**5. Combined approaches**

A recent improvement in DH technique was proposed by Coppola's group combining DH with Raman spectroscopy [26]. This combined approach allows to simultaneously study biochemical and morphological characteristics of human sperm cells irradiated with green laser radiation. The scheme of the combined phase imaging interferometer and Raman microscope system is illustrated in **Figure 9**.

**Figure 9.** Combined system allowing the simultaneous phase imaging and Raman spectroscopy measurements [26]. OBJ1: fiber-coupling objective; OBJ2: microscope objective; YF: single wavelength optical fiber; C1, C2: beam collimators; A: attenuator; L1–L4: lenses; λ/2: half-plate; BS: beam splitter; LPF: long-pass filter; M1, M2: mirrors; DM: dichroic mirror; CCD1: CCD camera for holographic imaging; CCD2: CCD camera of the monochromator; BS45: 45° dichroic beam splitter; NF0: 0° notch filter; S: monochromator.

The combined system consists of a holographic set up as described in Section 2 utilizing a red coherent laser source, combined with a Raman microscope where the probe source was a separate diode laser (green source). The same objective lens used for the holographic imaging was also used to focalize the Raman probe on the sample and to collect the backscattered light. The advantage offered by the holographic approach to numerically refocus the object under test is instrumental for the proposed combined approach. Indeed, in order to maximize the Raman signal, the Raman probe (green-laser) has to be focused on the specimen. However, due to chromatic aberration, the holographic image acquired by using the coherent red laser results out of its focal plane. By means of the refocusing algorithm, this problem can be overcome so that the Raman spectra and the holograms can be acquired simultaneously. **Figure 10(a)** represents the reconstructed amplitude map at the acquisition plane obtained by in a single-shot measurement, out of its focal plane, of the investigated spermatozoon. Then, the amplitude map in **Figure 10(b)** was numerically processed by using the refocusing algorithm [26], providing the focused amplitude map of the propagated object field reported in **Figure 10(b)**. A pseudo 3D representation of this phase map is reported in **Figure 10(c)**.

This anomalous behavior has been also confirmed by the retrieved motility parameters. In fact, the VSL measured for cell 1 (green) was about 10 μ/s, i.e., a value lower than those relating to other four cells (VSLmean = 20 μ/s) and describes effectively the inefficient cell movement. Moreover, the "normal" cells (cells 2, 3, 4 and 5) have a reduced oscillation around the average path; in fact, their wobble is pretty uniform around 0.97 and 0.99. On the other hand, this value is sensibly lower for the "abnormal" cell, providing a quantitative description of the

**Figure 8.** Multiple sperm cells tracking. Transversal (a) and reconstructed 3D path (b). Scale bar is 20 μm, data were

**Figure 7.** Transversal (a) and 3D path (b) of a sperm cell presenting a bent tail Ref. [42] (by permission of The Optical

A recent improvement in DH technique was proposed by Coppola's group combining DH with Raman spectroscopy [26]. This combined approach allows to simultaneously study biochemical and morphological characteristics of human sperm cells irradiated with green laser radiation. The scheme of the combined phase imaging interferometer and Raman microscope

wide fluctuation of the spermatozoon head.

acquired each 11 s. Ref. [42] (by permission of The Optical Society).

**5. Combined approaches**

Society).

344 Holographic Materials and Optical Systems

system is illustrated in **Figure 9**.

**Figure 10.** (a) Reconstructed amplitude map at the plane of acquisition; (b) Reconstructed amplitude map of the region of interest at the focus plane; (c) Pseudo 3D representation of the phase map of the region of interest at the focal plane. Ref. [26] (by permission of IEEE Society).

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 from 4.4 to 50 mW, corresponding to the fluences in the range of 13–165 MJ/cm<sup>2</sup> . The postacrosomal 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 s (green-light fluence of 10 MJ/cm<sup>2</sup> ); while, the red-light used for holographic measurements was set at 100 mW (red-light fluence of 100 mJ/cm<sup>2</sup> ). For the selected experimental conditions, 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 decreases already after 30 MJ/cm<sup>2</sup> when no morphological changes were detected. In the spectral range of 1200–1400 cm−1, the observed reduction in intensity of the peaks is at 1250 and 1375 cm−1, already for fluence of 30 MJ/cm<sup>2</sup> was due to possible alterations in the secondary 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 [47, 49]. At fluences higher than 150 MJ/cm<sup>2</sup> , the spermatozoon is completely disintegrated [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] (by permission of IEEE Society).

(**Figure 11(a)**). The arrows indicate the regions of the sperm cells where the most relevant morphological variations were observed.

In **Figure 11(b)** and **(c)** a progressive reduction in the height, and therefore in the volume, is well visible, suggesting a sort of "photoporation" near the exposed region. At fluences higher than 150 MJ/cm<sup>2</sup> , the sperm cell seemed to swell and an increase of the luminescence background of the Raman signal was observed confirming a local heating of the sample [47] due to the absorption at 532 nm [50–53].
