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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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2D intensity map of each Raman spectrum corresponding to different regions of the spermatozoon: tail, nucleus and acrosomal vesicle.
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biosensors-05-00141-f006: 2D intensity map of each Raman spectrum corresponding to different regions of the spermatozoon: tail, nucleus and acrosomal vesicle.

Mentions: By assigning a specific color to each of the described spectra, the false color image of the spermatozoon can be reconstructed. As for the DH, the Raman image reveals the “protuberance” on the post-acrosomal region of the head. This feature was visible only for a few (around 2%–3%) out of the dozens of analyzed cells. The protuberance can be better identified from the 2D intensity map reported in Figure 6, clearly showing a local concentration of material in the sperm region connecting the tail to the head. A detailed inspection into Figure 5B (blue line) demonstrated that the sharp vibrations originating from the presence of proteins in the spectral region at 1005 and 1450 cm−1, clearly less intense in the other spectra, together with the presence of mitochondria, correspond to the most important Raman markers of such “protuberance”. Our results suggest an intriguing correlation between the amount of protein and the presence of the “protuberance” that could be associated with the presence of centrioles in the sperm region connecting the tail to the head. Indeed, the centrioles are cylindrical cell structures composed essentially by tubulin, therefore affecting the local concentration of protein. However, in order to give a correct biological explanation to the origin of this formation, additional analyses and comparisons with traditional fluorescence confocal microscopy results are required.


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)

2D intensity map of each Raman spectrum corresponding to different regions of the spermatozoon: tail, nucleus and acrosomal vesicle.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00141-f006: 2D intensity map of each Raman spectrum corresponding to different regions of the spermatozoon: tail, nucleus and acrosomal vesicle.
Mentions: By assigning a specific color to each of the described spectra, the false color image of the spermatozoon can be reconstructed. As for the DH, the Raman image reveals the “protuberance” on the post-acrosomal region of the head. This feature was visible only for a few (around 2%–3%) out of the dozens of analyzed cells. The protuberance can be better identified from the 2D intensity map reported in Figure 6, clearly showing a local concentration of material in the sperm region connecting the tail to the head. A detailed inspection into Figure 5B (blue line) demonstrated that the sharp vibrations originating from the presence of proteins in the spectral region at 1005 and 1450 cm−1, clearly less intense in the other spectra, together with the presence of mitochondria, correspond to the most important Raman markers of such “protuberance”. Our results suggest an intriguing correlation between the amount of protein and the presence of the “protuberance” that could be associated with the presence of centrioles in the sperm region connecting the tail to the head. Indeed, the centrioles are cylindrical cell structures composed essentially by tubulin, therefore affecting the local concentration of protein. However, in order to give a correct biological explanation to the origin of this formation, additional analyses and comparisons with traditional fluorescence confocal microscopy results are required.

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