Apparatus for determining the presence of a characteristic in a sample, in particular for determining the sex of fertilized chicken eggs, use and method - Patents.com

Abstract

The invention relates to an apparatus (10) for determining the presence of a characteristic in a sample (12), comprising: a light source (14) emitting pulsed excitation radiation (16); a detection device (18) for detecting the characteristic fluorescence radiation (20) emitted by the sample (12); and a computer-based evaluation means (22), the detection device (18) configured for detecting the characteristic fluorescence radiation (20) of the sample (12) with time resolution by time-correlated single photon counting at different wavelengths and providing the evaluation means (22) with two-dimensional data comprising a wavelength dimension and a time dimension, the evaluation means (22) configured for classifying the provided data into classes by a classifier, the evaluation means (22) configured for identifying characteristic wavelengths to be prioritized during classification based on features formed in the time dimension of the data, the evaluation means (22) configured for preferentially considering data at the characteristic wavelengths for determining the presence of the characteristic in the sample (12). The invention further relates to a method for in-ovo sexing of fertilized chicken eggs (12). The invention further relates to the use of the above-mentioned apparatus (10).

Description

[Technical field]

[0001] The present invention relates to an apparatus for determining the presence of a characteristic in a sample, preferably for sexing fertilized chicken eggs.

[0002] The present invention further relates to an in ovo method for sexing fertilized chicken eggs.

[0003] The invention further relates to the use of the device described above. [Background technology]

[0004] Efforts are currently being made to make it possible to determine the sex of future chicks already in fertilized eggs.

[0005] In the chicken egg, different fluorophores arise in male and female chicks during development. The corresponding molecules have unpredictable fluorescence intensities due to their complex structures. In fluorescence, energy transfer from the excited state to the ground state of the molecule is observed. This event is time-dependent.

[0006] US Patent No. 5,399,633 describes an apparatus and method for optical in-ovo sexing of fertilized chicken eggs. The apparatus comprises a light source emitting excitation radiation for exciting fluorescence in a region inside the egg, spectroscopic means for time- and / or spectrally resolved analysis of the fluorescence radiation emitted from the region inside the egg, and an evaluation unit for sexing from data determined by the spectroscopic means. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2021 / 144420(A1) Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, an object of the present invention is to provide a means for improving the accuracy of in ovo sex determination using an apparatus with a simplified structure. [Means for solving the problem]

[0009] This object is achieved according to the invention by means of the features of the independent claims. Preferred embodiments of the invention are set out in the dependent claims, which can each alone or in combination represent aspects of the invention.

[0010] According to the invention, an apparatus for determining the presence of a characteristic in a sample, preferably for determining the sex of fertilized chicken eggs, comprises: - a light source emitting pulsed excitation radiation, - a detector for detecting the characteristic fluorescent radiation emitted by the sample; and - Computer-based assessment instruments the detection device is configured to detect intrinsic fluorescent radiation of the sample with time resolution by time-correlated single photon counting at different wavelengths and provide the evaluation means with two-dimensional data comprising a wavelength dimension and a time dimension; The evaluation means is configured to classify the data provided by the classifier into classes, at least one class representing a characteristic of the sample, the evaluation means is configured to identify a characteristic wavelength to be prioritized during classification based on features formed within a time dimension of the data, and the evaluation means is configured to give preferential consideration to data at this characteristic wavelength in order to determine the presence of the characteristic of the sample.

[0011] The present invention further relates to an in ovo method for sexing a fertilized chicken egg, - emitting pulsed excitation radiation with a light source to excite intrinsic fluorescence within an area within the chicken egg, on the egg membrane of the chicken egg, and / or on the egg shell of the chicken egg; - detecting with time resolution the intrinsic fluorescent radiation emitted by the inner region of the egg, the membrane and / or the shell of the egg at different wavelengths using a detection device by time-correlated single photon counting; - providing two-dimensional data comprising a wavelength dimension and a time dimension to an evaluation means by a detection device; - determining the sex of the fertilized chicken egg from the provided two-dimensional data using an evaluation means, classifying the data provided by the classifier into two classes, a first class representing male fertilized chicken eggs and a second class representing female fertilized chicken eggs, where during the classification, a characteristic wavelength to be prioritized is identified based on features formed within the time dimension of the data; and determining the sex of the fertilized chicken eggs by preferentially considering data at the characteristic wavelengths to determine the sex of the eggs.

[0012] The method is preferably carried out using the apparatus described above.

[0013] It has been demonstrated that classification of two-dimensional data into classes for the purpose of determining the presence of a property of a sample provides improved accuracy for data having a relatively high time resolution. Furthermore, it has been demonstrated that the information present in the decay behavior of the intrinsic fluorescent radiation is not equally relevant at all wavelengths, but that intrinsic wavelengths can be identified that are given priority consideration in classification, allowing for improved accuracy. Correspondingly, devices and methods for detection of intrinsic fluorescent radiation by single photon counting techniques and identification of intrinsic wavelengths provide improved accuracy.

[0014] With regard to in ovo sexing of fertilized chicken eggs, in other words besides the energy of the emitted photons (wavelength dimension of 2D data) for sexing of fertilized chicken eggs, knowledge of the lifetime and decay curves of excited molecular states (time dimension of 2D data) is relevant, the time dimension containing more explicit information at specific wavelengths compared to other wavelengths and correspondingly is taken into account preferentially during the evaluation.

[0015] With regard to the apparatus, it is intended that the apparatus comprises a light source emitting pulsed excitation radiation, a detection device for detecting intrinsic fluorescence radiation by time-correlated single-photon counting at a number of different wavelengths, and a computer-based evaluation means.

[0016] In order to detect the intrinsic fluorescence radiation with high time resolution, the intrinsic fluorescence radiation is detected here by time-correlated single photon counting (TCSPC). Instead of the prior art (Patent Document 1) which uses a method in which the full spectrum is captured by an ICCD camera at different times for each excitation pulse of the light source, in TCSPC the full spectrum is not recorded for each excitation pulse.

[0017] In TCSPC, single photons of a periodic light signal - here the intrinsic fluorescence radiation - are detected and the respective time between the excitation pulse of the pulsed excitation radiation and the arrival of the photon in the detection device is determined. In other words, fluorophores in the test sample, preferably a chicken egg, are excited with the pulsed excitation radiation of the light source. The time measurement is started by an excitation pulse and stopped by the photon emitted on the transition from the excited state to the ground state. This measurement is repeated many times and single time-correlated photons (with respect to the excitation pulse) are sorted according to their measurement time in a so-called TCSPC histogram. The TCSPC histogram represents the time course of the intrinsic fluorescence radiation after excitation. Preferably, the TCSPC histogram generated by the detection device has a class width, also called bin width or container width, for the histogram classes, between 1 ps and 50 ps, ​​preferably between 10 ps and 20 ps. Preferably, the class width of the TCSPC histogram is adapted to the device and / or to the test sample. More preferably, the time resolution of the entire device is taken into account when fitting the class width of the TCSPC histogram - and particularly preferably the full width at half maximum (FWHM) of the instrument response function (IRF), which depends primarily on the light source and the pulse length arising from the light source and / or detector elements of the detection device.

[0018] In other words, instead of measuring the full spectrum (wavelength dimension of the two-dimensional data) at different times, a TCSPC histogram, which represents the time course of the intrinsic fluorescence radiation - i.e. the time dimension of the two-dimensional data - is calculated over many measurement cycles - typically 10 6, and each measurement cycle begins with an excitation pulse of the light source.

[0019] The detection device is configured to provide the evaluation means with two-dimensional data comprising a wavelength dimension and a time dimension. The two-dimensional data preferably represents the decay behavior of the intrinsic fluorescence (time dimension) at different wavelengths (wavelength dimension). The two-dimensional data is passed on to the evaluation means, for example in the form of an m×n matrix. Preferably, the two-dimensional data has more data points in the time dimension than in the wavelength dimension. This has proven to be particularly advantageous for determining the presence of a characteristic in the sample, in particular for determining the sex of chicken eggs.

[0020] Based on the two-dimensional data, the evaluation means can identify specific wavelengths that should be prioritized. This is achieved by a classifier classifying the provided data into classes. For each sample and each possible property of the sample, there can be a different number of classes, which represent the possible properties of the sample.

[0021] Preferably, the identification of the characteristic wavelengths is based on the principles of supervised machine learning, the characteristic wavelengths being calculated by the evaluation means from two-dimensional data representative of the corresponding classes of samples. According to a preferred development, the evaluation means is capable of and / or is configured to calculate from the decay behavior of the characteristic fluorescence of the wavelengths - i.e. from the time dimension of the two-dimensional data - one or more features and separation properties specific to the classes to which they belong. The features can be calculated from a time series representative of the individual wavelengths.

[0022] Alternatively, features can be calculated from a linear combination of the time series representing the individual wavelengths.

[0023] Further preferably, the evaluation means is configured to identify wavelengths having relatively high separation properties as characteristic wavelengths. Preferably, only a number of characteristic wavelengths are identified until a sufficient classification rate is reached for the desired and / or observed sample. Non-preferred wavelengths preferably do not contribute to class separation and are therefore ignored and not further considered for classification.

[0024] It has been shown that classifications without wavelength preference, as well as classifications in which all wavelengths are equally prioritized, have a lower hit rate than classifications with wavelength preference. More preferably, a separation characteristic analysis metric is taken into account when identifying the unique wavelengths. In connection with the in-ovo sexing method, preferably Linear Discriminant Analysis (LDA) is to be used as the metric. LDA is particularly suitable for data with a Gaussian distribution.

[0025] Preferably, the evaluation means is configured to take into account the data preferentially at a characteristic wavelength, in particular taking into account features formed in the time dimension of the data preferentially at a characteristic wavelength, in order to determine the presence of the characteristic property of the sample. With respect to this method, sexing of the eggs is preferably performed by taking into account the data preferentially at a characteristic wavelength, in particular taking into account features formed in the time dimension of the data preferentially at a characteristic wavelength.

[0026] The device for determining the presence of a characteristic in a sample is not only used for determining the sex of fertilized chicken eggs. The device is also suitable for classifying samples with respect to other characteristics. For this purpose, the sample does not have to be specially prepared. In particular, the described device can be used when authenticating samples and / or objects with respect to a certain characteristic, i.e. when establishing whether a claimed characteristic of a sample is relevant, for example for counterfeit authentication in banknotes or for authentication of a claimed origin characteristic of food. In particular, the device is suitable for authentication when a sample with a claimed characteristic, i.e. for example a genuine banknote, closely resembles a sample without the claimed characteristic, i.e. in this case a counterfeit banknote.

[0027] According to a further preferred development, the detection device is configured to detect the intrinsic fluorescence radiation of the sample with time resolution as unique wavelengths preferentially identified by time-correlated single photon counting and to provide the evaluation means with two-dimensional data including a wavelength dimension and a time dimension, the wavelength dimension corresponding to the number of unique wavelengths, and the evaluation means is configured to classify the data provided by the classifier into classes. In other words, the evaluation means determines whether or not a characteristic is present in the sample based on the two-dimensional data. This is preferably achieved by the detection device determining a TCSP histogram exactly at the unique wavelengths and providing the evaluation means with a time series only at the unique wavelengths. In this way, a high hit rate is achieved in determining the characteristic of the sample without having to detect the intrinsic fluorescence at all possible wavelengths.

[0028] According to a preferred development of the invention, the device is provided with a light source configured as a pulsed excitation laser system or a pulsed LED. Particularly preferably, the light source is configured to excite the sample with a wavelength in the UV range, particularly preferably in the range around and / or at 266 nm. Even more preferably, the light source is configured to emit a wavelength in the range from ≧250 nm to ≦520 nm, in particular in the range from ≧280 nm to ≦400 nm, as excitation radiation.

[0029] With regard to the pump laser system, it is further preferred that the pump laser system is configured as a solid-state laser with a frequency conversion stage and / or an optical parametric oscillator (OPO). Preferably, a Ti:Sapphire laser with a frequency conversion stage and / or an optical parametric oscillator (OPO) is used. Alternatively, the pump laser system is preferably configured as a frequency-doubled Nd:YAG laser, preferably with an optical parametric oscillator. Further preferred, the pump laser system is a diode laser system, preferably in a MOFA arrangement. Further preferred, the pump laser system is configured as a master oscillator power amplifier (MOPA) and / or a master oscillator fiber amplifier (MOFA). MOPAs and / or MOFAs are laser systems that use a seed laser as a master oscillator to set the characteristics of the emission radiation with high coherence in the amplification unit. Particularly preferred, infrared and / or near infrared laser diodes are used as master oscillators or seed lasers to set the laser emission characteristics in a multi-stage fiber amplifier in a MOFA arrangement. The second, third and / or fourth harmonic wavelengths can be further generated by nonlinear frequency conversion using birefringent crystals. This allows for particularly compact and transportable measurement structures.

[0030] Alternatively, the light source can be configured as a pulsed LED. LEDs have the advantage of being inexpensive. Furthermore, the high spectral bandwidth of the LED emission compared to excitation laser systems has the advantage that a relatively large number of absorption processes are induced in the different fluorophores in the sample by the LED as light source. Preferred LEDs used as light sources emit at wavelengths in the UV range, particularly preferably at 265 nm, 285 nm, 310 nm and / or >310 nm, each with a spectral bandwidth of ±10 nm. Furthermore, this spectral bandwidth can be reduced by corresponding interference filters.

[0031] In order to allow the highest possible time resolution in the detection of the intrinsic fluorescence radiation, it is advantageous if the length of the excitation pulse of the pulsed excitation radiation is as short as possible. In this connection, the light source is preferably configured to emit pulsed excitation radiation with a pulse length of ≦500 ps, ​​preferably ≦200 ps, ​​particularly preferably ≦100 ps. This is made possible in particular by the embodiment of the light source as a pulsed excitation laser system. Alternatively, the light source is configured to emit pulsed excitation radiation with a pulse length of ≦5 ns, preferably ≦2 ns, particularly preferably ≦1 ns. This is made possible in particular by the embodiment of the light source as an LED. This short pulse length increases the time resolution and thus the accuracy in determining the presence of a property of the sample.

[0032] Since the intrinsic fluorescence radiation detected by the TCSPC undergoes a large number of measurement cycles to generate a TCSPC histogram, it is further preferred that the light source is configured to emit pulsed excitation radiation with a pulse repetition rate of ≧10 MHz, which significantly reduces the time consumption for two-dimensional data detection.

[0033] Furthermore, in a TCSPC for detecting intrinsic fluorescence radiation, the excitation intensity is preferably kept so small that the probability of detection of one fluorescence photon per excitation cycle is ≦1, i.e. at most one photon is detected within each excitation cycle. In this connection, according to a further preferred development of the invention, the device comprises an optical attenuator in the optical path between the light source and the sample, which adjusts the energy of the excitation radiation. Particularly preferably, it is a variable attenuator, in particular a variable laser beam attenuator. Even more preferably, this attenuator is such that the energy per pulse for exciting the sample is so small that the probability of detection of one fluorescence photon per excitation cycle is ≦1. Single-photon statistics are usually possible if the counting pulses at the detector elements are caused on average only by 20-100 excitation pulses. In other words, this means that the count rate at the detector elements is preferably in the range of 1-5% of the excitation rate. This means, for example, that at a pulse repetition rate of 80 MHz, the attenuator is adjusted so that the average count rate of the detector elements does not exceed 4 MHz. That way it is easily guaranteed that the TCSPC histogram does not contain systematic measurement errors due to the so-called pile-up effect.

[0034] As already mentioned, the detection device is configured for detecting the intrinsic fluorescence radiation of the sample with time resolution by time-correlated single photon counting at different wavelengths. According to a preferred development of the invention, in this connection, the detection device for detecting the intrinsic fluorescence radiation at different wavelengths comprises a monochromator, a spectrograph and / or a spectrometer. This significantly facilitates the detection of the intrinsic fluorescence radiation at different wavelengths. The detection device can, for example, comprise a stepwise rotatable diffraction grating and a monochromator. This has the advantage that a wide spectral range, for example from 370 nm to 700 nm, can be scanned stepwise and a TCSPC histogram can be detected for each scanning wavelength.

[0035] In an alternative embodiment of the present invention, the device preferably comprises an interchangeable interference filter between the sample and the detector element of the detection device for spectral separation. This interference filter can be replaced by another interference filter for each detected wavelength. This is particularly advantageous for the time-resolved detection of the intrinsic fluorescence radiation of the sample by time-correlated single-photon counting exactly as the preferentially identified intrinsic wavelength. In a further preferred alternative, the detection device comprises a number of detector elements and one or more beam splitters that split the intrinsic fluorescence radiation into a number of partial beams. In this way, a TCSPC histogram can be recorded for each partial beam using a preferably interchangeable interference filter and a detector element.

[0036] Further with reference to the detection device, it is provided that the detection device preferably includes a hybrid photomultiplier tube (hybrid PMT) - also called hybrid photodetector - as a detector element. Preferably, the detector element is a combination of a front-end PMT and an avalanche photodiode (APD) as an additional amplification stage. More preferably, the detector element is configured to replicate the photoelectrons induced in the latter amplification stage by a factor of 50000 to 150000. More preferably, the detector element is configured to achieve a time resolution of approximately 120 ps (FWHM of the detector response function (IRF)).

[0037] The detector element is further preferably configured as a multi-channel detector, in particular a multi-channel plate PMT (MC-PMT), so that several different wavelengths can be detected simultaneously. For example, a multi-channel detector has 16 channels for detection. The embodiment of the detection device as a combination of a spectrograph with a fixedly arranged diffraction grating and an MCP-PMT allows for simultaneous multispectral detection with an equidistant division of the entire detected wavelength range. The division and width of the wavelength intervals are according to the number of channels of the MCP-PMT.

[0038] Preferably, the detector is configured to receive intrinsic fluorescent radiation having a wavelength in the range of 200 nm or more and 700 nm or less.

[0039] In connection with the TCSPC histogram, according to a preferred development, the device for generating an electrical trigger signal which can be generated simultaneously with the excitation pulse comprises a pulse signal generator. In this way, an electrical trigger signal generated simultaneously with the excitation pulse by means of a time-to-amplitude converter can trigger a fast voltage ramp which is stopped by measuring the photons of the intrinsic fluorescence radiation. As a pulse signal generator, for example, a direct excitation laser system can be used, for which an electrical trigger signal is available. Alternatively, the electrical trigger signal can be generated by a trigger photodiode, for which a portion of the excitation radiation is decoupled via a beam splitter.

[0040] Moreover, in this connection, it is further preferred that the detector element generates an electrical output pulse for each detected photon, which is preferably formed into a standard pulse in a fast discriminator. In this way, the detector standard pulse can stop the voltage ramp initiated by the electrical trigger signal of the excitation laser system. A voltage is assigned to each stopping point, and thus a TCSPC histogram can be generated in this way.

[0041] According to a further preferred development of the invention, the device preferably comprises a long-pass edge filter in the optical path between the sample and the detection device for filtering the wavelength of the excitation radiation, in such a way that the excitation radiation scattered by the sample can be easily filtered.

[0042] According to a further preferred development, the device includes an optical component in the optical path between the sample and the detection device, which focuses the intrinsic fluorescence radiation emitted by the sample onto the detection device. This optical component can be configured, for example, as a lens, in particular as a focusing lens. In particular when using an LED as the light source, the device can further include an optical component between the light source and the sample, which focuses the excitation radiation.

[0043] According to a further preferred development of the invention with respect to the illumination of the sample, the device is configured to illuminate the sample with excitation radiation in free space and to deflect the illuminated intrinsic fluorescence radiation in free space at an angle different from zero with respect to the excitation radiation to the detection device. Free space in this context means that the light is not transmitted through a fiber-based light guide system but propagates freely in space. In this preferred development, the device does not comprise a fiber-based light guide system for guiding the light, for example an optical fiber. The omission of the fiber-based light guide system prevents losses of pulse energy in the light guide system and / or broadening of the excitation pulse, which can be problematic especially for excitation wavelengths in the UV range. Furthermore, the device without a fiber-based light guide system can be configured very simply, since a measurement head for illuminating the sample and / or for receiving the intrinsic fluorescence radiation from the sample can be omitted.

[0044] In connection with the propagation in free space, according to a further preferred development, the device has a shielding means for shielding against ambient light. Particularly preferably, the shielding means is a sample chamber, which is configured so that the excitation radiation from the light source can propagate to a sample located in the sample chamber shielded from ambient light and that the intrinsic fluorescence radiation emitted by the sample can propagate shielded from ambient light to the detection device.

[0045] According to an alternative development of the invention, the device preferably comprises a measuring head, a) the measurement head is configured to deliver excitation radiation into and / or to the sample, or b) the measurement head is configured to receive intrinsic fluorescent radiation from and / or by the sample, or c) A measurement head is configured to transmit excitation radiation into and / or to the sample and to receive intrinsic fluorescence radiation from and / or by the sample.

[0046] Preferably, the measurement head may be connected to a light guide system, more preferably, especially in case c), a Y-shaped light guide system comprising two light guide skeins brought together at the side of the measurement head.

[0047] In this case, in particular, the light guide skein is formed as a light guide bundle, the individual light guides of which are twisted together, for example, on the sides of the head end of the measurement head. Alternatively, the individual light guides can also be distributed evenly over an approximately circular cross section. Still alternatively, the light guides guiding the excitation radiation can be arranged, for example, in a circular manner in the inner region of the cross section, and the light guides guiding the intrinsic fluorescence radiation can be arranged, for example, concentrically around the outer periphery. In this way, the same measurement head can easily realize the emission of excitation radiation into and / or to the sample and the reception of intrinsic fluorescence radiation from and / or by the sample.

[0048] As already described with respect to the computer-based evaluation means, the evaluation means is configured to classify the provided data into classes using a classifier, at least one class representing a characteristic of the sample. In this connection, preferably, the classifier is a linear classifier. A linear classifier divides the classes along a linear hyperplane. More preferably, the classifier is designed by linear discriminant analysis based on the training data using feature selection. In other words, this is preferably an evaluation means based on machine learning. Preferably, the evaluation means learns from examples - the training data - and can generalize them after the learning phase. To that end, in machine learning, the algorithm builds a statistical model based on the training data.

[0049] According to a further preferred embodiment of the present invention, the evaluation means is an evaluation means based on so-called feature engineering. This feature engineering is a form of data processing and refers to the selection and processing of features to be considered for the creation of the machine learning model. In this connection, it is further preferred that the features formed in the time dimension of the data include first central moment (mean), second central moment (standard deviation) and third central moment (skewness) and / or that the features formed in the time dimension of the data are histogram-based features, signal sequence-based features and / or transformation-based features. Preferably, the evaluation means is further configured to remove features with weak separation properties, preferably using Fisher's Linear Discriminant Analysis (LDA).

[0050] Furthermore, according to a further preferred development, the evaluation means is configured to identify characteristic wavelengths using machine learning, such that two-dimensional data having little resolution in the wavelength dimension can achieve good accuracy in determining the presence of a characteristic of the sample despite low spectral resolution.

[0051] According to a further preferred development of the invention, the evaluation means is configured to determine the sex of the fertilized chicken egg taking into account the two-dimensional data. In other words, the device is preferably used for determining the sex of the fertilized chicken egg. In this connection, the evaluation means is further configured to classify the provided data by means of a classifier into two classes, the first class representing a male characteristic of the sample chicken egg and the second class representing a female characteristic.

[0052] As already mentioned, the present invention further relates to an in ovo method for sexing fertilized chicken eggs, - emitting pulsed excitation radiation with a light source to excite intrinsic fluorescence within an area within the chicken egg, on the egg membrane of the chicken egg, and / or on the egg shell of the chicken egg; - detecting with time resolution the intrinsic fluorescent radiation emitted by the inner region of the egg, the membrane and / or the shell of the egg at different wavelengths using a detection device by time-correlated single photon counting; - providing two-dimensional data comprising a wavelength dimension and a time dimension to an evaluation means by a detection device; - determining the sex of the fertilized chicken egg from the provided two-dimensional data using an evaluation means, classifying the data provided by the classifier into two classes, a first class representing male fertilized chicken eggs and a second class representing female fertilized chicken eggs, where during the classification, a characteristic wavelength to be prioritized is identified based on features formed within the time dimension of the data; and determining the sex of the fertilized chicken eggs by preferentially considering data at the characteristic wavelengths to determine the sex of the eggs.

[0053] The in-ovo sexing method for fertilized chicken eggs is provided for by exciting the intrinsic fluorescence in an area inside the egg, the egg membrane and / or the egg shell with a light source. Preferably, intrinsic fluorescence is excited on the egg membrane and / or on the egg shell. Furthermore, the area inside the egg is preferably the blood passage area and / or the embryonic structure area. Furthermore, it is provided for not having to puncture the egg for sexing. Instead, it is possible to sex the egg directly on, at and / or through the egg shell with the described device and / or the described method. In other words, it is provided for preferably directing the emission of the pulsed excitation radiation towards the egg shell. This has the advantage that the method is very easy and fast to carry out and the risk of infection of the egg is significantly reduced. Which area is selected depends in particular on the development stage in the fertilized chicken egg. Furthermore, it is not necessary to hatch the egg. Sexing can also be done on unhatched chicken eggs.

[0054] Alternatively, it is also possible to puncture the chicken eggs for sexing. In this connection, a hole is preferably made in the eggshell of the chicken egg. The hole preferably has a dimension or hole diameter D in the range of 0.5 mm≦D≦3 mm. In particular, the hole is made without puncturing the egg membrane underneath the eggshell and / or without puncturing the shell membrane and / or egg membrane underneath the eggshell. The measurement of the intrinsic fluorescence can be carried out in the amniotic membrane, the shell membrane and / or the egg membrane.

[0055] According to a further alternative embodiment of the method of the present invention, the interior of the egg and / or an interior region of the egg is removed from the eggshell and process steps can be carried out outside the eggshell accordingly.

[0056] According to a further preferred development of the invention, the method further comprises the steps of: - detecting characteristic fluorescent radiation emitted from an internal region of the chicken egg, the egg membrane and / or the egg shell with a detection device at a characteristic wavelength by time-correlated single photon counting with time resolution; - providing two-dimensional data comprising a wavelength dimension and a time dimension to the evaluation means by the detection device, the wavelength dimension corresponding to a number of characteristic wavelengths; It is supposed to include:

[0057] That is, as soon as the evaluation means has identified a preferred specific wavelength, the specific fluorescence can be detected precisely at the specific wavelength for sexing in further samples, and this further wavelength does not have to be taken into further consideration.

[0058] As already mentioned, the device is not only used for determining the sex of fertilized chicken eggs. In this regard, the present invention relates to - to determine the ageing of fuels and / or raw materials for industrial production, such as immersion baths, hydraulic oils and / or lubricants, - In particular to control the quality of food and / or medicines, - To determine the origin characteristics of samples, especially food products; - To determine the degree of contamination of the sample, especially the surface, - To authenticate counterfeits, and / or - to identify changes in cellular metabolism, The present invention relates to the use of the device described above.

[0059] The identification of an alteration in cellular metabolism is preferably the identification of an alteration in cellular metabolism for non-therapeutic purposes, more preferably the identification of an alteration in cellular metabolism for non-diagnostic and / or non-therapeutic purposes.

[0060] The present invention will now be described in detail with reference to the accompanying drawings, in which: The features listed below can each represent an aspect of the present invention either alone or in combination. [Brief description of the drawings]

[0061] [Figure 1] 1 is a schematic diagram showing the configuration of a chicken egg and an in ovo sex-determining device for this chicken egg according to a preferred embodiment of the present invention. [Diagram 2] 2 is a schematic diagram showing two alternatives for directing excitation radiation and emitted fluorescent radiation into the structure of a chicken egg according to a preferred embodiment of the present invention shown in FIG. 1. [Diagram 3] 2 is a schematic diagram illustrating a light source of the configuration shown in FIG. 1. [Figure 4] 7 is a schematic diagram showing a TCSPC histogram obtained by the apparatus of FIG. 1, FIG. 5 or FIG. 6 according to a preferred embodiment of the present invention. [Diagram 5] 2 is a schematic diagram showing an alternative configuration to that of FIG. 1 of an in-ovo sexing device according to a preferred embodiment of the present invention. [Figure 6] 1 is a schematic diagram illustrating a further alternative configuration of an in-ovo sexing device according to a preferred embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] 1 shows a schematic diagram of an apparatus 10 for sexing a fertilized chicken egg 12, according to one preferred embodiment of the present invention. The apparatus 10 includes a light source 14 for emitting pulsed excitation radiation 16, a detection device 18 for detecting intrinsic fluorescent radiation 20 emitted from the chicken egg 12, and a computer-based evaluation means 22.

[0063] The detection device 18 is configured to detect the characteristic fluorescence radiation 20 of the chicken eggs 12 with time resolution by time-correlated single photon counting (TCSPC) at different wavelengths and provide two-dimensional data comprising a wavelength dimension and a time dimension to the evaluation means 22. The evaluation means 22 is configured to classify the data provided by the classifier into two classes, a first class representing male fertilized chicken eggs 12 and a second class representing female fertilized chicken eggs 12, during which features formed in the time dimension of the data are taken into preferential consideration at the characteristic wavelength.

[0064] The egg 12 is here fixed in a sample holder 24 and positioned in the optical path such that the free propagating excitation radiation 16 of the light source 14 impinges on the egg 12. A variable laser optical attenuator 26 is further provided between the light source 14 and the egg 12 to reduce the excitation energy to an excitation energy suitable for TCSPC.

[0065] The intrinsic fluorescence radiation 20 emitted from the interior region of the egg 12 is detected by the detector 18. For this purpose, the detector 18 is arranged in relation to the egg 12 such that the emitted intrinsic fluorescence radiation 20 strikes the detector 18 in free propagation at an angle of approximately 90 degrees. In order to focus the intrinsic fluorescence radiation 20 onto the detector 18, the device 10 has a lens 28 in the optical path between the egg 12 and the detector 18. Furthermore, a long-pass filter 30 between the egg 12 and the detector 18 is used to filter out the wavelengths of the excitation radiation 16 scattered by the egg 12. Before the intrinsic fluorescence radiation 20 strikes the detector 18, it is further attenuated by an aperture 32.

[0066] The device 10, i.e. in the embodiment shown in Figure 1, does not have a fiber-based light guide system 34 for directing light to the eggs 12 and / or the detection device 18. Figure 2 shows two parts of the device 10 in an alternative embodiment, where the device 10 includes a light guide system 34 with a measurement head 36.

[0067] In the variant shown in fig. 2a), the light guide system 34 is configured in a Y-shape and comprises two light guide skeins 38, 40, a light guide skein 38 for the excitation radiation and a light guide skein 40 for the intrinsic fluorescence radiation 20. Both light guide skeins 38, 40 are grouped together in the measurement head 36, so that the measurement head 36 is configured both to emit the excitation radiation 16 to the egg 12 and to receive the intrinsic fluorescence radiation 20 emitted by the egg 12.

[0068] In the variant shown in FIG. 2b), the light guide system 34 is configured as a single light guide system and comprises only a light guide skein 40 for the intrinsic fluorescence radiation 20. The excitation radiation 16 propagates further freely from the light source 14 (not shown in FIG. 2) to the chicken egg 12. The measuring head 36 of the light guide system 34 is accordingly configured to receive the intrinsic fluorescence radiation 20 from the chicken egg 12.

[0069] FIG. 3 shows a schematic diagram of the light source 14 in the configuration shown in FIG. 1. The light source 14 is realized here as a laser system 14 and generates pump pulses with a pulse length of approximately 80 ps. The laser system 14 includes an infrared laser diode 42 that emits laser radiation at 1064 nm and is used as a master oscillator or seed laser to set the laser emission characteristics of a multi-stage fiber amplifier 44. A birefringent crystal 46 and a dichroic mirror 48 present in the optical path are used to generate the second harmonic 50a (532 nm), the third harmonic 50b (355 nm) and the fourth harmonic 50c (266 nm) wavelengths by nonlinear frequency conversion. In the configuration shown in FIG. 1, the fourth harmonic 50c at 266 nm is used as the pump radiation for the sample.

[0070] In connection with the detection arrangement 18 shown in Fig. 1, Fig. 4 shows a schematic diagram of a TCSPC histogram 52 determined during the detection of the intrinsic fluorescence radiation 20, which represents the time course of the intrinsic fluorescence radiation after excitation. As already described, the detection arrangement 18 is configured for the time-resolved detection of the intrinsic fluorescence radiation 20 of the chicken egg 12 by means of TCSPC at a number of different wavelengths. To this end, the detection arrangement 18 comprises a spectroscopic device with a monochromator 54 in the embodiment shown in Fig. 1. As detector element 56, the detection arrangement 18 comprises a hybrid photomultiplier tube 56a. The monochromator 54 consists essentially of a diffraction grating which can be rotated in stages. By rotating the diffraction grating in stages, the spectral range to be examined can be scanned.

[0071] 5 shows an alternative embodiment of the device 10, in which the detector element 56 is configured here as an MCP-PMT 56b with 16 channels. In contrast to FIG. 1, the diffraction grating of the monochromator 54 is additionally arranged stationarily. This embodiment of the device 10 allows the spectral range to be examined to be "resolved" down to 16 WL subintervals (detection channels).

[0072] 6 shows a further alternative embodiment of the device 10, in which a number of detector elements 56 are used. Both detector elements 56 are configured as hybrid photomultiplier tubes 56a, similar to FIG. 1. Instead of a diffraction grating, a beam splitter 57 is used. Between the beam splitter 57 and both hybrid PMTs 56a, an interference filter 59 is respectively interposed, which transmits one specific wavelength each.

[0073] In TCSPC, single photons 58a, 58b of the intrinsic fluorescence radiation 20 are detected and the respective times 62 between the excitation pulse 60 of the pulsed excitation radiation 16 and the arrival of each photon 58 in the detection device 18 are determined. To this end, the detection device includes TCSPC electronics 61, which are shown in schematic form in Figs. 1, 5 and 6. With reference to Fig. 4, a time measurement is started by an excitation pulse 60a, which stops the measurement with an emitted photon 58a on the transition from the excited state to the ground state (Fig. 4a). The process is repeated with the next excitation pulse 60b and the next photon 58b (Fig. 4b). Repeating this measurement multiple times results in the TCSPC histogram 52 shown in Fig. 4c as a function of the measurement times 62 of the single photons 58. As further shown in Figs. 1, 5 and 6, the device 10 has an electrical connection 64 for measuring the time intervals 62, which transmits an electrical trigger signal coincident with the excitation pulse 60 to the detection device 18. The TCSPC electronics 61, shown diagrammatically as a rectangle in Figures 1, 5 and 6 and which may for example be physically configured as a PC insert card, evaluates the signal and creates a TCSPC histogram, which is provided to the evaluation means 22 as two-dimensional data including wavelength and time dimensions. The detector 18 is configured to detect intrinsic fluorescence radiation 20, i.e., by TCSPC, at a number of different wavelengths and provide two-dimensional data to the evaluation means 22, where the data is represented by a mathematical matrix A∈R m×n where the matrix contains m data points in the wavelength dimension, 74 data points in this example. In the time window, the matrix contains n data points that allow high time resolution for the required hit rate, 250 data points in this example.

[0074]

number

[0075] In this case, the row of A, that is, (a j1 , …, a jn ), j=1,...,m correspond respectively to a TCSPC histogram and thus to a physical, primarily time-resolution measurement for a particular fixed wavelength.

[0076] For sexing of the eggs 12, a classifier classifies the provided data into two classes, a first class representing male fertilized eggs 12 and a second class representing female fertilized eggs 12, during which a characteristic wavelength is identified based on features formed in the time dimension of the data, and sexing of the eggs 12 is performed by preferentially considering the data at the characteristic wavelength. As a classifier, a linear classifier is used here, which separates the data along a hyperplane. The features formed in the time dimension of the data are here a j :=(a j1 , …, a jn ), the first three moments of the central moment for j=1, namely the mean μ, the standard deviation σ and the slope S. [Explanation of symbols]

[0077] 10 equipment 12. Chicken eggs 14 Light source 16 Excitation Radiation 18 Detection Device 20 Intrinsic Fluorescence Radiation 22 Evaluation methods 24 Sample holder 26 Variable Laser Optical Attenuator 28 Lenses 30 Longpass Filter 32 Aperture 34 Light Guide System 36 Measuring Head 38 Light Guide ▲ Skein ▼ 40 Light Guide ▲ Skein ▼ 42 Infrared laser diode 44 Multi-stage fiber amplifier 46 Birefringent Crystals 48 Dichroic Mirror 50 Second to fourth harmonics of laser wavelength 1064 nm 52 TCSPC histogram 54 Monochromator 56a Detector element, hybrid PMT 56b Detector element, MCP-PMT 57 Beam splitter 58 photon 59 Interference Filters 60 Excitation Pulses 61 TCSPC Electronics 62 Time between excitation pulse and photon detection 64 Electrical Connections

Claims

1. A device (10) for determining the presence of a characteristic in a sample (12), preferably for sexing a fertilized chicken egg (12), said device (10) comprising: a light source (14) emitting pulsed excitation radiation (16), - a detection device (18) for detecting the intrinsic fluorescent radiation (20) emitted by said sample (12), and - a computer-based evaluation means (22), the detection device (18) is configured to detect the intrinsic fluorescence radiation (20) of the sample (12) with time resolution by time-correlated single photon counting at different wavelengths and to provide the evaluation means (22) with two-dimensional data comprising a wavelength dimension and a time dimension, The evaluation means (22) is configured to classify the provided data into classes by a classifier, at least one class representing a characteristic of the sample (12); The evaluation means (22) is configured to identify characteristic wavelengths to be prioritized during the classification based on features formed within the time dimension of the data, and the evaluation means (22) is configured to preferentially consider the data at the characteristic wavelengths to determine the presence of a characteristic of the sample (12).

2. The apparatus (10) of claim 1, wherein the light source (14) is configured as a pulsed excitation laser system or a pulsed LED.

3. 3. The apparatus (10) according to claim 1, wherein the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse repetition rate of ≧10 MHz and / or the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse length of ≦500 ps and / or the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse length of ≦5 ns.

4. The apparatus (10) of any one of claims 1 to 3, further comprising an optical attenuator (26) in an optical path between the light source (14) and the sample (12) for adjusting the energy of the excitation radiation (16).

5. The apparatus (10) according to any one of claims 1 to 4, wherein the detection device (18) for detecting the intrinsic fluorescence radiation (20) at different wavelengths comprises a monochromator (54), a spectrograph, a beam splitter (57) having a plurality of interference filters (59) and / or a spectrometer, and / or the detection device (18) comprises a hybrid photomultiplier tube (56a) and / or a multichannel plate photomultiplier tube (56b) as detector element (56).

6. The apparatus (10) of any one of claims 1 to 5, further comprising a long-pass edge filter (30) in an optical path between the sample (12) and the detection device (18) for filtering the wavelength of the excitation radiation (16).

7. The apparatus (10) according to any one of claims 1 to 6, wherein the apparatus (10) is configured to irradiate the sample (12) with the excitation radiation (16) in free space, and to deflect the intrinsic fluorescence radiation (20) irradiated at an angle different from zero with respect to the excitation radiation (16) in free space to the detection device (18).

8. The apparatus (10) includes a measurement head (36), a) the measurement head (36) is configured to deliver the excitation radiation (16) into and / or to the sample (12), or b) the measurement head (36) is configured to receive the intrinsic fluorescent radiation (20) from and / or by the sample (12); or c) the apparatus (10) according to any one of claims 1 to 6, wherein the measurement head (36) is configured to transmit the excitation radiation (16) into and / or to the sample (12) and to receive the intrinsic fluorescence radiation (20) from and / or by the sample (12).

9. The apparatus (10) of any one of claims 1 to 8, wherein the evaluation means (22) is configured to identify the characteristic wavelengths by machine learning.

10. The device (10) according to any one of the preceding claims, wherein the evaluation means (22) is configured to determine the sex of the fertilized chicken egg (12) taking into account the two-dimensional data.

11. A method for in ovo sexing of a fertilized chicken egg (12), comprising the steps of: - emitting pulsed excitation radiation (16) for exciting intrinsic fluorescence using a light source (14) in an area inside the chicken egg (12), on the membrane of the chicken egg (12) and / or on the shell of the chicken egg (12); - detecting with time resolution said intrinsic fluorescent radiation (20) emitted by the internal region, the membrane and / or the shell of said chicken egg (12) at different wavelengths by time-correlated single photon counting with a detection device (18); - providing, by said detection device (18), two-dimensional data comprising a wavelength dimension and a time dimension to evaluation means (22); - determining the sex of the fertilized chicken egg (12) using the evaluation means (22) from the provided two-dimensional data, classifying said provided data by a classifier into two classes, a first class representing males of said fertilized chicken eggs (12) and a second class representing females of said fertilized chicken eggs (12); During classification, specific wavelengths that should be prioritized are identified based on features formed within the time dimension of the data. By classifying and sexing the fertilized egg (12) by preferentially considering data at characteristic wavelengths to sex the egg (12).

12. to determine the ageing of fuels and / or raw materials for industrial production, such as immersion baths, hydraulic oils and / or lubricants, - in particular to control the quality of food and / or medicines, - to determine the origin characteristics of samples, especially food products; - To determine the degree of contamination of the sample, especially the surface, - To authenticate counterfeits, and / or - to identify changes in cell metabolism, Use of a device (10) according to any one of claims 1 to 10.

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