Scanning near-field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly-L-lysine coated coverslips, fixed according to a standard protocol and imaged by aperture-SNOM in air. In the SNOM images, all peculiar sperm portions show well-resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high-resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.
