Combined Raman and polarization sensitive holographic imaging for a multimodal label-free assessment of human sperm function

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.

These values show a decrease of about fourfold of the mean value of Δϕ when the acrosome reaction occurs. Results are summarized in the following Regarding the PCA, this analysis was performed on the amplitude ratio and phase difference data sets obtained for control (0h in heparin) and reacted (4h in heparin) samples. Figure S3 shows the loadings of PC1, PC2, PC3 for both amplitude ratio (β) and phase difference (Δϕ) parameters. PC1 indicates that a large contribution to discrimination is related on part of cell that is not birefringent; this can be seen by the large contribution from the histogram bin at 0 radiant, which represents the masked pixels and those have Δϕ=0 and β=0. This is in good agreement with results of β and Δϕ maps reported in Fig. 3-a and 3-b. PC2 and PC3 can distinguish more subtle differences between different phase of cells capacitation.
Supplementary Figure 1. PCA Loading of the first three principal components for both amplitude ratio (β) and phase difference (Δϕ) parameters.

Raman Analysis of the sperm capacitation.
Raman spectra obtained from the AA and ES sperm head regions were preliminary analysed.
To identify the spectral variation differences between the spectra acquired from these cell regions, we performed the PCA. The score plot and the corresponding PC1, PC2 and PC3 loadings for the spectra acquired from the ES and AA cell regions are reported in Figure S2. A good separation can be achieved by using the first three PCs, suggesting that there is an inherent molecular difference between the two analyzed head regions. The ES region appears to have a higher level of nuclear material when compared to the AA region, as represented by PC1. The accuracy of the algorithm, determined using the leave-one-out cross validation approach, was about 95%. To identify Raman spectroscopic changes that correlate with different phases of the cell capacitation response, PCA was performed on the entire Raman spectra data set inclusive of all time points (0, 1, 2, 3, 4 hours) of heparin treatment. Figure S3 show the loadings of PC1, PC2, PC3 for both ES and AA cell region. The PC1 loadings show negative lipids features at 1445 cm −1 , indicating a lower contribution in capacitated cells towards positive PC1, which are the control spectra (0h). Negative features can be found in the glycoprotein region at 890 cm −1 . Positive features in the protein spectral regions characterize the loadings of PC2 and PC3.

Methods
Sample preparation. The Percoll density gradient was prepared as follows: 1 ml of 80\% Percoll (SYDNEY IVF, K-SISG-50-80) was deposited in a sterile test tube with a conical bottom (15 ml). Subsequently, gently 1 ml of 55\% Percoll (SYDNEY IVF, K-SISG-50-40) was deposited taking care not to disturb the contact surface between the two layers. Sperm was then deposited on the top of the gradient and then centrifuged (300g, 20 min). The pellet obtained was resuspended in 2 ml of Ham's F-10 medium and centrifuged (300g, 5 min) before resuspending, again, in 1-2 ml of Ham's F-10 medium, to obtain a sperm concentration of about 10 million sperm/ml.
Vitality and motility test. Before and after Percoll gradient and heparin treatment, sperm motility was assessed by using a Makler counting chamber (SAFI Medical Instruments). To assure that the treatment with heparin did not affect sperm cell viability, an eosin-nigrosin staining vitality test was performed before and after treatment. 20 μl of eosin (Carlo Erba -446632) and 30 μl of nigrosin (Carlo Erba -464853) were added to 10 μl of sperm sample for 30 s. Then, an aliquot of 10 μl of sperm was smeared and air-dried on a glass slide, and the sperm cells counted with a bright-field microscope equipped with a 100X objective. Spermatozoa with damaged membranes had a red or fark pink staining pattern over the entire head, while living spermatozoa (with intact membranes) showed a white staining pattern over the head. Vitality was evaluated as the percentage of spermatozoa with white fluorescence to the total of the observed sperm cells.