**4. Tracking analysis**

It is important to note that the volume estimation could not be performed by the DESA technique. Due the great influence of nuclear vacuoles in the sperm head on the fertilization capacitance of sperm cell [9], the volumetric analysis was performed to analyze vacuolated human spermatozoa. In **Figure 4(a)**, a conventional differential interference contrast (DIC) image of a vacuolated human sperm is reported. The holographic 3D reconstruction of the vacuolated spermatozoon is illustrated in **Figure 4(b)**. From this figure, it is clear that spermatozoa with vacuole had a reduced volume, and this reduction could be probably be due to

**Table 2.** Mean morphometric values of normal human sperm heads obtained by DESA and holographic techniques [36].

**Table 1.** Descriptive statistics for statistically significant (*ρ* < 0.001) phase shifts according to the NZ/OAT group [35].

**] Volume [μm3**

**]**

**Table 3** summarizes the volumetric analysis carried out for three different groups of spermatozoa defined by the length and width of the head. Mean values of the total volume of the

Memmolo et al. [37] proposed to use DH in order to identify and measure specific region-ofinterest of spermatozoa. **Figure 5** shows some steps of the applied method that starts from a filtered version of the reconstructed spermatozoon image and is able to identify the head

**Figure 4.** Differential interference contrast image (a) and pseudo 3D holographic reconstruction (b) of a vacuolated

variation of the inner structure of the sperm head with a loss of material.

**Sperm group Median Mean SD CI** NZ 2.90 2.91 0.61 2.94 OAT 2.00 2.10 0.38 2.13

340 Holographic Materials and Optical Systems

**Imaging Length [μm] Width [μm] Perimeter [μm] Area [μm2**

DESA 5.1 ± 0.6 3.5 ± 0.4 13.8 ± 1.4 14.1 ± 2.0 -

Holography 5.6 ± 0.3 2.9 ± 0.5 14.3 ± 1.2 13.0 ± 1.2 8.0 ± 0.8

spermatozoa minus the vacuoles volume are also reported.

sperm head. Ref [36] (by permission of Cambridge University Press).

region.

One of the main advantages of the holographic approach is the possibility to retrieve a quantitative 3D image by means of a numerical refocusing of only one bi-dimensional image at different object planes. Thus, the realigning of the optical imaging system with mechanical translation can be eliminated. This peculiarity enables the characterization of live specimen [38], and in particular, to track the 3D spatial motion of spermatozoa that quickly move out of focus. The tracking approach allows estimating many quantitative parameters useful for a semen analysis [39], such as:


However, traditional techniques provide all these data as in-plane parameters. Conversely, the holographic approach allows adding 3D information about the trajectory followed by the sperm cells in a volume. This additional information can provide a better understanding of the sperm behavior and its relation with male infertility [40]. The 3D trajectories of human sperms across a large volume have been dynamically tracked by the Ozcan's group using a lens-free holographic approach [41]. The employed method generates the hologram by the interference of two components of the same light beam (in-line configuration). In particular, the holographic setup is composed of two partially coherent lightemitting-diodes (LEDs) at two different wavelengths that illuminate the sperms vertically (red wavelength) and obliquely at 45° (blue wavelength) [41]. The combination of these two different images allows determining the 3D location of each sperm. In **Figure 6**, the 3D dynamic swimming patterns of human sperm evaluated by means of this approach are illustrated. In particular, the volume investigated was relative to a depth-of-field of about 0.5–1 mm and a field-of-view of >17 mm<sup>2</sup> . The results show that most part of sperm (90%) moves forward swiftly along a slightly curved axis. The remaining part of spermatozoa exhibit a helical trajectory with a noticeable movement along the *z*-axis (about 4–5%) or a hyperactivated 3D swimming with large lateral movements (<3%) or a hyperhelical pattern (about 0.5%).

By means of the observation of these trajectories on a large number of sperm cells (>1500), a statistic analysis on various parameters has been estimated; in particular, in **Table 4** some of these parameters are summarized.

Furthermore, thanks to the high accuracy of the technique, the authors observed that among the helical human sperms, a significant majority (approximately 90%) preferred right-handed helices over left-handed ones, with a helix radius of approximately 0.5–3 μm.

A different approach has been proposed by Di Caprio et al. [42]. In particular, the authors used an off-axis set-up and the capabilities of holographic technique to retrieve in-focus images independently of the focal plane of the acquire image. Thus, in order to estimate the swimming trajectories of human sperm, a set of holograms was recorded, keeping constant the distance between the sample and the microscope objective. Each retrieved image is used to evaluate the *X* and *Y* coordinates of sperm cells, whereas to obtain the *Z* position, Unlabeled Semen Analysis by Means of the Holographic Imaging http://dx.doi.org/10.5772/67552 343

• curvilinear velocity (VCL), i.e., the total distance that the sperm head covers in the observation

• straight-line velocity (VSL), i.e., determined from the straight-line distance between the first and last points of the trajectory and gives the net space gain in the observation period;

• average path velocity (VAP), i.e., the distance the spermatozoon has traveled in the average

However, traditional techniques provide all these data as in-plane parameters. Conversely, the holographic approach allows adding 3D information about the trajectory followed by the sperm cells in a volume. This additional information can provide a better understanding of the sperm behavior and its relation with male infertility [40]. The 3D trajectories of human sperms across a large volume have been dynamically tracked by the Ozcan's group using a lens-free holographic approach [41]. The employed method generates the hologram by the interference of two components of the same light beam (in-line configuration). In particular, the holographic setup is composed of two partially coherent lightemitting-diodes (LEDs) at two different wavelengths that illuminate the sperms vertically (red wavelength) and obliquely at 45° (blue wavelength) [41]. The combination of these two different images allows determining the 3D location of each sperm. In **Figure 6**, the 3D dynamic swimming patterns of human sperm evaluated by means of this approach are illustrated. In particular, the volume investigated was relative to a depth-of-field of about

moves forward swiftly along a slightly curved axis. The remaining part of spermatozoa exhibit a helical trajectory with a noticeable movement along the *z*-axis (about 4–5%) or a hyperactivated 3D swimming with large lateral movements (<3%) or a hyperhelical pattern

By means of the observation of these trajectories on a large number of sperm cells (>1500), a statistic analysis on various parameters has been estimated; in particular, in **Table 4** some of

Furthermore, thanks to the high accuracy of the technique, the authors observed that among the helical human sperms, a significant majority (approximately 90%) preferred right-handed

A different approach has been proposed by Di Caprio et al. [42]. In particular, the authors used an off-axis set-up and the capabilities of holographic technique to retrieve in-focus images independently of the focal plane of the acquire image. Thus, in order to estimate the swimming trajectories of human sperm, a set of holograms was recorded, keeping constant the distance between the sample and the microscope objective. Each retrieved image is used to evaluate the *X* and *Y* coordinates of sperm cells, whereas to obtain the *Z* position,

helices over left-handed ones, with a helix radius of approximately 0.5–3 μm.

. The results show that most part of sperm (90%)

direction of movement in the observation period;

• amplitude of lateral head displacement (ALH).

0.5–1 mm and a field-of-view of >17 mm<sup>2</sup>

these parameters are summarized.

(about 0.5%).

• linearity (LIN): a comparison of the straight-line and curvilinear paths;

• wobble (WOB): a comparison of the average and curvilinear paths; and

period;

342 Holographic Materials and Optical Systems

**Figure 6.** Swimming trajectories of human sperms evaluated by Ozcan's group Ref. [41] (by permission of the National Academy of Sciences). (A) The typical pattern. (B) The helical pattern. (C) The hyperactivated pattern. (D) The hyperhelical pattern. The inset in each panel represents the front view of the straightened trajectory of the sperm.


**Table 4.** Mean values of some parameters related to the motility of human sperm: curvilinear velocity (VCL), straight-line velocity (VSL), linearity, amplitude of lateral head displacement (ALH) [41].

a numerical self-focusing function was applied on the reconstructed images [43–45]. It is worth noting that the possibility to keep constant the distance sample-objective, allows an *in vitro* volumetric field reconstruction, i.e., not possible with the traditional optical techniques. Authors applied this approach to estimate the swimming pattern of a spermatozoon with a bent tail. As shown in **Figure 7**, this morphological anomaly causes a nonlinear out-of-plane motion.

Moreover, the authors reported the simultaneous tracking of five human spermatozoa, moving on a different focal plane (**Figure 8**). From the trajectories displayed in **Figure 8**, an anomalous one can be recognized (indicated by an arrow). In fact, while every other cell moves in parallel, the anomalous spermatozoon swims along a broken track and on a tilted direction.

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

**Figure 8.** Multiple sperm cells tracking. Transversal (a) and reconstructed 3D path (b). Scale bar is 20 μm, data were acquired each 11 s. Ref. [42] (by permission of The Optical Society).

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 wide fluctuation of the spermatozoon head.
