Device and method for determining the sex of a fertilized bird egg

Abstract

Device (10) for determining the sex of a fertilized bird egg (12), with - a light source (14) for emitting pulsed excitation radiation (16), - a detection device (18) for detecting an intrinsic fluorescence radiation (20) emitted by the bird's egg (12), and - a computer-based evaluation device (22), wherein the detection device (18) is designed to detect the intrinsic fluorescence radiation (20) of the bird's egg (12) in a time-resolved manner at different wavelengths by means of time-correlated single photon counting and to provide the evaluation device (22) with two-dimensional data with a wavelength dimension and a time dimension, wherein the evaluation device (22) is designed to classify the provided data into classes by means of a classifier, wherein a first class represents a male sex of the fertilized bird egg (12) and a second class represents a female sex of the fertilized bird egg (12), wherein the evaluation device (22) is designed to identify specific wavelengths to be prioritized during the classification on the basis of features formed in the time dimension of the data by means of machine learning, and wherein the evaluation device (22) is designed to prioritize the data at the specific wavelengths in order to determine the sex of the bird's egg (12).

Description

[0001] The invention relates to a device for determining the sex of a fertilized bird egg.

[0002] The invention further relates to a method for in-ovo sex determination of a fertilized bird egg.

[0003] There is currently an effort to be able to determine the sex of a future chick while the egg is fertilized.

[0004] In the bird egg, different fluorophores develop during development in male and female chicks. Due to their complex structure, the corresponding molecules possess unpredictable fluorescence properties. Fluorescence involves energy transitions from the excited state to the ground state of the molecule. This process is time-dependent.

[0005] Document WO 2021 / 144420 A1 describes a device and method for optical in-ovo sex determination of a fertilized bird's egg. The device comprises a light source for emitting excitation radiation to excite fluorescence in a region inside the bird's egg, a spectroscopic device for time- and / or spectrally resolved analysis of fluorescence radiation emitted from the region inside the bird's egg, and an evaluation unit for sex determination from the data obtained by the spectroscopic device.

[0006] The document WO 2018 / 001 437 A1 describes a device for incubating biological material, wherein the device comprises a FLIM unit (fluorescence lifetime microscopy unit).

[0007] The publication Optics Express, Vol 26, No.3, 2270-2279 (2018) (DOI: 10.1364 / OE.26.002270) describes time- and frequency-resolved fluorescence measurement with a single TCSPC detector using a Fourier transform approach.

[0008] Based on this, the object of the invention is to provide means in which the accuracy of in-ovo sex determination is increased with a simplified structure of the device.

[0009] According to the invention, the object is achieved by the features of the independent claims. Preferred embodiments of the invention are specified in the subclaims, each of which may represent an aspect of the invention individually or in combination.

[0010] According to the invention, a device for determining the sex of a fertilized bird egg - hereinafter also referred to as sample - is provided with - a light source for emitting pulsed excitation radiation, - a detection device for detecting an intrinsic fluorescence radiation emitted by the sample, and - a computer-based evaluation device, wherein the detection device is designed to detect the intrinsic fluorescence radiation of the sample in a time-resolved manner at different wavelengths by means of time-correlated single photon counting and to provide the evaluation device with two-dimensional data with a wavelength dimension and a time dimension, wherein the evaluation device is designed to classify the provided data into classes by means of a classifier, wherein a first class represents a male sex of the fertilized bird's egg and a second class represents a female sex of the fertilized bird's egg, wherein the evaluation device is designed to identify specific wavelengths to be prioritized during the classification on the basis of features formed in the time dimension of the data, and wherein the evaluation device is designed to prioritize the data at the specific wavelengths to determine the presence of the property of the sample.

[0011] Furthermore, the invention relates to a method for in-ovo sex determination of a fertilized bird egg, comprising the steps - Emission of pulsed excitation radiation for excitation of autofluorescence in an area inside the bird's egg, on an egg membrane of the bird's egg and / or on the eggshell of the bird's egg by means of a light source, - time-resolved detection of the intrinsic fluorescence radiation emitted from the interior of the egg membrane and / or the eggshell of the bird's egg using a detection device at different wavelengths by time-correlated single photon counting, - Providing two-dimensional data with a wavelength dimension and a time dimension by the detection device to an evaluation device, - Sex determination of the fertilized bird egg from the provided two-dimensional data by means of the evaluation device, by

[0012] Classification of the provided data by means of a classifier into two classes, wherein a first class represents a male sex of the fertilized bird egg and a second class represents a female sex of the fertilized bird egg, wherein during the classification specific wavelengths to be prioritized are identified on the basis of features formed in the time dimension of the data, and sex determination of the bird egg by prioritizing consideration of the data at the specific wavelengths.

[0013] The method is preferably carried out by means of the above device.

[0014] It was found that when classifying two-dimensional data into classes for the purpose of sexing a bird's egg, improved accuracy is achieved with data with higher temporal resolution. Furthermore, it was determined that information contained in the decay behavior of the autofluorescence radiation is not equally relevant at all wavelengths, but that specific wavelengths can be identified, giving priority to which in the classification allows for improved accuracy. Accordingly, the device and method exhibit improved accuracy by detecting the autofluorescence radiation using single-photon counting and by identifying the specific wavelengths.

[0015] In other words, with regard to in-ovo sex determination of a fertilized bird's egg, knowledge of the lifetime and the decay profile of excited molecular states (time dimension of the two-dimensional data) in addition to the energy of the emitted photons (wavelength dimension of the two-dimensional data) is relevant for the identification of the sex of the fertilized bird's egg, whereby the time dimension at the specific wavelengths contains more meaningful information than other wavelengths and is therefore prioritized in the evaluation.

[0016] With regard to the device, it is provided that the device comprises the light source for emitting the pulsed excitation radiation, the detection device for detecting the self-fluorescence radiation at several different wavelengths by means of time-correlated single photon counting and the computer-based evaluation device.

[0017] For the purpose of detecting the self-fluorescence radiation with high temporal resolution, the self-fluorescence radiation is recorded using time-correlated single photon counting (TCSPC). Unlike the prior art (WO 2021 / 144420 A1), which uses a method in which the complete spectrum is recorded at different times after each excitation pulse from the light source using an ICCD camera, TCSPC does not record the complete spectrum after each excitation pulse.

[0018] In TCSPC, individual photons of a periodic light signal – in this case, the autofluorescence radiation – are detected, and the respective times between the excitation pulse of the pulsed excitation radiation and the photon's arrival at the detection device are determined. In other words, the fluorophores in the sample under investigation – preferably the bird's egg – are excited by the pulsed excitation radiation from the light source. The time measurement is started by the excitation pulse, and the photon emitted during the transition from the excited state to the ground state stops the measurement. The measurement is repeated multiple times, and the individual temporally correlated photons (relative to the excitation pulse) are sorted into a so-called TCSPC histogram according to their measured time. The TCSPC histogram represents the temporal progression of the autofluorescence radiation after excitation.The TCSPC histogram generated by the detection device preferably has a class width, also called bin width, for the histogram classes from 1 ps to 50 ps, ​​preferably from 10 ps to 20 ps. The class width of the TCSPC histogram can preferably be adapted to the device and / or the sample to be examined. Further preferably, when adapting the class width of the TCSPC histogram, a temporal resolution of the entire device—and particularly preferably a full width at half maximum (FWHM) of the instrument response function (IRF)—is taken into account. The full width at half maximum (FWHM) of the IRF depends essentially on the light source and a pulse length generated by the light source and / or on a detector element of the detection device.

[0019] In other words, the complete spectrum (wavelength dimension of the two-dimensional data) is not measured at different times, but the TCSPC histogram representing the temporal course of the self-fluorescence radiation - i.e. the time dimension of the two-dimensional data - is measured using a large number of measurement cycles - typically in the range of 10 6 - determined, whereby each measurement cycle is started by an excitation pulse from the light source.

[0020] The detection device is designed to provide the evaluation device with two-dimensional data with a wavelength dimension and a time dimension. The two-dimensional data preferably represent the decay behavior of the intrinsic fluorescence (time dimension) at the different wavelengths (wavelength dimension). The two-dimensional data can be transferred to the evaluation device, for example, in the form of an m × n matrix. The two-dimensional data preferably has more data points in the time dimension than in the wavelength dimension. This has proven particularly advantageous for determining the presence of a property of the sample and, in particular, for determining the sex of the bird's egg.

[0021] Based on the two-dimensional data, the evaluation device can identify the specific wavelengths to prioritize. This is achieved by the classifier classifying the provided data into classes. Depending on the sample and its possible properties, a varying number of classes may be present, with the classes representing possible properties of the sample.

[0022] Preferably, the identification of the specific wavelengths is based on the principle of supervised machine learning, wherein the specific wavelengths are calculated from the two-dimensional data representing the samples of corresponding classes by means of the evaluation device. According to a preferred development, the evaluation device is designed such that one or more characteristic features and associated class-specific separation properties can be calculated and / or are calculated from the decay behavior of the intrinsic fluorescence of the wavelengths—i.e., from the time dimension of the two-dimensional data. The characteristic features can be calculated from time series representing individual wavelengths. Alternatively, the characteristic features can be calculated from linear combinations of time series representing individual wavelengths.

[0023] Further preferably, the evaluation device is designed such that those wavelengths with relatively higher separation properties are identified as specific wavelengths. Preferably, only as many specific wavelengths are identified until the desired and / or sufficient classification rate for the sample under consideration is achieved. The non-prioritized wavelengths preferably make no contribution to class separation and are therefore neglected and not further considered for classification.

[0024] It has been shown that classification without wavelength prioritization, which is equivalent to a classification in which all wavelengths are prioritized equally, has a lower hit rate than classification with wavelength prioritization. Furthermore, a metric of discrimination analysis is preferably considered when identifying specific wavelengths. In connection with the method for in-ovo sex determination, linear discriminant analysis (LDA) is preferably used as the metric. LDA is particularly suitable for data exhibiting a Gaussian distribution.

[0025] Preferably, the evaluation device is configured to prioritize the data at the specific wavelengths to determine the presence of the sample property, and in particular to prioritize features formed in the time dimension of the data at the specific wavelengths. With regard to the method, the sex determination of the bird's egg is thus preferably carried out by prioritizing the data at the specific wavelengths, and in particular by prioritizing features formed in the time dimension of the data at the specific wavelengths.

[0026] The device for determining the presence of a property in a sample can be used not only to determine the sex of a fertilized bird's egg. The device is also suitable for classifying samples based on other properties. This does not require any special sample preparation. The described device can be used particularly when samples and / or objects are being authenticated with respect to a property, i.e., when determining whether a claimed property of the sample is correct, such as in the authentication of counterfeit banknotes or in the authentication of a claimed origin of a food product.The device is particularly suitable for authentication when the sample that has the claimed property - for example, the original banknote - is very similar to the sample that does not have the claimed property - in this example, the counterfeit banknote.

[0027] According to a further preferred development, the detection device is configured to detect the sample's intrinsic fluorescence radiation in a time-resolved manner at the specific wavelengths identified as prioritized using time-correlated single-photon counting and to provide the evaluation device with two-dimensional data with the wavelength dimension and the time dimension, wherein the wavelength dimension corresponds to the number of specific wavelengths, and wherein the evaluation device is configured to classify the provided data into classes using the classifier. In other words, the evaluation device determines whether or not the property is present in a sample based on the two-dimensional data.This is preferably achieved by having the detection device specifically determine the TCSP histogram at specific wavelengths and thus making time series available to the evaluation device only at those specific wavelengths. This allows for a high accuracy rate in determining the sample's properties without having to record the intrinsic fluorescence at all possible wavelengths.

[0028] According to a preferred development of the invention, with respect to the device, the light source is configured as a pulsed excitation laser system or as a pulsed LED. Particularly preferably, the light source is configured such that the sample is excited with a wavelength in the UV range, particularly preferably in a range around 266 nm and / or at 266 nm. Further preferably, the light source is configured to emit, as excitation radiation, radiation with a wavelength in a range from greater than or equal to 250 nm to less than or equal to 520 nm, preferably greater than or equal to 280 nm to less than or equal to 400 nm.

[0029] With regard to the excitation laser system, it is further preferably provided that the excitation laser system is designed as a solid-state laser with a frequency conversion stage and / or optical parametric oscillator (OPO). Preferably, a titanium-sapphire laser with a frequency conversion stage and / or optical parametric oscillator (OPO) is used. Alternatively, the excitation laser system is preferably designed as a frequency-quadrupled Nd:YAG laser with an optional optical parametric oscillator. Further preferably, the excitation laser system is a diode laser system, preferably in MOFA configuration. Further preferably, the excitation laser system is designed as a master oscillator power amplifier (MOPA) and / or as a master oscillator fiber amplifier (MOFA). MOPA and / or MOFA are laser systems that use a seed laser as a master oscillator in order to specify the properties of the emission radiation to an amplification unit with high coherence.Particularly preferred is an infrared laser diode and / or a near-infrared laser diode as the master oscillator or seed laser, respectively, to specify the laser emission properties for a multi-stage fiber amplifier in the MOFA arrangement. Using birefringent crystals, the wavelengths of the second, third, and / or fourth harmonics can also be generated through nonlinear frequency conversion. This allows for a particularly compact and portable measurement setup.

[0030] Alternatively, the light source can be configured as a pulsed LED. LEDs have the advantage of being cost-effective. Furthermore, the increased spectral bandwidth of the LED emission compared to the excitation laser system can have the advantage that the LED as a light source induces a larger number of absorption processes for different fluorophores in the sample. Preferred LEDs for use as a light source emit at wavelengths in the UV range, particularly preferably at 265 nm, 285 nm, 310 nm, and / or > 310 nm, with a spectral bandwidth of ± 10 nm each. Furthermore, the spectral bandwidth can be reduced using appropriate interference filters.

[0031] In order to enable the highest possible temporal resolution in the detection of the autofluorescence radiation, it is advantageous if the length of the excitation pulse of the pulsed excitation radiation is as short as possible. In this context, it is preferably provided that the light source is designed to emit pulsed excitation radiation with a pulse length of ≤ 500 ps, ​​preferably ≤ 200 ps, ​​particularly preferably ≤ 100 ps. This is possible in particular by designing the light source as a pulsed excitation laser system. Alternatively, it is provided that the light source is designed to emit pulsed excitation radiation with a pulse length of ≤ 5 ns, preferably ≤ 2 ns, particularly preferably ≤ 1 ns. This is possible in particular in connection with the light source being designed as an LED. The short pulse length increases the temporal resolution and thus the accuracy when determining the presence of a property of the sample.

[0032] Since the autofluorescence radiation detected by TCSPC requires a large number of measurement cycles to generate the TCSPC histogram, it is also preferred that the light source be configured to emit pulsed excitation radiation with a pulse repetition rate of ≥ 10 MHz. This significantly reduces the time required to acquire the two-dimensional data.

[0033] In addition, in TCSPC, the excitation intensity for detecting the intrinsic fluorescence radiation is preferably kept so low that the detection probability for a fluorescence photon per excitation cycle is less than or equal to 1, i.e., at most one photon is detected during each excitation cycle. In this context, according to a further preferred development of the invention, the device comprises an optical attenuator in the beam path between the light source and the sample for adjusting the energy of the excitation radiation. This is particularly preferably a variable attenuator, in particular a variable laser beam attenuator. Further preferably, the attenuator is such that the energy per pulse for exciting the sample is so low that the detection probability for a fluorescence photon per excitation cycle is less than or equal to 1.Typically, single-photon statistics can be achieved if a counting pulse at the detector element is triggered, on average, by only one of 20 to 100 excitation pulses. In other words, this means that the counting rate at the detector element is preferably in the range of 1-5% of the excitation rate. For example, at a pulse repetition rate of 80 MHz, the attenuator is adjusted so that the average counting rate of the detector element does not exceed 4 MHz. This provides a simple way to ensure that the TCSPC histogram is not affected by systematic measurement errors caused by the so-called pile-up effect.

[0034] As already mentioned, the detection device is designed to detect the sample's intrinsic fluorescence radiation in a time-resolved manner at different wavelengths using time-correlated single-photon counting. According to a preferred development of the invention, the detection device for detecting the intrinsic fluorescence radiation at different wavelengths comprises a monochromator, a spectrograph, and / or a spectrometer. This greatly simplifies 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 broad spectral range, for example, from 370 nm to 700 nm, can be scanned stepwise, and a TCSPC histogram can be acquired for each scanned wavelength.

[0035] In an alternative embodiment of the invention, the device for spectral separation preferably comprises an exchangeable interference filter between the sample and a detector element of the detection device. The interference filter can be exchanged for a different interference filter depending on the wavelength to be detected. This is particularly advantageous if the intrinsic fluorescence radiation of the sample is detected in a time-resolved manner at the specific wavelengths identified as prioritized using time-correlated single-photon counting. In a further preferred alternative, the detection device comprises several detector elements and one or more beam splitters that split the intrinsic fluorescence radiation into several partial beams. In this way, a TCSPC histogram can be recorded for each partial beam using a preferably exchangeable interference filter and the detector element.

[0036] Furthermore, in connection with the detection device, it is provided that the detection device preferably comprises a hybrid photomultiplier (hybrid PMT)—also called a hybrid photodetector—as the detector element. The detector element is preferably a combination of a front-end PMT with an avalanche photodiode (APD) as an additional amplification stage. Further preferably, the detector element is designed to multiply a photoelectron triggered in the latter amplification stage by a factor of 50,000 to 150,000. Further preferably, the detector element is designed to achieve a time resolution of approximately 120 ps (FWHM of the Instrument Response Function (IRF)).

[0037] The detector element is further preferably designed as a multi-channel detector, preferably a multi-channel plate PMT (MC-PMT), so that several different wavelengths can be detected simultaneously. For example, the multi-channel detector has 16 channels for detection. The design of the detection device as a combination of a spectrograph with a rigidly positioned diffraction grating and an MC-PMT enables simultaneous, multi-spectral detection with equidistant subdivision of the total detected wavelength range. The subdivision and the width of the wavelength intervals depend on the number of channels of the MC-PMT.

[0038] Preferably, the detection device is designed to receive the self-fluorescence radiation with a wavelength in a range of greater than or equal to 200 nm to less than or equal to 700 nm.

[0039] In connection with the TCSPC histogram, according to a preferred development, the device for generating an electrical trigger signal that can be generated synchronously with the excitation pulse comprises a pulse signal generator. Thus, a time-amplitude converter can be used to initiate a rapid voltage ramp using the electrical trigger signal generated synchronously with the excitation pulse. This rapid voltage ramp is stopped by measuring a photon of the intrinsic fluorescence radiation. For example, the excitation laser system, which provides the electrical trigger signal, can be used directly as the pulse signal generator. Alternatively, the electrical trigger signal can be generated using a trigger photodiode, for which a fraction of the excitation radiation is coupled out via a beam splitter.

[0040] In this context, it is further preferred that the detector element preferably generates an electrical output pulse for each detected photon, which is then 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 thus assigned to each stop point, allowing the TCSPC histogram to be generated.

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

[0042] According to a further preferred development, the device comprises an optical component in the beam path between the sample and the detection device for focusing the intrinsic fluorescence radiation emitted by the sample onto the detection device. The optical component can be designed, for example, as a lens, in particular as a converging lens. Particularly when using LEDs as the light source, the device can further comprise optical components between the light source and the sample for focusing the excitation radiation.

[0043] With regard to the irradiation of the sample, according to a further preferred development of the invention, the device is designed to irradiate the sample with the excitation radiation in free space and is designed such that the self-fluorescence radiation emitted at a non-zero angle to the excitation radiation is directed in free space onto the detection device. In this context, "free space" means that the light is not transported via a fiber-based light guide system, but rather propagates freely in space. In this preferred development, the device therefore does not have a fiber-based light guide system by means of which the light is guided, such as an optical fiber.By omitting a fiber-based light guide system, losses of pulse energy in the light guide system and / or broadening of the excitation pulse can be prevented, which can be particularly problematic for excitation wavelengths in the UV range. Furthermore, the device without a fiber-based light guide system has the advantage of eliminating the need for a measuring head for irradiating the sample and / or receiving the sample's intrinsic fluorescence radiation, thus resulting in a very simple device design.

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

[0045] According to an alternative development of the invention, it is preferably provided that the device comprises a measuring head, a) wherein the measuring head is designed to emit the excitation radiation into and / or onto the sample, or b) wherein the measuring head is designed to receive the intrinsic fluorescence radiation from and / or from the sample, or c) wherein the measuring head is designed for jointly emitting the excitation radiation into and / or onto the sample and for receiving the self-fluorescence radiation from and / or from the sample.

[0046] Preferably, the measuring head can be connected to a fiber optic system. More preferably, particularly in case c), this can be a Y-shaped fiber optic system with two fiber optic strands that are combined on the measuring head side.

[0047] In particular, it is provided that fiber optic strands are designed as fiber optic bundles, the individual fiber optics of which are intertwined, for example twisted, on the side of one end of the measuring head. Alternatively, the individual fiber optics can be evenly distributed across the generally circular cross-section. Further alternatively, the fiber optics carrying the excitation radiation can be arranged, for example, in a circular arrangement in the inner region of the cross-section, and the fiber optics carrying the autofluorescence radiation can be arranged, for example, in a concentric ring around the outside. In this way, the excitation radiation can be emitted into and / or onto the sample and the autofluorescence radiation can be received from and / or by the sample using the same measuring head.

[0048] With regard to the computer-based evaluation device, as already mentioned, it is provided that the evaluation device is designed to classify the provided data into classes by means of the classifier, wherein at least one class represents the property of the sample. In this context, it is preferably provided that the classifier is a linear classifier. A linear classifier separates the classes along a linear hyperplane. It is further preferably provided that the classifier is constructed by means of feature selection with the aid of linear discriminant analysis on the basis of training data. In other words, it is preferably an evaluation device based on machine learning. The evaluation device preferably learns from examples - the training data - and can generalize these after the learning phase has ended.To do this, machine learning algorithms build a statistical model based on the training data.

[0049] According to a further preferred embodiment of the invention, the evaluation device is an evaluation device based on so-called feature engineering. Feature engineering is a form of data processing and describes the selection and processing of features that are used to create a machine learning model. In this context, it is also preferably provided that the features formed in the time dimension of the data include the central moments of the 1st order (mean), 2nd order (standard deviation), and 3rd order (skewness), and / or that the features formed in the time dimension of the data are histogram-based features, signal series-based features, and / or transformation-based features.Preferably, it can also be provided that the evaluation device is designed to eliminate features with weak separation properties, preferably with the aid of Fischer's Linear Discriminant Analysis (LDA).

[0050] Furthermore, the evaluation device is designed to identify the specific wavelengths using machine learning. This allows for high accuracy in determining the presence of a sample property using two-dimensional data with low resolution in the wavelength dimension, despite the low spectral resolution.

[0051] Furthermore, it is provided that the evaluation device is configured to determine the sex of the fertilized bird's egg taking into account the two-dimensional data. In other words, the device is used to determine the sex of the fertilized bird's egg. In this context, it is further provided that the evaluation device is configured to classify the provided data into two classes by means of the classifier, wherein a first class represents the male sex characteristic and a second class represents the female sex characteristic of the bird's egg as a sample.

[0052] As already mentioned, the invention further relates to the method for in-ovo sex determination of the fertilized bird egg, comprising the steps - Emission of pulsed excitation radiation for excitation of autofluorescence in an area inside the bird's egg, on an egg membrane of the bird's egg and / or on the eggshell of the bird's egg by means of a light source, - time-resolved detection of the intrinsic fluorescence radiation emitted from the interior of the egg membrane and / or the eggshell of the bird's egg using a detection device at different wavelengths by time-correlated single photon counting, - Providing two-dimensional data with a wavelength dimension and a time dimension by the detection device to an evaluation device, - Sex determination of the fertilized bird egg from the provided two-dimensional data by means of the evaluation device, by Classification of the provided data by means of a classifier into two classes, wherein a first class represents a male sex of the fertilized bird egg and a second class represents a female sex of the fertilized bird egg, wherein during the classification specific wavelengths to be prioritized are identified on the basis of features formed in the time dimension of the data, and sex determination of the bird egg by prioritizing consideration of the data at the specific wavelengths.

[0053] In the method for in-ovo sex determination of a fertilized bird's egg, it is provided that the intrinsic fluorescence in a region inside the bird's egg, of the egg membrane of the bird's egg and / or of the eggshell of the bird's egg is excited by means of a light source. Preferably, the intrinsic fluorescence is excited on the egg membrane of the bird's egg and / or on the eggshell of the bird's egg. Furthermore, it is provided that the region inside the bird's egg is preferably a bloodstream region and / or a region of embryonic structures. It is further preferably provided that the bird's egg does not have to be opened for sex determination. Instead, it is possible to determine the sex directly on, at, and / or through the eggshell of the bird's egg using the described device and / or the described method. In other words, it is therefore preferably provided that the emission of pulsed excitation radiation is directed onto the eggshell of the bird's egg.This has the advantage that the procedure is very simple and quick to perform, and the risk of infection of the bird's egg is greatly reduced. Which area is chosen can depend, in particular, on the stage of development in the fertilized bird's egg. Furthermore, it is not necessary for the bird's egg to be incubated. The sex can also be determined from an unincubated egg.

[0054] Alternatively, the bird's egg can be opened for sex determination. For this purpose, a hole is preferably made in the eggshell. The hole preferably has a dimension or diameter D in the range 0.5 mm ≤ D ≤ 3 mm.

[0055] In particular, it is intended that the hole be created without perforating the membrane beneath the eggshell and / or without perforating the shell membrane and / or egg membrane beneath the eggshell. The measurement of autofluorescence can be performed on the membrane, the shell membrane, and / or the egg membrane.

[0056] According to a further alternative embodiment of the method according to the invention, it can be provided that an interior of the bird's egg and / or the area inside the bird's egg is removed from the eggshell and the method steps are carried out accordingly outside the eggshell.

[0057] According to a further preferred development of the invention, it is also provided that the method comprises the steps - time-resolved detection of the self-fluorescence radiation emitted from the interior region, from the egg membrane and / or from the eggshell of the bird's egg by means of the detection device at the specific wavelengths by time-correlated single photon counting, and - Providing two-dimensional data with a wavelength dimension and a time dimension by the detection device to the evaluation device, wherein the wavelength dimension corresponds to the number of specific wavelengths, includes.

[0058] Once the evaluation system has identified the specific wavelengths to be prioritized, the autofluorescence of subsequent samples can be specifically recorded at those specific wavelengths for sex identification. The other wavelengths do not need to be considered further.

[0059] The invention will be explained below by way of example with reference to the accompanying drawings using preferred embodiments, wherein the features presented below can represent an aspect of the invention both individually and in combination. They show: Fig. 1 a schematic representation of a structure with a bird's egg and a device for in-ovo sex determination of this bird's egg according to a preferred embodiment of the invention, Fig. 2 a schematic representation of two alternatives regarding the guidance of the excitation radiation and the emitted self-fluorescence radiation to the Fig. 1 shown structure with bird's egg according to a preferred embodiment of the invention, Fig. 3 a schematic representation of a light source of the Fig. 1 shown structure, Fig. 4 a schematic representation of the device in Fig. 1, Fig. 5 or Fig. 6 obtained TCSPC histogram, according to a preferred embodiment of the invention, Fig. 5 a schematic representation of a Fig. 1 alternative construction of the device for in-ovo sex determination according to a preferred embodiment of the invention, and Fig. 6 a schematic representation of a further alternative construction of the device for in-ovo sex determination according to a preferred embodiment of the invention.

[0060] Fig. Figure 1 shows a schematic representation of a device 10 for determining the sex of a fertilized bird's egg 12 according to a preferred embodiment of the invention. The device 10 comprises a light source 14 for emitting pulsed excitation radiation 16, a detection device 18 for detecting an intrinsic fluorescence radiation 20 emitted by the bird's egg 12, and a computer-based evaluation device 22.

[0061] The detection device 18 is configured to detect the intrinsic fluorescence radiation 20 of the bird's egg 12 in a time-resolved manner at different wavelengths using time-correlated single photon counting (TCSPC) and to provide the evaluation device 22 with two-dimensional data with a wavelength dimension and a time dimension. The evaluation device 22 is configured to classify the provided data into two classes using a classifier, wherein a first class represents a male sex of the fertilized bird's egg 12 and a second class represents a female sex of the fertilized bird's egg 12. During the classification, features formed in the time dimension of the data are prioritized at specific wavelengths.

[0062] In this case, the bird's egg 12 is mounted on a sample holder 24 and positioned in the beam path such that the freely propagating excitation radiation 16 from the light source 14 strikes the bird's egg 12. A variable laser beam attenuator 26 is also provided between the light source 14 and the bird's egg 12 to reduce the excitation energy to an excitation energy suitable for TCSPC.

[0063] The autofluorescence radiation 20 emitted from an area inside the bird's egg 12 is detected by the detection device 18. For this purpose, the detection device 18 is arranged relative to the bird's egg 12 such that the autofluorescence radiation 20, emitted at an angle of approximately 90 degrees, impinges freely on the detection device 18. For the purpose of focusing the autofluorescence radiation 20 onto the detection device 18, the device 10 has a lens 28 in the beam path between the bird's egg 12 and the detection device 18. In addition, the wavelength of the excitation radiation 16 scattered by the bird's egg 12 is filtered out by means of a long-pass filter 30 between the bird's egg 12 and the detection device 18. Before the autofluorescence radiation 20 impinges on the detection device 18, it is also attenuated by means of an aperture 32.

[0064] The device 10 therefore has in the Fig. 1 does not have a fiber-based light guide system 34 by means of which the light is directed onto the bird's egg 12 and / or onto the detection device 18. Fig. 2 shows two sections of the device 10 in alternative embodiments in which the device 10 comprises a light guide system 34 with a measuring head 36.

[0065] In the Fig. 2a), the light guide system 34 is Y-shaped and comprises two light guide strands 38, 40, one light guide strand 38 for the excitation radiation and one light guide strand 40 for the autofluorescence radiation 20. The two light guide strands 38, 40 are brought together in the measuring head 36, so that the measuring head 36 is designed to jointly emit the excitation radiation 16 onto the bird's egg 12 and to receive the autofluorescence radiation 20 emitted by the bird's egg 12.

[0066] In the Fig. 2b), the light guide system 34 is designed as a simple light guide system and comprises only one light guide strand 40 for the self-fluorescence radiation 20. The excitation radiation 16 continues to propagate freely from the light source 14 (in Fig. 2 not shown) to the bird's egg 12. The measuring head 36 of the light guide system 34 is designed accordingly to receive the self-fluorescence radiation 20 from the bird's egg 12.

[0067] Fig. 3 shows a schematic representation of the light source 14 of the Fig. 1. The light source 14 is implemented here as a laser system 14 that generates excitation pulses with a pulse length of approximately 80 ps. The laser system 14 comprises an infrared laser diode 42 that emits laser radiation at 1064 nm and is used as a master oscillator or seed laser to specify the properties of the laser emission to a multi-stage fiber amplifier 44. By means of the birefringent crystals 46 and the dichroic mirrors 48 present in the beam path, the wavelengths of the second harmonic 50a (532 nm), the third harmonic 50b (355 nm), and the fourth harmonic 50c (266 nm) can be generated by means of nonlinear frequency conversion. Fig. In the setup shown in Figure 1, the fourth harmonic 50c at 266 nm is used as excitation radiation of the sample.

[0068] In connection with the Fig. 1 shown detection device 18 represents Fig. 4 shows a schematic representation of the TCSPC histogram 52 determined during the detection of the intrinsic fluorescence radiation 20, which represents the temporal progression of the intrinsic fluorescence radiation after excitation. As already mentioned, the detection device 18 is designed to detect the intrinsic fluorescence radiation 20 of the bird's egg 12 in a time-resolved manner at several different wavelengths using TCSPC. For this purpose, the detection device 18 has, in the Fig. 1, a spectrometric device with a monochromator 54 is used. The detection device 18 has a hybrid photomultiplier 56a as the detector element 56. The monochromator 54 essentially consists of a stepwise rotatable diffraction grating. The spectral range to be examined can be scanned by stepwise rotating the diffraction grating.

[0069] Fig. Figure 5 shows an alternative embodiment of the device 10, in which the detector element 56 is designed as an MCP-PMT 56b, which in this case comprises 16 channels. In contrast to Fig. 1, the diffraction grating of the monochromator 54 is also fixedly positioned. With this embodiment of the device 10, the spectral range to be examined can be "broken down" into up to 16 WL subintervals (detection channels).

[0070] Fig. Figure 6 shows a further alternative embodiment of the device 10, in which multiple detector elements 56 are used. Both detector elements 56 are analogous to Fig. 1 is designed as a hybrid photomultiplier 56a. Instead of a diffraction grating, a beam splitter 57 is used. An interference filter 59, each of which transmits a specific wavelength, is inserted between the beam splitter 57 and each of the two hybrid PMTs 56a.

[0071] In TCSPC, individual photons 58a, 58b of the self-fluorescence radiation 20 are detected and the respective times 62 between an excitation pulse 60 of the pulsed excitation radiation 16 and the arrival of the respective photon 58 in the detection device 18 are determined. For this purpose, the detector device comprises TCSPC electronics 61, which are schematically shown in the Fig. 1, Fig. 5, and Fig. 6. In relation to Fig. 4, the time measurement is started by the excitation pulse 60a and the photon 58a emitted during the transition from the excited state to the ground state stops the measurement ( Fig. 4a). With the next excitation pulse 60b and the next photon 58b, the process is repeated ( Fig. 4b). By repeating the measurement several times, the measured times 62 of the individual photons 58 are Fig. 4c shown TCSPC histogram 52. As in the Fig. 1, Fig. 5 and Fig. 6, the device 10 has an electrical connection 64 for measuring the time interval 62 in order to transmit an electrical trigger signal generated synchronously with the excitation pulse 60 to the detection device 18. The TCSPC electronics 61, which is shown in the Fig. 1, Fig. 5 and Fig. 6 is schematically shown as a box, and can, for example, be physically designed as a PC plug-in card, evaluates the signals and creates the TCSPC histogram, which is provided to the evaluation device 22 as two-dimensional data with a wavelength dimension and a time dimension.

[0072] The detection device 18 is thus designed to detect the self-fluorescence radiation 20 at several different wavelengths by means of TCSPC and to provide the evaluation device 22 with two-dimensional data. In the present case, the data are in the form of mathematical matrices A ∈ ℝ m×nwhere the matrix in the wavelength dimension contains m data points, in this example there are 74 data points. In the time dimension, the matrix has n data points, which enable the high temporal resolution required for the hit rate; in this example there are 250 data points: A=(a11a12⋯a1na21a22⋯a2n⋮⋮⋯⋮am1am2⋯amn), where the rows of A, i.e. (a j1 , ..., a jn ),j = 1, ... ,m each correspond to a TCSPC histogram and thus physically essentially to the time-resolved measurements for certain fixed wavelengths.

[0073] For the purpose of determining the sex of the bird egg 12, a classifier classifies the provided data into two classes, with a first class representing a male sex of the fertilized bird egg 12 and a second class representing a female sex of the fertilized bird egg 12. During the classification, specific wavelengths to be prioritized are identified on the basis of features formed in the time dimension of the data, and a sex determination of the bird egg 12 is carried out by prioritizing the data at the specific wavelengths. A linear classifier is used as the classifier in this case, which separates the data along a hyperplane. The features formed in the time dimension of the data are the first three moments of the central moments of a j := (a j1 , ..., a jn ), j = 1, namely mean µ, standard deviation σ and skewness S. Reference symbol 10 Device 12 bird eggs 14 Light source 16 Excitation radiation 18 Detection device 20 Autofluorescence radiation 22 Evaluation device 24 sample holders 26 variable laser beam attenuator 28 lens 30 long-pass filters 32 aperture 34 Fiber optic system 36 measuring head 38 light guide strand 40 light guide strands 42 infrared laser diode 44 multi-stage fiber amplifier 46 birefringent crystal 48 dichroic mirrors 50 second to fourth harmonics of the laser wavelength 1064nm 52 TCSPC histogram 54 Monochromator 56a Detector element, hybrid PMT 56b Detector element, MCP-PMT 57 beam splitters 58 photons 59 interference filters 60 excitation pulses 61 TCSPC Electronics 62 Time between excitation pulse and detection of the photon 64 electrical connection

Claims

[1] Device (10) for determining the sex of a fertilized bird egg (12), with - a light source (14) for the emission of pulsed excitation radiation (16), - a detection device (18) for detecting autofluorescence radiation (20) emitted by the bird egg (12), and - a computer-based evaluation unit (22), wherein the detection device (18) is designed to detect the intrinsic fluorescence radiation (20) of the bird egg (12) with time resolution at different wavelengths by means of time-correlated single-photon counting and to provide the evaluation device (22) with two-dimensional data with a wavelength dimension and a time dimension, wherein the evaluation device (22) is designed to classify the provided data into classes using a classifier, wherein a first class represents a male sex of the fertilized bird egg (12) and a second class represents a female sex of the fertilized bird egg (12), wherein the evaluation device (22) is designed to identify specific wavelengths to be prioritized during classification based on features formed in the time dimension of the data using machine learning, and wherein the evaluation device (22) is designed to prioritize the data at the specific wavelengths in order to determine the sex of the bird egg (12). [2] Device (10) according to claim 1, wherein the light source (14) is designed as a pulsed excitation laser system or as a pulsed LED. [3] Device (10) according to one of the preceding claims, wherein the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse repetition rate of ≥ 10 MHz and / or wherein the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse length of ≤ 500 ps and / or wherein the light source (14) is configured to emit pulsed excitation radiation (16) with a pulse length of ≤ 5 ns. [4] Device (10) according to one of the preceding claims, wherein the device (10) comprises an optical attenuator (26) in the beam path between the light source (14) and the bird's egg (12) for adjusting the energy of the excitation radiation (16). [5] Device (10) according to one of the preceding claims, wherein the detection device (18) for detecting the autofluorescence radiation (20) at different wavelengths comprises a monochromator (54), a spectrograph, a beam splitter (57) with several interference filters (59) and / or a spectrometer and / or wherein the detection device (18) comprises a hybrid photomultiplier (56a) and / or a multichannel plate photomultiplier (56b) as a detector element (56). [6] Device (10) according to one of the preceding claims, wherein the device (10) comprises a long-pass edge filter (30) in the beam path between the bird's egg (12) and the detection device (18) for filtering one wavelength of the excitation radiation (16). [7] Device (10) according to one of the preceding claims, wherein the device (10) is configured to irradiate the bird egg (12) in free space with the excitation radiation (16) and is configured such that the autofluorescence radiation (20) emitted at an angle other than zero to the excitation radiation (16) is directed in free space onto the detection device (18). [8] Device (10) according to any one of claims 1 to 6, wherein the device (10) comprises a measuring head (36), and a) wherein the measuring head (36) is designed to emit the excitation radiation (16) into and / or onto the bird's egg (12), or b) wherein the measuring head (36) for receiving the autofluorescence radiation (20) is designed from and / or of the bird egg (12), or c) wherein the measuring head (36) is configured to jointly emit the excitation radiation (16) into and / or onto the bird egg (12) and to receive the autofluorescence radiation (20) from and / or the bird egg (12). [9] Device (10) according to one of the preceding claims, wherein the evaluation device (22) is configured to determine the sex of the fertilized bird egg (12) taking into account the two-dimensional data. [10] Method for in-ovo sex determination in a fertilized bird egg (12), comprising the steps - Emission of pulsed excitation radiation (16) for excitation of autofluorescence in a region inside the bird egg (12), on an egg membrane of the bird egg (12) and / or on the eggshell of the bird egg (12) by means of a light source (14), - time-resolved detection of the intrinsic fluorescence radiation (20) emitted from the interior, the egg membrane and / or the eggshell of the bird egg (12) by means of a detection device (18) at different wavelengths by time-correlated single-photon counting, - Providing two-dimensional data with a wavelength dimension and a time dimension by the detection device (18) to an evaluation unit (22), - Sex determination of the fertilized bird egg (12) from the provided two-dimensional data using the evaluation device (22), by Classification of the provided data into two classes using a classifier, where a first class represents a male sex of the fertilized bird egg (12) and a second class represents a female sex of the fertilized bird egg (12), where, in the classification based on features formed in the time dimension of the data, specific wavelengths to be prioritized are identified using machine learning, and sex determination of the bird egg (12) by prioritizing consideration of the data at the specific wavelengths.

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