APPARATUS FOR DETERMINING THE PRESENCE OF A CHARACTERISTIC OF A SAMPLE, AND IN PARTICULAR FOR SEX DETERMINATION OF A FERTILISED BIRD EGG, USE, AND METHOD

Abstract

A device for determining the presence of a characteristic of a sample includes a light source for emitting pulsed excitation radiation, a detection device for detecting an autofluorescence radiation emitted by the sample, and a computer-based evaluation device. The detection device is configured to detect the autofluorescence 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 dimenion. The evaluation device is configured to classify the provided data into classes by means of a classifier. The evaluation device is configured to identify specific wavelengths to be prioritized during the classification on the basis of features formed in the time dimension of the data.

Claims

1. Device for determining the presence of a characteristic of a sample, and preferably for determining the sex of a fertilized bird's egg, comprising: a light source for emitting pulsed excitation radiation; a detection device for detecting autofluorescence radiation emitted by the sample; and a computer-based evaluation unit, wherein the detection device is configured to detect the autofluorescence radiation of the sample in a time-resolved manner at different wavelengths via time-correlated single photon counting and to provide the evaluation unit with two-dimensional data having a wavelength dimension and a time dimension, wherein the evaluation unit is configured to classify the provided data into classes via a classifier, wherein at least one class represents the characteristic of the sample, wherein the evaluation unit is configured 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 unit is configured to take into consideration the data at the specific wavelengths in a prioritized manner in order to determine the presence of the characteristic of the sample.

2. Device according to claim 1, wherein the light source is configured as a pulsed excitation laser system or as a pulsed LED.

3. Device according to claim 1, wherein the light source is configured to emit pulsed excitation radiation with a pulse repetition rate of 10 MHz, and/or wherein the light source is configured to emit pulsed excitation radiation having a pulse length of 500 ps, and/or wherein the light source is configured to emit pulsed excitation radiation having a pulse length of 5 ns.

4. Device according to claim 1, wherein the device comprises an optical attenuator in the beam path between the light source and the sample for adjusting an energy of the excitation radiation.

5. Device according to claim 1, wherein the detection device for detecting the autofluorescence radiation at different wavelengths comprises a monochromator, a spectrograph, a beam splitter with a plurality of interference filters and/or a spectrometer, and/or wherein the detection device comprises as detector element a hybrid photomultiplier and/or a multichannel plate photomultiplier.

6. Device according to claim 1, wherein the device 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.

7. Device according to claim 1, wherein the device is configured to irradiate the sample in free space with the excitation radiation and is configured such that the autofluorescence radiation emitted at an angle not equal to zero to the excitation radiation is directed in the free space onto the detection device.

8. Device according to claim 1, wherein the device comprises a measuring head, wherein a) the measuring head is configured to emit the excitation radiation into and/or onto the sample, or b) the measuring head is configured to receive the autofluorescence radiation out of and/or from the sample, or c) the measuring head is configured to transmit the excitation radiation into and/or onto the sample and to receive the autofluorescence radiation out of and/or from the sample.

9. Device according to claim 1, wherein the evaluation unit is configured to identify the specific wavelengths via machine learning.

10. Device according to claim 1, wherein the evaluation unit is configured to determine the sex of the fertilized bird's egg by taking into consideration the two-dimensional data.

11. Method for in-ovo sex determination in a fertilized bird's egg, the method comprising the steps of: emitting pulsed excitation radiation for excitation of autofluorescence in an area in the interior of the bird's egg, on an egg membrane of the bird's egg and/or on the egg shell of the bird's egg by means of via a light source; time-resolved detection of the autofluorescence radiation emitted from the area inside, from the egg membrane and/or from the egg shell of the bird's egg via a detection device at different wavelengths by time-correlated single photon counting; providing an evaluation device with two-dimensional data with a wavelength dimension and a time dimension by the detection device; determining the sex of the fertilized bird's egg from the provided two-dimensional data via the evaluation device by classifying the provided data via a classifier into two classes, 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 specific wavelengths to be prioritized are identified in the classification on the basis of features formed in the time dimension of the data; and sex determination of the bird's egg by prioritized consideration of the data at the specific wavelengths.

12. Use of the device according to claim 1 for determining a degree of aging of fuels and/or industrial operating materials, such as immersion baths, hydraulic oils and/or lubricants; for quality control, in particular of food and/or medicines; for determining a property of origin of the sample, in particular of food; for determining a degree of contamination of the sample, in particular of a surface; for detecting a falsification; and/or for detecting a change in the cell metabolism.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0081] In the following, the disclosure is explained with reference to the accompanying drawings by way of exemplary embodiments, wherein the features presented below may each individually and in combination represent an aspect of the disclosure. In the drawings:

[0082] FIG. 1 is a schematic representation of a setup with a bird's egg and a device for in-ovo sex determination for this bird's egg according to an embodiment;

[0083] FIG. 2 is a schematic representation of two alternatives with regard to the guidance of the excitation radiation and the emitted autofluorescence radiation to the setup with bird's egg shown in FIG. 1 according to an embodiment;

[0084] FIG. 3 is a schematic representation of a light source of the setup shown in FIG. 1;

[0085] FIG. 4 is a schematic representation of the TCSPC histogram obtained by means of the device in FIG. 1, 5 or 6 according to an embodiment;

[0086] FIG. 5 is a schematic representation of an alternative setup to FIG. 1 of the device for in-ovo sex determination according to an embodiment; and

[0087] FIG. 6 is a schematic representation of a further alternative setup of the device for in-ovo sex determination according to an embodiment.

DETAILED DESCRIPTION

[0088] FIG. 1 shows a schematic representation of a device 10 for determining the sex of a fertilized bird's egg 12 according to an embodiment. The device 10 comprises a light source 14 for emitting pulsed excitation radiation 16, a detection device 18 for detecting autofluorescence radiation 20 emitted by the bird's egg 12, and a computer-based evaluation unit 22.

[0089] The detection device 18 is configured to detect the autofluorescence radiation 20 of the bird's egg 12 in a time-resolved manner at different wavelengths by means of time-correlated single photon counting (TCSPC) and to provide the evaluation device 22 with two-dimensional data with a wavelength dimension and a time dimen-sion. The evaluation device 22 is configured to classify the provided data into two classes by means of 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, and wherein features formed in the time dimension of the data are prioritized and taken into consideration at specific wavelengths during the classification.

[0090] In the present case, the bird's egg 12 is attached to a sample holder 24 and placed in the beam path in such a way that the freely propagating excitation radiation 16 from the light source 14 hits on the bird's egg 12. In addition, a variable laser beam attenuator 26 is provided between the light source 14 and the bird's egg 12 in order to reduce an excitation energy to an excitation energy suitable for TCSPC.

[0091] The autofluorescence radiation 20 emitted from an area inside the bird's egg 12 is detected by means of the detection device 18. For this purpose, the detection device 18 is arranged in relation to the bird's egg 12 in such a way that the autofluorescence radiation 20, which is emitted at an angle of approximately 90 degrees, hits onto the detection device 18 in a freely propagating manner. In order to focus the autofluorescence radiation 20 on the detection device 18, the device 10 comprises 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 that is scattered at 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 hits onto the detection device 18, it is also attenuated by means of an aperture 32.

[0092] In the embodiment shown in FIG. 1, the device 10 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.

[0093] In the variant shown in FIG. 2a), the light guide system 34 is configured 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 merged in the measuring head 36, so that the measuring head 36 is configured to 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.

[0094] In the variant shown in FIG. 2b, the light guide system 34 is configured as a simple light guide system and comprises only one light guide strand 40 for the autofluorescence radiation 20. The excitation radiation 16 continues to propagate freely from the light source 14 (not shown in FIG. 2) to the bird's egg 12. The measuring head 36 of the light guide system 34 is designed to receive the autofluorescence radiation 20 from the bird's egg 12.

[0095] FIG. 3 shows a schematic representation of the light source 14 of the setup shown in FIG. 1. The light source 14 is realized in the present case as a laser system 14 that generates excitation 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 specify the characteristics of the laser emission of a multi-stage fiber amplifier 44. The birefringent crystals 46 and dichroic mirrors 48 present in the beam path allow the wavelengths of the second harmonic 50a (532 nm), the third harmonic 50b (355 nm) and the fourth harmonic 50c (266 nm) to be generated by means of nonlinear frequency conversion. In the setup shown in FIG. 1, the fourth harmonic 50c at 266 nm is used as the excitation radiation of the sample.

[0096] In conjunction with the detection device 18 shown in FIG. 1, FIG. 4 shows a schematic representation of the TCSPC histogram 52 determined during the detection of the autofluorescence radiation 20, which represents the time course of the autofluorescence radiation after excitation. As already mentioned, the detection de-vice 18 is configured to detect the autofluorescence radiation 20 of the bird's egg 12 in a time-resolved manner at several different wavelengths by means of TCSPC. To this end, the detection device 18 in the embodiment shown in FIG. 1 comprises a spectrometric device with a monochromator 54. As a detector element 56, the detection device 18 comprises a hybrid photomultiplier 56a. The monochromator 54 essentially consists of a diffraction grating that can be rotated in steps. The spectral range to be examined can be scanned by rotating the diffraction grating in steps.

[0097] FIG. 5 shows an alternative embodiment of the device 10, in which the detector element 56 is designed as an MCP-PMT 56b, which in the present case comprises 16 channels. In contrast to FIG. 1, moreover, the diffraction grating of the monochromator 54 is fixed in position. With this embodiment of the device 10, the spectral range to be examined can be split into up to 16 WL sub-intervals (detection channels).

[0098] FIG. 6 shows a further alternative embodiment of the device 10, in which several detector elements 56 are used. Both detector elements 56 are configured as hybrid photomultipliers 56a, as in FIG. 1. Instead of a diffraction grating, a beam splitter 57 is used. Between the beam splitter 57 and the two hybrid PMTs 56a, an interference filter 59 is respectively interconnected which respectively transmits a specific wavelength.

[0099] In TCSPC, individual photons 58a, 58b of the autofluorescence radiation 20 are de-tected 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 de-vice 18 are determined. For this purpose, the detection device comprises TCSPC electronics 61, which is schematically shown in FIGS. 1, 5, and 6. With reference 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). The process is repeated with the next excitation pulse 60b and the next photon 58b (FIG. 4b). By repeating the measurement many times, the TCSPC histogram 52 shown in FIG. 4c is obtained according to the measured times 62 of the individual photons 58. As indicated in FIGS. 1, 5 and 6, for measuring the time interval 62 the device 10 has an electrical connection 64 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 schematically represented as a box in FIGS. 1, 5 and 6 and may, for example, be physically configured as a PC plug-in card, evaluates the signals and creates the TCSPC his-togram, which is output to the evaluation device 22 as two-dimensional data with a wavelength dimension and a time dimension.

[0100] The detection device 18 is therefore configured to detect the autofluorescence radia-tion 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 present as mathematical matrices A mn, wherein the matrix comprises m data points in the wavelength dimension, in the present example there are 74 data points. In the time dimension, the matrix comprises n data points, which enable the high time resolution necessary for the required accuracy, in the present example there are 250 data points:

wherein the rows of A, i.e. (aj1, . . . , ajn), j=1, . . . , m, each correspond to a TCSPC histogram and thus physically correspond essentially to the time-resolved measurements for certain fixed wavelengths.

[0101] In order to determine the sex of the fertilized bird's egg 12, a classifier classifies the provided data into two classes, 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, wherein specific wavelengths to be prioritized are identified in the classification on the basis of features formed in the time dimension of the data, and a sex determination of the bird's egg 12 is carried out by prioritized consideration of the data at the specific wavelengths. In the present case, as classifier a linear classi-fier is used which separates the data along a hyperplane. The features formed in the time dimension of the data are in the present case the first three moments of the central moments of

aj:=(aj1, . . . , ajn), j=1, namely mean value , standard deviation and skewness S.

[0102] As used herein, the terms general, generally, and approximately are intended to account for the inherent degree of variance and imprecision that is often attributed to, and often accompanies, any design and manufacturing process, including engineering tolerances, and without deviation from the relevant functionality and intended outcome, such that mathematical precision and exactitude is not implied and, in some instances, is not possible.

[0103] All the features and advantages, including structural details, spatial arrangements and method steps, which follow from the claims, the description and the drawing can be fundamental to the invention both on their own and in different combinations. It is to be understood that the foregoing is a description of one or more preferred exemplary embodiments of the invention. The invention is not limited to the particular embodiment(s) disclosed herein, but rather is defined solely by the claims below. Furthermore, the statements contained in the foregoing description relate to particular embodiments and are not to be construed as limitations on the scope of the invention or on the definition of terms used in the claims, except where a term or phrase is expressly defined above. Various other embodiments and various changes and modifications to the disclosed embodiment(s) will become apparent to those skilled in the art. All such other embodiments, changes, and modifications are intended to come within the scope of the appended claims.

[0104] As used in this specification and claims, the terms for example, for instance, such as, and like, and the verbs comprising, having, including, and their other verb forms, when used in conjunction with a listing of one or more components or other items, are each to be construed as open-ended, meaning that the listing is not to be considered as excluding other, additional components or items. Other terms are to be construed using their broadest reasonable meaning unless they are used in a context that requires a different interpretation.

LIST OF REFERENCE NUMERALS

[0105] 10 device [0106] 12 bird's egg [0107] 14 light source [0108] 16 excitation radiation [0109] 18 detection device [0110] 20 autofluorescence radiation [0111] 22 evaluation device [0112] 24 sample holder [0113] 26 variable laser beam attenuator [0114] 28 lens [0115] 30 long-pass filter [0116] 32 aperture [0117] 34 light guide system [0118] 36 measuring head [0119] 38 light guide strand [0120] 40 light guide strand [0121] 42 infrared laser diode [0122] 44 multilevel fiber amplifier [0123] 46 birefringent crystal [0124] 48 dichroic mirror [0125] 50 second to fourth harmonic of the laser wavelength 1064 nm [0126] 52 TCSPC histogram [0127] 54 monochromator [0128] 56a detector element, hybrid PMT [0129] 56b detector element, MCP-PMT [0130] 57 beam splitter [0131] 58 photon [0132] 59 interference filter [0133] 60 excitation pulse [0134] 61 TCSPC electronics [0135] 62 time between excitation pulse and detection of the photon [0136] 64 electrical connection