ranges;
• 25 : Fluorescence spectra of curcumin in acetone when excited at 266 nm and 355 nm;
• 26 : time-dependent fluorescence signals of curcumin in acetone from four measurement channels corresponding to four wavelength ranges;
• 27 : Fluorescence spectra of NADH and tryptophan in water when excited at 266 nm and 355 nm;
• 28 : time-dependent fluorescence signals of NADH and tryptophan in water from four measurement channels corresponding to four wavelength ranges;
• 29 : Fluorescence spectra of bacillus thuringiensis at 266 nm and 355 nm excitation;
• 30 : time-dependent fluorescence signals of bacillus thuringiensis from four measuring channels corresponding to four wavelength ranges;
• 31 : a sample construction;
• 32 an internal clocking of the charge integrator as a function of the laser pulses of different wavelengths;
• 33 : an experimental setup;
• 34 a schematic representation of the optical delay path;
• 35 a delay of two consecutive pulses of different wavelengths;
• 36 : Tables a) to c);
• 37 a schematic representation of the experimental setup; the laser beams of the 266 nm and 355 nm outputs are first passed through a wave plate and a Glan-Taylor prism for power matching; Abbreviations: GT: Glan-Taylor prism;
• 38 : Image of the delay range; and
• 39 : a program interface of the experiment control program.

1 schematically shows an embodiment of a system 10 for the classification of chemical and / or biological substances. It includes a radiation source 12 in the form of a laser 14 for generating electromagnetic excitation pulses at a total of four wavelengths, namely 266 μm, 355 nm, 532 nm and 1064 nm. The laser is an Nd: YAG laser.

The excitation pulses with an excitation wavelength of 266 nm are passed to the sample via an optical arrangement with mirror and Glan-Taylor prisms (GT) and λ / 2 plates (λ / 2). The laser 14 generated excitation pulses at the wavelength 355 nm are first via a delay device 18 with two mutually inclined by an angle β mirrors 20 and 22 to separate the excitation pulse at 355 nm in time from the excitation pulse at 266 nm. The delay can be changed by changing the angle β, as shown schematically in FIG. Depending on the angle of inclination β, more or fewer reflections are produced for the pulses with wavelength 355 nm between the two mirrors 20 and 22 ,

2 shows schematically the operation of the delay device 18 with the two mirrors 20 and 22 , With every reflection of one of the two mirrors 20 respectively 22 the reflection angle α _{i of} the beam path becomes smaller and smaller until the direction of propagation reverses and the excitation pulse with wavelength 355 nm causes the delay device 18 leave on the side where they entered.

The excitation pulses having a wavelength of 355 nm which are delayed in time relative to the excitation pulses at 266 nm become overlapping with their beam paths to form the sample 16 cleverly.

fluorescence radiation 24 from the sample 16 is with a proof optics 26 comprising a parabolic mirror 28 collected and in two fiber bundles 30 and 32 coupled.

With the fiber bundle 30 a part of the fluorescence radiation becomes a first spectroscopy device 34 comprising a grating spectrometer 36 directed.

From the grating spectrometer 36 The data is sent over a signal line 38 to a data collection device 40 passed, in turn, over a data line 42 to a computer 44 connected is.

The second fiber bundle 32 is with a photomultiplier tube (PMT) 46 connected via a signal line 48 electrical signals to an oscilloscope 50 forwards. The oscilloscope 50 is over another signal line 52 also with the computer 44 connected.

3 shows exemplary time intervals of 266 nm and 355 nm excitation pulses, the angle of the delay device β 18 depend. The time profiles shown from top to bottom differ by four reflections from each other, so that a total of time intervals of up to 120 nanoseconds with the delay device 18 can be adjusted.

4 shows wavelength-dependent fluorescence spectra of three bacterial samples, namely Bacillus thuringiensis (dotted line), Micrococcus luteus (dashed line) and Oligella urethralis (solid line). The samples were excited with excitation pulses with a wavelength of 266 nm.

5 shows wavelength-dependent spectra of the three samples of bacteria when excited with laser pulses at 355 nm.

In 6 are time-dependent fluorescence spectra of the three samples when excited with excitation pulses at a wavelength of 266 nm.

7 shows time-resolved spectra of the three samples with excitation pulses at a wavelength of 355 nm.

In particular, the in 7 The spectra shown clearly differ from each other for the three samples. Similarly, the wavelength-dependent spectra, which in 5 are shown. The temporal courses of the spectra, which in 6 are shown for the three samples are virtually indistinguishable. The wavelength - dependent spectra of Bacillus thuringiensis and Micrococcus luteus in 4 are also difficult to distinguish from each other, but distinct from the spectrum of Oligella urethralis.

8th shows tables of classification results, wherein in table a) only the wavelength-dependent fluorescence data were taken into account, in table b) the time-resolved fluorescence spectroscopy data, and in table c) the combined data sets. The classification was done using a C5.0 decision tree algorithm. For the wavelength-dependent data, an accuracy of approximately 97% was achieved for the assignment, for the time-resolved data and the combined data an accuracy of approximately 98%.

9 schematically shows another embodiment of a system 10 for classifying chemical and / or biological substances. It includes a laser 14 for generating excitation pulses with wavelengths of 266 nm and 355 nm. The simultaneously generated, the laser 14 Spatially separated excitation pulses of different excitation wavelengths become on the one hand, namely the excitation pulses with wavelength 266 nm, directly to the sample 16 passed through an optical arrangement with mirrors, Glan-Taylor prism (GT) and λ / 2-plate (λ / 2), the excitation pulses with wavelength 355 nm via an optical arrangement comprising a delay device 18 with two tilted by a small angle β plan mirror 20 and 22 , The beam paths for the two different excitation pulses are brought to coincide and hit the sample 16 in spatially identical areas.

The one from the sample 16 emitted fluorescence radiation 24 is via a parabolic mirror 28 in a fiber bundle 30 coupled and the first spectroscopy device 34 in the form of a grating spectrometer 36 directed. Via the signal line 38 are those of a detection device 54 the spectroscopy device 34 in the form of a 32-channel photomultiplier tube array (PMT array) 56 to the data acquisition device 40 in the form of a Vertilon Photoniq meter.

Part of the over the signal line 38 supplied electronic signals is in front of the data acquisition device 40 capacitively coupled and via a signal line 58 to the oscilloscope 50 directed. The signal line 58 comprises a total of four signal lines to dissolve four spectral ranges of time in their course. This part of the system forms part of a second spectroscopy device for measuring time-dependent intensity profiles of the emitted fluorescence radiation 24 ,

10 shows by way of example a measured laser power at the output of the laser 14 with wavelength 355 nm for different transverse positions of a metal wedge at a distance of 2.1 m in front of the laser output. The measurement was based on the so-called Knife-Edge method. The laser power during the translation of a sharp anodized metal wedge is measured by the laser beam for different transverse positions of the wedge.

In 11 a beam diameter is shown for different distances of the metal wedge relative to the output of the laser. From the measurements were the beam diameter at the output of the laser 14 to about 7.3 mm and a divergence of the radiation field to 0.35 mrad. Due to the small divergence is the use of the delay device 18 with the two mirrors 20 and 22 This is because the radiation field does not expand too much even after covering a distance of several 10 m.

A pulse length of the laser pulses was determined with a fast photodiode at about 500 ps.

12 shows by way of example the delay time in ns as a function of the number of reflections of the excitation pulses with wavelength 355 nm in the delay device 18 that is, between the mirrors 20 and 22 , As expected, this results in a linear dependence. As described, the number of reflections can be varied by varying the angle β between the two mirrors 20 and 22 be changed, resulting in different maturities.

In 13 is a schematic construction of a part of an embodiment of a system 10 for the classification of chemical and / or biological substances, in particular that part, with which through the fiber bundle 30 guided fluorescence radiation from the sample 16 spectrally and temporally analyzed.

The fluorescence radiation 24 is via a telescope in the fiber bundle 30 coupled and to the grating spectrometer 36 directed. The in the grating spectrometer 36 split fluorescence radiation 24 is converted into electronic signals with the 32-channel PMT array 56.

Via signal lines 38 are the ones from the PMT array 56 generated signals on a total of four memory banks of the data acquisition device 40 directed.

The data acquisition device 40 includes a processor with 32 parallel data preprocessing channels, which are then forwarded to a 16-bit digital signal processor as well as to a processor expansion interface. These communicate with each other and with an SDRAM.

The control is triggered by a trigger pulse of the laser control 60 , in turn, from the computer 44 is controlled. The in the data collection device 40 Recorded measurement data are transmitted via the data line 42 to the computer 44 directed.

The timing of the integration of the measurement signals in the data acquisition device 40 is schematic in 15 shown. At time t ₀ , an excitation pulse at 266 nm is applied to the sample 16 cleverly. With a propagation delay corresponding to t ₀ + t _propagation , the fluorescence _radiation hits 24 at the spectroscopy device 34 on. The one with the PMT array 56 generated measuring signals are in the banks 1 and 2 the data acquisition device 40 saved.

At the time t _delay , an excitation pulse 355 nm to the sample 16 cleverly. The delay time t _delay corresponds to the duration of the excitation pulse by the delay device 18 , Once again offset by the transit time t _propagation hits the fluorescent radiation 24 in response to the excitation pulse at wavelength 355 nm at the spectroscopy device 34 on. The electrical signals of the PMT array 56 are in the data collection device 40 in the benches 3 and 4 stored.

In 16 schematically is a sequence of a computer program for controlling the experiment, so the fluorescence measurement for characterization of the sample 16 shown. The program is based on the LabVIEW graphical programming system and is used to control the experiment and data storage.

Through a transistor-transistor-logic (TTL) signal generated by a National Instruments (NI) multifunction data acquisition card, the computer program controls a laser shutter which holds the sample 16 protects against unwanted laser radiation. Furthermore, the above-described components of the data acquisition device, namely the Vertilon PhotoniQ and the oscilloscope, are initialized and read out. The "state machine program structure" used makes it possible to wait for events after initialization of the program and then to go through different loops depending on the event. This makes it possible to change parameters for data acquisition and to control the laser shutter. After starting a measurement, depending on the setting of measuring sequence variables, the in the 17 to 19 go through the measuring procedures shown schematically.

In the 17 schematically illustrated continuous measurement is used primarily for adjusting the system. After the start, successive individual measurements are carried out and graphically displayed.

At the schematic in 18 shown single measurement only a spectral information is read and, if desired, also stored.

The measuring procedure for a spectral and temporal measurement is schematically in 19 shown. In doing so, both the oscilloscope 50 as well as the charge integrator of the data acquisition device 40 read out and graphically display the results. Optionally, the data obtained can be stored in two different files.

In the 21 to 30 are wavelength-dependent and time-resolved measurements on a total of five samples.

In the 21 . 23 . 25 . 27 and 29 spectrally resolved spectra for the different excitation wavelengths 266 nm and 355 nm are shown, depending on the respective channel of the PMT array 56 ,

In the 22 . 24 . 26 . 28 and 30 the time dependence of the fluorescence of four channels is plotted as a function of the measuring time. After about 20 ns in each case the fluorescence signal can be seen, which is generated by the excitation of the laser pulse with a wavelength of 266 nm. The signal at 140 ns is generated by the laser pulse with a wavelength of 355 nm. The time shift of about 120 ns between the signals is due to the length of the delay line of the delay device 18 as described above.

Five samples were tested, namely fluorescein in water ( 21 and 22 ), Diesel in water ( 23 and 24 ), Curcumin in acetone ( 25 and 26 ), NADH and tryptophan in water ( 27 and 28 ) as well as Bacillus thuringiensis ( 29 and 30 ).

Different time constants for the two excitation wavelengths were determined for the five investigated samples. The cooldowns derived from a simple exponential fit are shown in the table in 20 represented for the respective samples. In the 20 given values do not correspond directly to those which correspond in particular by means of time-correlated single photon counting of certain fluorescence lifetimes, as in the present method carried out other factors must be considered. Here, the measured courses are the convolution of the excitation pulse, the average fluorescence lifetime and the temporal device function of the data acquisition device 40 and the detection device 54 together.

As shown in particular by the different wave-dependent fluorescence spectra and the significant differences in the time-resolved spectra, different substances can be used with the described system 10 and the method described can be distinguished reliably. All this is possible especially with measurement times of a few seconds with a hit probability close to 100%.

A database is provided that is stored in a storage device of the system 10 can be stored, the measured spectral and time-dependent data with the computer 44 be automatically compared with reference spectra of certain chemical and / or biological substances and, for example via an output device, in particular one with the computer 44 connected screen, an operator to be displayed. So it is possible in particular, the system 10 to use for the detection and classification of hazardous substances. For example, with such a system samples can be examined in real time, in particular localized aerosols or contaminants on remote surfaces, for example of cars, can be characterized and classified. Thus, known hazardous substances with a very high probability of occurrence can be assigned to defined substance classes in real time without complicated chemical detection methods.

Such systems can be used in particular in security research, where hazardous substances must be detected from greater distances. Systems can also be used in laser-induced fluorescence spectroscopy and microscopy as well as for medical diagnostics and process monitoring. The described systems and methods can also be used for material characterization.

DESCRIPTION OF OTHER EMBODIMENTS OF THE INVENTION

EMBODIMENT 1

In particular, the invention serves to improve the non-contact and long-range early detection of chemical and biological hazardous substances by means of a combination of two laser-induced fluorescence methods. Laser-induced fluorescence spectroscopy is a proven method for analyzing substance concentrations of gases and liquids and can be used for substance classification. Using adapted experimental setups, fluorescence lifetimes can be determined by means of time-resolved or frequency-modulation techniques, which also facilitate a substance differentiation. The developed system combines these two methods. In a process run, electronic signals are measured, which can be assigned to the spectral and temporal behavior of the laser-induced fluorescence (LIF), which is excited by two or more laser pulses of electromagnetic radiation of different wavelengths. As a result, a large amount of non-redundant information for differentiating different hazardous substances can be provided in a very short period of time.

• • Relativ geringer Informationsgehalt.
• • Es muss überprüft werden in wie weit man mit dem Informationsgehalt eine große Menge an Stoffen unterscheiden kann.

Fluorescence spectroscopy is an established technique used in biochemistry, chemistry and medicine for the analysis of organic matter. For this purpose, the sample is irradiated with electromagnetic radiation, usually in the ultraviolet spectral range, and the backscattered light spectrally analyzed. Compared to other spectroscopic methods, such as Raman spectroscopy, the method is very well suited for non-contact, long-range remote detection (Lakowicz, JR, Principles of Fluorescence Spectroscopy, 3rd Edition, Springer Science + Business Media, New York, 2006). The disadvantage is:

• • Relatively low information content.
• • It has to be checked to what extent one can distinguish a large amount of substances with the information content.

• • Für Frequenzdomänen-Messungen aufwendige Verfahren benötigt, so dass das Signal nicht in Echtzeit erfasst werden kann.
• • Bei der zeitkorrelierten Einzelphotonenzählung ist eine große Statistik erforderlich, was zu einer sehr langen Messzeit führt.

Fluorescence lifetime establishments can be divided into two classes, the time domain and the frequency domain measurements. In the former, the sample is excited by means of short laser pulses and the decay time is extracted directly from the measured time course of the fluorescence, for example by the method of time-correlated single-photon counting. In the second class, the intensity of the continuously exciting radiation is modulated in time and the decay time from the phase shift of the emitted to the exciting radiation is determined. Both methods were used for the identification of biological and chemical samples (Lakowicz, JR, Principles of Fluorescence Spectroscopy, 3rd Edition, Springer Science + Business Media, New York, 2006; Kinkennon, AE & McGown, Fluorescence Lifetime Characterization of Bacteria Using Total Lifetime Distribution Analysis with the Maximum Entropy Method, LB Journal of Fluorescence 7: 201 (1997) doi: 10.1007 / BF02758220). The disadvantage is:

• • For frequency domain measurements consuming procedures needed, so that the signal can not be detected in real time.
• • Time-correlated single-photon counting requires large statistics, resulting in a very long measurement time.

• • Für direkte Messung aufwendige und teure Taktung der Kamera zur Zeitmessung nötig.
• • Keine Messung in Echtzeit möglich.

In addition, the measurement of the temporal behavior of the fluorescence signal over a nanosecond clocked iCCD camera can be done (T. Fischbach, F. shower, A. Hausmann, C. Pargmann, V. Aleksejev, L. Poryvkina, I. Sobolev, S. Babichenko, and J.Handke.Standoff detection and classification procedure for bioorganic compounds by hyperspectral laser-induced fluorescence, may 2015). The disadvantage is:

• • For direct measurement expensive and expensive clocking of the camera for time measurement necessary.
• • No real-time measurement possible.

• • Geringe Signal Intensität und daraus resultierende lange Messzeiten.

Other spectroscopic methods such as Raman spectroscopy can be used for the detection of hazardous substances ( US 201113150757 Standoff explosive detector using deep-uv raman spectroscopy). The disadvantage is:

• • Low signal intensity and resulting long measurement times.

Advantages of the invention

• Increasing the information content by measuring the spectral and temporal fluorescence signals with multiple laser pulses at a different carrier frequency.
• • Measurement with high repetition rate, by measuring the spectral and temporal fluorescence signals within one process run.
• • The spectral and temporal fluorescence signals are generated and read out in a single process so that the signal generated by individual laser pulses of different wavelengths can be recorded.
• • To increase the information content and the resulting fast data acquisition, fluorescence spectroscopy and determination of the fluorescence lifetime are combined.

method

First, two or more laser pulses simultaneously provided by a laser source, each having different wavelengths, are separated in time. In the case of two used laser pulses of different wavelengths, a laser pulse is coupled into a delay line consisting of two mirrors tilted slightly to each other, so that the pulses are reflected back and forth between the mirrors and leave on the side of the delay line on which they were coupled. As a result, the laser pulses have a time interval which should be greater than the fluorescence decay time to be measured. This is usually less than 50 nanoseconds, which corresponds to a distance of about 15m. After the time separation, the beam paths of the pulses of different wavelengths are superimposed, so that the two laser pulses hit each other successively at the same spatial position on the sample. The fluorescence signal generated in the sample is coupled by means of a parabolic mirror into a spectrometer and here spectrally dissected over an optical grating, before depending on is detected by the wavelength by means of different channels of a detector. The electrical signal generated by the detector is passed to an integrator, which can be read by a computer. The idea is that part of the signal is capacitively decoupled from the detector before the integrator for the direct measurement of the time course. This signal is forwarded to a fast oscilloscope with the help of which it can be measured time-resolved and the data can also be read out by a computer.

In 31 an embodiment of the described invention is shown schematically. The short laser pulses (<1ns) having a wavelength λ ₂ output by a pulsed laser are coupled into a delay path. After passing through the path, the beam path of this laser pulse is superimposed with the beam path of the laser pulse with a wavelength λ ₁ , so that a pulse train of two laser pulses of different wavelengths are delayed relative to each other by 50-100 ns (see 31 ). The two laser pulses hit the sample one after the other and generate an excitation, which leads to fluorescence. The fluorescence radiation is emitted from the sample in all directions and a part is coupled via a detection mirror into a glass fiber with the aid of which the fluorescence radiation is conducted into the spectrometer. In the spectrometer, the fluorescence radiation is spectrally dissected by a diffraction grating and guided wavelength-specifically on a 32-channel photomultiplier array in which the measured photons are converted into electronic signals. These signals are forwarded to a charge integrator, which has been developed by the company Vertilon so that it has an internal clocking. This allows the control of the time interval of successive integrations in the nanosecond range with integration times of at least 50 ns. The 32 inputs of the integrator are assigned to 4 so-called banks, whereby the data of each input are forwarded to two banks and each 16 inputs are assigned to a bank. As in can be seen, the banks 1 & 2 are driven so that the signal of the fluorescence spectrum is integrated after excitation with laser radiation of wavelength 266nm at a time t ₀ + t _propagation over 50 ns. After a delay of t _delay which can be set as a function of the adjustment of the tilt angle of the delay path, the integration of banks 3 & 4 is then started, in which the signal of the fluorescence spectrum is integrated after excitation with laser radiation of wavelength 355 nm over 50 ns. In the respective banks, the signals are digitized and forwarded to the experiment control computer.

Before the integrator, a small part of the electronic signals is capacitively decoupled. The decoupled signals are used to directly measure the temporal behavior of the fluorescence with a fast oscilloscope. The measurements are controlled by a computer to which the measurement data are forwarded for further processing and storage.

This measurement method allows the simultaneous measurement of the spectral and temporal course of the fluorescence, so that here an increased information content can be obtained from the excitation of two successive laser pulses of different wavelengths. The system can be extended to a plurality of excitation pulses, in which case several pulses would have to be coupled into the delay path at different angles, so that a pulse train results from the utilized pulses of different wavelengths. Alternatively, a system of laser sources could be used in which the laser are electronically clocked so that the desired pulse train is formed.

Areas of commercial applications

• • Stand-off detection of hazardous substances
• • Laser-induced fluorescence spectroscopy and microscopy
• • process monitoring
• • Material characterization

EMBODIMENT 2

publication

New long-range detection system for the classification of chemical and biological hazardous substances by combining temporal and spectral laser-induced fluorescence techniques

introduction

There are different scenarios in which people can come into contact with chemical or biological hazardous substances. Depending on the respective scenarios, the dangerous substances can reach people in different ways, for example via the water supply and aerosol clouds [1, 2]. In order to be able to take rapid action against negative impacts on humans, fast and long-range detection, and hence a safe distance, and an identification system for sources of biological solutions are essential. Different optical techniques can be used as a basis for such a detection system. Some of these have been studied in references [3, 4].

One of the most commonly used techniques for long-range detection of biological and chemical samples from a safe distance is laser-induced fluorescence (LIF) spectroscopy. This provided high signal strength with fast detection times and large detection ranges [5-9]. For aerosol samples, LIF spectroscopy is often combined with so-called LIDAR measurements ("light detection and ranging"). LIDAR measurements are based on photon transit time measurements. These techniques are capable of rapidly locating a threat and mapping the distribution of clouds and their spread, as shown for example in various field trials [3, 7, 9]. Depending on the particular scenario, a promising approach is the combination of long-range detection systems with point sensors to increase detection reliability [10].

Here is presented a novel construction in which temporal and spectral LIF techniques are combined to enhance the specificity of the classification algorithms used to distinguish bacterial samples. For wavelength dependent resolved LIF measurements, the rather broad spectral properties of the examined samples allow a reduction of the spectroscopic resolution of 1024 spectral channels in reference [5] to 32 spectral channels without reduction of the classification performance as presented in reference [11]. But compared to other techniques, the low information content only allows limited identification. Laser pulses of different center wavelengths can be used to excite different fluorophores [5, 12], for example amino acids tryptophan and tyrosine, using a laser wavelength of, inter alia, 266 nm, or molecules that occur in the metabolism of living cells, such as reduced nicotine amide Adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), using a laser wavelength of, inter alia, 355 nm. The different spectral shapes of the fluorescence signals after excitation with these different laser pulses provide unique information for the subsequent classification procedure. By combining data sets of both excitation processes, the classification performance can be significantly improved [5, 11, 13]. In order to further improve the specificity in the presented system, temporally resolved LIF signals were simultaneously acquired for each excitation pulse using fast electronics. Since the temporal behavior of fluorescence depends on the components involved, and in particular their microenvironments, the results may even vary between substances of similar chemical composition [12, 14, 15]. As shown below, this variation provides sufficient diversity to distinguish three selected bacterial suspensions that were excited and measured from a distance of 3.5 m with an accuracy of 98%. The distinctness of further different bacterial samples will be investigated in the future. The combination of time-resolved fluorescence data from bacterial samples determined from a given distance was used for the first time for the following classification. The classification performance is comparable to that when simultaneously spectrally resolved LIF data is acquired and with the combination of both data sets.

Experimental construction

In schematically an embodiment of the structure is shown. A Nd: YAG laser system of the type Innolas Picolo Magna EVO III is used, which provides laser pulses with four different center wavelengths with a repetition rate of 100 Hz. The pulse durations of all laser pulses are below 0.7 ns. For LIF excitation, pulses in the ultraviolet spectral range were at 266 nm and 355 nm used. After passing through an arrangement for controlling the intensity of the pulses, the pulses having a wavelength of 355 nm were delayed in time using an optical delay line [16]. This comprises two nearly parallel mirrors, which are tilted against each other by a small angle β, as shown in FIG .a) is shown. Therefore, the reflection angles α _{i of} the laser beam path become smaller for each subsequent reflection, so that after a certain number of reflections, depending on the inclination angle β, the laser pulse changes its propagation direction and returns to the side of the delay path where it has entered. In this way, the extent of the mirror can be used twice, which reduces the size of the structure. After spatially overlapping the beam paths of the laser pulses with the wavelengths 266 nm and 355 nm, these are sent to the sample.

The combined beam path is directed to a fast photodiode and the resulting measurements are in .b). A delay of up to 120 ns is achieved, which corresponds to a running distance of 32 m with a mirror distance d of approximately 0.8 m. The experiment uses a delay of approximately 100 ns between two pulses and a repetition rate of the pulse trains of 100 Hz.

33 : shows an experimental setup: A Nd: YAG laser simultaneously generates laser pulses with two different wavelengths in the ultraviolet spectral range. After delaying the pulses with wavelength 355 nm, their beam paths are combined with the laser pulses with a wavelength of 266 nm and sent to the sample. The fluorescence response of the sample is measured with a setup comprising optical filters and photomultiplier tubes (PMT) and a spectrometer with a PMT array. Subsequently, the electronic signals are read out with a data acquisition system and a fast oscilloscope (λ / 2: (λ / 2 plate; GT: Glan-Taylor prism).

34 shows a schematic representation of the optical delay path, 35 a delay of two consecutive pulses of different wavelengths measured with a fast photodiode: from top to bottom, the angle β was decreased to allow four additional reflections for each subsequent measurement.

The fluorescence response is focused on two approximately 2 mm diameter fiber bundles using a 101.6 mm diameter parabolic mirror. One of the fiber bundles directs the radiation into a compact grating spectrometer (Hamamatsu A10766) combined with a 32-channel photomultiplier tube (PMT) array (Hamamatsu H7260). The electrical signals are read out by an adapted data acquisition system (Vertilon Photoniq IQSP482). The data acquisition system provides fast internal gating to allow collection of spectral fluorescence data from successive 266 nm and 355 nm laser pulses. In other words, it includes two so-called banks and each bank is connected to the 32 PMT channels. After a settable time equal to the duration of the radiation to travel from the sample to the detector, the first bank is triggered and starts the integration for 50 ns. After a time delay resulting from the delay of the two successive laser pulses, the second bank is triggered and also integrates the fluorescence signals of the second excitation for 50 ns. The second fiber bundle directs the radiation to an array of optical filters and photomultiplier tubes connected to a fast oscilloscope (Keysight DSO-X 6004a) to acquire the temporal fluorescence data at a resolution of 0.05 ns. The time base of the data acquisition is a trigger pulse generated by the laser control. The data acquisition parameters are controlled by a self-developed LabView [17] program.

Experimental results and discussion

The identification possibilities using the described system were investigated for bacterial samples by measurements of three different bacterial solutions. The measurements were performed at a distance of 3.5 m and with optical pulse energies of approximately 30 μJ and 50 μJ for laser pulses at 266 nm and 355 nm. The samples were prepared as described in reference [5], but an agar 1 broth (Sifin, Berlin, Germany) was used without blood instead of blood agar plates. The bacterial solutions were diluted with phosphate buffered saline (PBS) to give a concentration of the order of 10 ⁸ to 10 ⁹ colony forming units per ml.

Measurements were taken within two hours of preparation as decreasing food supply may affect the spectral shape of the bacterial samples, as examined in reference [18]. In 4 and 5 For each three samples, one hundred consecutive single excitation spectra and their mean were plotted. For excitation at energies corresponding to a wavelength of 266 nm, the spectral properties of Bacillus thuringiensis and Micrococcus Luteus almost similar, so that a clear assignment is impossible. However, the data for Oligella urethralis show a different spectral behavior, as in 4 to recognize. For excitation at 355 nm (see 5 ), the distinctness of the fluorescence signals is more pronounced for the three samples. By using the complete spectral data sets, the specificity of the classification is improved and thus a nearly unique assignment of the bacterial samples is possible.

4 to 7 : Spectral ( 4 and 5 ) and time ( 6 and 7 ) resolved LIF signals after excitation with laser pulses at wavelengths of 266 nm ( 4 and 6 ) and 355 nm ( 5 and 7 ). In order to obtain time-resolved LIF signals, optical bandpass filters were used at 310 nm ( 6 ) and 460 nm ( 7 ) used. For better visibility of the differences within the data sets, the opaque lines show the mean of 100 spectra acquired within one second and the transparent lines show 100 single excitation spectra to illustrate excitation variations. Dotted line: Bacillus thuringiensis; dashed line: Micrococcus luteus; solid line: Oligella urethralis.

FIGS. 6 and 7 show the corresponding time-resolved fluorescence data. For the measurements, different optical filters were placed at 310 nm and 460 nm with an optical bandwidth of 10 nm in front of the photomultiplier tubes. The time-dependent fluorescence signal of the different samples, excited at 266 nm and measured at 310 nm, can not be distinguished. When excited at 355 nm, the time signals show distinguishable shapes.

The identification performance of the system was evaluated using the C5.0 Discrimination Tree algorithm. One set contains 500 single-pulse, spectral and temporal LIF measurements for each bacterial sample that was analyzed. Within the dataset, 75% of the data was used for training purposes and 25% for test purposes. The results of the classification are given in Tables a) to c) of 36 shown.

Table of 36 : Classification results using a) the LIF spectral data, b) the time-resolved LIF data, and c) the combined data set. Columns: predictions; Rows: References.

For the spectral data an accuracy of 97% was achieved, whereas the temporal data provided an accuracy of 87%. For the combined data set, the accuracy is 98%, indicating that the fluorescence decay determines the performance of the classification when the combined data set is used for the three selected samples.

literature

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EMBODIMENT 3

report

introduction

At the present time, the early detection of chemical and biological hazardous substances as well as explosives is due to the increasing number of u.a. Industrial accidents and terrorist attacks is a very important issue. In particular, the non-contact detection of hazardous substances is becoming increasingly important, as this can avoid the risk of contamination or contact of people with the respective hazardous substances. Here, laser-based remote detection is one of the most promising methods, as it allows the targeted, long-range propagation of relatively high energies, which allows an information query at a great distance. In this regard, a demonstration experiment for the non-contact detection and classification of hazardous substances was established at the German Aerospace Center (DLR) in Lampoldshausen (4).

Based on this experiment, it was proposed to carry out further experiments on a new experimental set-up. In these experiments, the possibility of identifying or classifying different hazardous substances using spectrally and temporally resolved fluorescence signals is investigated. This new structure is presented in this report.

The decay time of the fluorescence in the responsible chemical compositions, called fluorophores, is usually in the low nanosecond range (7). However, it can also deviate greatly and be chemically influenced, so that lifetimes from a few picoseconds to milliseconds were measured (2, 5). For mixtures, the time courses of the fluorescence can vary greatly spectrally due to the different contributions of the contained fluorophores. This has been demonstrated, for example, for diesel blends where lifetimes in the range of 7 ns to 100 ns were measured in a spectral range of 300 nm-423 nm (3). For bacteria, fluorescence lifetimes ranging from 0.5 ns to 15 ns were measured (6). Here, the cooldown is strongly dependent on the microenvironment of the fluorophores in the bacteria, allowing a distinction to be made between a small number of bacteria tested and the measured tryptophan fluorescence lifetimes (1).

It will be investigated to what extent the measurement of the fluorescence lifetime as additional information for the spectral fluorescence measurement, can be used in the differentiation of different chemical and biological hazardous substances. In addition, it will be investigated whether the classification can be realized with the help of a more compact design compared to the existing demonstrator experiment. It became the in 38 planned and realized experimental setup shown.

For the classification of the different samples, high resolution spectra were recorded in the demonstrator experiment by means of a camera based on an intensified charge coupled device (ICIC), after which the fluorescence signal was integrated and analyzed over a certain spectral range to reduce the amount of data. To make the setup more compact in the experiment under study, a 32 channel photon multiplier tube array ("Photomultiplier Tube PMT, Hammamatsu 7260a) is used instead of a high resolution iCCD camera to capture the fluorescence signals. This provides 32 channels instead of the 1024 channels of the iC-CD and represents a great simplification of the system. However, this structure allows the real-time measurement of the fluorescence behavior with a resolution in the nanosecond range due to the very high sensitivity of the PMTs (single-photon detection) after excitation by a single laser pulse. In order for the fluorescence signal to be resolved in time, the excitation laser pulse should be shorter than the fluorescence lifetime. Therefore, a new short-pulse laser system was procured, which provides laser pulses with a pulse duration of <500 ns. This system is described in more detail in Chapter 2. During the demonstrator experiment it has been shown that the fluorescence signals differ when excited with laser pulses of different wavelengths in the ultraviolet spectral range, whereby additional information can be used for the characterization. Therefore, two excitation wavelengths are also used in the setup under investigation. The frequency-tripled and frequency-quadrupled output of an Nd: YAG laser from Innolas is used, whereby two laser pulses with output wavelengths of 266 nm and 355 nm are simultaneously provided. For the investigations of the time dependence of the fluorescence, the laser pulses have to hit each other with a time delay to the sample. In order to separate the laser pulses in time, a delay line was planned, constructed and examined. These studies are presented in Chapter 3. After the construction and the characterization of the excitation laser and the delay line, the detection system was set up and an experiment control program developed with the aid of which the measured signals can be recorded and stored. The detection setup is described in Chapter 4 and the operation of the computer program is described in Chapter 5. After completion of the experimental setup, the first spectral and temporal traces of fluorescence were recorded, which are presented in Chapter 6. At the end of the report in Chapter 7, the previous results are summarized and future perspectives of the experiment are discussed.

laser Setup

• • Innolas picolo Magna DPSS:
• • Wellenlängen: 266 nm und 355 nm
• • Pulsenergie: >30 mJ (266 nm); >30 mJ (355 nm)
• • Pulsdauer: <700 ps
• • Energiestabilität bei 266 nm <1.5 %
• • Energiestabilität bei 355 nm <1.5 %
• • Divergenz: <0.5 mrad bei 355 nm
• • Strahldurchmesser: ca. 6 mm

For the investigations, the laser system with the following specifications was tendered and procured.

• • Innolas picolo Magna DPSS:
• • Wavelengths: 266 nm and 355 nm
• Pulse energy:> 30 mJ (266 nm); > 30 mJ (355 nm)
• • Pulse duration: <700 ps
• Energy stability at 266 nm <1.5%
• Energy stability at 355 nm <1.5%
• • Divergence: <0.5 mrad at 355 nm
• • Beam diameter: approx. 6 mm

After installing the laser system, the energy stability of the different outputs was checked and the pulse duration was measured. The standard deviation of energy stability was 1.29% for the 266 nm output and 1.85% for the 355 nm, which slightly exceeds the requirement limit, but should not adversely affect the measurements since the stability is significantly better than in the demonstrator experiment. Furthermore, the beam size and the divergence (determinable from the beam size at two separate positions) of the laser beam were measured by the so-called Knife Edge method. Here, the laser power during translation of a sharp-edged metal wedge was measured by the laser beam for different transverse positions of the wedge. With the aid of the measured power, the beam diameter can be extracted from the adaptation of the parameters of the following function to the measured values: $$P_{measured}=\frac{\mathrm{<figure-callout\; id="1"\; label="P"\; filenames="DE102018129807A1\_0005.png,DE102018129807A1\_0006.png"\; state="\{\{state\}\}">P</figure-callout>}1}{2}\left[1\pm erf\left(\frac{\sqrt{2}\left(X-P2\right)}{P3}\right)\right]$$

Here P1 corresponds to the maximum measured power, P2 to a beam position parameter and P3 to the 1 / e beam radius to be determined. If the measurement is carried out at two or more positions, the divergence angle, ie the widening of the laser beam relative to the beam propagation, can be determined from the change in the beam diameter. The results of the measurement are in the 10 and 11 shown.

10 shows the measured laser power of the 355 nm output of the laser for different transverse positions of the metal wedge, measured at a distance of 2.1 m from the laser output.

11 shows a beam diameter for different distances of the metal wedge relative to the laser output.

From the measurements, the beam diameter at the output of the laser was determined to be about 7.3 mm and the divergence to 0.35 mrad. The very small divergence makes it possible to use the delay line described in Chapter 3, since this does not cause the beam to expand too much even after covering a distance of several 10 m. In a further measurement to characterize the laser system, the laser pulse length was determined by means of a very fast photodiode to about 500 ps. This value is below what is required and clearly reduces the influence of the laser excitation on the measurement of the fluorescence lifetime compared to the demonstrator experiment.

delay path

38 shows a picture of the delay line. The laser beam was visualized by liquid nitrogen. In the background you can see the detection setup (black enclosure), the spectrometer (gray on the black enclosure) and the Vertilon PhotoniQ measurement device (on the laser).

A delay line has been designed and constructed, which allows the simultaneously emitted laser pulses with wavelengths of 266 nm and 355 nm to be separated in time by up to 120 ns. It consists of two slightly tilted planar mirrors, the incident laser beam by tilting the mirror to each other first at each successive reflection has a smaller angle of reflection. After a number of reflections determined by the tilt angle, a point occurs at which the beam reverses and the reflection angle increases again in the following reflections until it leaves the delay line. In addition, it can be selected via the tilt angle, in which direction the beam is coupled out of the line. This is in illustrated and in presented together with parts of the experimental setup.

In order to integrate the mirrors into the experimental setup and to be able to adjust each other, two mirror holders were designed and manufactured in our institute. These were adapted to the dimensions and adjustment requirement, wherein in particular the angle of the two mirrors to each other had to be very precisely adjustable. For example, theoretical preliminary investigations showed that for the necessary separation of the laser beam after the last reflection, the angle of the mirrors to each other in the horizontal axis must be adjustable to about 10 μm, which corresponds to a positioning of the mirror edges with an accuracy of 2.5 μm. To meet the adjustment requirement, the two mirror holders were designed so that a mirror holder allows the horizontal alignment of the two mirrors to each other very accurately and the other mirror holder allows the vertical alignment of the mirror to each other.

After the integration of the line into the optical setup, the beam path of the laser pulses of the 266 nm and the 355 nm output was superimposed after passing through the delay line and their power was measured in time-resolved fashion by means of a photodiode (bandwidth> 7 GHz). The measurement is in 3 shown.

3 shows the measured laser power of the laser pulses at 266 nm (first pulse) and 355 nm (second pulse). From the measured power, the time delay for a different number of passes through the delay line was determined.

12 shows the time delay of the laser pulses as a function of the number of passes through the delay line.

From the measurement, the delay was determined depending on the number of passes through the delay line caused by the different path length. With a mirror spacing of approximately 85 mm, it was possible to achieve up to 40 reflections at the mirror surfaces, which leads to a delay of the second laser pulse by approximately 120 ns compared with the first laser pulse.

Detection setup and timing

In 13 the structure for the detection of the fluorescence signal is shown schematically.

The light scattered by the sample is transmitted through a parabolic mirror into the in 13 coupled fiber bundles coupled and directed by this in the spectrometer. Here it is spectrally decomposed via optical grids and detected as a function of the wavelength by means of different channels of the PMT array. The electrical signals generated there by the fluorescence signal are forwarded to a charge integrator from Vertilon (PhotoniQ IQSP482), which will be described in more detail in the next paragraph, whereby in each case part of the signal from four channels is capacitively decoupled. The decoupled signals are used to directly measure the temporal behavior of the fluorescence with a fast oscilloscope. The measurements are controlled by a computer to which the measurement data are forwarded for further processing and storage. The construction used for charge integration is in 14 shown in more detail.

The 32 signals received by the PMT array are routed to one cable (SBC090P) via one interface (SIB323) and to the 32 inputs of the charge integrator via another custom built interface (CIB3264). The individual inputs are assigned to 4 banks, each with 16 inputs assigned to one bank and the data of each input being relayed to two banks. As in 14 can be seen, the banks 1 & 2 are driven so that the signal of the fluorescence spectrum is integrated after excitation with laser radiation of wavelength 266 nm at a time t ₀ + t _propagation over 50 ns. After a delay of t _delay = 120 ns (this time is set depending on the adjustment of the tilt angle of the delay path) then the integration of the banks 3 & 4 is started, in which the signal of the fluorescence spectrum is integrated after excitation with laser radiation of wavelength 355 nm over 50 ns , In the respective banks, the signals are digitized and then forwarded via a 32-channel parallel processor and a 16-bit signal processor to the experiment control computer.

14 is a schematic representation of the charge integration unit vertilon photoniq and control. 15 shows the timing of the charge integration.

Experiment Control Program

A program was written using the Lab-VIEW graphical programming system for experiment control and data storage. A schematic representation of the functioning of the program is in 16 shown.

16 shows a schematic representation of the computer program for controlling the experiment.

Through a transistor-transistor-logic (TTL) signal generated by a National Instruments (NI) multifunction data acquisition card, the computer program controls a laser shutter that protects the sample from unwanted laser radiation. Furthermore, the data acquisition devices described in Chapter 4 (Vertilon PhotoniQ and Oscilloscope) are initialized and read out. A so-called "state-machine program structure" was used to wait for events after the program was initialized and then loop through different loops depending on the event, such as changing parameters for data acquisition and controlling the laser shutter , depending on the setting of the "measurement sequence variables" in go through the measuring procedures described in more detail.

The 17 to 19 show schematic representations of the measurement procedure.

The continuous measurement is mainly used for the adjustment of the system, here after the start of successive individual measurements are performed and graphically displayed. In the single measurement, only the spectral information is read out and, if desired, stored. The "spectral and temporal measurement" reads both the oscilloscope and the charge integrator, graphs the results and, if desired, saves the data in two different files.

In 39 the program interface for controlling the experiment is shown.

In the upper left corner the parameters for the measurement can be set and the measuring procedure can be selected. The next two subdivisions are used to set the parameters of the charge integrator and the oscilloscope. In the last subdivision of the first line, the parameters such as u.a. the file name can be set for storage. In the larger subdivision on the left, general settings such as the shutter position can still be checked and the current system state is indicated here. In the main display, in the center, the measurement results are displayed graphically, with the four smaller diagrams representing the time-dependent measurements of the decoupled channels and the larger the spectral measurement. The program can be stopped in the lower right corner. If individual measurements or continuous measurements are carried out, the program changes the tab in the central field and the spectral measurement is displayed larger.

Preliminary results

First measurements were made on five different samples for a distance of 3 m between sample and parabolic mirror or laser. During the measurements, the pulse energy of the laser pulses was not changed manually, with the lowest pulse energy that could be measured with our energy meter being about 10 μJ. Since at this pulse energy the PMTs were saturated with fluorescence, a neutral density filter (ND) of thickness ND2, ie with a permeability of 1%, was incorporated into the beam path in front of the sample and the pulse energy measured in front of this filter. For the laser pulses of the 266 nm laser output, a pulse energy of 40 μJ was measured, which corresponds to a pulse energy of 400 nJ on the sample. For the laser pulses of the 355 nm laser output, a pulse energy of 15 μJ was measured, which corresponds to a pulse energy of 150 nJ on the sample.

In the spectra several signals are noticeable. On the one hand, a drop in the signal on channel 8 of the PMT array can be seen in the fluorescence measurements at an excitation wavelength of 266 nm. This is achieved by a notch filter located in front of the fiber input. In addition, a strong signal can be seen on channel 21, which is caused by stray light of the frequency-doubled output and a residual portion of the frequency-doubled light in the frequency-quadrupled output of the laser. This should be reduced in the future by further enclosure of the optical structure. Furthermore, burglaries on the channels 4, 12, 20, 28 can be seen for both excitation wavelengths. These come through the capacitive Decoupling of the time signal to conditions. These burglaries will be reduced in the future by improving the electronic components and by revising the decoupling mechanisms.

In the 21 to 30 For the time dependence of the fluorescence, the four channels are plotted over the measuring time. After about 20 ns, the fluorescence signal can be seen, which is generated by the excitation of the laser pulse with a wavelength of 266 nm. The signal at 140 ns is generated by the laser pulse with a wavelength of 355 nm, wherein the two laser pulses were shifted in time by the delay line by about 120 ns to each other. For the five samples, different time constants for the two excitation wavelengths can be determined. The cooldowns obtained from a simple exponential fit are shown in Table 1. The values given do not correspond directly to those determined by, for example, time-correlated single-photon counting of fluorescence lifetimes, since other factors must be taken into account in our measurements. The measured profiles are composed of the convolution of the laser pulse, the average fluorescence lifetime, and the temporal device function of the detection unit. Other effects that affect the measurements are, for example, saturation of the PMT signals (seen in the measurement of fluorescine), crosstalk of the PMT channels and, as we suspect, a so-called ringing effect of the electronics of the detection system The saturation of the PMTs and the crosstalk of the channels leads to a change in the measured time dependencies with the laser power, so that these effects as good as possible avoided by a judicious choice of laser power for as many samples as possible and However, it must also be said that the fluorescence lifetime does not have to be measured accurately to distinguish the samples from the measured time behavior of the fluorescence, but reproducibly distinguishable time histories are used for the identification Overall, the diversity of the measured time dependencies shows that an information gain for the detection and the classification of the investigated substances by the recording of the time course of the fluorescence is present. The extent to which this distinctiveness can be used for the classification must be examined by reproducibility measurements and an extension of the investigated substances. Table 1: Cooldown determined from simple exponential fit sample fluorescein diesel curcumin Tryptophan & NADH Bacillus thuringiensis 266nm Channel 1 - 9.1 - - 5.1 Channel 2 - 10.5 3.8 6.1 - Channel 3 9.6 11.8 - 1.2 - Channel 4 8.6 - - - - 355nm Channel 1 - - - - - Channel 2 - 10.4 3.9 1.9 7.8 Channel 3 7.7 12.1 2.7 1.9 6.4 Channel 4 7.9 - - - -

21 and 22 : Fluorescein in water. 21 shows measured spectra, 22 measured time dependencies.

23 and 24 : Diesel in water. 23 shows measured spectra, 24 measured time dependencies.

25 and 26 : Curcumin in acetone. 25 shows measured spectra, 26 measured time dependencies.

27 and 28 : NADH and tryptophan in water. 27 shows measured spectra, 28 measured time dependencies.

29 and 30 : Bacillus thuringiensis. 29 shows measured spectra, 30 measured time dependencies.

Summary and Outlook

In the previous sections 1 to 6 of the report, the established and characterized experimental setup for the identification and classification of hazardous substances was presented. For this purpose, the entire optical structure and the characterization of the procured laser were first described. After that, the self-developed delay section and its mounting were presented and measurements of the function of the section were described (Section 3). Thereafter, in section 4, the optoelectronic structure used for the detection of the spectral and temporal fluorescence signals was presented and the experimental timing described in more detail. In the following section 5, the self-made program for controlling the experiment was described. In the last chapter first measurements for five different substances were presented and it could be shown that for these substances an information gain can be obtained by recording the time dependence of fluorescence. After an investigation of the possibilities for improvement of the detection electronics, the reproducibility of the temporal measurements will be investigated in the future. In addition, a larger amount of hazardous substances will be investigated and a classification algorithm for these substances will be implemented, so that this system can be used as a hazardous substance detection unit.

literature

[1] F. Awad, C. Ramprasath, N. Mathivanan, PR Aruna, and S. Ganesan. Steady-state and fluorescence lifetime spectroscopy for identification and classification of bacterial pathogens. Biomedical Spectroscopy and Imaging, 3: 381-391, 2014 ,

[2] J. Chen and PR Selvin. Lifetime and color-tailored fluorophores in the micro-to-millisecond time regime. Journal of the American Chemical Society, 122 (4): 657-660, Feb 2000 ,

[3] Z. Fan, OJ Schröder, and J. Krahl. Analysis of diesel fuels / biodiesel blends and identification of biodiesel using time-resolved laser-induced fluorescence spectroscopy (trlfs). Agricultural research - applied agricultural and forestry research, (1): 1-14, 2015 ,

[4] T. Fischbach, F. Duschek, A. Hausmann, C. Pargmann, V. Alekseev, L. Poryvkina, I. Sobolev, S. Babichenko, and J. Handke. Hyperspectral laser-induced fluorescence, May 2015 ,

[5] U. Kaiser, N. Sabir, C. Carrillo-Carrion, P. del Pino, M. Bossi, W. Heimbrodt, and WJ Parak. Förster resonance mediated enhancement of the fluorescence lifetime of organic fluorophores to the millisecond range by coupling to mn-doped cds / zns quantum dots. Nanotechnology, 27: 055101, 2016 ,

[6] AE Kinkennon and LB McGown. Fluorescence lifetime characterization of bacteria using the maximum and entropy method. Journal of Fluorescence, 7 (3), 1997 ,

[7] JR Lakowicz. Principles of Fluorescence Spectroscopy. Springer, third edition edition, 2006 ,

LIST OF REFERENCE NUMBERS

10

system

12

radiation source

14

laser

16

sample

18

delay means

20

mirror

22

mirror

24

fluorescence radiation

26

detection optics

28

parade

30

fiber bundles

32

fiber bundles

34

spectroscopy device

36

grating spectrometer

38

signal line

40

Data acquisition device

42

data distribution

44

computer

46

PMT

48

signal line

50

oscilloscope

52

signal line

54

detection device

56

PMT array

58

signal line

60

laser control

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

• US 6272376 B1 [0003]
• US 8743356 B1 [0003]
• WO 2014/145786 A1 [0003]
• US 2010/0067003 A1 [0003]
• US8665433 B2 [0004]
• US 201113150757 [0090]

Cited non-patent literature

• Burrows, W.D., Renner, S.E .: Biological warfare agents as threats to potable water. Environmental Health Perspectives. 107, 975-984 (1999) [0109]
• Henderson, D.A .: The looming threat of bioterrorism. Science. 283, 1279-1282 (1999). doi: 10.1126 / science.283.5406.1279 [0109]
• Buteau, S., Ho, J., Lahaie, P., Rowsell, S., Simard, J.-R., Kadlcak, J., Sgarzi, G., Tonini, P., Heuvel, J.C. van den, Blatny, JM, Farsund, ∅., Gran, H.-C., S. tian Lovold, Rustad, G., Sletmoen, M., Baxter, K., Castle, M., Foot, EVJ, Swim, C., Vanderbeek, R .: Laser based stand-off detection of biological agents. North Atlantic Treaty Organization, Research and Technology Organization (2010) [0109]
• Gaudio, P., Gelfusa, M., Murari, A., Pizzoferrato, R., Carestia, M., Cenciarelli, O., Parracino, S., Ludovici, G., Gabriele, J., Gabbarini, V., Giovanni, DD, Rossi, R., Ciparisse, JF, Bellecci, C., Malizia, A .: Application of optical techniques to detect chemical and biological agents. In: Defense S & T Technical Bulletin. pp. 1-13. Defense S & T Technical Bulletin (2017) [0109]
• Duschek, F., Fellner, L., Gebert, F., Grünewald, K., Köhntopp, A., Kraus, M., Mahnke, P., Pargmann, C., Tomaso, H., Walter, A .: Standalone detection and classification of bacteria by multispectral laser-induced fluorescence. Advanced Optical Technologies. 6, 75-83 (2017). doi: 10.1515 / aot-2016-0066 [0109]
• Farsund, ∅., Rustad, G., Skogan, G .: Stand-off detection of biological agents using laser-induced fluorescence-a comparison of 294 nm and 355 nm excitation wavelengths. Biomedical Optics Express. 3, 2964 (2012). doi: 10.1364 / boe.3.002964 [0109]
• Simard, J.R., Roy, G., Mathieu, P., Larochelle, V .: Standoff integrated bioaerosol active hyperspectral detection (Sindbahd): Final report. Defense R & D Canada (2003) [0109]
• Buteau, S., Rowsell, S .: Biological material detection identification and monitoring: Combining point and stand-off sensors technologies. In: Burgess, D., Owen, G., Bouma, H., Carlysle-Davies, F., Stokes, R.J., and Yitzhaky, Y. (eds.) Optics and Photonics for Counterterrorism, Crime Fighting, and Defense XII. SPIE 9995 (2016) [0109]
• Kraus, M., Fellner, L., Gebert, F., Grünewald, K., Pargmann, C., Walter, A., Duschek, F .: Comparison of Classification Models of Spectral Data of Laser Induced Fluorescence. In: Is Scientific International Conference on CBRNe. Springer (2017) [0109]
• Lakowicz, J.R .: Principles of Fluorescence Spectroscopy. Springer-Verlag New York Inc. (2006) [0109]
• Sivaprakasam, V., Lin, H.-B., Huston, A.L., Eversole, J.D .: Spectral characterization of biological aerosol particles using two-wavelength excited laser-induced fluorescence and elastic scattering measurements. Optics Express. 19, 6191 (2011). doi: 10.1364 / oe.19.006191 [0109]
• Kinkennon, A.E., McGown, L .: Fluorescence lifetime characterization of bacteria using the maximum entropy method. Journal of Fluorescence. 7, 201-210 (1997). doi: 10.1007 / bf02758220 [0109]
• Awad, F., Ramprasath, C., Mathivanan, N., Aruna, P.R., Ganesan, S .: Steady-state and fluorescence lifetime spectroscopy for identification and classification of bacterial pathogens. Biomedical Spectroscopy and Imaging. 3, 381-391 (2014). doi: 10.3233 / BSI-140095 [0109]
• Herriott, D.R., Schulte, H.J .: Folded Optical Delay Lines. Applied Optics. 4, (1965) [0109]
• Elliott, C., Vijayakumar, V., Zinc, W., Hansen, R .: National Instruments LabVIEW: A Programming Environment for Laboratory Automation and Measurement. Journal of the Association for Laboratory Automation. 12, 17-24 (2007). doi: 10.1016 / j.jala.2006.07.012 [0109]
• Fellner, L., Gebert, F., Walter, A., Grünewald, K., Duschek, F .: Variations in fluorescence spectra of a bacterial population during different growth phases. In: Is Scientific International Conference on CBRNe. Springer (2017) [0109]
• Gelfusa, M., Murari, A., Lungaroni, M., Malizia, A., Parracino, S., Peluso, E., Cenciarelli, O., Carestia, M., Pizzoferrato, R., Vega, J., Gaudio, P .: A support vector machine approach to the automatic identification of fluorescence spectra by biological agents. In: Burgess, D., Owen, G., Bouma, H., Carlysle-Davies, F., Stokes, R.J., and Yitzhaky, Y. (eds.) Optics and Photonics for Counterterrorism, Crime Fighting, and Defense XII. SPIE 9995 (2016) [0109]
• F. Awad, C. Ramprasath, N. Mathivanan, P.R. Aruna, and S. Ganesan. Steady-state and fluorescence lifetime spectroscopy for identification and classification of bacterial pathogens. Biomedical Spectroscopy and Imaging, 3: 381-391, 2014 [0148]
• J. Chen and P.R. Selvin. Lifetime and color-tailored fluorophores in the micro-to-millisecond time regime. Journal of the American Chemical Society, 122 (4): 657-660, Feb 2000 [0149]
• Z. Fan, O.J. Schröder, and J. Krahl. Analysis of diesel fuels / biodiesel blends and identification of biodiesel using time-resolved laser-induced fluorescence spectroscopy (trlfs). Landbauforschung - applied agricultural and forestry research, (1): 1-14, 2015 [0150]
• T. Fischbach, F. Duschek, A. Hausmann, C. Pargmann, V. Alekseev, L. Poryvkina, I. Sobolev, S. Babichenko, and J. Handke. Hyperspectral laser-induced fluorescence, may 2015 [0151]
• U. Kaiser, N. Sabir, C. Carrillo-Carrion, P. del Pino, M. Bossi, W. Heimbrodt, and W.J. Parak. Förster resonance mediated enhancement of the fluorescence lifetime of organic fluorophores to the millisecond range by coupling to mn-doped cds / zns quantum dots. Nanotechnology, 27: 055101, 2016 [0152]
• A.K. Kinkennon and L.B. McGown. Fluorescence lifetime characterization of bacteria using the maximum and entropy method. Journal of Fluorescence, 7 (3), 1997 [0153]
• J.R. Lakowicz. Principles of Fluorescence Spectroscopy. Springer, third edition edition, 2006 [0154]

Claims (20)

Method for classifying chemical and / or biological substances, in particular hazardous substances, in which method a comprehensive or carrying sample (16) containing at least one chemical and / or biological substance with at least one excitation pulse of electromagnetic radiation, in particular a laser pulse, of an excitation wavelength for fluorescence it is excited in which method an intensity of fluorescent radiation (24) emitted by the sample (16) due to the excitation with the at least one excitation pulse is measured as a function of wavelength and in which method a decrease in the intensity of the fluorescence radiation (24) emitted by the sample (16) ) is measured time-dependent for at least one wavelength and / or one wavelength range, in which method at least one reference spectrum in the form of at least one wavelength-dependent reference fluorescence spectrum of at least one chemical and / or biological substance and in Fo At least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance is provided, the measured wavelength-dependent intensity and the measured time-dependent intensity of the fluorescence radiation (24) emitted by the sample (16) having the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent fluorescence spectrum Reference fluorescence spectrum are compared to determine whether the sample (16) contains the at least one chemical and / or biological substance of a class or not. Method according to Claim 1 , Characterized in that a) the wavelength-dependent measurement of the intensity of the (from the sample 16) emitted fluorescence radiation (24) and the measurement of time-dependent decay of the light emitted from the sample fluorescence radiation (24) simultaneously or substantially be carried out simultaneously and / or b ) the sample (16) is excited to fluorescence with at least two excitation pulses of electromagnetic radiation in a short time interval from one another. Method according to one of the preceding claims, characterized in that the sample (16) is excited with at least two excitation pulses of electromagnetic radiation of different excitation wavelength in a short time interval from each other to the fluorescence. Method according to one of the preceding claims, characterized in that the at least one excitation pulse of electromagnetic radiation of an excitation wavelength with at least one excitation laser (14) is generated. Method according to one of Claims 2 to 4 , characterized in that the at least two excitation pulses are generated by a single radiation source (12). Method according to one of Claims 2 to 5 , characterized in that the at least two excitation pulses of different wavelengths are generated with a monochromatic radiation source (12) in cooperation with a nonlinear optics. Method according to Claim 5 or 6 , characterized in that the at least two excitation pulses from the radiation source (12) are generated simultaneously and then separated in time. Method according to one of Claims 2 to 7 , characterized in that a) the at least two excitation pulses are separated in time such that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample (16) and / or b) with the at least two excitation pulses the same spatial region of the sample ( 16) is excited. Method according to one of the preceding claims, characterized in that the fluorescence radiation (24) emitted by the sample (16) on the basis of the at least one excitation pulse is measured with a spectrometer (34) as a function of wavelength, with electronic measuring signals being generated as a function of the wavelength, in particular with the spectrometer (34) be further, in particular a) a part of the wavelength-dependent generated electronic measurement signals coupled and an intensity profile of the same time-dependent measured, in particular with an oscilloscope (50) or a fast analog-to-digital converter card for a computer, and / or b) the wavelength-dependent generated electronic measurement signals are averaged over a measurement period. System (10) for classifying chemical and / or biological substances, in particular hazardous substances, which system (10) at least one electromagnetic radiation source (12), in particular in the form of a laser (14), for generating at least one excitation pulse for exciting at least one chemical and / or biologically comprising, comprising or supporting sample (16) for fluorescence, which system (10) comprises first spectroscopy means (34) for wavelength dependent measuring of an intensity emitted by the sample (16) due to the excitation with the at least one excitation pulse Fluorescence radiation (24) and a second spectroscopy device (34) for measuring an intensity of the emitted fluorescence radiation (24) for at least one wavelength and / or wavelength range over time, which system (10) comprises a storage device for storing at least one reference spectrum in the form of at least a well and at least one time-dependent reference fluorescence spectrum of the at least one chemical and / or biological substance and which system (10) comparator (44) for comparing the measured wavelength-dependent intensity and the measured time-dependent intensity of the Sample (16) emitted fluorescence radiation (24) with the at least one wavelength-dependent reference fluorescence spectrum and the at least one time-dependent reference fluorescence spectrum comprises for determining whether the sample (16) contains the at least one chemical and / or biological substance of a class or not. System after Claim 10 , characterized in that the system (10) is designed for simultaneously or substantially simultaneously carrying out the wavelength-dependent measurement of the intensity of the fluorescence radiation (24) emitted by the sample with the first spectroscopy device (34) and the measurement of the time-dependent drop of that from the sample emitted fluorescence radiation (24) with the second spectroscopy device (34). System after Claim 10 or 11 , characterized in that a) the system (10) is designed to excite the sample (16) for fluorescence with at least two excitation pulses of electromagnetic radiation at a short time interval from each other and / or b) that the system (10) is designed to generate at least two excitation pulses electromagnetic radiation of different excitation wavelength in a short time interval from each other and / or c) the at least one radiation source (12) in the form of an excitation laser (14) is formed. System after Claim 12 , characterized in that the system (10) is designed to generate the at least two excitation pulses with a single radiation source (12), in particular with a different wavelength. System after Claim 12 or 13 , characterized in that the system (10) comprises at least one monochromatic radiation source (12) and a non-linear optics cooperating therewith for generating at least two excitation pulses of different wavelengths. System according to one of Claims 12 to 14 , characterized in that the system (10) is designed for simultaneously generating the at least two excitation pulses and that the system (10) comprises a delay device (18) for temporally separating the simultaneously generated at least two excitation pulses, wherein in particular the delay device (18) is formed is for temporally separating the at least two excitation pulses such that a time interval between two successive excitation pulses is greater than a fluorescence decay time of the sample (16). System according to one of Claims 12 to 15 , characterized in that the system (10) is designed to excite the same spatial area of the sample (16) with the at least two excitation pulses. System according to one of Claims 10 to 16 , characterized in that the first spectroscopy device (34) comprises a wavelength-dependent detection device (54), in particular in the form of an electron multiplier array (56). System after Claim 17 , characterized in that the detection device (54) is designed for the wavelength-dependent generation of electronic measurement signals. System after Claim 18 , characterized in that the system comprises a signal processing device (40) for decoupling a portion of the wavelength-dependent generated electronic measurement signals and for measuring a time-dependent intensity profile of the decoupled measurement signals, wherein in particular the signal processing means (40) a) an oscilloscope (50) or a fast analog Digital-converter card for a computer and / or b) the signal processing device (40) comprises an integration device for averaging the wavelength-dependent generated electronic measurement signals over a measurement period. Use of a system according to one of Claims 10 to 19 to carry out a method according to one of Claims 1 to 9 ,

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