The application of scanning near field optical imaging to the study of human sperm morphology
- Laura Andolfi1,
- Elisa Trevisan2,
- Barbara Troian3,
- Stefano Prato3,
- Rita Boscolo4,
- Elena Giolo4,
- Stefania Luppi4,
- Monica Martinelli4,
- Giuseppe Ricci†4, 5 and
- Marina Zweyer†5Email author
© Andolfi et al.; licensee BioMed Central. 2015
Received: 25 July 2014
Accepted: 18 December 2014
Published: 16 January 2015
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.
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.
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.
Conventional semen parameters such as sperm concentration, motility and morphology are generally used to assess male fertility [1-3]. However, a significant percentage of males with normal semen parameters, according to WHO guidelines , fails to conceive . In these cases, the presence of ultrastructural defects could be hypothesized [6-11]. Several morphological studies have been carried out to define the normal form of sperm (see reference  for review) and the last WHO semen manual provides objective criteria to assess the sperm morphology . However, there are many factors that may influence the results of the morphology assessment, including the technician’s concept of the definition of normality [12,13] and the staining procedures [14,15]. The most common technique to assess sperm morphology is conventional optical microscopy (OM), usually performed on fixed and stained specimens. In this case, sample preparation is rather easy but the resolution is limited to micron resolution (0.2 μm: Abbe diffraction limit). Moreover, it has been shown that different staining techniques may cause not uniform changes in sperm head dimensions . To reduce the subjectivity of sperm morphology assessment, computer-assisted sperm morphometry analysis (CASMA or ASMA) have been developed in the ‘90s [16,17]. Subsequently, this analysis has been improved by using special objectives for relief contrast observation . A further progress has been achieved by combining fluorescence microscopy and image analysis with open-access software [19,20]. This method allows automatic determination of sperm morphometry in a reduced time [19,20]. To avoid artefacts induced by fixation and staining, a new optical technique (Trumorph system) has been recently described . This technique allows the analysis of living unmodified spermatozoa that are immobilized in narrow chambers and examined by negative contrast phase microscopy . In the last decades, other fundamental studies have been carried out by transmission and scanning electron microscopies (TEM and SEM), which have enabled to detect ultrastructural details and have provided a huge amount of information concerning morphological organization of spermatozoa [22-25]. However, SEM imaging usually needs harsh sample preparation as drying at critical point, metal coating and vacuum environment (during coating procedure and measurements) that may modify some structural features of cells. Analogously, complex preparation of specimens (as resin embedding and sectioning by ultramicrotomy) are required for performing TEM imaging, which provides two-dimensional view of cell internal arrangement. Recently scanning probe microscopy (SPM) techniques have been exploited to improve the knowledge of spermatozoa morphology. Particularly, atomic force microscopy (AFM) has allowed investigating the topography of spermatozoa with nanometric resolution and without needing invasive sample preparation [26-29]. Moreover, either fixed or living cells can be imaged by this technique. Among scanning probe microscopies (SPM), scanning near-field optical microscope (SNOM) has been demonstrated to be a powerful and versatile tool for the observation of biological samples [30,31]. As in AFM, a sharp probe scans the sample surface to generate a topographic image with similar resolution and sensitivity . The SNOM probe consists of a tapered optical fibre that scans the surface and acts as a point light source with a subwavelength-aperture at the terminal part [32,33], which creates a near-field light that decays exponentially with the distance of 100 nm and carries highly localized information . As result, subdiffraction resolution optical images (reflection, transmission, back-reflection and fluorescence) and high-resolution (nanometer level) topographic image are simultaneously generated. Such technique has been successfully applied for the investigation of cell membranes at molecular level [35-38], subcellular structures [39,40], thin-sections  and chromosomes . In the present work, we exploit the advantage of SNOM technique to describe simultaneously different superficial and internal morphological details of human spermatozoa from normozoospermia and teratozoospermia, without needing invasive or expensive sample preparation.
Results and discussion
Morphological analysis of the sperm structures provides relevant information, necessary part to clinical data to evaluate the ability to fertilization. Numerous cellular structures characterizing the spermatozoa have been elucidated thanks to SEM and TEM studies at ultrastructural level, and more recently by AFM at nanometer scale. In this study, the potentialities of SNOM have been exploited to investigate the morphology of fixed human spermatozoa deriving from samples of patients with normozoospermia and teratozoospermia.
Topography: it is an accurate real three-dimensional image of the sample surface. The acquisition of signals in x,y,z axes allows measuring surface details at nanometric level. Differently from traditional microscopies (2D methods), SNOM produces a real 3D topography image, since z dimensions are directly measured point by point on the vertical axis, analogously to AFM images.
Optical reflection: the SNOM reflection image derives from local interactions between near field light and the surface of the specimen, in particular is due to the light scattered from a thin layer within 30–100 nanometers below the sample surface (depending on tip aperture). Hence this image provides mainly morphological information regarding structures immediately below the sample surface.
Optical transmission: the light transmitted through the whole thickness of the specimen and not adsorbed by the sample produces the SNOM transmission image. In conventional optical microscopy the entire sample is illuminated. On the contrary, in near-field the light interacts locally, producing signals point by point to reconstruct a whole image of the scanned area. Hence, SNOM imaging is quite similar to traditional optical transmission imaging, but its lateral resolution is more than 10 times higher.
SNOM optical reflection and transmission images can put into evidence the organization of cellular structures positioned at cell surface (i.e. membrane and structures immediately below) or internal ones (i.e. cytoskeleton, nucleus, mitochondria). Therefore together with the topography they can provide a complete picture of the cell morphology.
Before SNOM imaging the semen was purified from other cells and agglomerated proteins to reduce the amount of impurities that can deposit on glass cover slip together with sperm cells. This is an important aspect to get high quality SNOM images and avoid that tip gets dirty during scanning the surface. Moreover, the sequence of purification, fixation and drying procedures, used for SNOM imaging, did not cause remarkable cell damaging that could lead to the formation of artefacts.
As a matter of fact, sperm were firstly fixed, then partially dehydrated with alcohol and dried just before imaging. This procedure should minimize the morphological artefacts, since they mainly appear when cells are dried before fixation [43,44].
The analysis of the topographical image is shown in Figure 3D and E. Figure 3D shows a longitudinal profile of the midpiece, between the head and anulus along the red arrow direction of Figure 3A. The profile points into evidence four bumps (indicated by the black arrows), which likely are due to the presence of mitochondria helicoidally surrounding axonema (Figure 2), as also observed in previous AFM work . The total length of this region is found to be 4.5 μm in accordance with the range of values obtained in literature . In Figure 3E horizontal cross section profiles taken on the different part of the sperm are shown (white arrows in Figure 3A). These data display the difference in height of the tail: profile 1, made in the initial part of the midpiece provides height of 450 nm; profile 2, in midpiece region height of 340 nm; profile 3, in the anulus region height of 220 nm. The rest of the flagellum in three points is measured as shown in Figure 3A: profile 4, height of 120 nm; profile 5, height of 90 nm; profile 6, height of 70 nm. These results, in agreement with literature reported values, point out that the scanning of the spermatozoa by SNOM probe does not introduce artifacts and does not damage cell integrity [26,29].
In the case of coiled tail sperm, the SNOM topography (Figure 7G) shows that the midpiece region consists of repetitive structures. Such arrangement can be also detected in the SNOM optical reflection image likely related to an arrangement of the membrane and the layer immediately below (Figure 7H). Whereas in the SNOM optical transmission image, due to the overlapping of the coiled tail, it is difficult to detect and describe single components (Figure 7I).
In summary, we demonstrate that SNOM technique enables to describe the cell surface and the arrangement of the spermatozoa internal structures both in healthy and anomalous sperm by acquiring topographic and optical (transmission and reflection) images simultaneously. These structural details of spermatozoa can be detected by using rapid, inexpensive and non-harsh procedures for sample preparation. Specifically, the acrosome region located around the head is revealed and evaluated by SNOM optical transmission images. Analogously, the surface of midpiece region can be observed together with the organization of the mitochondria below the plasma membrane, also in comparison with cell presenting anomalies as the spermatozoon with two tails. Indeed in such case SNOM optical images suggest different organization of mitochondria. Finally the organization and arrangement of circumferential ribs and longitudinal column could be followed below the cell surface thanks to SNOM optical transmission images. This capability can be considered very interesting and useful also for the study of morphological and structural characteristics related to other anomalies as for instance: round acrosomeless sperm head that lead to infertility [48,49]; pathologies that involve the mitochondria (number and assembling), which usually lead to reduced sperm motility , flagellum abnormalities. [51-54]. Moreover, SNOM may be very helpful also in male contraception investigations, when sperm damages are intentionally provoked [55-57].
In conclusion SNOM can represent a very promising tool to analyze at high magnification (down to nanometer scale) the morphology of cell membrane and underlying structures both in normozoospermia and teratozoospermia without specific staining or harsh sample preparation procedures.
Ten semen samples from men undergoing routine infertility investigations were obtained. Semen samples were collected by masturbation into sterile containers after 3–6 days of sexual abstinence. After complete liquefaction, routine morphological semen analysis was performed using an optical microscope according to World Health Organization guidelines  and the morphology was evaluated by precoloured slides Testsimplets® Waldeck-Muenster/Germany. Five samples showed normozoospermia and five samples displayed teratozoospermia, according to WHO 2010 criteria . In order to test semen quality a leukocyte count was carried out by using standard peroxidase test, as described in the WHO laboratory manual. Leukocytospermia was defined as the presence of >1 × 106 leukocytes per milliliter of semen. For the detection of antisperm antibodies the reagent kit SpermMar® IgG Test from FertiPro N.V. (Sint-Martens-Latem, Belgium) was used.
A very clean sample is fundamental in all kinds of microscopies to obtain high quality images. Particularly in SPM techniques, it is important to avoid that debris attach to the tip during scanning. This could lead to the probe damaging and/or the formation of topographical artifacts. In the case of spermatozoa, the raw semen was processed and purified (washing steps) to get a clean sperm solution free from agglomerated proteins and other cell populations.
Semen preparation was performed by Swim-up technique. Such methodology allows obtaining both a spermatozoa selection and a specimen purification. One or more aliquots of 0.5 ml of semen was washed with 1 ml of medium (Quinn’s Advantage Medium w/HEPES, SAGE BioPharma™, Bedminster, NJ, USA, supplemented with 0.5% human serum albumin, SAGE Assisted Reproduction Products™, CooperSurgical, Trumbull, CT, USA) into a 5 ml Falcon conical tube (Becton Dickinson Labware, Meylan, France) and then centrifuged at 300×g for 10 min. The excess of supernatant was discarded and the pellet was resuspended in 0.5 ml of medium. Then, 0.5 ml of medium was gently layered on sperm suspension and the tube was inclinated at an angle of 45° and incubated at 37°C for at least 45 min. The tube was gently set upright and the upper interface was then gently aspirated with a Pasteur pipette. A small aliquot was examined for sperm concentration and sperm motility, the remaining part used for the experiments.
Sample preparation for SNOM imaging
For further sample cleaning, sperm was washed two times with Dulbecco’s modified phosphate buffered saline (PBS) (Sigma-Aldrich, USA) by centrifugation at 500×g for 5 min and resuspended in PBS (3 × 106 cell/ml). One aliquot of the cell suspension was deposited on poly-L-lysine coated coverslips (18 mm diameter) (Menzler-Gläser) and incubated at 37°C, 5% CO2 atmosphere for 1 h to improve the spermatozoa adherence to the substrate. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, washed three times in PBS, two times in water, partially dehydrated (up to 70% ethanol) and allowed to dry before SNOM imaging.
Near-field measurements were performed by using a TriA-SNOM microscope (A.P.E. Research, Trieste, Italy), equipped with a flexure scanning stage with a maximum xyz scan range of 100 μm × 100 μm ×10 μm, with a strain gauge sensors to obtain an absolute positioning of the probe. The TriA-SNOM setup was provided with interchangeable laser sources, coupled with a single mode optical fibre. In this work a laser wavelength of 532 nm was used. This laser source power is 20 mW, but after coupling into the fiber, the radiation power emitted by the aperture of SNOM tip is reduced to 2 μW- 20nW on the sample depending on the aperture diameter and shape of SNOM tip . Beside topography images, SNOM optical reflection and optical transmission signals were simultaneously detected with two photomultipliers (R74000, Hamamatsu Photonics Italia S.r.l., Milano, Italy). For these measurements we used a narrow band-pass interference filter at 532 nm wavelength (full width-half maximum = 10 ± 2 nm). SNOM probe was an aluminum-coated tapered pulled optical fiber with a nominal tip aperture of 50 nm (Lovalite, Troyes, France). Two optical view systems were integrated within the SNOM head to control the probe position and select the scan area. An upper optical vision system was used to monitor probe approach to the sample, and one transmission camera (with interchangeable achromatic objectives) was utilized for a bottom view of the sample. The SNOM images are acquired with: 256×256 pixel and 0.1-0.4 msec acquisition time for each point.
SNOM images were processed by A.P.E. Research SPM control software (A.P.E. Research, Trieste, Italy), and analysed by Gwyddion (free software).
This work was supported by a grant from the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy (RC 35/08). We are very grateful to Giovanna Baldini and Roberta Bortul for technical support.
We acknowledge Piccin publisher for gentle permission to use and modify picture shown in Figure 2A of this paper. The picture has been taken out from textbook “Embriologia Umana” Morfogenesi - Processi molecolari - Aspetti clinici di M. De Felici et al., ed. Piccin.
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