**2. Principle of the holographic imaging**

indicator for general health. On the other hand, in the zoo-technic field, animal semen analysis

The main requirements for the development of techniques used for an accurate semen analy-

• obtain results independent on the technician's experience and/or the laboratory environ-

The sperm cell is almost transparent in conventional bright field microscopy, as its optical proprieties differ slightly from the surrounding liquid, generating little contrast. On the other hand, a light beam that passes through a spermatozoon undergoes a phase change, in comparison with the surrounding medium, the amplitude of which depends on the light source, the thickness, and the integral refractive index of the object itself. A qualitative visualization of this phase contrast may be obtained by contrast interference microscopy (phase contrast or Nomarski/Zernicke interferential contrast microscopy). However, it is difficult and timeconsuming to obtain a quantitative morphological imaging. In fact, a fine z-movement of the biological sample is required in order to acquire a collection of different planes in focus. This collection of acquired images is used in postelaboration to produce a 3D image of the object under investigation [12]. The same approach has been used to obtain information about sperm motility. Nevertheless, this 2D intrinsic analysis implies a partial in-plane representation of the motility features due to difficulty to track the 3D spatial motion of spermatozoa that quickly move out of focus. In order to overcome these intrinsic limitations, several approaches have been recently developed. In this contest, the optical approaches are deeply investigated. In particular, over the last few years, holographic imaging in microscopy has been established as a valid noninvasive, quantitative, label-free, high-resolution, and phase-contrast imaging technique. So this chapter tries to summarize the state-of-art on the semen analysis and recent achievements obtained by a holographic imaging [13–17]. We will show that the unique potentialities of the holographic imaging have been used to provide structural information on both the morphology and the motility of sperm cells [18]. Moreover, the combination of the holographic technique with others approaches, such as the Raman spectroscopy, will be described, too. In fact, spermatozoa from infertile men could present a variety of alterations (such as alterations of chromatin organization [19], aneuploidy [20], and DNA fragmentation [21]) that can decrease reproductive capacity of men. Current methods of DNA assessment are mainly based on fluorescence microscopy, and thus samples are unusable after the analysis [22–25]. Therefore, the ability to simultaneously analyze, in a nondestructive and noninvasive way, both the morphology and biochemical functionalities of the spermatozoa could bring greater understandings [26]. Thus, the chapter will allow a bird's-eye view into the potentiality of the semen analysis performed by means of the holographic imaging, showing that this approach is extremely important for the intracytoplasmic sperm injection (ICSI) procedure, where it is highly required the development of a method that allows characterizing and directly select the

is commonly used in animal production laboratories and reproductive toxicology.

• use a label-free approach to eliminate all adverse effects of the probe labeling;

• avoid any alteration of the health of the spermatozoa under test;

mental conditions (such as temperature, humidity, and duration).

best spermatozoon to inject into the oocytes [9, 27].

sis are the following:

336 Holographic Materials and Optical Systems

An optical field consists of amplitude and phase distributions; if this field interacts with the object under test, the morphology of the object alters the phase distribution. The holographic approach employs the interference between two optical beams to transform the phase information (i.e., the morphological information) into a recordable intensity distribution [28]. A sketch of an experimental set up for holographic imaging is shown in **Figure 1**. It consists in a coherent laser beam splits into a reference and an object beam.

The object beam intensity is always set well below the level for causing any damage to the spermatozoa structure and functionality. A microscope objective lens is used to collect the object beam. The reference and the object beam are then recombined by a beam splitter onto a CCD (Charge-Coupled Device) or CMOS (Complementary Metal-Oxide Semiconductor) detector, which acquires the interference pattern. According to the angle *θ* between the reference and object beams, either on-axis (*θ* = 0°) or off-axis configuration can be adopted. Besides, the hologram of the sample under investigation, a second hologram is acquired on a reference surface in proximity to the object in order to numerically compensate all the aberrations introduced by the optical components, including the defocusing due to the microscope objective. The image reconstruction

**Figure 1.** Sketch of the principle of hologram formation (L: lens, MO: microscope objective, BS: beam splitter).

procedure allows retrieving a discrete version of the complex optical wavefront (amplitude and phase) present on the surface of the specimen under test. This optical wavefront is obtained multiplying the recorded hologram by a numerical replica of the reference beam and numerically backpropagating this product. Actually, this product generates three diffraction terms: zeroth order, real image, and conjugate image. In an on-axis configuration, unless a partially coherent light is adopted that allows reducing the speckle and multi-reflection interference noise [29], all terms are superimposed. Thus, in order to recover only the real image, i.e., an exact replica of the object wavefront, either a spatial or temporal phase-shifting methods has to be employed. However, in this way the complexity and capture time increase [13]. On the other hand, by introducing a small angle between the object and reference beam (off-axis configuration), a spatial separation between the three terms is obtained, at the expense of suboptimal use of camera sensor space-bandwidth product. Thus, this separation allows selecting and retrieving only the real image. The possibility offered by DH to numerically retrieve the phase distribution of the object wavefront allows not only the possibility to evaluate the object morphology but also to remove and/or compensate the unwanted wavefront variation (such as optical aberrations and slide deformations) [30–32].
