Excitation and measurement of fluorescence lifetimes using optical 3D measurement

Abstract

The present invention relates to a method for excitation and measurement of fluorescence lifetimes by means of optical 3D measurement and the use of a system for optical 3D measurement for measuring fluorescence lifetimes of fluorescent dyes.

Description

The present invention relates to a method for exciting and measuring fluorescence lifetimes by means of optical 3D measurement and to the use of a system for optical 3D measurement for measuring fluorescence lifetimes of fluorescent dyes.Background ArtFluorescence lives are used in the different (industrial) sectors, for example in the product protection industry, as a forensic security feature or in the medical sector as cancer markers. Fluorescence lives are difficult to read out and require a complicated laboratory construction which comprises, inter alia, a fluorescence spectrophotometer. With this present invention message, reading of fluorescence lifetimes is significantly facilitated and mobile.The fluorescence lifetime is currently determined in complicated, non-portable laboratory setups by means of time-correlated single photon counting. In this method, the sample to be examined is excited periodically / pulsed with photons. These photons are emitted by a laser source and the distances between two laser pulses are found in the range of nanoseconds or shorter. The wavelength of the fluorescence of the sample to be examined must be greater than the wavelength of the excitation light.After the emission of the (excitation) photons, the time measurement is started and ends with a signal of the fluorescence detector. After many excitation / measurement cycles have been passed through, a time-resolved histogram is obtained, from which the fluorescence lifetime can then be determined. The distribution is a function of the natural logarithm with the basis of e (Euler number). The fluorescence lifetime is the inverse function with the exponential function y=e·x.In the present invention, a completely novel way of determining fluorescent lifetimes is provided. A system for optical 3D measurement can be used in this case.For this purpose, photons are first emitted from a pulsed light source into a 3-dimensional space. After interaction of the photons with the surfaces of objects within this 3-dimensional space, they are detected, usually by means of a sensor. The time between photon emission and photon detection is used to calculate the distance traveled. This process is usually repeated millions of times per second, thus creating a precise 3D real-time map of the environment.It has surprisingly been found that the 3D real-time maps accessible thereby can be used for determining the fluorescence lives of fluorescent dyes.The interaction of the photons is delayed by the excited fluorescent dyes, which acts in calculating the distance travelled as a longer distance travelled compared to the distance travelled of photons that have interacted with surfaces from the environment, which distance is directly dependent on the fluorescence lifetime of the respective excited fluorescent dye. This longer distance traveled appears as depth in the 3D real-time maps. The greater the fluorescence lifetime, the greater will be the depth displayed in the real time 3D maps.In addition, the 3D real-time maps can be used to determine and link together the location of the fluorescent dye and its fluorescence lifetime at the same time.The present invention enables the measurement of fluorescence lives and the simultaneous determination of the location of the fluorescent dyes with easily accessible means, such as LiDAR systems, preferably portable LiDAR systems. First smartphones and tablets already contain LiDAR systems that can be used for the present invention.SUMMARY OF THE INVENTIONIn a first aspect, the present invention relates to a method for measuring fluorescence lives of fluorescent dyes, comprising the following steps:• providing a sample containing one or more fluorescent dyes in a 3-dimensional spatial domain;• emitting photons by means of a pulsed light source into the 3-dimensional spatial region containing the sample;• Detection of the photons after their interaction with the surfaces of the 3-dimensional dimenional region and the sample;• Measurement of the time between photon emission and detection of photons, wherein the measured time of photons that have interacted with the surface of the sample depends directly on the distance of the pulsed light source to the sample and the fluorescence lives of the one or more fluorescent dyes in the sample;• Calculation of the fluorescence lives caused by the one or more fluorescent dyes from the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source to the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source to the sample.In a further aspect, the present invention relates to the use of a system for optical 3D measurement comprising a) a unit for emitting photons by means of a pulsed light source; b) a unit for detecting photons; and c) a unit for measuring the time between the photon emission and the detection of the photons for measuring fluorescence lives of one or more fluorescent dyes in a sample arranged in a 3-dimensional space as the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source from the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source from the sample.DESCRIPTION OF THE INVENTIONIn a first aspect, the present invention relates to a method for measuring fluorescence lives of fluorescent dyes, comprising the following steps:• providing a sample containing one or more fluorescent dyes in a 3-dimensional spatial domain;• emitting photons by means of a pulsed light source into the 3-dimensional spatial region containing the sample;• Detection of the photons after their interaction with the surfaces of the 3-dimensional dimenional region and the sample;• Measurement of the time between photon emission and detection of photons, wherein the measured time of photons that have interacted with the surface of the sample depends directly on the distance of the pulsed light source to the sample and the fluorescence lives of the one or more fluorescent dyes in the sample;• Calculation of the fluorescence lives caused by the one or more fluorescent dyes from the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source to the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source to the sample.The pulsed light source is preferably a pulsed laser.The emitted photons preferably have a wavelength in the range from 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 nm to 1800 nm.The spacing of the pulses of the pulsed photons, preferably of the laser pulses, is preferably in the range of 0.2 to 1000 ns, preferably 0.5 to 50 ns, more preferably 1 to 20 ns, most preferably 1.0 to 10 ns.The method of the invention is used to measure the fluorescence lifetime of fluorescent dyes which are part of the sample to be tested.The sample to be examined may be any type of article containing one or more fluorescent dyes.The fluorescent dyes are preferably contained in the sample such that they are capable of being excited by photons and emitting fluorescent radiation.It is preferred that at least one surface of the sample contains the fluorescent dyes.The sample may contain one or more fluorescent dyes, such as 2-10 fluorescent dyes.Preferably, the plurality of fluorescent dyes differ at least in their emission wavelength and / or their fluorescence lifetime. The fluorescent dyes particularly preferably differ at least in their fluorescence lifetime.The sample is either liquid or solid in aggregate state.The sample may be an organic or inorganic substance.In one embodiment, the sample may contain, for example, a fibrous surface which is preferably printable. Examples are natural, cellulose-based substrates, synthetic polymer-based substrates and mixtures thereof, in particular bank notes, identification cards, passports, birth documents, travel identification cards, entry cards and other tickets. Some further examples are listed below: a check, a receipt, a bank card, a credit card, a check card, a currency, a cash card, an identification item, an identity item, an access item, an item for issuing permission, a personal identification card, a social insurance credential, a driver's license, a vaccination pass, a test certificate, a health card, a insurance card, a personalized item, a passport, a document, a paper document, a security document, a postage stamp, a personalized document, an adhoc document, a certificate, a stock certificate, a school certificate, a contract, an insurance policy, a testament, a parking ticket, a transport ticket or a ticket for admission to an event.Further examples of suitable samples are packages, labels of packages or other articles or objects on the surface of which fluorescent dyes can be applied.Such objects or objects are, for example, products and their intermediates or commercial products. Some examples of products and their intermediates or commercial products are listed in the following by way of example: a branded product, a consumer product, a pharmaceutical product, a health care product, an automobile, an aircraft, a nutritional product, a component, a hardware component, an electronic component, a computer chip, a book, a handbook.Preferably, the sample is printed with a printing composition containing the one or more fluorescent dyes.The term "printing composition" includes any composition which can be used, for example, as an ink formulation or toner in the printing methods mentioned below. The printing composition may be a liquid printing composition such as an ink formulation or a solid or powdered printing composition such as a toner.The term "ink formulation" includes any solvent and combinations thereof, as well as typical additives suitable for preparing a printable liquid.The term "toner" includes any solid or powder composition, as well as combinations thereof, as well as typical additives suitable for preparing a printable solid or powder.The term "printing" includes the deposition of pigments onto or into a solid substrate. Typical examples include, but are not limited to, offset printing, digital printing, inkjet printing, screen printing, transfer printing, stamp printing, roll-to-roll, non-contact printing, laser printing, spray printing, spray printing, thermal printing, thermal transfer printing, and other methods.The printing composition(s) is / are preferably commercially available printing composition(s) suitable for the deposition of pigments onto or into a solid substrate. Suitable printing compositions may be liquid printing compositions such as ink formulations or solid or powdery printing compositions such as toners, depending on the type of printing. Typical examples include, but are not limited to, offset printing, digital printing, inkjet printing, screen printing, transfer printing, stamp printing, roll-to-roll, non-contact printing, laser printing, spray printing, spray methods, and other methods.The printing composition may also contain color pigments in addition to one or more fluorescent dyes. This may result in the printed surface of the sample printed with the printing composition being visible to the human eye.In another embodiment, the printing composition contains no further color pigments besides the one or more fluorescent dyes. In this embodiment, the printed area of the printing composition on the surface of the sample may not be visible to human eyes due to concentration and print dissolution effects of the printing composition.In another embodiment, the sample may comprise a cellular as well as extracellular matrix. It would also be conceivable for the sample to have blood and lymph components.Examples are blood or tissue samples of human, animal or vegetable bodies.The sample is placed in a 3-dimensional space. The 3-dimensional space serves to create a standardized environment in which the photons interacting with the sample can be unambiguously distinguished from the photons interacting with the surfaces of the environment. Usually, such a 3-dimensional space is a measurement space.In order to distinguish the photons interacting with the sample from the photons interacting with the surfaces of the 3-dimensional space, a measurement of the 3-dimensional space without a sample is usually carried out. The time between the photon emission and the detection of the photons thus measured is subtracted from the measured time between the photon emission and the detection of the photons of the latter measurement after measuring the sample in the 3-dimensional space.Fluorescent dyes are dyes which emit fluorescent radiation after photon excitation. Excitation raises an electron to a higher energy state. When it falls back to a lower energy state, this energy is released again in the form of photons. In fluorescence, the electron falls from a higher singlet state back to the lower energy state. The wavelength of the emitted fluorescent radiation is generally higher (since of lower energy) than that of the absorbed photon radiation.The fluorescence lifetime in this case indicates the mean time that the excited electron remains in the higher energy state before it falls back into the lower energy state again with emission of the fluorescent radiation.The fluorescent dyes of the sample preferably have an absorption wavelength in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 nm to 1800 nm.The fluorescent dyes are therefore suitable for absorbing the emitted photons.The emission wavelength of the fluorescent dyes is preferably in the range from 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 to 1800 nm. The emission wavelength is generally longer-wavelength than the absorption wavelength of the fluorescent dye / s.The fluorescence lives are preferably independently in the range of 1 ns to 100 ms, preferably 10 ns to 1000 μs, more preferably 20 ns to 100 μs.The fluorescence lifetime of the fluorescent dye / dyes is usually greater than the distance of the pulses of the pulsed photons, preferably of the laser pulses. This is necessary in order to be able to measure changes in the fluorescence with the aid of the 3D optical measurement within a plurality of repetitions of the measurements at the distance of the pulses of the pulsed photons, preferably of the laser pulses.Suitable fluorescent dyes are fluorescent dyes which meet the above-mentioned criteria with respect to absorption wavelength, emission wavelength and fluorescence lifetime.Fluorescent dyes are usually selected from organic fluorescent dyes and inorganic fluorescent dyes or mixtures thereof.Organic dyes can be selected from the classes of proteins and peptides, small organic molecules, synthetic oligomers and polymers, and multicomponent systems.Typical examples of polymers and peptides are green fluorescent protein (GFP), yellow fluorescent protein (YFP) or red fluorescent protein (RFP). Non-proteinaceous fluorescent organic dyes are usually classified as xanthene derivatives, cyanine derivatives, squaraine derivatives, squaraine-rotaxane derivatives, naphathalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, tetrapyrrole derivatives, and dipyrromethane derivatives. Organic fluorescent dyes are usually commercially available in all emission spectral colors from blue (from 380 nm) to red (to 1800 nm).Suitable organic dyes having emission spectral colors above 800 nm are described, for example, in EP 0 933 407, U.S. Pat. No. 5,282,894, U.S. Pat. No. 5,665,151, WO 1998 / 018871, WO 2003 / 03803, U.S. Pat. No. 10,119,071 and U.S. Pat. No. 5,542,971.Suitable inorganic dyes are preferably semiconducting inorganic nanocrystals.Preferably, the fluorescent dyes comprise semiconducting inorganic nanocrystals. It is particularly preferred that the fluorescent dyes consist of semiconducting inorganic nanocrystals.Examples of suitable semiconducting inorganic fluorescent (core) nanocrystals include Ag 2 S, Ag 2 Se, Ag 2 Te, CdS, CdSe, CdTe, PbS, PbSe, PbTe, SnTe, ZnS, ZnSe, ZnTe, InP, InAs, Cu 2 S, In 2 S 3, InSb, GaP, GaAs, GaN, InN, InGaN, ZnSSe, ZnSeTe, ZnSTe, CdNBe, CdSeTe, HgNBe, HgSeTe, HgSTe, ZnNbS, ZnNbSe, ZnNbTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnNbNBe, ZnHgNBe, ZnNbSeTe, ZnHgSeTe, CdHgNBe, CdHgSeTe, CuInS 2, CuInSe 2, CuInGaSe 2, CuInZnS 2, CuZnSnSe 2, CuIn(S,Se) 2, CuInZn(S,Se) 2, AgIn(S,Se) 2.Further suitable examples, but not exclusively, are perovskite materials having the general formula ABX 3 or A 4 BX 6, wherein X may be selected from Cl, Br, I, O and / or mixtures thereof, wherein A may be selected from Cs, CH 3 NH 3, CH(NH 2)2, Ca, Sr, Bi, La, Ba, Mg and / or mixtures thereof, wherein B may be selected from Pb, Sn, Sr, Ge, Mg, Ca, Bi, Ti, Mn, Fe and / or mixtures thereof.Furthermore, core / shell and / or core / multishells of semiconducting inorganic nanocrystal architectures of II-VI, III-V, IV-VI, I-VI, I-III-VI semiconductors or mixtures thereof, and core / shell and / or core / multishells of perovskite materials, are further suitable examples.The crystal lattice of the semiconducting inorganic nanocrystals may additionally, but not exclusively, be doped with one or more metal ions, such as Cu +, Mg 2+, Co 2+, Ni 2+, Fe 2+, Mn 2+ and / or with one or more rare earth metals, such as ytterbium, praseodymium or neodymium.The semiconducting inorganic nanocrystals preferably have an average particle size of from 1 nm to 100 nm, more preferably from 2 nm to 50 nm, and most preferably from 3 nm to 15 nm in at least one dimension, preferably in all dimensions.The average particle size can be further increased / modified by various methods. Typical examples include, but are not limited to, a silica shell, a titanium oxide shell, a halogen shell, and other methods of increasing stability, masking, biocompatibility, water solubility, and / or cladding.An interesting property of the preferred semiconducting inorganic nanocrystals is that their excitation and emission spectrum is dependent, among other things, on their particle size.The printing composition(s) may contain one or more, such as 1-10, fluorescent dyes.To measure the fluorescence lifetime, photons are first emitted as described above.These emitted photons interact with the fluorescent dyes of the sample by being initially absorbed by the fluorescent dyes of the sample.Thereafter, photons are emitted from the fluorescent dyes in the form of fluorescent radiation as described above.This fluorescent radiation is detected along with the photons interacting with other portions of the surface of the sample as well as the 3-dimensional space. The photons usually interact with the other parts of the surface of the sample as well as the 3-dimensional space in which they are reflected from these surfaces.In addition, the time between the emission of the photons, preferably of a laser pulse of the laser, and the detection of the photons is measured.The photons-including the fluorescent radiation emitted by the one or more fluorescent dyes-are preferably measured by means of a sensor.The sensor can preferably detect photon radiation in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 to 1800 nm.In this region, the sensor is thus particularly suitable for detecting the fluorescent radiation emitted by the one or more fluorescent dyes. The selection of the sensor is thereby adjusted in particular to this property.The sensor may be a CMOS sensor, CCD sensor or PMD sensor.The sensor is preferably arranged in direct spatial proximity to the pulsed light source.This has the advantage that when calculating the fluorescence lives of the one or more fluorescent dyes, the distance between the sample and the sensor can be considered to be equal to the distance of the pulsed light source and the sample.Due to the nature of the fluorescent radiation, in which an electron of the fluorescent dye is first raised to a higher energy state and falls back to a lower energy state only after a certain time-the fluorescent lifetime-while emitting the fluorescent radiation, the time between photon emission and detection of the fluorescent radiation emitted by the one or more fluorescent dyes delays directly as a function of the fluorescent lifetime of the respective fluorescent dye.From the measured times between the photon emission and the detection of the photons, the fluorescence lives of the one or more fluorescent dyes can be calculated as the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source from the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source from the sample.The measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source from the sample, is composed of the time in which the photons travel the distance between light source and sample and between sample and detection location, for example sensor, and additionally of the time of the fluorescence radiation emitted with a delay by the one or more fluorescent dyes, depending on the respective fluorescence life.The time between the photon emission and the detection of the photons is preferably measured by means of optical 3D measurement.From the measured time between the emission of the photons, preferably a laser pulse of the laser, and the detection of the photons, preferably by the sensor, the distance covered by the photons between the location of the emission and the sample can be calculated.Usually, the distance travelled by the photons is calculated according to the formulad=the distance travelled by the photons, andc = Speed of Lightt=measured time between the emission of the photons, preferably of a light pulse of the light source, particularly preferably of a laser pulse of the laser, and the detection of the photons.The distance covered by the photons can be represented as a three-dimensional surface profile in the X-Y-Z direction. Here, from the three-dimensional surface profile of the entire 3-dimensional space with sample, the three-dimensional surface profile of the 3-dimensional space without sample is usually subtracted, so that a three-dimensional surface profile of the sample is obtained.This three-dimensional surface profile usually generally shows the contours of the surface of the sample, which is calculated by the photons interacting with the surface of the sample.The exception here is the fluorescent radiation emitted by the one or more fluorescent dyes. Due to the emission of the fluorescent radiation delayed directly as a function of the fluorescence lifetime, the detected fluorescent radiation appears as a depth in the Z direction within the three-dimensional surface profile.The depth in the Z direction, i.e. the distance of the calculated point from the surrounding points of the three-dimensional surface profile in the Z direction, is directly dependent on the respective fluorescence lifetime of the fluorescent dye. This means the longer the respective fluorescence lifetime, the greater the depth in the Z direction.Thus, the fluorescence lifetime of a fluorescent dye can be determined from the depth in the Z direction.By determining the point in the X-Y direction deviating in the Z direction in the three-dimensional surface profile, it is preferably also possible to determine the location of the excited fluorescent dye in the sample, preferably on the surface of the sample.Thus, by linking the determination of the deviation in the Z direction to the determination of the position in the X-Y direction, the fluorescence lifetime of a fluorescent dye can preferably be linked to its position in the sample, preferably on the surface of the sample.The measurement is preferably repeated many times at the distance of the pulsed photons, preferably the laser pulses. Time-dependent three-dimensional surface profiles are thereby generated.In a preferred embodiment, the time between the photon emission and the detection of the photons is measured by means of time of flight measurement.Particular preference is given here to pulse transit time methods, preferably LiDAR, more preferably time of flight (ToF)-LiDAR or frequency modulated continuous wave (FMCW) LiDAR.It is preferred that the time between the photon emission and the detection of the photons is measured by means of pulse transit time methods and the photons are emitted by means of pulsed laser beams.Here, the distance of the laser pulses is preferably in the range of 0.2 to 100 ns, preferably 0.5 to 50 ns, more preferably 1 to 20 ns, most preferably 1.0 to 10 ns.The fluorescence lifetime of the one or more fluorescent dyes is preferably greater than the distance of the laser pulses.Preferably, a system for optical 3D measurement is used for applying the method according to the invention as described herein.The 3D optical measurement system includes a) a unit for emitting photons by means of a pulsed light source; b) a unit for detecting photons; and c) a unit for measuring the time between the photon emission and the detection of the photons.Suitable units for emitting photons are, for example, lasers, preferably lasers for emitting pulsed laser beams.Suitable units for detecting photon radiation are, for example, sensors such as CMOS sensors, CCD sensors or PMD sensors.Suitable units for measuring the time are, for example, systems for measuring light transit times. These are usually connected to a unit for detecting photons, preferably a subunit of the unit for detecting photons.Furthermore, the system for optical 3D measurement usually has units for calculating the distance covered by the photons from the measured time between the photon emission and the detection of the photons and for representing the calculated distance covered in the form of a three-dimensional surface profile in the X-Y-Z direction.Data processing programs and graphic displays are usually available for these units.A time of flight (ToF) LiDAR system or a frequency modulated continuous wave (FMCW) LiDAR system is preferred, particularly preferably a time of flight (ToF) LiDAR system.The system for optical 3D measurement, preferably the time of flight (ToF) LiDAR system or the frequency modulated continuous wave (FMCW) LiDAR system, particularly preferably the time of flight (ToF) LiDAR system, is preferably a portable system.The portable system is preferably integrated into an electronic smart device, such as a smartphone or tablet.The currently common time of flight (ToF) LiDAR systems usually operate in a wavelength range from 850 to 905 nm.Therefore, when using a time of flight (ToF) LiDAR system, the emitted photons preferably have a wavelength in the range from 400 nm to 1500 nm, preferably 750 nm to 1250 nm, more preferably 800 nm to 1000 nm.The currently common frequency modulated continuous wave (FMCW) LiDAR systems usually operate in a wavelength range of around 1550 nm.Therefore, when using a frequency modulated continuous wave (FMCW) LiDAR system, the emitted photons preferably have a wavelength in the range from 800 nm to 3000 nm, preferably 1000 nm to 2000 nm, more preferably 1250 nm to 1800 nm.In a further aspect, the present invention relates to the use of a system for optical 3D measurement comprising a) a unit for emitting photons by means of a pulsed light source; b) a unit for detecting photons; and c) a unit for measuring the time between the photon emission and the detection of the photons for measuring fluorescence lives of one or more fluorescent dyes in a sample arranged in a 3-dimensional space as the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source from the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source from the sample.Suitable units for emitting photons are, for example, lasers, preferably lasers for emitting pulsed laser beams.Suitable units for detecting photon radiation are, for example, sensors such as CMOS sensors, CCD sensors or PMD sensors.Suitable units for measuring the time are, for example, systems for measuring light transit times. These are usually connected to a unit for detecting photons, preferably a subunit of the unit for detecting photons. Furthermore, the system for optical 3D measurement usually has units for calculating the distance covered by the photons from the measured time between the photon emission and the detection of the photons and for representing the calculated distance covered in the form of a three-dimensional surface profile in the X-Y-Z direction.Data processing programs and graphic displays are usually available for these units.A time of flight (ToF) LiDAR system or a frequency modulated continuous wave (FMCW) LiDAR system is preferred, particularly preferably a time of flight (ToF) LiDAR system.The system for optical 3D measurement, preferably the time of flight (ToF) LiDAR system or the frequency modulated continuous wave (FMCW) LiDAR system, particularly preferably the time of flight (ToF) LiDAR system, is preferably a portable system.The portable system is preferably integrated into an electronic smart device, such as a smartphone or tablet.In this case, all aspects of the method according to the invention as described herein are preferably applied to the use of optical 3D measurement.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedEP 0 933 407

[0051] U.S. Pat. No. 5,282,894

[0051] U.S. Pat. No. 5,665,151

[0051] WO 1998 / 018871

[0051] WO 2003 / 03803

[0051] U.S. Pat. No. 10,119,071

[0051] U.S. Pat. No. 5,542,971

[0051]

Claims

A method for measuring fluorescent lifetimes of fluorescent dyes, comprising the steps of: • providing a sample containing one or more fluorescent dyes in a 3-dimensional spatial region; • emitting photons by means of a pulsed light source into the 3-dimensional spatial region containing the sample; • detecting the photons after their interaction with the surfaces of the 3-dimensional spatial region and the sample; • measuring the time between the photon emission and the detection of the photons, wherein the measured time of the photons that have interacted with the surface of the sample depends directly on the distance of the pulsed light source to the sample and the fluorescent lifetimes of the one or more fluorescent dyes in the sample; • Calculation of the fluorescence lives caused by the one or more fluorescent dyes from the difference of the measured time, which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source to the sample, and the measured time, which is exclusively directly dependent on the distance of the pulsed light source to the sample.The method of claim 1, wherein the pulsed light source is a pulsed laser.The method according to claim 1 or 2, wherein the emitted photons have a wavelength in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 nm to 1800 nm.The method according to any one of the preceding claims, wherein the fluorescent dyes have an absorption wavelength in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 nm to 1800 nm, an emission wavelength in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 to 1800 nm and fluorescence lifetimes independently of one another in the range of 1 ns to 100 ms, preferably 10 ns to 1000 μs, more preferably 20 ns to 100 μs.The method of any preceding claim, wherein the sample is printed with a printing composition containing the one or more fluorescent dyes.The method of any preceding claim, wherein the fluorescent dyes comprise semiconducting inorganic nanocrystals.The method according to claim 6, wherein the semiconducting inorganic nanocrystals are selected from the group of perovskites, I-VI semiconductors, II-VI semiconductors, III-V semiconductors, IV-VI semiconductors, I-III-VI semiconductors, carbon dots and mixtures thereof.The method according to any one of the preceding claims, wherein time between the photon emission and the detection of the photons is measured by means of a sensor that detects photon radiation in the range of 400 nm to 3000 nm, preferably 750 nm to 2000 nm, more preferably 800 to 1800 nm.The method of claim 8, wherein the sensor is a CMOS sensor, CCD sensor, or PMD sensor.The method of claim 8 or 9, wherein the sensor is disposed in direct proximity to the pulsed light source.The method according to any one of the preceding claims, wherein the time between the photon emission and the detection of the photons is measured by means of time of flight measurement.The method according to any one of the preceding claims, wherein the time between the photon emission and the detection of the photons is measured by means of pulse transit time methods, preferably LiDAR, more preferably time of flight (ToF)-LiDAR or frequency modulated continuous wave (FMCW) LiDAR.The method according to claim 12, wherein the time between the photon emission and the detection of the photons is measured by means of pulse transit time methods and the photons are emitted by means of pulsed laser beams.The method according to any one of the preceding claims, wherein the distance of the light pulses, preferably the laser pulses, is in the range of 0.2 to 100 ns, preferably 0.5 to 50 ns, more preferably 1 to 20 ns, most preferably 1.0 to 10 ns, and the fluorescence lifetime of the one or more fluorescent dyes is greater than the distance of the light pulses, preferably the laser pulses.The method according to any one of the preceding claims, wherein the distance covered by the photons is calculated from the measured time between the photon emission and the detection of the photons according to the formula d = c ⋅ t 2 where d = distance covered by the photons c = speed of light t = measured time between the emission of the photons, preferably a light pulse of the light source, particularly preferably a laser pulse of the laser, and the detection of the photons.The method of claim 15, wherein the distance travelled by the photons is plotted as a three-dimensional surface profile in the X-Y-Z direction, in which the fluorescence lifetimes of the one or more fluorescent dyes due to the delay through the fluorescence lifetimes are shown as depth in the Z direction as a direct function of the respective fluorescence lifetime in the surface profile.The method according to any one of the preceding claims, wherein for the photon emission, the detection of the photons and the measurement of the time between the photon emission and the detection of the photons a portable system is provided, which is preferably integrated into an electronic smart device, such as a smartphone or tablet.Use of a system for optical 3D measurement comprising a) a unit for emitting photons by means of a pulsed light source; b) a unit for detecting photons; and c) a unit for measuring the time between the photon emission and the detection of the photons for measuring fluorescence lives of one or more fluorescent dyes in a sample arranged in a 3-dimensional space as the difference of the measured time which is directly dependent on the fluorescence lives of the one or more fluorescent dyes in the sample and the distance of the pulsed light source from the sample and the measured time which is exclusively directly dependent on the distance of the pulsed light source from the sample.