4. Combined optical approach for the noninvasive analysis of single spermatozoa

As seen in the previous sections, Raman spectroscopy and quantitative phase microscopies have been separately developed for assessing spermatozoa from a biochemical and morphological perspective, respectively. The two photonic techniques, employing intrinsic contrast mechanisms, allow noninvasively selecting the fertile spermatozoon according to its normal morphology as well as its DNA integrity. Kang et al. [47] first proposed a combined system where quantitative phase microscopy and Raman imaging allowed correlating morphological parameters with molecular information, i.e. the red blood cell thickness was correlated to the hemoglobin distribution. Huang and colleagues in 2014 published a study that evaluated the possibility to combine micro-Raman spectroscopy with image analysis for label-free identification of normal spermatozoa [48]. Recently, our group proposed a similar system, which combines Raman spectroscopy/imaging and digital holography microscopy as a potential tool to rapidly and objectively identify the healthy spermatozoa [36, 49, 50].

#### 4.1. The optical setup

In 2013, Merola et al. [44] provided an evaluation of the biovolume of spermatozoa (about

Figure 4. Some potentialities of DHM. Box 1: morphological analysis of semen carried out by DHM; top panels: sperm cells with distinct morphological defects; center and bottom panels: difference in height denotes a reduced volume in spermatozoon head with vacuoles respect to the normal spermatozoon [41, 43]. Box 2: examples of some physical

Another important semen parameter, the dry mass of the cell (i.e. the average mass of the proteins, carbohydrates, lipids, and so on within the cell), can be obtained by DHM. Indeed,

and the production of a tomographic 3D model, as showed in the box 2 of Figure 4.

parameters obtained by DHM [31, 44]. Box 3: 4D tracking of clinical sperm samples [45].

). The authors used optical tweezers to trap and rotate the cells; meanwhile, they flow through a microchannel, enabling recording digital holograms of the sperm at different angles

55 μm<sup>3</sup>

228 Spermatozoa - Facts and Perspectives

The setup used in our works for the simultaneous Raman and holographic analysis essentially consists of a Raman microscope coupled to an interferometer (Figure 5) [36, 49, 50]. We used two different laser sources: a green laser at 532 nm for the Raman excitation and a long coherence (>100 m) red laser at 660 nm for the holographic experiments. The red laser beam was split into two beams: the object beam passing through the sample and the reference beam

1. correlative analysis of morphological and biochemical alterations [49];

3. sex classification of bovine spermatozoa [49, 51].

Raman/holographic analysis of single sperm cells [49].

2. morphological and biochemical analysis of photo-damaged spermatozoa [36];

With the aim of demonstrating the potential applicability of the proposed multimodal imaging approach in identifying the fertile spermatozoa for IVF, we have first performed a qualitative

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Raman imaging can be performed scanning the laser over a region of interest of the sperm cell or on the entire cells, acquiring in that way a point-by-point spectrum. Similar spectral features correspond to similar molecular structures; consequently, we can identify separate regions of the spermatozoon (acrosome, nucleus, and tail) according to their different chemical composition (Figure 6a and b). Pseudo-color Raman map of the sperm is shown in Figure 6b, in which each color is arbitrarily associated to specific Raman spectral patterns. Therefore, the Raman map delineates not only the distribution of DNA and protein in the nucleus, acrosome and tail but also detects, in a label-free manner, small biochemical discrepancies correlated with the presence of morphological defects, highlighted by digital holography (Figure 6c). We found that the peculiar protuberance in the region of the spermatozoon connecting the head to the tail, the so-called middle piece, well correlated with the biochemical alteration detected in the Raman map. Indeed, by analyzing the typical Raman bands of the spectra acquired in that specific cell region, it was possible to correlate the morphological alteration to an increased

amount of proteins in the middle-piece region, where mitochondria are localized [49].

configuration (Figure 7a–c). Importantly, at laser fluences (30 MJ/cm2

cell morphology [49].

Recently, we applied the multimodal imaging tool for the online evaluation of the damages induced by green laser radiation for studying in which dose and how it affects the irradiated sperm cells [36]. Severe spermatozoa variations associated with a topological redistribution of the sample and a gradual decrease in the Raman signal intensity were detected in a label-free

alterations were detected by digital holography, high specific spectral variations were monitored to evaluate the cell photodegradation. More specifically, Raman analysis provided precise information on the most affected biochemical structures, finding that DNA phosphate

Figure 6. (a) Raman spectra and (b) false-color Raman map of different regions of the sperm cell head: middle piece (yellow), nucleus (red), acrosome (blue), and membrane (green). (c) 3D digital holographic reconstruction of the sperm

) where no morphological

Figure 5. Experimental set up of the combined Raman and holographic system used in our works [36, 49, 50].

that directly goes to the detector. Importantly, the reference beam was controllable in intensity and polarization enabling us to improve signal intensity and contrast. The collimated object beam of the holographic pathway is recombined to the reference beam by a beam splitter (BS). The recombined beams are filtered and sent to the CCD camera (CCD1) for the holograms recording.

The green light is focused on the sample by a high numerical aperture objective lens. The high N.A. of the objective and the wavelength chosen for the Raman beam allows to reaching spatial resolutions on the order of less than 0.5 μm, particularly suitable for cell imaging application. The back-scattered light from the sample is collected by the same objective lens (OBJ) and separated from the holographic radiation by a long pass dichroic mirror (DM) reflecting wavelength below 600 nm. The scattered light, consisting of Rayleigh and Raman radiation, is filtered through a dichroic beam splitter (BS45) that rejects the Rayleigh light at 532 nm. The Raman signal is further filtered using a laser-blocking filter (NF0) to eliminate the residual Rayleigh scattering and then focused onto the entrance slit of a monochromator. The Raman signal is finally detected using a cooled CCD camera (CCD2).

#### 4.2. Morphological and biochemical analysis of single sperm cells

In this paragraph, some of the most interesting results we have obtained by applying the Raman/holographic microscope for the sperm cell characterization will be discussed. Our investigations mainly focused on:


that directly goes to the detector. Importantly, the reference beam was controllable in intensity and polarization enabling us to improve signal intensity and contrast. The collimated object beam of the holographic pathway is recombined to the reference beam by a beam splitter (BS). The recombined beams are filtered and sent to the CCD camera (CCD1) for the holograms

Figure 5. Experimental set up of the combined Raman and holographic system used in our works [36, 49, 50].

The green light is focused on the sample by a high numerical aperture objective lens. The high N.A. of the objective and the wavelength chosen for the Raman beam allows to reaching spatial resolutions on the order of less than 0.5 μm, particularly suitable for cell imaging application. The back-scattered light from the sample is collected by the same objective lens (OBJ) and separated from the holographic radiation by a long pass dichroic mirror (DM) reflecting wavelength below 600 nm. The scattered light, consisting of Rayleigh and Raman radiation, is filtered through a dichroic beam splitter (BS45) that rejects the Rayleigh light at 532 nm. The Raman signal is further filtered using a laser-blocking filter (NF0) to eliminate the residual Rayleigh scattering and then focused onto the entrance slit of a monochromator. The

In this paragraph, some of the most interesting results we have obtained by applying the Raman/holographic microscope for the sperm cell characterization will be discussed. Our

Raman signal is finally detected using a cooled CCD camera (CCD2).

4.2. Morphological and biochemical analysis of single sperm cells

investigations mainly focused on:

recording.

230 Spermatozoa - Facts and Perspectives

With the aim of demonstrating the potential applicability of the proposed multimodal imaging approach in identifying the fertile spermatozoa for IVF, we have first performed a qualitative Raman/holographic analysis of single sperm cells [49].

Raman imaging can be performed scanning the laser over a region of interest of the sperm cell or on the entire cells, acquiring in that way a point-by-point spectrum. Similar spectral features correspond to similar molecular structures; consequently, we can identify separate regions of the spermatozoon (acrosome, nucleus, and tail) according to their different chemical composition (Figure 6a and b). Pseudo-color Raman map of the sperm is shown in Figure 6b, in which each color is arbitrarily associated to specific Raman spectral patterns. Therefore, the Raman map delineates not only the distribution of DNA and protein in the nucleus, acrosome and tail but also detects, in a label-free manner, small biochemical discrepancies correlated with the presence of morphological defects, highlighted by digital holography (Figure 6c). We found that the peculiar protuberance in the region of the spermatozoon connecting the head to the tail, the so-called middle piece, well correlated with the biochemical alteration detected in the Raman map. Indeed, by analyzing the typical Raman bands of the spectra acquired in that specific cell region, it was possible to correlate the morphological alteration to an increased amount of proteins in the middle-piece region, where mitochondria are localized [49].

Recently, we applied the multimodal imaging tool for the online evaluation of the damages induced by green laser radiation for studying in which dose and how it affects the irradiated sperm cells [36]. Severe spermatozoa variations associated with a topological redistribution of the sample and a gradual decrease in the Raman signal intensity were detected in a label-free configuration (Figure 7a–c). Importantly, at laser fluences (30 MJ/cm2 ) where no morphological alterations were detected by digital holography, high specific spectral variations were monitored to evaluate the cell photodegradation. More specifically, Raman analysis provided precise information on the most affected biochemical structures, finding that DNA phosphate

Figure 6. (a) Raman spectra and (b) false-color Raman map of different regions of the sperm cell head: middle piece (yellow), nucleus (red), acrosome (blue), and membrane (green). (c) 3D digital holographic reconstruction of the sperm cell morphology [49].

sex-associated membrane proteins in X- and Y-bearing sperm cells. In order to quantify the efficiency and the accuracy of the Raman spectroscopy in discriminating the two sperm cell populations, we have analyzed the data using a multivariate statistical method, known as principal component analysis, that allows to visualizing the separation of the two classes of cells into two well distinct clusters (Figure 8c), only on the base of the differences in their Raman fingerprint. The sorting accuracy resulted of 90.6%, which is comparable to the accuracy (90–94%) achievable with standard sorting techniques such as fluorescence-activated cell sorting (FACS). Then, the spectroscopic results have been correlated with the morphological analysis, where the sperm head volume has been evaluated by applying the Otsu's method [51] on the holographic phase map, a procedure generally used to indirectly measure morphological properties of the region under investigation (results not reported, see [49, 51]). As the main differences between X and Y chromosomes are the size (X chromosome is bigger than Y one) and the total DNA content, these major differences can be reflected in the head sperm volume, representing therefore a fast and automated way to identify the chromosome type [49, 51].

Figure 8. (a) Average Raman spectra of 900 X- (purple line) and 900 Y- (blue line) sperm cell spectra acquired in the

Advanced Label-Free Optical Methods for Spermatozoa Quality Assessment and Selection

content measured for X and Y sperm cells, ΔA = 4.2 0.9%. (c) PCA score plots of PC2, PC3 and PC4 showing the

, highlighting the different DNA

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233

"fingerprint" spectral region. (b) Measured peak area of the bands at 726 and 785 cm<sup>1</sup>

separation of data corresponding to X- and Y- sperm cells [49, 51].

Therefore, by combining holographic with Raman microscopy provides label-free, quantitative morphological and chemical information from unfixed sperm cells. The combination offers an excellent system for a complete, morphological and physiological, monitoring of the sperm cell

Despite the feasibility of Raman spectroscopy and holography microscopies has been successfully demonstrated in many medical and biological applications [14, 16, 30, 52–55], there is still a significant lack of translation and implementation of such innovative techniques into clinical practice. Indeed, while thanks to the technological advances the capability and information

5. Towards clinical applications: a demanding path

achievable are quickly expanding, there are some concerns to consider [56].

quality.

Figure 7. (Top) Reconstructed phase map of the (a) nonirradiated and (b) irradiated region of interest at the focus plane. The star (\*) indicates the cell position where the Raman spectrum is acquired that corresponds to the irradiated area. (c) Raman spectra of the sperm cell at three different selected laser fluences (0, 61 and 107 MJ/cm<sup>2</sup> ). (d) Zoom of four selected Raman spectra acquired at laser fluences of 0, 30, 61 and 107 MJ/cm<sup>2</sup> in the spectral region between 700–800 cm�<sup>1</sup> , 1050– 1120 cm�<sup>1</sup> , 1200–1400 cm�<sup>1</sup> and 1550–1720 cm�<sup>1</sup> [36].

backbone (900–1100 cm�<sup>1</sup> ) and lipids and proteins (1200–1400 cm�<sup>1</sup> ) are very sensitive to photoxidative denaturation (Figure 7d) [36].

To test the biochemical/morphological ability of the proposed multimodal approach, we additionally tested bovine sperm cells. The sex preselection of the offspring reveals a high significant impact on animal production management as well as genetic improvement programs. Since a noninvasive method for sex predetermination in animals is still not available, we used our multimodal approach for identifying and separating X and Y-bearing sperm cells. The key parameters estimated in our works [49, 51], on hundreds bovine spermatozoa of different bulls, were the Raman bands correlated to DNA content and head volume.

Indeed, specific peaks related to the vibrational modes of the DNA bases (726 and 785 cm�<sup>1</sup> ) can be used to sort X- and Y-bovine sperm cells (Figure 8a and b). However, additional significant spectral variations can be observed in the Raman bands mainly associated with the presence of lipids and proteins (1400–1600 cm�<sup>1</sup> , Figure 8a and b), due to the different composition of the

Advanced Label-Free Optical Methods for Spermatozoa Quality Assessment and Selection http://dx.doi.org/10.5772/intechopen.71028 233

Figure 8. (a) Average Raman spectra of 900 X- (purple line) and 900 Y- (blue line) sperm cell spectra acquired in the "fingerprint" spectral region. (b) Measured peak area of the bands at 726 and 785 cm<sup>1</sup> , highlighting the different DNA content measured for X and Y sperm cells, ΔA = 4.2 0.9%. (c) PCA score plots of PC2, PC3 and PC4 showing the separation of data corresponding to X- and Y- sperm cells [49, 51].

sex-associated membrane proteins in X- and Y-bearing sperm cells. In order to quantify the efficiency and the accuracy of the Raman spectroscopy in discriminating the two sperm cell populations, we have analyzed the data using a multivariate statistical method, known as principal component analysis, that allows to visualizing the separation of the two classes of cells into two well distinct clusters (Figure 8c), only on the base of the differences in their Raman fingerprint. The sorting accuracy resulted of 90.6%, which is comparable to the accuracy (90–94%) achievable with standard sorting techniques such as fluorescence-activated cell sorting (FACS).

Then, the spectroscopic results have been correlated with the morphological analysis, where the sperm head volume has been evaluated by applying the Otsu's method [51] on the holographic phase map, a procedure generally used to indirectly measure morphological properties of the region under investigation (results not reported, see [49, 51]). As the main differences between X and Y chromosomes are the size (X chromosome is bigger than Y one) and the total DNA content, these major differences can be reflected in the head sperm volume, representing therefore a fast and automated way to identify the chromosome type [49, 51].

Therefore, by combining holographic with Raman microscopy provides label-free, quantitative morphological and chemical information from unfixed sperm cells. The combination offers an excellent system for a complete, morphological and physiological, monitoring of the sperm cell quality.

#### 5. Towards clinical applications: a demanding path

backbone (900–1100 cm�<sup>1</sup>

232 Spermatozoa - Facts and Perspectives

1120 cm�<sup>1</sup>

photoxidative denaturation (Figure 7d) [36].

, 1200–1400 cm�<sup>1</sup> and 1550–1720 cm�<sup>1</sup> [36].

lipids and proteins (1400–1600 cm�<sup>1</sup>

) and lipids and proteins (1200–1400 cm�<sup>1</sup>

To test the biochemical/morphological ability of the proposed multimodal approach, we additionally tested bovine sperm cells. The sex preselection of the offspring reveals a high significant impact on animal production management as well as genetic improvement programs. Since a noninvasive method for sex predetermination in animals is still not available, we used our multimodal approach for identifying and separating X and Y-bearing sperm cells. The key parameters estimated in our works [49, 51], on hundreds bovine spermatozoa of different

Figure 7. (Top) Reconstructed phase map of the (a) nonirradiated and (b) irradiated region of interest at the focus plane. The star (\*) indicates the cell position where the Raman spectrum is acquired that corresponds to the irradiated area. (c)

Raman spectra acquired at laser fluences of 0, 30, 61 and 107 MJ/cm<sup>2</sup> in the spectral region between 700–800 cm�<sup>1</sup>

Indeed, specific peaks related to the vibrational modes of the DNA bases (726 and 785 cm�<sup>1</sup>

be used to sort X- and Y-bovine sperm cells (Figure 8a and b). However, additional significant spectral variations can be observed in the Raman bands mainly associated with the presence of

, Figure 8a and b), due to the different composition of the

bulls, were the Raman bands correlated to DNA content and head volume.

Raman spectra of the sperm cell at three different selected laser fluences (0, 61 and 107 MJ/cm<sup>2</sup>

) are very sensitive to

). (d) Zoom of four selected

, 1050–

) can

Despite the feasibility of Raman spectroscopy and holography microscopies has been successfully demonstrated in many medical and biological applications [14, 16, 30, 52–55], there is still a significant lack of translation and implementation of such innovative techniques into clinical practice. Indeed, while thanks to the technological advances the capability and information achievable are quickly expanding, there are some concerns to consider [56].

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 fluorescence background [57–59].

Author details

References

Annalisa De Angelis<sup>1</sup>

Anna Chiara De Luca<sup>1</sup>

\*, Maria Antonietta Ferrara2

1 Institute of Protein Biochemistry - National Research Council, Naples, Italy

2 Institute for Microelectronics and Microsystems - National Research Council, Naples, Italy

[1] Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HWG, Behre HM, et al. World Health Organization reference values for human semen characteristics. Human Repro-

[3] Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, et al. Andrology: Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. Journal of Assisted Reproduction

[4] Zaini A, Jennings MG, Baker HWG. Are conventional sperm morphology and motility assessments of predictive value in subfertile men? International Journal of Andrology.

[5] Avendaño C, Oehninger S. DNA fragmentation in morphologically normal spermatozoa: How much should we be concerned in the ICSI era? Journal of Andrology. 2011 Jul 8;32

[6] Sharma R, Masaki J, Agarwal A. Sperm DNA fragmentation analysis using the TUNEL

[7] Simon L, Lutton D, McManus J, Lewis SE. Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization

[8] Fernández JL, Muriel L, Goyanes V, Segrelles E, Gosálvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin

[9] Evenson DP. Sperm chromatin structure assay (SCSA®). In: Spermatogenesis: Methods

[10] Sánchez V, Wistuba J, Mallidis C. Semen analysis: Update on clinical value, current needs

assay. In: Spermatogenesis: Methods and Protocols. 2013;927:121-136

success. Fertility and Sterility. 2011;95(2):652-657

dispersion test. Fertility and Sterility. 2005;84(4):833-842

and future perspectives. Reproduction. 2013;146(6):R249-R258

[2] Mortimer ST. CASA—Practical aspects. Journal of Andrology. 2000;21(4):515-524

\*Address all correspondence to: a.deangelis@ibp.cnr.it

duction Update. 2009;00(0):1-15

and Genetics. 2001;18(4):215-220

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(4):356-363

, Giuseppe Coppola<sup>2</sup> and

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Advanced Label-Free Optical Methods for Spermatozoa Quality Assessment and Selection

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

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 Raman database is still required.

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.
