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Metal deposition and shape reproduction at biological temperatures on cell-level samples

Non-heat damage Al film coating on bovine sperm

Sperm in good condition were separated from the bovine semen preserved in a frozen state using a special microfluidic channel16. The adsorption of the separated sperm on the amino group-modified glass substrate was confirmed, and this adsorption was then compared with that of the sperm on a normal glass substrate using light microscopy (Extended Data Fig. 1). After adsorption, the sperm heads were immediately analyzed using laser microscopy, and at approximately 10 µm, the diameter was consistent with that from data reported in a previous paper.17.

Furthermore, the sperm was deposited on the Al thin film and then similarly analyzed using laser microscopy. There was no significant change in the sperm head size and clear images were observed, suggesting that the formation of the Al thin film did not cause thermal degeneration (Fig. 2a). To confirm reproducibility of this process, another sperm adsorption substrate was prepared, and sperm before and after deposition were observed using a laser microscope (Extended Data Fig. 2). There was no change in sperm surface shape before and after deposition. At least, the protein structure did not change due to thermal denaturation, indicating that the deposition was reproducible. To clarify the effects of heat on the bovine sperm, the motility of the sperm under different temperature conditions was compared and the temperature increase during high-power sputtering was analyzed (Fig. 2b). In the motility performance analysis conducted at 37 °C for 10 min, most sperm within the viewing range showed active flagellar motility (Extended Data Table 1).

Figure 2

Deposition of Al thin film using non-heat damage technique. (a) Glass after sperm adsorption and deposition of Al thin film; laser microscopy imaging was conducted using high magnification. Inset graphs show the result of optical analysis of the sperm head size from tip to neck. (b) Motility of spermatozoa was analyzed after incubation at 37 °C and 45 °C for 10 min. Films were deposited under temperature conditions conforming to respective results, and the effect of thermal denaturation on sperm shape was observed. (c) Scanning electron microscopy images showing sperm from head to tail after Al thin film deposition. (d) Elemental analysis of the sperm-glass and sperm heads for Al-sperm-glass.

In contrast, at 45 °C for 10 min, none of the sperm showed flagellar movement, and thermal denaturation of the tail was observed. The optimal deposition temperature of Al that is essential to maintaining the shape of the sperm with active flagellar motility has been shown to be ≤ 40 °C. The film was deposited with a Temperature-label ® attached to the back of the glass. The cathode, which can generate plasma even at a low radio frequency (RF) power of 30 W, deposited the Al thin film without discolouring the Temperature-label ®. Laser microscopy images of the spermatozoa did not reveal any microstructural changes.

Furthermore, the optical height analysis result illustrated in the inset image clearly shows the difference in the nanoscale thickness of the sperm head, midpiece, and tail. Sputtering at 100 W discolored the label on the back side of the glass at 45 °C. The adsorbed sperm on the substrate showed a change in the surface structure due to thermal denaturation. High vacuum during sputtering is also associated with structural changes, whereas air-drying of sperm has been proven to have little effect on their morphology18.

A scanning electron microscope (SEM), which can be used to clearly observe the whole image of the sperm, was used to observe the surface of the Al thin film samples in more detail (Fig. 2c). Fine ridges on the surface and neck were clearly observed in the middle and head of the sperm. The Al thin film, which was shown to be deposited on the sperm surface with high adhesiveness, was analyzed and confirmed to be Al using energy-dispersive X-ray spectroscopy (EDS, Fig. 2d). The spectra obtained from the elemental analysis clearly showed the deposition of Al in contrast to the test conducted on the uncoated substrate, which showed no deposition (Extended Data Table 1). In our study, the substrates were pre-treated to become hydrophilic surfaces during the sperm adsorption process, and therefore, the binding and cohesive energies of adatoms are weaker than the cohesive energy to the substrate surface and the resulting layer-by-layer stacking. Thus, interfacial structures of sperm are preserved during our sputter-deposition process.

Mold fabrication for shape reproduction and observation

Biomimetic metal nano-moulds (BMNMs) were fabricated using a simple method where the Al film was peeled off the glass using tapes. Optical analysis of the exfoliated Al thin film surface using a laser microscope revealed numerous indentations that were similarly shaped to the sperm (Fig. 3a). The heights of the sperm observed on the BMNM were analyzed focusing on those with abnormal shapes of the midpiece and tail.

Figure 3
figure 3

Shape reproduction analysis and observation of fabricated mould. (a) Schematic diagram shows stripping method for producing Al thin film from substrate using tapes; the surface of stripped film was analyzed using a laser microscope (Red box). Height analysis of sperm with abnormally shaped middle pieces and tails. (b) Dimethylpolysiloxane (polydimethylsiloxane, PDMS) was poured into biomimetic metal nano-moulds (BMNM) to reproduce sperm surface shape. PDMS surface separated from the mold was optically analyzed using laser microscopy (Black box). Height analysis of sperm with abnormally shaped middle pieces and tails. (c) Surface of the first and last PDMS bases; BMNMs were examined using laser microscopy after five replications using the same BMNM. Height of optical head surface was analyzed using laser microscopy.

The optical height analysis suggested that the abnormalsperm shape was clearly concave, suggesting that the deposited Al thin film was formed with a high degree of adhesion to the sperm. The surface morphology was highly preserved even with BMNMs fabricated using a simple procedure. In contrast, the presence of microscale wave-like shapes on the fabricated BMNMs was a problem caused by the tape used for peeling. There are several possible solutions to this problem, such as metal plating of a microscale thickness on the thin films.

The shape of the bovine sperm surfaces was reproduced using PDMS because of its high shape recognition ability.19.20. The PDMS molding method was performed by pouring a mixture containing a degassed crosslinker and heating at approximately 70 °C for 4 h. When the molded PDMS was separated from the mould, inverted sperm with the same shape as the mold were observed on the surface (Fig. 3b). The optical height analysis showed a convex area on PDMS that mimics the sperm shape, and the height analysis of the surrounding area showed a severe height difference. This was likely because the PDMS surface was affected by micro-scale irregularities of the tape surface, which was the base of the mould. The shape was reproduced five times using the same BMNM (Fig. 3c). The PDMS surface analysis using laser microscopy successfully showed that the sperm had the same shape as those in the first and fifth PDMS bases. The surface shape of the mold showed high preservation in the performance evaluation. For the BMNM, the shape of the sperm was clearly observed on the surface after the fifth round of shape reproduction. This observation suggests that the thin Al film did not damage the mold at a temperature of 70 °C, required to rapidly form PDMS.

In addition, height analysis of the same sperm head of each sample using laser microscopy showed a high degree of agreement in the characteristics of irregularities. The same analysis was performed for different sperm after sputtering the Al thin film, and the difference in height was smoother than that of PDMS. This suggests that PDMS reproduced the adhesive surface at the microscale level between the sputtered film and the sperm surface because the pattern of irregularities on the sperm head surface was similar to that on the Al-sperm-glass.

Detailed structural surface analysis of sperm

The shape of Al-coated sperm was clearly analyzed using atomic force microscopy (AFM) for surface characterization (Fig. 4a). A more detailed analysis focusing on the sperm head showed a difference in height towards the tip, similar to the results of the optical height analysis. The increase in height from the head to the midpiece was also consistent with the optical analysis results. By focusing on the tail, it was possible to analyze the fine structure of the tail tip, which was not observed using laser microscopy. At the tip of the tail, fine fibers were separate, indicating that the sperm tail was composed of fibrous tubulin proteins. The normal sperm tail is known to consist of a 9 + 9 + 2 fiber pattern21.22. The number of fibers observed in this target was not confirmed, but the related findings suggest that the exposed microstructure maintained its shape after Al sputtering at a biological temperature. AFM has already been established for microbial observation23; however, biological temperature sputtering demonstrated superiority in long-term preservation of AFM samples, as it does not require consideration of shape changes due to thermal denaturation.

Figure 4
figure 4

Analysis of various structures of sperm surface. (a) Atomic force microscopy analysis of sperm from head to tail after Al thin film deposition. White squares show areas analyzed in greater detail. (b and c) Examination of surface morphology of the sperm stored in biomimetic metal nano-moulds (BMNM) and shape reproduced on dimethylpolysiloxane (polydimethylsiloxane, PDMS) using AFM. Laser microscopy image focusing on sperm shown in black frame. (d) Height analysis of AFM image and three-dimensional height mapping. Triangles show microstructure of the sperm head. Black = acrosome, yellow = equatorial segment (EqS), white = necklace, red = midpiece.

The BMNM and reproduced PDMS were also analyzed using AFM (Fig. 4b and c). The same sperm were analyzed from the field of view of the laser microscope described in the previous section. In BMNM, it was possible to analyze the shape of the sperm using laser microscopy. This was evidenced by the microstructure of the exposed tubulin tail. In the PDMS sperm, high-resolution images from the head to tail were successfully captured and examined, as in the Al-sperm-glass, but tubulin at the tip of the tail was not clearly observed. The rubbery nature of PDMS made it difficult to observe the submicron soft parts.

Analysis of the three-dimensional maps of AFM images showed distinct microstructures (Fig. 4d). An equatorial segment, membrane pits, and a necklace in the middle were observed in the heads of the Al sperm, and the height of the midpiece from the head was clear24. These structures have already been confirmed by direct observation of the raw adsorbed sperm on the substrate using AFM25.26. These microstructures were found on both the BMNM and PDMS that reproduced the shape. This proves that the BMNM preserved the biological surface structures by sputtering with high adhesion at the nanoscale.

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