The optimal thickness of gold coating of cilia for scanning electron microscopy was studied using respiratory mucosa obtained from pigs. We tested 8 different coating times, from 10 seconds to 4 minutes, which resulted in gold layer thicknesses varying from 16 ± 1 nm to 100 ± 3 nm. The thickness of the gold layer with a coating time of 60 seconds and voltage of 2.5 kV was 43 ± 5 nm. This thickness of gold layer gave good image quality without causing any electric charging. With thinner gold films, the amount of electric charging increased. When the coating time was longer, the gold layer was thicker but image quality did not improve. The thicknesses of the gold layers were measured using transmission electron microscopy (TEM)

Nuutinen, J.

Pelttari, A.

Rautiainen, M.

Toskala, E.

English

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Scanning Microscopy Volume 9 Number 1 Article 23 3-31-1995 Gold Coating of Respiratory Cilia for Scanning Electron Microscopy E. Toskala University of Kuopio J. Nuutinen University of Kuopio M. Rautiainen University of Tampere A. Pelttari University of Kuopio Follow this and additional works at: https://digitalcommons.usu.edu/microscopy  Part of the Biology Commons Recommended Citation Toskala, E.; Nuutinen, J.; Rautiainen, M.; and Pelttari, A. (1995) "Gold Coating of Respiratory Cilia for Scanning Electron Microscopy," Scanning Microscopy: Vol. 9 : No. 1 , Article 23. Available at: https://digitalcommons.usu.edu/microscopy/vol9/iss1/23 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Scanning Microscopy, Vol. 9, No. 2, 1995 (Pages 303-306) 0891-7035/95$5 .00 + .25 Scanning Microscopy International, Chicago (AMF O'Har e), IL 60666 USA GOLD COATING OF RESPIRATORY CILIA FOR SCANNING ELECTRON MICROSCOPY E. Toskala'"1,3 , J. Nuutinen 1, M. Rautiainen3, A. Pelttari 2 Departments of 1Otorhinolaryngology and 2Electron Microscopy,University of Kuopio, SF-72110 Kuopio, Finland Department of 3Clinical Medicine, Otorhinolaryngology , University of Tampere, SF-33101 Tampere, Finland (Received for publication June 27, 1994 and in revised form March 31, 1995) Abstract The optimal thickness of gold coating of cilia for scanning electron microscopy was studied using respira-tory mucosa obtained from pigs. We tested 8 different coating times, from 10 seconds to 4 minutes, which re-sulted in gold layer thicknesses varying from 16 ± 1 nm to 100 ± 3 nm. The thickness of the gold layer with a coating time of 60 seconds and voltage of 2.5 kV was 43 ± 5 nm. This thickness of gold layer gave good image quality without causing any electric charging. With thinner gold films, the amount of electric charging increased. When the coating time was longer, the gold layer was thicker but image quality did not improve. The thicknesses of the gold layers were measured using transmission electron microscopy (TEM). Key Words: Respiratory cilia, gold coating, scanning electron microscopy, ciliary ultrastructure . ,. Address for correspondence Elina Toskala Department of Clinical Medicine, Otorhinolaryngology, University of Tampere, P.O.Box 607, 33101 Tampere, Finland Telephone number : 358 31 2156111 FAX number: 358 31 2156164 303 Introduction Non-conductive specimens examined by scanning electron microscopy (SEM) must be coated with a thin film of conductive material. The coating layer is pri-marily used to eliminate or reduce the electric charge which builds up rapidly in a non-conducting specimen when it is scanned by a beam of high energy electrons (Jazbi and Sayegh , 1978; Echlin, 1981). Sputter coating is used routinely for SEM studies (Bell et al., 1987). However, very few investigations have been performed regarding the suitable thickness of the gold coating layer for SEM of respiratory mucosa and cilia. From a clini-cal standpoint, the correlation of ciliary ultrastructure and respiratory diseases , such as immotile cilia syn-drome, is of current interest (Afzelius, 1981; Herzon , 1981; Rautiainen et al., 1990). In the present study, the correlation between the im-age quality and the thickness of the coating layer was examined using eight different coating times with gold in a diode sputter-coating system. The aim was to deter-mine the optimal thickness of the gold layer which would provide good image quality and contrast without any charging effects. Materials and Methods Eighteen specimens of respiratory mucosa from two healthy pigs were used in this study. Specimens were cut from the tracheae, above the bifurcation and random-ly divided into 9 different groups (each group containing two specimens). Eight different coating times were used to study the structures and the thicknesses of coating lay-ers: 10 seconds, 30 seconds, 1 minute, 1 minute 30 sec-onds, 2 minutes, 2 minutes 30 seconds, 3 minutes, and 4 minutes. In addition, two samples, which had no metal coating, were also examined. To avoid mechanical artefacts, the specimens were taken from the trachea immediately after the animal had been killed by electric shock . The material was obtained from a local slaughterhouse. The specimens were first washed in 0. 9 % NaCl and then fixed in 1 % glutaralde-hyde and 4% formalin cocktail fixative with phosphate E. Toskala et al. 1a --Figure 1. The thicknesses of gold layers and corre-sponding coating times studied by TEM. (a) 16 ± 1 nm, 10 seconds; (b) 43 ± 5 run, 1 minute; and (c) 100 ± 3 run, 4 minutes. Bars = 100 run. buffer (1300 mOsmol/kg, pH 7.0) (McDowell and Trump 1976; Rautiainen et al., 1987). The specimens were then handled according to modified thiocarbohy-drazide procedure (OTOTO-method) as described by Malick et al. (1975), dehydrated in graded ethanol series (50 % , 60 % , 70 % , 80 % , 90 % , 94 % , absolute ethanol) at 10 minutes for each step. They were then dried with CO2 in a critical point dryer (Balzers , Liechtenstein). Specimens were mounted by conductive carbon adhesive (Hydro-Collage, Balzers) on aluminum stubs. Specimens were coated with gold in a sputter coater (Polaron II E51002). The distance of the gold source from the specimen was 40 mm, and the voltage used for coating was 2.5 kV. Coating was carried out in an argon atmosphere with a current of 20 mA; the pressure in the chamber was 5 Pa. To measure the thickness of these eight different gold layers, small epoxy (Ladd LX 112) sticks were 304 2 a .. Figure 2. The surface structure and corresponding thicknesses of gold layers obtained with different coating times and studied by TEM . (a) Ten second coating time, 16 ± 1 nm thick, the size of gold grains is 1.3 ± 0.2 nm. (b) One minute coating time, 43 ± 5 run thick, the size of gold grains is 2. 7 ± 0.5 run. (c) Four min-ute coating time, 100 ± 3 nm thick, the size of the gold grains is 5 .1 ± 0. 7 nm. Bars = 100 nm. coated at the same time as the specimens. The sticks were then embedded in epoxy resin, and after polymeri-sation, ultrathin cross-sections were cut. The structures of the gold layers were studied from copper grids cov-ered with formvar film; these were coated at the same time as the specimens. The structures and the thicknes-ses of the gold layers were studied in a JEOL JEM 1200 EX (Peabody, MA, USA) transmission electron micro-scope (TEM) operated at an acceleration voltage of 80 kV. Specimens were photographed at primary magnifi -cations of 25000x. The thicknesses of gold layers and the size of the grains were measured from micrographs using a measuring magnifier (Polaron 7x). The results were expressed as mean values of ten measurements. Specimens of respiratory mucosa were examined in a scanning electron microscope (JEOL JSM-35) operated at an accelerating voltage of 15 kV . From ciliated areas of the mucosa, 10 randomly selected areas were photo-graphed at the primary magnifications of 600x and 6000x . The final magnifications of the micrographs were 800x and 8000x. Results and Discussion The thicknesses of the gold layers ranged from 16 ± 1 nm (Fig. la; 10 second coating time) to 100 ± 3 nm (4 minute coating time). The structure of the metal coating film in TEM was continuous when the gold layer was 43 ± 5 nm (1 minute coating time) or thicker . The structures of thinner films were no longer homogenous. The metal grains were larger when the gold layer was Gold coating of respiratory cilia thicker. With 10 second coating time, the size of the gold grain was 1.3 ± 0.2 nm; with 1 minute coating time, it was 2. 7 ± 0.5 nm; and with 4 minute coating time, it was 5. 1 ± 0. 7 nm (Fig. 2) . The best result was achieved with a 43 nm thick gold film which was obtained with a coating time of 60 seconds (Fig. lb). This resulted in good image quality without any electric charging (Fig. 3a). Specimens coat-ed with a gold layer thicker than 43 nm had no electric charge. However, the image quality did not improve, and the diameter of cilia increased significantly when the 305 Figure 3. (a) Pig respiratory mucosa coated with a 43 ± 5 nm thick gold film. Image quality is good, and there was no electric charging. (b) Respiratory mucosa coated with a 100 ± 3 nm thick gold film . Cilia are significantly thicker than with a 43 nm layer. (c) Respi-ratory mucosa without gold coating . Cilia are transpar-ent and the cell membranes are unclear (arrows). Bars 1 µ.m. coating time was longer (Fig. 3b). With a 90 nm gold layer , the mean diameter of cilia was measured at 280 ± 10 nm, but when there was only a 43 nm layer, it was 240 ± 10 nm. When there was no coating at all, the mean diameter of cilia was 230 ± 20 nm. The measurements were made from the micrographs about l µ.m from the tip of ten cilia. In percentage terms, the diameter of cilia increased from the uncoated sample value by 4 % with a 43 nm gold layer and by 22 % with a 100 nm layer (Fig. 3). The resolution was significantly worse when the coating film was thinner than 43 nm because electric charging made it difficult to see cilia and their fine details, such as cilia tips. The samples, which had no metal coating, also had an electric charge which made electron microscopic examination of their cilia difficult. In these specimens, cilia appear "transparent ," and the cell membrane and its outlines are unclear (Fig. 3c) . In recent years, the ultrastructure of cilia has been studied intensively. However, most of these studies are based on TEM observations (Satir, 1961; Rautiainen et al., 1987, 1991). A major advantage of SEM is the possibility of obtaining three-dimensional images of large E. Toskala et al. cilia populations . In our experience, SEM is very useful in studying ciliary structure and should be used more frequently (Toskala et al., 1994). The OTOTO-procedure on its own without any metal coating has been used for SEM studies of organ tissues. The advantage of this technique is that it should eliminate charging without having metals which can ob-scure the specimens (Malick et al., 1975; Murphy, 1978). Nonetheless, in the present study, while the preparation of a specimen by the OTOTO-method alone decreased electric charging, it did not eliminate it com-pletely, and a metal coating was required to obtain a better image quality. In conclusion, gold coating of specimens is advantageous in SEM of respiratory cilia compared to the OTOTO-method alone. A thickness of 43 nm result-ed in a homogenous gold layer, and with the sputter coater used in this study, this was obtained within one minute. This allowed the examination of specimen with a good image resolution and good contrast, but without any electric charging. Platinum , which has finer grains than pure gold, is used as a coating for high resolution SEM. However, platinum is very expensive, and for conventional SEM, which still is used in many laboratories, gold is the routine coating material when low resolution will suffice. References Afzelius BA (1981) The "immotile cilia syndrome" and other ciliary abnormalities induced by infection and injury. Am Rev Respir Dis 124: 107-109. Bell P , Lindroth M , Fredriksson B-A (1987) Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scan-ning transmi ssion electron microscopy. J Electron Microsc Tech 7: 49-159. Echlin P (1981) Recent advances in specimen coat-ing techniques. Scanning Electron Microsc 1981; I: 79-90. Herzon F (1981) Upper respiratory tract ciliary ultrastructural pathology. Ann Otol Rhino! Laryngol 90: 1-12. Jazbi B, Sayegh F (1978) Scanning and transmission electron microscopy of nasal mucosa . Otolaryngol Clin North Am 10: 167-175. Malick L, Wilson RB, Stetson D (1975) Modified thiocarbohydrazide procedure for scanning electron microscopy : Routine use for normal, pathological and experimental tissues. Stain Tech 50: 265-269. McDowell EM, Trump BF (1976) Histological fixa-tive suitable for diagnostic light and electron nucros-copy. Arch Pathol Lab Med 100: 405-414 . 306 Murphy JA (1978) Noncoating techniques to render biological specimens conductive. Scanning Electron Microsc 1978; II: 175-194. Rautiainen M, Collan Y, Klirjli J, Nuutinen J (1987) Artefacts in ultrastructure of respiratory cilia by various fixation procedures and different types of handling. ORL J Oto-Rhino-Laryngol 49: 193-198 . Rautiainen M, Collan Y, Nuutinen J, Afzelius BA (1990) Ciliary orientation in the "immotile cilia syn-drome ." Eur Arch Otorhinolaryngol 247: 100-103. Rautiainen M, Nuutinen J, Collan Y (1991) Short nasal respiratory cilia and impairment of the mucociliary function. Eur Arch Otorhinolaryngol 248: 271-274. Satir P ( 1961) Cilia. Scient Am 204 (2): 108-118. Toskala E , Rautiainen M, Nuutinen J (1994) Scan-ning and transmission electron microscopic findings in cilia from human nasal turbinate and sinus mucosa fol-lowing respiratory infection . Eur Arch Otorhinolaryngol 251: 76-79 . Discussion with Reviewers G.M. Roomans: Did you compare OTOTO-prepared , coated specimens to conventionally prepared (no OTOTO), coated specimens? Authors: Yes we did , but our experience is that OTOTO improve s image quality; however , it alone is not sufficient, and therefore , also needs coating . Specimens without OTOTO preparation also need a thicker gold layer. M. Lindroth: Are there osmotic artefacts that look like blebbing at the tips of the cilia? Authors: Blebbing s can be caused by osmotic changes, but that is still only a hypothetical explanation because the origin of blebbings is not clear. M. Lindroth: How can measurements of grain size be of importance? After all, there is a high degree of dec-oration artefact s due to the properties of gold; i.e. , the grain size varies a lot. Authors: Even though the size of the gold grains varied, clearly , the size was larger at longer coating times. This explains why cilia were thicker in SEM micrographs with longer coating times . 

Gold Coating of Respiratory Cilia for Scanning Electron Microscopy

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Gold Coating of Respiratory Cilia for Scanning Electron Microscopy

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