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Label-free imaging and biochemical characterization of bovine sperm cells.

Ferrara MA, Di Caprio G, Managò S, De Angelis A, Sirleto L, Coppola G, De Luca AC - Biosensors (Basel) (2015)

Bottom Line: A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination.In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS).We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microelectronics and Microsystems, National Research Council, Via P. Castellino, 111, 80131 Naples, Italy. antonella.ferrara@na.imm.cnr.it.

ABSTRACT
A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

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(A) Raman image (9 × 12 µm2) of a spermatozoon acquired on Xplora inverted Raman microscope of HORIBA Jobin Yvon; (B) Raman spectra (integration time: 10 s) acquired from tail (blue line), nucleus (green line) and acrosomal vesicle (magenta line). The colors of the spectra correspond to the colors in the image.
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biosensors-05-00141-f005: (A) Raman image (9 × 12 µm2) of a spermatozoon acquired on Xplora inverted Raman microscope of HORIBA Jobin Yvon; (B) Raman spectra (integration time: 10 s) acquired from tail (blue line), nucleus (green line) and acrosomal vesicle (magenta line). The colors of the spectra correspond to the colors in the image.

Mentions: To obtain complementary information on the biochemical structure of spermatozoa, Raman imaging technology is used. Figure 5A shows a 9 × 12 μm2 Raman image of a spermatozoon acquired by raster scanning the sample under the microscope (mapping step 0.2 μm). Spectra were recorded at each image pixel with an integration time of 10 s at a laser power of 15 mW measured at the microscope objective. Figure 5B shows the Raman spectra acquired from distinct region of the spermatozoon. The nucleus Raman spectrum (green line) is characterized by strong Raman bands assigned to the nucleic acids and DNA (726, 785, 1095 and 1581 cm−1). Pronounced bands corresponding to proteins and lipids (1200–1300, 1480 and 1600–1680 cm−1) identify the acrosomal vesicle spectrum (magenta line). Finally, the tail Raman spectrum (blue line) is characterized by a sharp peak around 751 cm−1 previously assigned to mitochondria and strong peaks at 1005 and 1450 cm−1 consistent with the presence of proteins in this spectral region. The band assignment of the Raman peaks is summarized in Table 1.


Label-free imaging and biochemical characterization of bovine sperm cells.

Ferrara MA, Di Caprio G, Managò S, De Angelis A, Sirleto L, Coppola G, De Luca AC - Biosensors (Basel) (2015)

(A) Raman image (9 × 12 µm2) of a spermatozoon acquired on Xplora inverted Raman microscope of HORIBA Jobin Yvon; (B) Raman spectra (integration time: 10 s) acquired from tail (blue line), nucleus (green line) and acrosomal vesicle (magenta line). The colors of the spectra correspond to the colors in the image.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493542&req=5

biosensors-05-00141-f005: (A) Raman image (9 × 12 µm2) of a spermatozoon acquired on Xplora inverted Raman microscope of HORIBA Jobin Yvon; (B) Raman spectra (integration time: 10 s) acquired from tail (blue line), nucleus (green line) and acrosomal vesicle (magenta line). The colors of the spectra correspond to the colors in the image.
Mentions: To obtain complementary information on the biochemical structure of spermatozoa, Raman imaging technology is used. Figure 5A shows a 9 × 12 μm2 Raman image of a spermatozoon acquired by raster scanning the sample under the microscope (mapping step 0.2 μm). Spectra were recorded at each image pixel with an integration time of 10 s at a laser power of 15 mW measured at the microscope objective. Figure 5B shows the Raman spectra acquired from distinct region of the spermatozoon. The nucleus Raman spectrum (green line) is characterized by strong Raman bands assigned to the nucleic acids and DNA (726, 785, 1095 and 1581 cm−1). Pronounced bands corresponding to proteins and lipids (1200–1300, 1480 and 1600–1680 cm−1) identify the acrosomal vesicle spectrum (magenta line). Finally, the tail Raman spectrum (blue line) is characterized by a sharp peak around 751 cm−1 previously assigned to mitochondria and strong peaks at 1005 and 1450 cm−1 consistent with the presence of proteins in this spectral region. The band assignment of the Raman peaks is summarized in Table 1.

Bottom Line: A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination.In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS).We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microelectronics and Microsystems, National Research Council, Via P. Castellino, 111, 80131 Naples, Italy. antonella.ferrara@na.imm.cnr.it.

ABSTRACT
A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

Show MeSH
Related in: MedlinePlus