The application of scanning near field optical imaging to the study of human sperm morphology

Abstract

Background: The morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures. Results: In this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols. Conclusions: Such findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together.

Ultra‐structural analysis of human spermatozoa by aperture scanning near‐field optical microscopy

“…Since, as discussed in the introduction, transmission SNOM intensity is proportional to light scattering at large angles, these images highlight the structure of subcellular and subwavelength organelles. The observed features have an organization similar to that reported in TEM images : the nucleus occupies the major part of the sperm head and contains condensed chromatin; the acrosome region covers two‐third of the head ; the midpiece region shows a granular sequential organization which recalls the coiled helix of the mitochondria that surround the axoneme and the dense fibers, similarly to what observed in our previous work . These data confirm that these optical features are reproducible and can be observed in general on the healthy spermatozoa.…”

“…An AFM study of mammalian spermatozoa guided in understanding cell structure–function relationship, which is expected to have a significant application in human reproduction . In our previous work we have demonstrated the promising capability of SNOM to provide detailed information about the inner cellular structures of spermatozoa, without any specific labeling .…”

Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches

“…Post-thaw scanning electron microscopy has revealed an increase in the number of loose sperm head and tail defects. New nanoscopic techniques, such as scanning near-field optical microscopy (Andolfi et al, 2015), may help to elucidate the topographic images and subcellular structural details of spermatozoa without special staining or sample preparation, including the assessment of different cytoplasmic organs and the structure of the mitochondria (Di Santo et al, 2012).…”

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research

“…As shown in Figure 3 , the helicoidal organization of mitochondria in the midpiece region is well discernible in normal spermatozoa [ 94 ], and other structural features of healthy and anomalous spermatozoa are detected, all well resembling those observed in transmission electron microscopy images [ 95 ]. These works demonstrate the ability of SNOM to visualize inner cellular compartments with a typical contrast (transmission and reflection) for standard microscopy but with a higher resolution [ 94 , 95 ]. The ability to detect differences in the optical contrast of cellular structures and avoid standard staining protocols is very appealing, as staining can mask structural features in some cases at very high resolutions.…”