2.1. Basic principles of Raman spectroscopy

infertility. For soft/moderate infertility, usually homologous intrauterine insemination (HIUI) or in vitro fertilization (IVF) is carried out. HIUI consists in transferring a small volume of selected and isolated motile and morphologically normal spermatozoa directly into the uterus a few hours before the ovulation. IVF is used after continued failures of HIUI due, for example, to an impervious uterine tube, and the fertilization of the oocytes takes place outside the

Parameter Reference value 95% confidence index

Sperm volume 1.5 mL 1.4–1.7 Sperm concentration 15 million sperm/mL 12–16 Total sperm number 39 million sperm per ejaculate 33–46 Morphology 4% normal forms 3–4 Vitality 58% live 55–63 Progressive motility 32% 31–34 Total motility (progressive + nonprogressive) 40% 38–42

In case of severe male infertility, the intracytoplasmic sperm injection (ICSI) is preferred. In this technique, a single spermatozoon is directly injected into an oocyte, bypassing the physiological selection naturally performed by the female tract. Although ICSI has drastically reduced the number of viable sperm required for fertilization, giving hope of conception to extremely severe cases of oligospermia, the rate of successful pregnancy still remains low (<30%), due to the lack of accurate and reliable methods for selecting the spermatozoon that able to fertilize the oocyte. The first selection is based on motility and morphology. The sperm cells preparation procedure includes a density gradient centrifugation (morphology-based selection) followed by swim-up method (motility-based selection) in order to mimic the physiological selection of the spermatozoa made by the female genital tract. In most laboratories, the sperm quality assessment is relied on the expertise and the subjective skills of the operator. To increase precision and reproducibility, numerous automated computer-aided sperm analysis (CASA) systems have been introduced, and they can automatically view multiple fields in a shallow specimen chamber to capture images of 500 to >2000 sperm in <2 minutes [2]. By using CASA, it is also possible to retrieve some morphometrical parameters, such as ellipticity and regularity,

whereas the measurement of the acrosome area generally requires staining.

principle, incapable to fertilize the oocyte [4, 5].

Moreover, CASA has difficulty in distinguishing spermatozoa from particulate debris, leading to some extent of inaccuracy. In addition, several studies have shown that the correlation between male fertility and the percentage of morphologically normal or motile sperm cells in semen sample is relatively low [3, 4]. Indeed, in infertile men, a high percentage of spermatozoa with a normal morphology or motility could have a damaged DNA, and therefore, they are, in

woman body.

Table 1. WHO's parameters for semen analysis (2010).

220 Spermatozoa - Facts and Perspectives

Raman spectroscopy is an optical technique based on the scattering of the light interacting with a sample. Light scattering originates from the elastic or inelastic collision between an incident photon and a molecule of the sample. When the collision occurs, the molecule undergoes an excitation to a virtual state followed by a nearly simultaneous de-excitation towards the initial energy level (elastic) or a vibrational level different from the initial one

Figure 1. Jablonski diagram representing quantum energy transitions for Rayleigh, Raman scattering (Stokes and anti-Stokes), and fluorescence emission.

(inelastic) (Figure 1). The virtual state is an energy level created only when photons interact with electrons and its energy is determined by the frequency itself of the incident photons. When molecules return back to their initial energy state, photons are emitted (scattered). In the elastic process, named Rayleigh scattering, the emitted photons have the same energy of the incident ones. Whereas in the inelastic scattering, there are two possibilities (Figure 1): 1. molecules decay from the virtual state to an excited vibrational level emitting photons with a lower energy than the incident ones (Stokes scattering) and 2. when molecules in a vibrational state were excited in a virtual state, they can decay in the ground state, producing photons with energy higher than the incident ones (anti-Stokes scattering). Since the number of molecules in an excited state decreases very fast, anti-Stokes scattering is much less probable than the Stokes scattering (typically by a factor around 1000). For this reason, Raman analysis is usually limited to the observation of the Stokes scattering.

The energy difference between the initial and final vibrational levels (Raman shift) expressed in wavenumbers (cm�<sup>1</sup> ) is given through the relation:

$$
\hbar \nu = \hbar \left( \frac{1}{\lambda\_{inc}} - \frac{1}{\lambda\_{scat}} \right) \tag{1}
$$

more intense than the Raman scattering. Using the notch filter, disperse the spectral components of the light by means of the diffraction grating and image the light onto the detector

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In a confocal configuration (micro-RS), a spatial resolution of ~300 nm in the transverse x-y plane and of ~1.2 μm in the axial direction can be achieved. Moreover, for extended samples, it is possible to acquire the Raman spectra on a two- or three-dimensional array of points, scanning the sample with a step comparable with the spatial resolution (Raman imaging). In this way, 2D images (or 3D profiles) are obtained, reporting the spatial variation of a given Raman parameter. This parameter is usually the intensity of a particular Raman band or sometimes it derives from a more complicated analysis of the whole Raman spectrum [18, 19].

Interestingly, due to the cited characteristics, RS has been successfully employed for the study of several living/fixed cells with subcellular resolution [18]. The first Raman-based single cell analyses were conducted just on individual living salmon sperm cells due to their relative simple structure [20]. Since this pioneer study, no other Raman experiments on sperm cells have been reported until the 2009 when Huser and colleagues [21] examined the spectra obtained from human spermatozoa with different nuclear shapes (box 1 in Figure 2) in order to determine if there was a correlation between DNA-protein complex in sperm chromatin and the morphology of the nucleus. RS results showed that while the DNA packaging of normal and abnormal shaped nuclei was different, the nature and the efficiency of DNA packaging in normal head spermatozoa appeared to vary greatly [21]. Therefore, by selecting sperm cells solely based on morphology, a fraction of the normal considered sperm cells used for in vitro fertilization will contain improperly packaged DNA, thus resulting infertile. In this study, fixed membrane-free cells were used in

order to minimize the potentially interfering contributions from membrane proteins.

The results of this study are also relevant from an epigenetic point of view. Indeed, they show the possibility to use RS for detecting epigenetic alterations (DNA packaging and spatial conformation, methylation and histone modification) in those spermatozoa that, if used in assisted reproductive techniques, may increase the incidence of imprinting disorders and have

In other studies, RS was applied on whole and fixed sperm cells in order to demonstrate the efficiency of the technique in identifying DNA-damaged sperm cells (box 2 and panels A and Z in the box 3 of Figure 2). Usually, the DNA fragmentation as a consequence of oxidative stress is induced by UV radiation [23, 24] or Fenton's treatment [25]. All the studies found that the PO2 backbone of DNA was significantly affected by the exposure to radiation or treatment, and thus, its corresponding Raman band could represent a significant biomarker of DNA fragmentation [23–25]. Mallidis et al. previously identified characteristic spectral changes indicative of nuclear DNA damage of single fixed human spermatozoa and using Raman

In the following study [25], the same group determines the possibility to use Raman microspectroscopy for identifying different levels of sperm nuclear DNA damage induced by oxidative

(high sensitive charge-coupled device (CCD) camera).

2.2. Raman spectroscopy analysis of sperm cells quality

a deleterious impact on embryonic development [21, 22].

mapping, they localized the most damaged sites [24].

in which λinc and λscat are the wavelengths of the incident and Raman scattered photons, respectively. Therefore, the energy (or frequency) shifts in the scattered radiation provide a direct measure of the vibrational frequencies of the molecule (ν). Since different molecules are characterized by defined vibrational modes, by collecting the photons scattered at different frequencies allows reconstructing a sort of "chemical fingerprint" of the sample.

Therefore, RS turns out to be an extremely powerful tool for interdisciplinary researches, involving physicists, chemists, and biologists, as it allows the characterization of molecules based on properties of chemical bonds. It is a noninvasive and nondestructive technique providing molecular-level information, allowing the investigation of functional groups, bonding types, and molecular conformations.

From an experimental point of view, to observe a Raman spectrum, it is required to suppress the light scattered at the same frequency of the incident radiation (Rayleigh scattering), often more intense than the Raman scattering. Using the notch filter, disperse the spectral components of the light by means of the diffraction grating and image the light onto the detector (high sensitive charge-coupled device (CCD) camera).

In a confocal configuration (micro-RS), a spatial resolution of ~300 nm in the transverse x-y plane and of ~1.2 μm in the axial direction can be achieved. Moreover, for extended samples, it is possible to acquire the Raman spectra on a two- or three-dimensional array of points, scanning the sample with a step comparable with the spatial resolution (Raman imaging). In this way, 2D images (or 3D profiles) are obtained, reporting the spatial variation of a given Raman parameter. This parameter is usually the intensity of a particular Raman band or sometimes it derives from a more complicated analysis of the whole Raman spectrum [18, 19].

#### 2.2. Raman spectroscopy analysis of sperm cells quality

(inelastic) (Figure 1). The virtual state is an energy level created only when photons interact with electrons and its energy is determined by the frequency itself of the incident photons. When molecules return back to their initial energy state, photons are emitted (scattered). In the elastic process, named Rayleigh scattering, the emitted photons have the same energy of the incident ones. Whereas in the inelastic scattering, there are two possibilities (Figure 1): 1. molecules decay from the virtual state to an excited vibrational level emitting photons with a lower energy than the incident ones (Stokes scattering) and 2. when molecules in a vibrational state were excited in a virtual state, they can decay in the ground state, producing photons with energy higher than the incident ones (anti-Stokes scattering). Since the number of molecules in an excited state decreases very fast, anti-Stokes scattering is much less probable than the Stokes scattering (typically by a factor around 1000). For this reason, Raman analysis is

Figure 1. Jablonski diagram representing quantum energy transitions for Rayleigh, Raman scattering (Stokes and anti-

The energy difference between the initial and final vibrational levels (Raman shift) expressed

λinc

in which λinc and λscat are the wavelengths of the incident and Raman scattered photons, respectively. Therefore, the energy (or frequency) shifts in the scattered radiation provide a direct measure of the vibrational frequencies of the molecule (ν). Since different molecules are characterized by defined vibrational modes, by collecting the photons scattered at different

Therefore, RS turns out to be an extremely powerful tool for interdisciplinary researches, involving physicists, chemists, and biologists, as it allows the characterization of molecules based on properties of chemical bonds. It is a noninvasive and nondestructive technique providing molecular-level information, allowing the investigation of functional groups, bond-

From an experimental point of view, to observe a Raman spectrum, it is required to suppress the light scattered at the same frequency of the incident radiation (Rayleigh scattering), often

� <sup>1</sup> λscat

(1)

) is given through the relation:

<sup>ℏ</sup><sup>ν</sup> <sup>¼</sup> <sup>ℏ</sup> <sup>1</sup>

frequencies allows reconstructing a sort of "chemical fingerprint" of the sample.

usually limited to the observation of the Stokes scattering.

in wavenumbers (cm�<sup>1</sup>

Stokes), and fluorescence emission.

222 Spermatozoa - Facts and Perspectives

ing types, and molecular conformations.

Interestingly, due to the cited characteristics, RS has been successfully employed for the study of several living/fixed cells with subcellular resolution [18]. The first Raman-based single cell analyses were conducted just on individual living salmon sperm cells due to their relative simple structure [20]. Since this pioneer study, no other Raman experiments on sperm cells have been reported until the 2009 when Huser and colleagues [21] examined the spectra obtained from human spermatozoa with different nuclear shapes (box 1 in Figure 2) in order to determine if there was a correlation between DNA-protein complex in sperm chromatin and the morphology of the nucleus. RS results showed that while the DNA packaging of normal and abnormal shaped nuclei was different, the nature and the efficiency of DNA packaging in normal head spermatozoa appeared to vary greatly [21]. Therefore, by selecting sperm cells solely based on morphology, a fraction of the normal considered sperm cells used for in vitro fertilization will contain improperly packaged DNA, thus resulting infertile. In this study, fixed membrane-free cells were used in order to minimize the potentially interfering contributions from membrane proteins.

The results of this study are also relevant from an epigenetic point of view. Indeed, they show the possibility to use RS for detecting epigenetic alterations (DNA packaging and spatial conformation, methylation and histone modification) in those spermatozoa that, if used in assisted reproductive techniques, may increase the incidence of imprinting disorders and have a deleterious impact on embryonic development [21, 22].

In other studies, RS was applied on whole and fixed sperm cells in order to demonstrate the efficiency of the technique in identifying DNA-damaged sperm cells (box 2 and panels A and Z in the box 3 of Figure 2). Usually, the DNA fragmentation as a consequence of oxidative stress is induced by UV radiation [23, 24] or Fenton's treatment [25]. All the studies found that the PO2 backbone of DNA was significantly affected by the exposure to radiation or treatment, and thus, its corresponding Raman band could represent a significant biomarker of DNA fragmentation [23–25]. Mallidis et al. previously identified characteristic spectral changes indicative of nuclear DNA damage of single fixed human spermatozoa and using Raman mapping, they localized the most damaged sites [24].

In the following study [25], the same group determines the possibility to use Raman microspectroscopy for identifying different levels of sperm nuclear DNA damage induced by oxidative

cytometric analysis of DNA fragmentation, which represents the most statistically robust, reproducible, and standardized procedure available for the determination of sperm nDNA

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In 2015, Edengeiser et al. [26] analyzed spermatozoa under near-physiological conditions using confocal Raman microspectroscopy. The spermatozoa are immobilized on pre-treated object slides. More in detail, CaF2 slides were coated with concanavalin A and overlaid with preheated Ringer's solution just before putting a drop of isolated spermatozoa which, after the analysis, can be easily removed from the substrate. The study demonstrated for the first time the possibility to image and analyze several tens to hundreds individual cells with a rate of one cell per minute with submicrometer resolution (panels B–Y in the box 3 of Figure 2). This opens up possibilities to investigate different physical and biochemical parameters under

Quantitative phase microscopy (QPM) is a label-free imaging technique, which allows reconstructing both the amplitude and the phase information of an optical field that passes through the sample, and it is particularly interesting in case of transparent biological cells. Respect to differential interference contrast (DIC) microscopy [29] or Nomarski/Zernike's phase contrast [30], the QPM gives a quantitative measure of the optical path difference (OPD) at each point in the sample. OPD in each position (x, y) of the acquisition plane is defined as the refractive

where nc and ns are the refractive index of the cell and the surrounding medium (assumed to be homogeneous), respectively. The resulting OPD map of the cell is reconstructed by recording the interference fringes pattern, the so called "hologram," of two superimposed coherent beams, one that interacts with an object under test and another that does not come in contact with the object and acts as a reference beam, and calculating the phase difference between them [31]. If the hologram is acquired by a digital sensor array, typically a charge-coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) device, digital holographic microscopy (DHM) technique is implemented. A typical interferometric setup for

The acquired hologram is then mathematically analyzed, allowing obtaining the complex field of the object beam that can be reconstructed at different distances, too. Therefore, numerical refocusing of a digital hologram, that is a 2D image, at different object planes, without any zscan of the optical system, allows to retrieve a 3D quantitative imaging [31]. This makes digital holography a very powerful method for metrology applications, particularly attractive in the field of biology as it is noninvasive, noncontact, and label-free, allowing the characterization of

OPD xð Þ¼ ; y t xð Þ ; y ð Þ nc � ns (2)

physiological conditions, leaving the assessed spermatozoa functional.

3. Quantitative phase imaging for sperm analysis

3.1. Basic principles of quantitative phase microscopy

index variation across the cell thickness, t(x, y):

DHM is reported in Figure 3.

live specimen.

damage [28].

Figure 2. Overview of the most representative studies on Raman spectroscopy and imaging for the label-free analysis of sperm cells. Box 1: the highlighted variations in peak intensities in the Raman spectra correspond to different sperm head shapes [21]. Box 2: the peaks at 1095 and 1050 cm�<sup>1</sup> represent biomarkers of fragmented DNA in the sperm nucleus [24, 25]. Box 3: panel A shows the chemical Raman reconstruction of distinct sperm regions [23]; panels B–Y represent the biochemical composition of individual immobilized, living human sperm cells [26]; and panel Z shows the efficiency of Raman imaging in revealing small irregularities in the sperm head such as vacuoles (yellow circles) distinguishable based solely on the presence of differing spectra [24].

stress and corroborated the findings using an established assay and an alternative but complementary spectroscopic technique (Fourier-transform infrared (FTIR) spectroscopy). The results of this last work confirm that RS is able to reveal different levels of oxidative DNA fragmentation, especially associated to alterations within the 1050–1095 cm�<sup>1</sup> spectral range (Raman spectra in the box 2 of Figure 2), which includes the band associated with the DNA phosphate backbone, changes that were confirmed by similar shifts in the corresponding FTIR peaks (not shown) [25]. Also, the Raman bands associated to protein and lipid content (1400–1600 cm�<sup>1</sup> ) showed some alterations induced by UV radiation, consistent with protein denaturation and lipid peroxidation that are well-known markers of oxidative damage [27]. Raman-based identification of DNA-damaged sperm cells linearly correlated with the findings from the flow cytometric analysis of DNA fragmentation, which represents the most statistically robust, reproducible, and standardized procedure available for the determination of sperm nDNA damage [28].

In 2015, Edengeiser et al. [26] analyzed spermatozoa under near-physiological conditions using confocal Raman microspectroscopy. The spermatozoa are immobilized on pre-treated object slides. More in detail, CaF2 slides were coated with concanavalin A and overlaid with preheated Ringer's solution just before putting a drop of isolated spermatozoa which, after the analysis, can be easily removed from the substrate. The study demonstrated for the first time the possibility to image and analyze several tens to hundreds individual cells with a rate of one cell per minute with submicrometer resolution (panels B–Y in the box 3 of Figure 2). This opens up possibilities to investigate different physical and biochemical parameters under physiological conditions, leaving the assessed spermatozoa functional.
