**6. Conclusion**

By using this combined approach, the effect of the green-laser light (Raman probe) by irradiating a specific region of the sperm cell was investigated for an increasing laser power

crosomal sperm cell region was irradiated for 3 s, and after each exposure, a single hologram and a Raman spectrum were simultaneously acquired. Raman measurements were performed using a green-laser power of 0.5 mW on the sample and an integration time of 20

any possible degradation effect associated with the red-laser light can be neglected [46]. Indeed, no adverse effects on the cells were observed even after hours of irradiation with red-laser light [47]. The biochemical characterization highlighted that the Raman bands related to localized vibration of the DNA bases (700–800 cm−1) remains almost invariant when irradiated by the green-laser light, while the Raman bands associated with O-P-O (Oxygen-Phosphorus-Oxygen) backbone (900–1100 cm−1) are subjected to photoinduced oxidation [47] and the peak at 1095 cm−1 decreases in intensity proportionally to the break of the double-helical structure with the fluences increase [48]. More precisely, it was observed that intensity

spectral range of 1200–1400 cm−1, the observed reduction in intensity of the peaks is at 1250

ary and tertiary conformation of proteins. Finally, all native nucleic acids exhibiting a broad and intense band near 1668 cm−1, which originates from coupled C=O stretching and N–H deformation modes, are highly sensitive to disruption of Watson-Crick hydrogen bonding

[26]. Regarding the simultaneous morphological analysis, **Figure 11** shows the phase profile variations associated with the irradiating fluences, along the line SS′ (**Figure 11(b)**) and PP′ (**Figure 11(c)**), representing two different directions along the spermatozoa structure

**Figure 11.** (a) Reconstructed phase map of the region of interest at the focus plane; the lines along which the profile is monitored during the exposition are highlighted. The Raman spectrum is acquired in the postacrosomal region. (b) Phase profile of the irradiated sperm cell at three different selected laser fluences along the lines SS′ and (c) PP′. Ref. [26]

); while, the red-light used for holographic measurements

when no morphological changes were detected. In the

was due to possible alterations in the second-

, the spermatozoon is completely disintegrated

). For the selected experimental conditions,

. The posta-

from 4.4 to 50 mW, corresponding to the fluences in the range of 13–165 MJ/cm<sup>2</sup>

s (green-light fluence of 10 MJ/cm<sup>2</sup>

346 Holographic Materials and Optical Systems

decreases already after 30 MJ/cm<sup>2</sup>

(by permission of IEEE Society).

and 1375 cm−1, already for fluence of 30 MJ/cm<sup>2</sup>

[47, 49]. At fluences higher than 150 MJ/cm<sup>2</sup>

was set at 100 mW (red-light fluence of 100 mJ/cm<sup>2</sup>

In this chapter, an overview of the recent achievements in holography imaging applied to both morphological and motility characterizations of sperm cells has been reported. Results obtained by means of digital holography have demonstrated the possibility to provide 3D information on both the morphology and motility of sperm cells; this information could be used to better emphasize the differences between normal and abnormal sperm morphology. Moreover, the DH approach is a noninvasive technique, allowing the analysis of live spermatozoa, such as 3D tracking of the spatial motion, in order to select normal sperm cells. In particular, the possibility offered by digital holography to add the third dimension in the sperm analysis will give information useful both to relate the sperm anomalies with male infertility and to enable differentiation of the spermatozoa in good health. Finally, DH can be easily combined with other techniques allowing different simultaneous characterization. Indeed, it was demonstrated that a promising optical approach, based on digital holography and Raman spectroscopy technologies can be used for the quality assessment of spermatozoa. Applying this combined approach for analyzing the sperm cells, high-resolution images, and Raman spectra have been obtained, clearly highlighting some morphological and biochemical alterations. In particular, DH and Raman spectroscopy simultaneous approach was used for studying the photodamage induced by visible green light in the spermatozoa structure.
