EP3074750A1 - Method and apparatus for discriminating x and y sperms based upon raman spectroscopy - Google Patents

Method and apparatus for discriminating x and y sperms based upon raman spectroscopy

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Publication number
EP3074750A1
EP3074750A1 EP14815053.5A EP14815053A EP3074750A1 EP 3074750 A1 EP3074750 A1 EP 3074750A1 EP 14815053 A EP14815053 A EP 14815053A EP 3074750 A1 EP3074750 A1 EP 3074750A1
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European Patent Office
Prior art keywords
sperm
raman
raman spectroscopy
irradiation
analysis
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EP14815053.5A
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German (de)
French (fr)
Inventor
Anna Chiara De Luca
Donatella BALDUZZI
Giuseppe Coppola
Andrea Galli
Luigi SIRLETO
Roberto PUGLISI
Maria Antonietta FERRARA
Pietro Ferraro
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Consiglio Nazionale delle Richerche CNR
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Consiglio Nazionale delle Richerche CNR
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Publication of EP3074750A1 publication Critical patent/EP3074750A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes

Definitions

  • the present invention relates to a method and apparatus for discriminating X sperm and Y sperm of animal species, in particular bovine species, noninvasively and in a not destructive way by means of linear Raman spectroscopy.
  • the invention relates to the problem of the biochemical analysis of the X and Y sperms with approaches which make possible the subsequent use in fertilization techniques.
  • cytofluorimetry S. F. Ibrahim et al. Current Opinion in Biotechnology 14, 5 (2003). They cytofluorimeter is a powerful instrument allowing to separate, based upon the DNA content, two populations of sperms (X- and Y- cells) with a precision higher than 90% (D.L. Garner, Theriogenology 65, 943, (2006)). However, the cytofluorimetry usually characterizes and separates the cells by means of an outer marking, for example based upon the activation and the analysis of the fluorescence signals (FACS).
  • FACS fluorescence signals
  • Such technique requests the outer marking of the cell by means of a modification with a codifying gene for fluorescent proteins or by marking the cells with fluorescent dyes and probes which could kill or damage the cells themselves during the fastening process.
  • outer marking has hindered the study of the native cells which do not show auto-fluorescence in the natural state.
  • the fastening process can damage the sample.
  • the "sexed" sperms have a lower fertility than the controlling ones.
  • cytofluorimetry Another possible disadvantage of the cytofluorimetry is the use of specific markers of DNA detectable by excitation with UV radiation.
  • UV laser the risk of cytotoxic and/or mutagenic effects on the cells cannot be wholly excluded, even though several healthy animals are born after fecundation with sexed sperms with this technique. For this reason, the cytofluorimetry results to be a little suitable method to be used to discriminate between X sperm and Y sperm.
  • Raman spectroscopy could be a valid alternative for the discrimination of native cells.
  • Raman spectroscopy is a spectroscopic technique based upon Raman effect.
  • Raman effect or scattering consists in the inelastic scattering of photons by the effect of interaction between the incident light and the sample.
  • a beam of incident light on a sample in fact, can be scattered elastically, that is the scattered photons have the same frequency as the incident ones (Rayleigh effect).
  • a small light percentage (about one photon over a million of incident photons) is subjected to inelastic scattering (Raman effect), that is scattered with a higher or lower frequency than the original one.
  • the energy difference between the incident photons and those scattered inelastically corresponds to the vibrational energetic levels of the scattering molecule.
  • US 2012/0225474 describes the use of IR spectroscopy and a quantum cascade laser in Mid-IR for separating cells and in particular X sperm and Y sperm.
  • the IR spectroscopy is an absorbing spectroscopic technique, wherein when a IR photon is absorbed by a molecule, this passes from a fundamental vibrational status to an excited one.
  • the resulting IR spectrum is characterized by a series of peaks with variable height for each transaction.
  • US 2012/0225474 suggests the possible use of Raman spectroscopy as noninvasive vibrational technique as alternative to the absorption techniques.
  • the known state of art does not make possible an analysis of the sperms by means of Raman without damaging the latter to the purpose of a subsequent use thereof, for example, in fecundation techniques.
  • a first object of the present invention is: a method for discriminating noninvasively X sperm and Y sperm as defined by claim 1 .
  • a second object of the present invention is an apparatus for discriminating noninvasively X sperm and Y sperm as defined by claim 10.
  • the present invention provides some relevant advantages.
  • the main advantage consists in the fact that the discrimination between X sperm and Y sperm takes place noninvasively, that is without using markers or fluorophores which could alter the biochemical features of the analyzed cells, and in a not destructive way, that is by using a laser power and a wavelength which does not damage the sample, thus allowing a direct use thereof, for example, in artificial insemination or assisted fecundation techniques.
  • one of the advantages associated to the herein described invention is linked to the region chosen for the irradiation with the linear Raman spectroscopy.
  • the inventors have demonstrated that an irradiation involving the nucleus area adjacent to the tail of the sperm and the consequent acquisition of emission spectra related exclusively to such region allows discriminating effectively between X sperm and Y sperm since the spectra of such region characterize for a higher signal/noise ratio. Therefore, the herein described method as well as the apparatus allow discriminating between X sperm and Y sperm with greater accuracy with respect to the known methods and apparatuses. Additionally, the herein described invention even allows obtaining information related to the concentration variation in the DNA content between X sperm and Y sperm, by making possible then, apart from a qualitative analysis, even a quantitative analysis of the sperms.
  • FIG. 1 shows Raman spectrum acquired in different regions of the sperm X after an irradiation with a laser for linear Raman excitation; in particular, the spectrum (a) corresponds to the acrosomal vesicle, the spectra (b), (c) and (d) correspond to different regions of the nucleus and more precisely to the central, acrosomal regions and the one adjacent to the tail, and the spectrum (e) corresponds to the tail region; (B) shows an image at the optical microscope of an X sperm and the various regions wherein Raman spectra are acquired;
  • FIG. 2 shows Raman spectra (linear Raman spectroscopy) mediated on 900 spectra acquired by as many bovine X sperm cells (grey line) and 900 spectra acquired on as many bovine Y sperm cells (black line), (B) spectrum obtained by the difference between the X and Y sperm cells;
  • FIG. 3 shows the histograms related to the measurement of the area of Raman peaks (linear Raman spectroscopy) corresponding to the wave numbers 726, 785, 1095 and 1581 cm “1 , respectively, measured for X (grey) and Y (black) sperms;
  • Figures 4A and 4B show respectively an apparatus according to a preferred embodiment of the invention and a scheme for irradiating the sperm with a laser beam for Raman excitation (linear Raman spectroscopy) performed with such apparatus (legend: S-mirror; L-lens; BS-beam splitter; F-filter; FTB- cut band filter);
  • Figures 5A and 5B show a scheme respectively of microfluidic path and of double trap system combined to the laser beam for Raman excitation (linear Raman spectroscopy) based upon a preferred embodiment variant of the apparatus of figure 5A;
  • Figure 6 shows three bidimensional graphs (A) and a three-dimensional graph (B) obtained by means of PCA analysis (Principal Component Analysis) of 900 spectra acquired experimentally for each species of X (in grey) and Y (in black) sperms and coming from three different bulls corresponding to the three different shapes (square, circle, star).
  • PCA analysis Principal Component Analysis
  • the first portion of the study of the inventors has involved the analysis of Raman signals, obtained by means of applying the linear Raman spectroscopy, in different area of the bovine sperm and the selection of the region to be studied/analyzed to highlight the changes between bovine X and Y sperms.
  • the analysed sample is provided by Al Center Cogent (Cogent Breeding Ltd, UK).
  • the bovine semen was pre-separated and the X sperm and Y sperm are discriminated according to the "Beltsville Sperm Sorting Technology" [Seidel, E. G . and Garner D. L. (2002) Current status of sexing mammalian spermatozoa Reproduction 124, 733-743.]
  • the provided samples had a purity of 91 -94%.
  • the X sperm or Y sperm were diluted in PBS ⁇ Phosphate Buffer Saline) and a drop of 4 ⁇ _ was deposited inside the sample-bearing element.
  • the sample-bearing element (in the phase of preliminary study phase) was constituted by a microscope coverslip made of quartz (thickness 150 pm) and by a microscope slide (thickness 1 mm) still made of quartz. The sample drop was deposited between the two slides, then sealed with glaze.
  • the so-prepared sample was left to settle for about half an hour and arranged below Raman microscope (linear Raman spectroscopy) where the measurements were performed.
  • the bovine sperm has the shape designated in figure 1 and substantially it divides into two portions: the head and the tail.
  • the head occupies an area of about 10x5 pm 2 and, in turn, is constituted by two portions; the nucleus, containing strongly thickened chromatin and as equipment haploid and acrosome, the task thereof is to open a passage in the ovum's wall thanks to lytic enzymes contained therein and released in the right moment.
  • the sperm tail is constituted by a very long flagellum (about 40 pm) which has a filamentous core and the initial portion thereof, that is that linked to the head (the so-called neck), has a slightly larger diameter than any other portion of the tail and it includes a rolled-up chain of mitochondria around the central core.
  • figure 1 shows Raman spectra (linear Raman spectroscopy) acquired in the above-mentioned different regions of an X sperm, that is nucleus, tail and acrosome. The measurements were repeated on twenty different cells. The spectra acquired in the three different regions (nucleus, tail, acrosome) show intensity peaks and slightly different positions, by reflecting the biochemical differences which can be observed in each one of the different cell areas.
  • Raman spectra (linear Raman spectroscopy) were also acquired in different regions of the nucleus: acrosomal, central area and region near the tail (as defined in figure 1 ). As it can be observed in figure 1A, traces (b)-(d), the spectra are very similar in terms of relative intensities of Raman peaks (linear Raman spectroscopy) and position in the three different regions of the nucleus, but it is found a higher signal-noise ratio (SNR) in the region of the head adjacent to the tail.
  • SNR signal-noise ratio
  • the nucleus spectrum is substantially associated to the DNA content of the sperm. Therefore, this better SNR of the nucleus region adjacent to the tail can be associated to the presence of a higher DNA concentration.
  • This sperm region is the one which will show then the greatest differences between X sperm and Y sperm. In fact, the greatest differences between X sperm and Y sperm are due to the presence of X (in the X sperms) and Y chromosomes (in the Y sperms) which substantially are constituted by DNA. X chromosomes are bigger and include a higher percentage of DNA and such difference reflects in Raman spectra.
  • Raman spectra linear Raman spectroscopy
  • X sperm and Y sperm were acquired in the nucleus region near the tail (area (d) in figure 1 ) to highlight the differences between X sperm and Y sperm.
  • the inventors proceeded to acquire the spectra related to X sperm and Y sperm of three different bulls, wherein each spectrum is an average of 900 cells (300 cells for each donor) to evaluate the possible variability in the biochemical features from cell to cell and from bull to bull.
  • Figure 2 shows Raman average spectra (linear Raman spectroscopy) related to X sperm and Y sperm of three different donors acquired in the nucleus region near the tail. As it can be observed, the two spectra are very similar but show crucial differences (better highlighted in figure 2B with a dotted line) just linked to the DNA contribution. In general, the most evident difference between X sperm and Y sperm of animal species, in particular bovine species, are observed in the peaks of Raman spectrum (linear Raman spectroscopy) at one or more of the following frequency ranges: 720-730 cm “1 ; 780-790 cm “1 ; 1090-1 100 cm “1 ; 1576-1586 cm “ and 1395-1405 cm “1 .
  • the most evident differences are observed at the peaks 726 cm-1 , 785 cm-1 , 1095 cm-1 and 1581 cm-1 , which are almost exclusively due to the presence of DNA [J. M. Benevides "Characterization of DNA structure by Raman spectroscopy Nucleic Acids Res. 1983 August 25; 1 1 (16): 5747-5761 ]. More precisely, the spectrum related to Y sperms shows a reduced intensity for the previously mentioned peaks, by detecting a reduced concentration of the DNA content detected in the analysis volume with respect to the spectrum related to X sperms.
  • the average variation in the areas involving the peaks at 726 and 785 cm-1 confirms the previously acquired data which demonstrate that the difference in terms of DNA content between X sperm and Y sperm in the bovine species is 3.9% (Garner et al., Biol Reprod.;28(2):312-21 , 1983).
  • the average variation of the area related to the peaks at 1095 and 1581 cm-1 is higher, as these peaks are not exclusively linked to the presence of DNA.
  • PCA Principal Component Analysis
  • the main object of this technique is to reduce a higher or lower number of variables (representing as many features of the analyzed phenomenon, in our case the pixels of Raman spectrum (linear Raman spectroscopy)) in some latent variables ("feature reduction").
  • feature reduction takes place by means of a linear transformation of the variables which projects the original ones in a new Cartesian system wherein the new variable with the higher variance is projected on the first axis, the second by size of the variance on the second axis and so on.
  • the decrease in number of the initial variables is not performed by eliminating those which can seem uninfluential, but by constructing new synthetical variables, indeed the main components [Jackson, J. E. (1991 ) A User's Guide to Principal Components. New York: Wiley.].
  • Raman spectrum constituted by N dots (for example the pixels of acquisition means, such as a CCD camera) can be seen like a dot in a N- dimensional space. Therefore, to n spectra n dots will correspond in the N- dimensional space.
  • PCA rotates this cluster of dots until finding the size wherein there is the maximum deformation of the scattergram (maximum variability of the system). This size represents a new coordinate for the system and it corresponds to the first main component. By still rotating the system one will find the second main component, and so on. Therefore the spectra can be represented graphically as dots depending upon the main components and the width of these clusters of dots as well as the distance between the two clusters associated to two classes of cells will give information about the data classification quality.
  • the first main component PC1 appear mainly due to the variation between the several samples caused by the variation in the background (mainly due to the auto-fluorescence of the cell and the solvent), and it cannot be attributed directly to the biochemical changes distinguishing the different types of cells. Therefore, PC1 is not taken into consideration for the data classification.
  • 900 grey dots, in figure 6, are linked to 900 Raman spectra (linear Raman spectroscopy) acquired by bovine X sperms and the three different shapes associated to the dots (square, circle, star) correspond to three different bulls.
  • 900 black dots correspond to 900 Raman spectra (linear Raman spectroscopy) acquired by bovine Y sperms.
  • a clear separation between the two clusters of dots associated to X sperm and Y sperm can be seen.
  • the sperms directly collected by the sperm of an animal can be separated by seeing where the new spectra obtained by irradiation of the nucleus region adjacent to the tail by means of a laser for Raman excitation (linear Raman spectroscopy) arrange with respect to these two reference clusters obtained too by irradiation of the nucleus region adjacent to the tail of X sperm or Y sperm.
  • the powerful classification capabilities due to PCA do not limit to a graphic observation. In fact, it is possible using PCA analysis in predictive way by using the method of the so-called leave-one-ouf and constructing the so-called confusion matrix [Stehman, Stephen V. (1997). "Selecting and interpreting measures of thematic classification accuracy". Remote Sensing of Environment 62 (1 ): 77-89].
  • the obtained result can be evaluated by the analysis of a table, called “confusion matrix” or “classification matrix”, wherein the objects really belonging to each class (real class) and the objects assigned to each class by the model (assigned class) are visible. From this matrix the total percentages of correct classification, and inside each class and the corresponding classification error, are deduced.
  • cross-validation the method of the cross validation
  • the model predictive capabilities the method of the cross validation (“cross-validation”) can be used, which consists in calculating the model by excluding an object at a time (leave-one-ouf method) or an object of N objects (leave-more-ouf), by predicting the values of the reply for all objects excluded by the model. For example, if there are 10 samples (5 belonging to the class A and 5 to the class B) and the leave-one-ouf method is used to validate the obtained classification model, one proceeds as follows:
  • the model is then re-calculated 10 times, by obtaining a new series of parameters, called “cross-validated” (CV), for evaluating the quality of the predicting classification model.
  • CV cross-validated
  • PCA is repeated, by leaving a spectrum outside the classification (spectrum s) and only in a second moment the spectrum s is considered.
  • the distance of s from the two clusters of dots associated to the two classes is measured and one establishes to whom the spectrum s belongs. If the spectrum s is nearer the cluster X and it was a spectrum acquired by an X sperm, then it is classified correctly and it will populate the values of the main diagonal of the confusion matrix. If s is nearer to the cluster Y but corresponds to a spectrum acquired by an X sperm, then the classification is not correct and such spectrum will populate the values of the secondary diagonal of the confusion matrix.
  • each line of the confusion matrix gives the classification provided for a specific cell type.
  • the diagonal terms of the confusion matrix give the number of the correctly classified cells (true positive and true negative).
  • Table 2 shows the confusion matrix obtained by the inventors.
  • this set of data is used as reference for the classification and validation of the data/spectra acquired by sperms collected directly from animals, preferably bovine animals, and in particular bull.
  • each step is carried out according to the modes illustrated in details in the previous description, taken singularly or in combination.
  • FIG. 4A represents a scheme of an apparatus according to a preferred embodiment of the invention, such apparatus is designated as a whole with 1 .
  • the apparatus 1 comprises a laser source for linear Raman excitation 2 irradiating, through transmission/optical deviation means designated as a whole with 3, a sample of X or Y sperm trapped in a measurement volume 4.
  • the laser source for linear Raman excitation emits in a range of wavelength between 500-900 nm.
  • the herein described method as well as the apparatus use continuous laser sources with low power (few mW are applied to the sample) which make the technique developed by the inventors not destructive.
  • CARS coherent antistokes Raman scattering
  • the laser source for linear Raman excitation can have a wavelength of 416 nm, 460 nm, 514 nm, 532 nm, 594 nm, 633 nm, 650 nm, 694 nm, 750 nm, 780 nm, 800 nm.
  • a second laser source 5, emitting one or more trapping beams, is used indeed to the purpose of the optical retaining of the sperm in the measurement volume 4.
  • such entrapment is performed by irradiating bilaterally the sperm with two laser beams.
  • transmission/optical deviation means 6 are provided, interposed between the trapping source 5 and the measurement volume 4.
  • the above-mentioned optical trapping laser 5 thus avoids the need for pre- treating the sperm to limit the mobility thereof.
  • the trapping laser 5 can be used not only to retain the sperm, but even as manipulation means to expose the nucleus region adjacent to the tail of the sperm itself, by placing it under the laser beam used for the linear Raman analysis, still as shown in figure 5B.
  • the trapping laser 5 can be a laser in the spectral region of the infrados, in particular between 700-1200 nm, even so as to reduce the photo-damage or could even coincide with the laser for linear Raman excitation itself.
  • the laser source 5 and the transmission/optical deviation means 6, and/or the above-mentioned means for the optical manipulation can be implemented as means equivalent to those considered herein, for example of the type known as "optical pliers" [Woei Ming Lee' Peter J Reece, Robert F Marchington, Nikolaus K Metzger, Kishan Dholakia "Construction and calibration of an optical trap on a fluorescence optical microscope” Nature Protocols 2, 3226 - 3238 (2007)].
  • a so-called “galvo-mirro system is used, based thereupon a mirror 1 1 is mounted on a so-called “galvo” system, which allows rotating the mirror 1 1 at high frequencies (A.C. De Luca et al. Opt. Express, 16, 7943 (2008)).
  • a so-called “galvo” system which allows rotating the mirror 1 1 at high frequencies (A.C. De Luca et al. Opt. Express, 16, 7943 (2008)).
  • time sharing it is possible creating two traps in so-called “time sharing”, to entrap the sperm in two places and position it under the Raman microscope in the wished place (as already said, the nucleus area near the tail).
  • figure 5B shows a scheme of double trap system combined with the laser for linear Raman excitation.
  • the system of multiple traps can be implemented even by using a so-called "spatial light modulator” (SLM) as described in Mike Woerdemann et al 2009 J. Opt. A: Pure Appl. Opt. 1 1 034010 doi: 10.1088/1464-4258/1 1 /3/034010.
  • SLM spatial light modulator
  • means for the spectral analysis is provided, in the present example a monocromator 7, to detect and evaluate the scattering optical signal produced by the sperm.
  • transmission/optical deviation means 8 can be provided between the region of, or circumscribing, the measurement volume 4 and the monocromator 7 .
  • the laser beams coming from the sources 2 and 5 are inserted in an inverted microscope, which preferably uses an objective with high numerical aperture to illuminate the single cell.
  • the laser beams are focused by the objective 8, which indeed allows obtaining highly focused beams, particularly suitable to the entrapment.
  • a and a camera 10 are further provided, or equivalent means, to observe the sample image during acquisition.
  • the light scattered by the sperm sample that is Raman scattering (linear Raman spectroscopy)
  • the spectrometer 7 is equipped with a preferably backlighted and cooled CCD camera 9 or by equivalent detecting means.
  • the herein described apparatus actually comprises an analysis unit 7 configured to analyze in specific way Raman spectrum (linear Raman spectroscopy) emitted by the sperm resulting from irradiating only the nucleus portion adjacent to the tail of the sperm.
  • Raman spectrum linear Raman spectroscopy
  • the apparatus for example can comprise a software able to make a comparison between the reference spectra acquired as previously described and the spectra obtained by irradiating exclusively the nucleus region adjacent to the tail of the sperm to be discriminated.
  • the comparison is performed by means of the differences not only between the peaks around 726 cm “1 , 785 cm “1 , 1095 cm “1 and 1581 cm “1 , substantially due to a variation in the DNA content, but even the peaks around 1400 cm “1 associated to the presence of proteins and lipids.
  • the apparatus 1 can be further equipped with a microfluidic system, visible in magnified way in figure 5A and designated as a whole with 100. Such system 100 makes the sample to flow under Raman beam and it facilitates the separation of the two types of cells X and Y.
  • the system 100 can provide a channel 101 for inletting one or more sperms, a path 102 defining even the measurement volume 4 and a pair of outlet channels 103 and 104 wherein the sperms classified X and Y, respectively, are sent preferably automatically by means the optical trapping means or different means for the optical manipulation.
  • the microfluidic system can be made of polydimethylsiloxane (PDMS) by means of the so-called "soft lithography" technique.
  • PDMS is obtained by mixing a basis prepolymer and a cross-linking agent (for example those known with the tradenames of Sylgard 184, Daw Corwing), preferably in a ratio 10: 1.
  • the mixture is placed in a vacuum drier to eliminate possible air bubbles and then deposited on a mould made of SU8.
  • PDMS is thermally hardened on heating plate.
  • the so-hardened PDMS is detached gently from the master made of SU8 and pierced in order to inject the liquid inside the channels of the microfluidic system.
  • Such channels are sealed by treating PDMS and a microscope slide (made of quartz) with an oxygen plasma and by joining them therebetween.
  • the sperm directly collected from the animal can be diluted in a buffer solution (for example PBS) to preserve the vitality thereof.
  • a buffer solution for example PBS
  • a trap laser is used to immobilize in one or more places a single sperm, which is also properly positioned under the beam for Raman excitation.
  • the configuration is so that the laser for Raman excitation illuminates the nucleus are near the sperm neck and in this area a Raman spectrum of the sperm itself is acquired.
  • the acquired spectrum to one or more reference spectra (preferably by following the procedure reported above) it is possible to understand to which class (X or Y) the analyzed sperm belongs.
  • the trap laser, or different means for the optical manipulation can be used even to move the sperms under analysis in one of the two outlet microfluidic channels.

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Abstract

The invention relates to a method based upon the use of Raman spectroscopy to face the problem of the biochemical analysis and of the differentiation of the X sperm and Y sperm in the animal species, in particular bovine species, with approaches which make possible the subsequent use in fecundation techniques. A method addressed to the resolution of this problem is based upon the separation between X sperm and Y sperm before insemination. An apparatus is also described implementing such method which allows to distinguish and characterize the difference between X sperm and Y sperm by acquiring and analyzing Raman spectra noninvasively and in a not destructive way. By means of a statistical approach, such as for example the analysis of the main components and the linear discriminating analysis, the spectroscopic difference between X sperm and Y sperm is evaluated, processed and used to separate the two types of cells. The invention also relates to the development of a cytofluorimeter combining the Raman microscope with a system for trapping optically the sperms and a microfluidic chamber. More precisely, the optical trapping system, equipped with an objective with high numerical aperture, is used to immobilize the cells and, the same objective is used to excite and gather Raman spectra. At last, the microfluidic chamber is used to make the sample to flow, with the final purpose of providing a quick, precise and not invasive separation of X sperm and Y sperm, (figure 5A).

Description

METHOD AND APPARATUS FOR DISCRIMINATING BETWEEN X AND Y SPERMS BASED UPON RAMAN SPECTROSCOPY
DESCRIPTION
Technical field of the invention
The present invention relates to a method and apparatus for discriminating X sperm and Y sperm of animal species, in particular bovine species, noninvasively and in a not destructive way by means of linear Raman spectroscopy.
Background
The invention relates to the problem of the biochemical analysis of the X and Y sperms with approaches which make possible the subsequent use in fertilization techniques.
Currently, different approaches for discriminating and separating X and Y sperms have been proposed: the separation of the cells activated by fluorescence (FACS) (G. E. Seidel et al. Reproduction 124, 733, (2002); D.L. Garner, Theriogenology 65, 943, (2006); E. A. Howes et al. J Reprod Fertil 1 10 (2) 195 (1997)), the size evaluation (Z. Zavaczki, C. et al. Fertility and Sterility 85, 121 (2006); E. B. van Muster et al. Theriogenology 52, 1281 , (1999)), immunological techniques (P.J.M. Hendriksen Theriogenology 52, 1295 (1999)) and the electrophoretic separation (S. Kaneko et al. Biochem Biophys Res Commun 124, 950 (1984)). However, these techniques have still problems in terms of high costs and damage of the cells.
The techniques for selecting the cells have revolutionized the research in the cell biology and thereamong the most important one for classifying the cells is the cytofluorimetry (S. F. Ibrahim et al. Current Opinion in Biotechnology 14, 5 (2003)). They cytofluorimeter is a powerful instrument allowing to separate, based upon the DNA content, two populations of sperms (X- and Y- cells) with a precision higher than 90% (D.L. Garner, Theriogenology 65, 943, (2006)). However, the cytofluorimetry usually characterizes and separates the cells by means of an outer marking, for example based upon the activation and the analysis of the fluorescence signals (FACS). Such technique then requests the outer marking of the cell by means of a modification with a codifying gene for fluorescent proteins or by marking the cells with fluorescent dyes and probes which could kill or damage the cells themselves during the fastening process. Such outer marking has hindered the study of the native cells which do not show auto-fluorescence in the natural state. Furthermore, the fastening process can damage the sample. In fact, it has been demonstrated that the "sexed" sperms have a lower fertility than the controlling ones.
Another possible disadvantage of the cytofluorimetry is the use of specific markers of DNA detectable by excitation with UV radiation. By using UV laser the risk of cytotoxic and/or mutagenic effects on the cells cannot be wholly excluded, even though several healthy animals are born after fecundation with sexed sperms with this technique. For this reason, the cytofluorimetry results to be a little suitable method to be used to discriminate between X sperm and Y sperm.
Therefore, the development of a new separation system which can characterize and separate, noninvasively and in not destructive way, native cells and keep the cells in a not altered and vital state after separation remains an open matter.
Raman spectroscopy could be a valid alternative for the discrimination of native cells.
Raman spectroscopy is a spectroscopic technique based upon Raman effect. Raman effect (or scattering) consists in the inelastic scattering of photons by the effect of interaction between the incident light and the sample. A beam of incident light on a sample, in fact, can be scattered elastically, that is the scattered photons have the same frequency as the incident ones (Rayleigh effect). A small light percentage (about one photon over a million of incident photons) is subjected to inelastic scattering (Raman effect), that is scattered with a higher or lower frequency than the original one. The energy difference between the incident photons and those scattered inelastically corresponds to the vibrational energetic levels of the scattering molecule. By collecting the photons scattered at the different frequencies it is possible reconstructing a Raman spectrum which can then provide information about the chemical composition, the molecular structure, the intermolecular interactions of the sample under analysis (D. J. Gardiner "Practical Raman spectroscopy" Springer-Verlag 1989).
In fact, US 2012/0225474 describes the use of IR spectroscopy and a quantum cascade laser in Mid-IR for separating cells and in particular X sperm and Y sperm. The IR spectroscopy is an absorbing spectroscopic technique, wherein when a IR photon is absorbed by a molecule, this passes from a fundamental vibrational status to an excited one. The resulting IR spectrum is characterized by a series of peaks with variable height for each transaction. Furthermore, US 2012/0225474 suggests the possible use of Raman spectroscopy as noninvasive vibrational technique as alternative to the absorption techniques. However, US 2012/0225474 states explicitly that Raman spectroscopy has the problem of providing weak signals by suggesting, in particular, the use of the not linear technique "coherent antistokes Raman scattering" (CARS) as alternative to the linear Raman spectroscopy [Fouad El- Diasty "Coherent anti-Stokes Raman scattering: Spectroscopy and microscopy" Vibrational Spectroscopy 55, 1-37 (201 1 )].
Therefore, the known state of art does not make possible an analysis of the sperms by means of Raman without damaging the latter to the purpose of a subsequent use thereof, for example, in fecundation techniques.
Summary of the invention The technical problem placed and solved by the present invention is then to provide a method and apparatus allowing an alternate approach based upon the linear Raman spectroscopy for the chemical analysis and the separation of the sperms and allowing to obviate to the drawbacks mentioned above with reference to the known art. Such problem is solved by a method for discriminating noninvasively X sperm and Y sperm as defined by claim 1.
In particular, a first object of the present invention is: a method for discriminating noninvasively X sperm and Y sperm as defined by claim 1 . A second object of the present invention is an apparatus for discriminating noninvasively X sperm and Y sperm as defined by claim 10.
Preferred features of the present invention are subject of the depending claims.
The present invention provides some relevant advantages. The main advantage consists in the fact that the discrimination between X sperm and Y sperm takes place noninvasively, that is without using markers or fluorophores which could alter the biochemical features of the analyzed cells, and in a not destructive way, that is by using a laser power and a wavelength which does not damage the sample, thus allowing a direct use thereof, for example, in artificial insemination or assisted fecundation techniques. Furthermore, one of the advantages associated to the herein described invention is linked to the region chosen for the irradiation with the linear Raman spectroscopy. In fact, the inventors have demonstrated that an irradiation involving the nucleus area adjacent to the tail of the sperm and the consequent acquisition of emission spectra related exclusively to such region allows discriminating effectively between X sperm and Y sperm since the spectra of such region characterize for a higher signal/noise ratio. Therefore, the herein described method as well as the apparatus allow discriminating between X sperm and Y sperm with greater accuracy with respect to the known methods and apparatuses. Additionally, the herein described invention even allows obtaining information related to the concentration variation in the DNA content between X sperm and Y sperm, by making possible then, apart from a qualitative analysis, even a quantitative analysis of the sperms.
Other advantages, features and use modes of the present invention will result evident from the following detailed description of some embodiments, shown by way of example and not with limitative purpose.
Brief description of the figures The figures of the enclosed drawings will be referred to, wherein:
■ Figure 1 : (A) shows Raman spectrum acquired in different regions of the sperm X after an irradiation with a laser for linear Raman excitation; in particular, the spectrum (a) corresponds to the acrosomal vesicle, the spectra (b), (c) and (d) correspond to different regions of the nucleus and more precisely to the central, acrosomal regions and the one adjacent to the tail, and the spectrum (e) corresponds to the tail region; (B) shows an image at the optical microscope of an X sperm and the various regions wherein Raman spectra are acquired;
■ Figure 2: (A) shows Raman spectra (linear Raman spectroscopy) mediated on 900 spectra acquired by as many bovine X sperm cells (grey line) and 900 spectra acquired on as many bovine Y sperm cells (black line), (B) spectrum obtained by the difference between the X and Y sperm cells;
■ Figure 3: shows the histograms related to the measurement of the area of Raman peaks (linear Raman spectroscopy) corresponding to the wave numbers 726, 785, 1095 and 1581 cm"1, respectively, measured for X (grey) and Y (black) sperms;
■ Figures 4A and 4B show respectively an apparatus according to a preferred embodiment of the invention and a scheme for irradiating the sperm with a laser beam for Raman excitation (linear Raman spectroscopy) performed with such apparatus (legend: S-mirror; L-lens; BS-beam splitter; F-filter; FTB- cut band filter);
Figures 5A and 5B show a scheme respectively of microfluidic path and of double trap system combined to the laser beam for Raman excitation (linear Raman spectroscopy) based upon a preferred embodiment variant of the apparatus of figure 5A;
Figure 6 shows three bidimensional graphs (A) and a three-dimensional graph (B) obtained by means of PCA analysis (Principal Component Analysis) of 900 spectra acquired experimentally for each species of X (in grey) and Y (in black) sperms and coming from three different bulls corresponding to the three different shapes (square, circle, star).
Detailed description of preferred embodiments
Several embodiments and variants of the invention will be described hereinafter, and by referring to the above illustrated figures.
In the following detailed description, additional embodiments and variants with respect to embodiments and variants already treated in the same description will be illustrated by limiting to the differences with respect to what already shown. Furthermore, the different embodiments and variants described hereinafter can be used in combination, when they are compatible.
* * *
First of all, the base principles and preliminary studies will be illustrated which have lead to the method and apparatus of the invention.
The first portion of the study of the inventors has involved the analysis of Raman signals, obtained by means of applying the linear Raman spectroscopy, in different area of the bovine sperm and the selection of the region to be studied/analyzed to highlight the changes between bovine X and Y sperms. The analysed sample is provided by Al Center Cogent (Cogent Breeding Ltd, UK). The bovine semen was pre-separated and the X sperm and Y sperm are discriminated according to the "Beltsville Sperm Sorting Technology" [Seidel, E. G . and Garner D. L. (2002) Current status of sexing mammalian spermatozoa Reproduction 124, 733-743.] The provided samples had a purity of 91 -94%.
The X sperm or Y sperm were diluted in PBS {Phosphate Buffer Saline) and a drop of 4 μΙ_ was deposited inside the sample-bearing element. The sample-bearing element (in the phase of preliminary study phase) was constituted by a microscope coverslip made of quartz (thickness 150 pm) and by a microscope slide (thickness 1 mm) still made of quartz. The sample drop was deposited between the two slides, then sealed with glaze.
The so-prepared sample was left to settle for about half an hour and arranged below Raman microscope (linear Raman spectroscopy) where the measurements were performed.
The bovine sperm has the shape designated in figure 1 and substantially it divides into two portions: the head and the tail.
The head occupies an area of about 10x5 pm2 and, in turn, is constituted by two portions; the nucleus, containing strongly thickened chromatin and as equipment haploid and acrosome, the task thereof is to open a passage in the ovum's wall thanks to lytic enzymes contained therein and released in the right moment.
The sperm tail is constituted by a very long flagellum (about 40 pm) which has a filamentous core and the initial portion thereof, that is that linked to the head (the so-called neck), has a slightly larger diameter than any other portion of the tail and it includes a rolled-up chain of mitochondria around the central core.
At first, the inventors acquired Raman spectra from different cell area (the size of the spot laser was about 0.3x0.3 pm2), and the obtained results are shown in figure 1 . In particular, figure 1 (A) shows Raman spectra (linear Raman spectroscopy) acquired in the above-mentioned different regions of an X sperm, that is nucleus, tail and acrosome. The measurements were repeated on twenty different cells. The spectra acquired in the three different regions (nucleus, tail, acrosome) show intensity peaks and slightly different positions, by reflecting the biochemical differences which can be observed in each one of the different cell areas.
A synthesis of the main bands and of their classification is shown in Table 1 reported below. In particular, Raman spectrum (linear Raman spectroscopy) of the nucleus is characterize by very pronounced peaks at 726, 785, 1581 e 1091 cm-1 associated to the DNA presence. The acrosome spectrum has intense bands in the region of the Amide I (1600-1680 cm"1) proteins, Amide III (around 1200-1300 cm"1) proteins and of the lipids, with C-H vibration at 1480 cm-1. The tail spectrum is characterized by a peak around 751 cm"1 associated to the presence of mitochondria.
Raman spectra (linear Raman spectroscopy) were also acquired in different regions of the nucleus: acrosomal, central area and region near the tail (as defined in figure 1 ). As it can be observed in figure 1A, traces (b)-(d), the spectra are very similar in terms of relative intensities of Raman peaks (linear Raman spectroscopy) and position in the three different regions of the nucleus, but it is found a higher signal-noise ratio (SNR) in the region of the head adjacent to the tail.
The nucleus spectrum is substantially associated to the DNA content of the sperm. Therefore, this better SNR of the nucleus region adjacent to the tail can be associated to the presence of a higher DNA concentration. This sperm region is the one which will show then the greatest differences between X sperm and Y sperm. In fact, the greatest differences between X sperm and Y sperm are due to the presence of X (in the X sperms) and Y chromosomes (in the Y sperms) which substantially are constituted by DNA. X chromosomes are bigger and include a higher percentage of DNA and such difference reflects in Raman spectra.
For this reason, Raman spectra (linear Raman spectroscopy) were acquired in the nucleus region near the tail (area (d) in figure 1 ) to highlight the differences between X sperm and Y sperm. Once selected the area wherein the greatest differences between X sperm and Y sperm are observed, the inventors proceeded to acquire the spectra related to X sperm and Y sperm of three different bulls, wherein each spectrum is an average of 900 cells (300 cells for each donor) to evaluate the possible variability in the biochemical features from cell to cell and from bull to bull. Figure 2 shows Raman average spectra (linear Raman spectroscopy) related to X sperm and Y sperm of three different donors acquired in the nucleus region near the tail. As it can be observed, the two spectra are very similar but show crucial differences (better highlighted in figure 2B with a dotted line) just linked to the DNA contribution. In general, the most evident difference between X sperm and Y sperm of animal species, in particular bovine species, are observed in the peaks of Raman spectrum (linear Raman spectroscopy) at one or more of the following frequency ranges: 720-730 cm"1; 780-790 cm"1 ; 1090-1 100 cm"1 ; 1576-1586 cm" and 1395-1405 cm"1.
In particular, the most evident differences are observed at the peaks 726 cm-1 , 785 cm-1 , 1095 cm-1 and 1581 cm-1 , which are almost exclusively due to the presence of DNA [J. M. Benevides "Characterization of DNA structure by Raman spectroscopy Nucleic Acids Res. 1983 August 25; 1 1 (16): 5747-5761 ]. More precisely, the spectrum related to Y sperms shows a reduced intensity for the previously mentioned peaks, by detecting a reduced concentration of the DNA content detected in the analysis volume with respect to the spectrum related to X sperms.
In order to quantify these differences the areas of the above-mentioned peaks were measured and the obtained values are shown in figure 3. In particular a variation is observed between X sperm and Y sperm of the area related to Raman peaks 726, 785, 1095 and 1581 cm-1 equal to dA726=4±2%, dA785=4.3±1 .8%, dA1 095=9±3% and dA1 58i=10±3%, respectively.
As it can be observed, the average variation in the areas involving the peaks at 726 and 785 cm-1 , substantially due to a variation in DNA content, confirms the previously acquired data which demonstrate that the difference in terms of DNA content between X sperm and Y sperm in the bovine species is 3.9% (Garner et al., Biol Reprod.;28(2):312-21 , 1983). The average variation of the area related to the peaks at 1095 and 1581 cm-1 , is higher, as these peaks are not exclusively linked to the presence of DNA. In fact, it is to be noted that the difference between Raman spectra (linear Raman spectroscopy) related to X sperm and Y sperm is not exclusively due to the presence of X and Y chromosomes and therefore to a different content of DNA. The difference of smaller extent between the spectra related to X sperm and Y sperm can be observed even in the region around 1400 cm"1 and it is associated to the presence of proteins and lipids. In fact, with the experimental apparatus which was used to acquire the spectra (Raman microscope with laser source preferably at 532 nm and microscope objective 100x), not being a confocal system, the resolution which can be reached along the axis z is about 1 pm. Such experimental condition involves that together with the nucleus region which one wants to observe even a portion of the signal due to the cell membrane is acquired (see scheme shown in figure 4B). Previous studies (described in particular in US 5,021 ,244) demonstrate that the X sperm and Y sperm have a different composition of the membrane proteins thereto probably this difference observed in Raman spectra (linear Raman spectroscopy) is observed.
Analyzed according to the above-illustrated modes, the biochemical differences highlighted by Raman spectra (linear Raman spectroscopy) related to X sperm and Y sperm, these spectra were subsequently processed to discriminate effectively and noninvasively the two types of cells. In order to do it a statistical technique known as "Principal Component Analysis" (PCA) which is a technique for simplifying data used in the field of the multivariate statistics.
The main object of this technique is to reduce a higher or lower number of variables (representing as many features of the analyzed phenomenon, in our case the pixels of Raman spectrum (linear Raman spectroscopy)) in some latent variables ("feature reduction"). The decrease takes place by means of a linear transformation of the variables which projects the original ones in a new Cartesian system wherein the new variable with the higher variance is projected on the first axis, the second by size of the variance on the second axis and so on. The decrease in number of the initial variables, then, is not performed by eliminating those which can seem uninfluential, but by constructing new synthetical variables, indeed the main components [Jackson, J. E. (1991 ) A User's Guide to Principal Components. New York: Wiley.].
Briefly, Raman spectrum constituted by N dots (for example the pixels of acquisition means, such as a CCD camera) can be seen like a dot in a N- dimensional space. Therefore, to n spectra n dots will correspond in the N- dimensional space. PCA rotates this cluster of dots until finding the size wherein there is the maximum deformation of the scattergram (maximum variability of the system). This size represents a new coordinate for the system and it corresponds to the first main component. By still rotating the system one will find the second main component, and so on. Therefore the spectra can be represented graphically as dots depending upon the main components and the width of these clusters of dots as well as the distance between the two clusters associated to two classes of cells will give information about the data classification quality.
Therefore, by representing graphically the spectra depending upon the main components (the so-called "score plot') it is possible, from all biochemical variations associated to several spectra, to observe in a more immediate way the variability and the effectiveness of classifying our measurements.
At this point 300 spectra related to X sperm and Y sperm were analyzed for each one of the three different bulls (an average of 900 spectra per type of cell, 300 spectra for each donor) by using PCA. In this case the classification was of supervised type, in fact the set of classes (X and Y sperms) is defined in advance. The supervised method has the purpose of deciding to which belonging class (defined in advance) each sample (spectrum) has to be assigned, based upon the carrier of measurements (main components) provided by Raman measurement. At last, bidimensional graphs were constructed depending upon the second, third and fourth main component - PC2, PC3 and PC4, respectively - and a three-dimensional graph related to such main components. Such graphs are shown in figure 6. The first main component PC1 appear mainly due to the variation between the several samples caused by the variation in the background (mainly due to the auto-fluorescence of the cell and the solvent), and it cannot be attributed directly to the biochemical changes distinguishing the different types of cells. Therefore, PC1 is not taken into consideration for the data classification.
900 grey dots, in figure 6, are linked to 900 Raman spectra (linear Raman spectroscopy) acquired by bovine X sperms and the three different shapes associated to the dots (square, circle, star) correspond to three different bulls. Analogously, 900 black dots correspond to 900 Raman spectra (linear Raman spectroscopy) acquired by bovine Y sperms. Surprisingly, a clear separation between the two clusters of dots associated to X sperm and Y sperm can be seen.
Therefore, the sperms directly collected by the sperm of an animal can be separated by seeing where the new spectra obtained by irradiation of the nucleus region adjacent to the tail by means of a laser for Raman excitation (linear Raman spectroscopy) arrange with respect to these two reference clusters obtained too by irradiation of the nucleus region adjacent to the tail of X sperm or Y sperm. The powerful classification capabilities due to PCA do not limit to a graphic observation. In fact, it is possible using PCA analysis in predictive way by using the method of the so-called leave-one-ouf and constructing the so-called confusion matrix [Stehman, Stephen V. (1997). "Selecting and interpreting measures of thematic classification accuracy". Remote Sensing of Environment 62 (1 ): 77-89].
At the end of the classification process, the obtained result can be evaluated by the analysis of a table, called "confusion matrix" or "classification matrix", wherein the objects really belonging to each class (real class) and the objects assigned to each class by the model (assigned class) are visible. From this matrix the total percentages of correct classification, and inside each class and the corresponding classification error, are deduced. To know the model predictive capabilities the method of the cross validation ("cross-validation") can be used, which consists in calculating the model by excluding an object at a time (leave-one-ouf method) or an object of N objects (leave-more-ouf), by predicting the values of the reply for all objects excluded by the model. For example, if there are 10 samples (5 belonging to the class A and 5 to the class B) and the leave-one-ouf method is used to validate the obtained classification model, one proceeds as follows:
- eliminating sample Nr.1 ; - re-calculating the model by using only the 9 left samples;
- re-assigning the sample Nr. 1 to a class based upon the model re- calculated on the 9 left samples (if the sample Nr. 1 is closer to cluster A and it was a sample belonging to class A, then it is classified correctly and populates the values of the main diagonal of the confusion matrix; if the sample Nr. 1 is closer to cluster B and it was a sample belonging to class A then it is not classified correctly and it populates the values of the secondary diagonal of the confusion matrix);
- eliminating the sample Nr. 2;
- re-calculating again the model on 9 remained samples and proceed with the re-assignment of - sample Nr. 2;
- proceeding by eliminating one at a time all samples up to Nr. 10 and reassigning them each time based upon the model re-calculated without the eliminated sample.
At the end of the process the model is then re-calculated 10 times, by obtaining a new series of parameters, called "cross-validated" (CV), for evaluating the quality of the predicting classification model.
Briefly, in order to obtain one or more reference spectra even the following procedure can be used. PCA is repeated, by leaving a spectrum outside the classification (spectrum s) and only in a second moment the spectrum s is considered. The distance of s from the two clusters of dots associated to the two classes (in our case X and Y sperms) is measured and one establishes to whom the spectrum s belongs. If the spectrum s is nearer the cluster X and it was a spectrum acquired by an X sperm, then it is classified correctly and it will populate the values of the main diagonal of the confusion matrix. If s is nearer to the cluster Y but corresponds to a spectrum acquired by an X sperm, then the classification is not correct and such spectrum will populate the values of the secondary diagonal of the confusion matrix.
Practically, each line of the confusion matrix gives the classification provided for a specific cell type. The diagonal terms of the confusion matrix give the number of the correctly classified cells (true positive and true negative). Table 2 shows the confusion matrix obtained by the inventors.
The average of the values on the main diagonal of the confusion matrix gives information about the classification effectiveness of the herein described technique which is equal to Effectiveness= (Nr. of correctly classified class A samples + Nr. of correctly classified class B samples) / total Nr. of samples, that is
■B22 + s y
Ef fic tenia =———— = 9H6 %
By using samples of previously separated X and Y sperms with a purity between 91 -94%, a classification effectiveness of 90.6% is obtained.
In the herein considered example, this set of data is used as reference for the classification and validation of the data/spectra acquired by sperms collected directly from animals, preferably bovine animals, and in particular bull.
What sofar illustrated demonstrates the effectiveness of a method for discriminating noninvasively X sperm and Y sperm based upon the following main steps:
- optical trapping of a sperm in a volume of irradiation;
- irradiating with a laser for linear Raman excitation the sperm trapped in the volume of irradiation at a nucleus region adjacent to the tail;
- acquiring Raman spectrum (linear Raman spectroscopy) of the sperm resulting from the irradiation of only nucleus region adjacent to the tail;
- comparing the acquired spectrum to one or more reference spectra acquired, too, by irradiating exclusively the nucleus region adjacent to the tail of the reference sperms;
- identifying the X or Y nature of the sperm based upon the comparison of the spectra obtained by means of the linear Raman spectroscopy for said nucleus region adjacent to the tail, wherein each step is carried out according to the modes illustrated in details in the previous description, taken singularly or in combination.
* * *
Having demonstrated the operating principle of Raman technique, that is linear Raman spectroscopy for discriminating X sperm and Y sperm and the associated method for said discrimination, a preferred embodiment of an apparatus is described hereinafter which, by using the same principles considered above, really allows to discriminate and separate sperms. Figure 4A represents a scheme of an apparatus according to a preferred embodiment of the invention, such apparatus is designated as a whole with 1 .
The apparatus 1 comprises a laser source for linear Raman excitation 2 irradiating, through transmission/optical deviation means designated as a whole with 3, a sample of X or Y sperm trapped in a measurement volume 4. Preferably the laser source for linear Raman excitation emits in a range of wavelength between 500-900 nm. In particular, the herein described method as well as the apparatus use continuous laser sources with low power (few mW are applied to the sample) which make the technique developed by the inventors not destructive. To this purpose it is to be noted that the not linear technique "coherent antistokes Raman scattering" (CARS), on the contrary, as it is known to the person skilled in the art, requires pulsing lasers with high power.
In particular, the laser source for linear Raman excitation can have a wavelength of 416 nm, 460 nm, 514 nm, 532 nm, 594 nm, 633 nm, 650 nm, 694 nm, 750 nm, 780 nm, 800 nm. A second laser source 5, emitting one or more trapping beams, is used indeed to the purpose of the optical retaining of the sperm in the measurement volume 4. Preferably, as shown in figure 5B, such entrapment is performed by irradiating bilaterally the sperm with two laser beams. Even in this case, transmission/optical deviation means 6 are provided, interposed between the trapping source 5 and the measurement volume 4.
The above-mentioned optical trapping laser 5 thus avoids the need for pre- treating the sperm to limit the mobility thereof.
The trapping laser 5 can be used not only to retain the sperm, but even as manipulation means to expose the nucleus region adjacent to the tail of the sperm itself, by placing it under the laser beam used for the linear Raman analysis, still as shown in figure 5B.
The trapping laser 5 can be a laser in the spectral region of the infrados, in particular between 700-1200 nm, even so as to reduce the photo-damage or could even coincide with the laser for linear Raman excitation itself.
The laser source 5 and the transmission/optical deviation means 6, and/or the above-mentioned means for the optical manipulation can be implemented as means equivalent to those considered herein, for example of the type known as "optical pliers" [Woei Ming Lee' Peter J Reece, Robert F Marchington, Nikolaus K Metzger, Kishan Dholakia "Construction and calibration of an optical trap on a fluorescence optical microscope" Nature Protocols 2, 3226 - 3238 (2007)].
Preferably, a so-called "galvo-mirro system is used, based thereupon a mirror 1 1 is mounted on a so-called "galvo" system, which allows rotating the mirror 1 1 at high frequencies (A.C. De Luca et al. Opt. Express, 16, 7943 (2008)). In this way it is possible creating two traps in so-called "time sharing", to entrap the sperm in two places and position it under the Raman microscope in the wished place (as already said, the nucleus area near the tail). To this purpose see figure 5B, which shows a scheme of double trap system combined with the laser for linear Raman excitation. The system of multiple traps can be implemented even by using a so-called "spatial light modulator" (SLM) as described in Mike Woerdemann et al 2009 J. Opt. A: Pure Appl. Opt. 1 1 034010 doi: 10.1088/1464-4258/1 1 /3/034010. Then means for the spectral analysis is provided, in the present example a monocromator 7, to detect and evaluate the scattering optical signal produced by the sperm. Even in this case, transmission/optical deviation means 8 can be provided between the region of, or circumscribing, the measurement volume 4 and the monocromator 7 .
In the present example, the laser beams coming from the sources 2 and 5 are inserted in an inverted microscope, which preferably uses an objective with high numerical aperture to illuminate the single cell. The laser beams are focused by the objective 8, which indeed allows obtaining highly focused beams, particularly suitable to the entrapment.
A and a camera 10 are further provided, or equivalent means, to observe the sample image during acquisition.
The light scattered by the sperm sample, that is Raman scattering (linear Raman spectroscopy), is gathered by the same objective 8 and sent to the spectrometer 7, in particular on a slide for opening the monocromator. The latter is equipped with a preferably backlighted and cooled CCD camera 9 or by equivalent detecting means.
Generally, then, in order to allow discriminating between X sperm and Y sperm the herein described apparatus actually comprises an analysis unit 7 configured to analyze in specific way Raman spectrum (linear Raman spectroscopy) emitted by the sperm resulting from irradiating only the nucleus portion adjacent to the tail of the sperm.
In particular, then, the apparatus for example can comprise a software able to make a comparison between the reference spectra acquired as previously described and the spectra obtained by irradiating exclusively the nucleus region adjacent to the tail of the sperm to be discriminated.
As shown above, in an embodiment of the invention the comparison is performed by means of the differences not only between the peaks around 726 cm"1 , 785 cm"1, 1095 cm"1 and 1581 cm"1 , substantially due to a variation in the DNA content, but even the peaks around 1400 cm"1 associated to the presence of proteins and lipids. The apparatus 1 can be further equipped with a microfluidic system, visible in magnified way in figure 5A and designated as a whole with 100. Such system 100 makes the sample to flow under Raman beam and it facilitates the separation of the two types of cells X and Y. In particular, as outlined in figure 5A the system 100 can provide a channel 101 for inletting one or more sperms, a path 102 defining even the measurement volume 4 and a pair of outlet channels 103 and 104 wherein the sperms classified X and Y, respectively, are sent preferably automatically by means the optical trapping means or different means for the optical manipulation. By way of example, the microfluidic system can be made of polydimethylsiloxane (PDMS) by means of the so-called "soft lithography" technique. In particular, PDMS is obtained by mixing a basis prepolymer and a cross-linking agent (for example those known with the tradenames of Sylgard 184, Daw Corwing), preferably in a ratio 10: 1. The mixture is placed in a vacuum drier to eliminate possible air bubbles and then deposited on a mould made of SU8. Subsequently, PDMS is thermally hardened on heating plate. The so-hardened PDMS is detached gently from the master made of SU8 and pierced in order to inject the liquid inside the channels of the microfluidic system. Such channels are sealed by treating PDMS and a microscope slide (made of quartz) with an oxygen plasma and by joining them therebetween.
The sperm directly collected from the animal can be diluted in a buffer solution (for example PBS) to preserve the vitality thereof.
Based upon a possible synthesis of what illustrated sofar in relation to the apparatus and method of the invention, a trap laser is used to immobilize in one or more places a single sperm, which is also properly positioned under the beam for Raman excitation. In particular, the configuration is so that the laser for Raman excitation illuminates the nucleus are near the sperm neck and in this area a Raman spectrum of the sperm itself is acquired. By comparing, preferably in real time, the acquired spectrum to one or more reference spectra (preferably by following the procedure reported above) it is possible to understand to which class (X or Y) the analyzed sperm belongs. The trap laser, or different means for the optical manipulation, can be used even to move the sperms under analysis in one of the two outlet microfluidic channels.
The present invention has been sofar described by referring to preferred embodiments. It is to be meant that other embodiments may exist belonging to the same inventive core, as defined by the protection scope of the herebelow reported claims.

Claims

1. A method for discriminating X sperm and Y sperm noninvasively, which method comprises the following steps:
- trapping a sperm optically in un volume of irradiation; - irradiating with a laser for Raman excitation according to the linear Raman spectroscopy said sperm trapped in the volume of irradiation, wherein the irradiation involves the nucleus region adjacent to the tail of said sperm;
- acquiring Raman spectrum obtained by means of the linear Raman spectroscopy of the sperm resulting from irradiating exclusively said nucleus region adjacent to the tail;
- comparing the acquired spectrum with one or more reference spectra related to said nucleus region adjacent to the tail ;
- identifying X or Y nature of the sperm on the basis of said comparison.
2. The method according to any one of the previous claims, wherein said sperm is a sperm of bovine species, in particular bull.
3. The method according to any one of the previous claims, wherein said laser for Raman excitation according to the linear Raman spectroscopy has a wavelength comprised in a range of about 500-900 nm.
4. The method according to any one of the previous claims, wherein said comparing step is performed by evaluating the intensity and/or the areas under the peaks of Raman spectrum obtained by means of the linear Raman spectroscopy at one or more of the following frequency ranges: 720-730 cm"1 , preferably about 726 cm"1 ; 780-790, preferably about 785 cm"1; 1090-1 100 cm"1, preferably about 1095 cm"1 ; 1576-1586 cm"1, preferably about 1581 cm"1; and 1395-1405 cm"1 , preferably about 1400 cm"1.
5. The method according to any one of the previous claims, wherein said comparing step is performed in real time by means of automatic processing.
6. The method according to any one of the previous claims, wherein said reference spectrum or spectra are obtained by PCA ("Principal Component Analysis") analysis of spectra related to X and Y sperms.
7. The method according to any one of the previous claims, wherein said trapping step is performed by using at least a second laser beam, which emits in a spectral range between 700 nm and 1200 nm and/or said laser for Raman excitation according to the linear Raman spectroscopy.
8. The method according to any one of the previous claims, comprising a separation step, wherein the sperm is directed towards a respective microfluidic channel depending upon the X or Y class of membership.
9. The method according to claims 7 and 8, wherein said separation step is performed by using said second laser beam.
10. An apparatus (1 ) for discriminating X sperm and Y sperm noninvasively, in particular bovine sperms, comprising:
- optical means (5) for entrapping a sperm in a volume of irradiation (4);
- irradiating optical means for Raman excitation (2) according to the linear Raman spectroscopy of said sperm optically trapped in said volume of irradiation (4), suitable to focus the radiation at a nucleus portion of the sperm adjacent to the tail;
- an analysis unit (7) configured to analyze Raman spectrum obtained by means of the linear Raman spectroscopy of the sperm emitted resulting from irradiating exclusively at said portion of the nucleus adjacent to the tail, and apt to provide an output indication of the X or Y nature of the irradiated sperm;
- a microfluidic path (100) that contains said volume of irradiation (4) and also includes channels (103, 104) for separating X sperm and Y sperm as identified by said analysis unit (7).
11. The apparatus (1 ) according to the previous claim, wherein said trapping means (5) are of double trap type, providing an irradiation in two points for retaining the sperm.
12. The apparatus (1 ) according to claim 10 or 1 1 , wherein said trapping means (5) comprises a laser source of a wavelength comprised in a range of about 700-1200 nm.
13. The apparatus (1 ) according to anyone of claims 10 to 12, wherein said irradiation means for Raman excitation (2) according to the linear Raman spectroscopy emits at a wavelength comprised in a range of about 500-900 nm.
14. The apparatus (1 ) according to anyone of claims 10 to 13, wherein said analysis unit (7) comprises means apt to discriminate the X or Y nature of the sperm based upon the ray scattering analysis at one or more of the following frequency ranges: 720-730 cm"1 , preferably about 726 cm"1; 780-790 cm"1, preferably about 785 cm"1; 1090-1 100 cm"1, preferably about 1095 cm"1 ; 1576- 1586 cm"1, preferably about 1581 cm"1; and 1395-1405 cm"1, preferably about 1400 cm"1.
15. The apparatus (1 ) according to anyone of claims 10 to 14, wherein said analysis unit (7) comprises means of PCA ("Principal Component Analysis") statistical analysis.
16. The apparatus (1 ) according to anyone of claims 10 to 15, further comprising means (10) for acquiring the image of the sperm in said volume of irradiation (4).
17. The apparatus (1 ) according to anyone of claims 10 to 16, further comprising means for addressing automatically the sperms in the respective microfluidic channel (103, 104).
18. A method according to anyone of claims 1 to 9, which uses an apparatus according to anyone of claims 10 to 17.
EP14815053.5A 2013-11-25 2014-11-25 Method and apparatus for discriminating x and y sperms based upon raman spectroscopy Withdrawn EP3074750A1 (en)

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IT000648A ITRM20130648A1 (en) 2013-11-25 2013-11-25 METHOD AND APPARATUS FOR DISCRIMINATING BETWEEN SPERMATOZOES X AND Y BASED ON RAMAN SPECTROSCOPY
PCT/IB2014/066319 WO2015075698A1 (en) 2013-11-25 2014-11-25 Method and apparatus for discriminating x and y sperms based upon raman spectroscopy

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CN110579435B (en) 2012-10-15 2023-09-26 纳诺赛莱克特生物医药股份有限公司 Systems, equipment and methods for particle sorting
US11371941B2 (en) * 2016-07-04 2022-06-28 Celltool Gmbh Device and method for the determination of transfection
CN117470842B (en) * 2023-10-23 2025-10-17 中国科学院遗传与发育生物学研究所 Sperm posture and movement state control method

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US5021244A (en) 1988-12-06 1991-06-04 Cytogam, Inc. Sex-associated membrane antibodies and their use for increasing the probability that offspring will be of a desired sex
JP2004101323A (en) * 2002-09-09 2004-04-02 Suruga Seiki Kk Method and apparatus for selecting x - y spermatozoon
DE102007013107B4 (en) * 2007-03-15 2011-02-10 Friedrich-Schiller-Universität Jena Method for determining the sex of birds
WO2009149733A1 (en) * 2008-06-13 2009-12-17 Embl Heidelberg Next generation flow cytometer sorter
DE102010023099B3 (en) * 2010-06-09 2011-11-17 Celltool Gmbh Method and device for characterizing biological objects
US8941062B2 (en) * 2010-11-16 2015-01-27 1087 Systems, Inc. System for identifying and sorting living cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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