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) arevaluable analytical tools in biological and medical research, allowing the detection of both biochemicaland morphological variations of the sample without labels or long sample preparation. Here, usingthis multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reactioninduced by heparin. The multimodal microscopy provides morphofunctional information that can assessthe sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringenceanalysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holographyapplied for polarization imaging allows for revelation of the polarization state of the sample, showing atotal birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in thepost-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopiccharacterization of protein/lipid delocalization in the plasma and acrosomal membranes that canbe used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variationscan be correlated with diferent time phases of the cell capacitation response. Although furtherexperimentation is required, the proposed multimodal approach could represent a potential label-freediagnostic tool for use in reproductive medicine and the diagnosis of infertility.