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The Raman signals are generally low and often obscured by the presence of the cell autofluorescence. However, the fluorescence-free detection can be achieved using instruments working in the near-IR region of the spectrum, also reducing the cell photodamage. Alternatively, modulating or multiwavelengths approaches can be employed to eliminate the fluores-

Another crucial aspect for translational applications is the time required to collect the spectral data and reconstruct the Raman images. Indeed, for reproductive medicine applications, performing experiment in real-time is crucial. Recently, several approaches have been proposed providing a faster imaging modality and allowing investigation of several sperm cells simultaneously such as the Coherent anti-Stokes Raman scattering (CARS) and surface-enhanced Raman scattering (SERS) imaging [60, 61] or the use of structured illumination [62, 63]. Moreover, in our specific case, the sample is moving while measuring. This represents another problem that researchers are trying to overcome using the laser trapping capability [64, 65], slide functiona-

A further obstacle to the clinical success of these new methods is the complexity in interpreting the results. Indeed, the newer instruments for Raman/holographic imaging are fast, efficient, and reliable; however, they require specialized operators. An useful system should be easier to use, providing clear and automated answers to biomedical problems instead of spectra or holograms. The ongoing implementation of computer-assisted diagnosis algorithms is helping the interpretation of the holographic images, while further work on the creation of larger

The economical aspect has also to be considered. Raman spectroscopy and digital holography use precise equipment. If we consider that a holographic imaging system is sensitive to optical pathway differences on the nanoscale and has to be isolated to any kinds of vibrations for avoiding artifacts, we can well image that the precision required in the construction of such devices is more expansive than that of conventional microscopes currently in clinical use.

The path to clinical implementation of innovative multimodal imaging techniques for sperm cell assessment passes through the following milestones: 1. identification of the medical problem and the need for a new technique; 2. experimental tests of the technique, showing the proof of principle and the feasibility of the specific application; 3. closely collaboration between researchers and clinicians for evaluating the clinical relevance of the information the new technique provides; 4. optimisation of the technique for the specific application in order to improve its sensibility, specificity and robustness; 5. clinical trials; 6. industrial implementation of the system for making it clinician and patient friendly; and 7. Clinical implementation.

The proof of principle of Raman spectroscopy and holographic imaging as sperm selection techniques has been successfully demonstrated. Their use for detecting epigenetic alterations, including DNA packaging and spatial conformation, methylation, and histone modification, that could seriously affect the embryonic development has been showed [21]. Researches in this field are currently focusing on the points 3 and 4, as highlighted in this chapter. Our efforts aim to assess the feasibility and the reliability of the two techniques before initiating the clinical trials, filling in such a way the gap between experimentation and clinical implementation.

cence background [57–59].

234 Spermatozoa - Facts and Perspectives

Raman database is still required.

lization procedures [25, 66], or microfluidic devices [67, 68].

Annalisa De Angelis<sup>1</sup> \*, Maria Antonietta Ferrara2 , Giuseppe Coppola<sup>2</sup> and Anna Chiara De Luca<sup>1</sup>

