A Low Voltage Scanning Electron Microscopy Study ...

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Copyright © 2012 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Advanced Microscopy Research Vol. 7, 170–175, 2012

A Low Voltage Scanning Electron Microscopy Study of the Theca of Marine Dinoflagellates Yuri B. Okolodkov1 ∗ and Dora A. Huerta-Quintanilla2 1 Laboratorio de Botánica Marina y Planctología, Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana, Calle Hidalgo 617, Col. Río Jamapa, Boca del Río, 94290, Veracruz, México 2 Laboratorio Nacional para el Estudio de Nano y Biomateriales, Departamento de Física Aplicada, Centro de Investigación y Estudios Avanzados–Instituto Politécnico Nacional, Unidad Mérida, Carretera Antigua a Progreso Km 6, 97310, Mérida, Yucatán, México

A low voltage scanning electron microscopy regime is proposed for routine examination of the non-metalized thecae of marine dinoflagellates, a JEOL Delivered byusing Ingenta to:JSM-7600F Field Emission scanning electron microscope with a Schottky field-emission electron gun. The selected options were as Vladimir Basiuk follows: voltage 1–1.5 kV (sometimes up to 2 kV), working distance 15–20 mm, gentle beam, IP : 132.248.29.219 secondary electron detector, probe current 1–2 × 10−10 A, and specimen tilt 0 to 20 . Although the Fri, 11 2013 15:45:43 micrographs taken at 5 kV were of higher Jan quality, satisfactory resolution, contrast and magnification in the micrographs taken at 1.0–1.2 kV were obtained. Minute details of the dinoflagellate theca such as the apical pore complex, the sulcal area, the hypothecal pore, sutures and thecal ornamentation, that are considered among the most important diagnostic features, were observed at up to 11,000×. This indicates that images of thecate dinoflagellates can be obtained much less expensively and faster when necessary, due to a more rapid procedure that includes only washing and dehydration and does not include sputter coating with a metal. A long working distance helps the observations by increasing the depth of focus.

Keywords: Dinoflagellates, Identification, Low Voltage Scanning Electron Microscopy, Morphology, SEM, Taxonomy, Theca.

1. INTRODUCTION Scanning electron microscopy (SEM) is widely used in studies on the morphology of dinoflagellates, a group of mainly marine planktonic unicellular organisms that are known as causative agents of the so-called red tides or harmful algal blooms and bioluminescence in the sea. The taxonomy and systematics of dinoflagellates is based on the morphological characteristics of the thecal or amphiesmal plates, which are the visible part of the theca1 or amphiesma.2 3 Dinoflagellates are conventionally divided into thecate and athecate (unarmored or naked) forms, and the thecate ones have a theca visible by means of light microscopy (LM). The plates consist of cellulose, and they are located inside alveoli (sort of enclosed cisterns) under the external plasmatic membrane. The cell cover of dinoflagellates may contain dinosporin, a complex aromatic polymer similar to that in terrestrial plants but chemically different from it.4 More recent data indicate that ∗

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dinoflagellate cyst walls are composed of a suite of chemically different biopolymers that can be species specific.5 The thecal plates are closely attached to each other and form a continuous surface. The contact zones are called sutures, and are also known as intercalary bands or growth zones, due to the additional deposit of cellulose while the cell volume increases. In some species growth occurs only along one margin of the thecal plate; in others it occurs along the whole perimeter. The theca of dinoflagellates is characterized by tiny pores, under which trichocysts (offense or defense organelles) that have a hairlike filament capable of being ejected are situated in most genera and species. For a number of reasons, the LM remains the main tool for identification of dinoflagellate species. Balech6 stressed that there is no morphological detail of the dinoflagellate theca shown by SEM that had not been detected by those who used LM; however, he believed that some new details of the apical pore complex (APC), sulcal or cingular plates could be revealed by SEM. In addition to these parts of the theca, one should add the theca ornamentation and the size and shape of the trichocyst pores. 2156-7573/2012/7/170/006

doi:10.1166/jamr.2012.1111

Okolodkov and Huerta-Quintanilla

A Low Voltage Scanning Electron Microscopy Study of the Theca of Marine Dinoflagellates

(5) coating the cells with a metal (usually with gold, gold-palladium, palladium, sometimes with aluminum or copper); and (5) viewing with SEM in a vacuum or directly on a stub in the case of environmental scanning electron microscopes.7–12 Protocols may vary, especially when fragile unarmored species are under study, and may include post-fixation with osmium tetroxide. Since the early 1980s, hexamethyldisilazane (HMDS), an organosilicon compound, has been used in biological, medical and biomedical sciences as a method of chemical drying of dehydrated cells.13–20

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Fig. 1. Scanning electron micrographs taken of non-metalized specimens at 1.0–1.2 kV, lower secondary electron detector (LEI), with gentle beam (GB). (a) Prorocentrum rotundatum J. Schill., right valve view; 1.0 kV, 2300×, working distance (WD) = 19 mm. (b) Dinophysis caudata Saville-Kent var. pedunculata (Schmidt) Schröder, right side view; 1.2 kV, 1300×, WD = 21 mm. (c) Gonyaulax polygramma F. Stein, ventral-apical view; 1.0 kV, 1800×, WD = 18 mm. (d) Ceratocorys horrida F. Stein, antapical view; 1.2 kV, 1000×, WD = 20 mm. (e), (f) Diplopsalopsis bomba (F. Stein) Dodge: (e)—apical view of the cell (epitheca), 1.0 kV, 2000×, WD = 19 mm; (f)—the apical pore complex (APC) bordered by three apical plates (1 –3 ), 1.0 kV, 9000×, WD = 21 mm. Scale bars: 10 m in (a)–(e), 1 m in (f).

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Protocols for SEM observations usually include (1) washing the cells from fixative agents (paraformaldehyde, glutaraldehyde, Lugol’s solution or ethanol) and dissolved marine salts in distilled, deionized or nanopure water or cacodylate or phosphate buffer; (2) dehydration of specimens in a graded ethanol or acetone series; (3) replacement of 100% ethanol or acetone with liquid carbon dioxide by critical-point drying or sublimation drying in order to prevent cell shape distortion, or sometimes just air-drying; (4) mounting them on aluminum or copper stubs;

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During the 1980s, low-voltage SEM (1 to 4 kV) became routine in the semiconductor industry and then in both zoology and botany.21–23 In comparison to the high-voltage regime, more detailed information on the surface morphology and sometimes on the surface chemistry can be obtained from low-voltage images.24 The aim of this paper is to show that it is possible to study non-metalized (uncoated) and non-critical-pointdried biological samples in a high-resolution field emission SEM microscope at low accelerating voltages, using the r-filter and Gentle Beam facilities recently introduced in these types of microscopes.


2. MATERIALS AND METHODS For our study, two phytoplankton samples collected on 13 March 2007 were selected out of about a hundred samples taken in the National Park Sistema Arrecifal Veracruzana, the southwestern Gulf of Mexico because of (1) the almost total absence of detritus and diatoms that are frequently much more abundant in the coastal zone and therefore impede SEM observation and (2) the relatively high morphological and species diversity of dinoflagellates observed with an Olympus CKX41 inverted microscope in a 1-ml Sedgwick-Rafter counting chamber. The samples were rinsed in distilled water four times in Eppendorf


Fig. 2. Scanning electron micrographs taken of non-metalized specimens at 1.0–1.2 kV, LEI, with GB. (a) Blepharocysta splendor-maris (Ehrenb.) Ehrenb., ventral view; 1.0 kV, 1900×, WD = 19 mm. (b) Protoperidinium claudicans (Paulsen) Balech, ventral view; 1.2 kV, 1400×, WD = 21 mm. (c) Protoperidinium oviforme (P. A. Dang.) Balech, ventral view; 1.0 kV, 1700×, WD = 19 mm. (d), (e) Protoperidinium concinnum Faust: (d)—ventral view of the cell, 1.0 kV, 950×, WD = 19 mm; (e)—the apex without the APC, 1.0 kV, 10,000×, WD = 19 mm. (f) Protoperidinium venustum (Matzen.) Balech, ventral view, 1.0 kV, 1200×, WD = 19 mm. Scale bars: 10 m in (a)–(d) and (f), 1 m in (e).

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study (Figs. 1–3). For comparison, we took micrographs of the same species (of the same non-metalized cell) at different voltages with and without the gentle beam and also of another cell metalized with a 20–25 nm layer of goldpalladium (Fig. 4). The images obtained were not edited in any way. We used a JSM-7600F Field Emission SEM with a Schottky field-emission electron gun, which easily gives a stable and high probe current, as compared with traditional cold field-emission electron guns.25 The Gentle Beam method slows down the electron beam just in front of the specimen, reducing the electron beam energy to


Fig. 3. Scanning electron micrographs taken of non-metalized specimens at 1.0–1.2 kV. (a), (b) Protoperidinium divergens (Ehrenb.) Balech: (a)— apical view of the cell (epitheca), 1.2 kV, 1200×, WD = 21 mm; (b)—the apical pore complex, 1.2 kV, 11,000×, WD = 21 mm. (c), (d) Protoperidinium pellucidum Bergh: (c)—ventral view of the cell, 1.0 kV, 2000×, WD = 21 mm; (d)—a fragment of the theca showing the first postcingular plate (1 ) with the hypothecal pore (arrow head), and the anterior part of the sulcus: the anterior sulcal (S.a.) and right sulcal (S.d.) plates, and the transitional plate (t). (e), (f) Protoperidinium divergens (Ehrenb.) Balech: (e)—ventral view of the cell, 1.0 kV, 1500×, WD = 19 mm; (f)—sulcal area, 1.0 kV, 3300×, WD = 19 mm. Scale bars: 10 m in (a)–(c) and (e), 1 m in Figures 2(e) and (f).

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1.5-ml vials (water was decanted every 1.5–2 hours). Then they were dehydrated in a grade ethanol series (30, 50, 70, 90% and twice in 100%) and stirred before depositing 3 to 4 drops of the cell suspension on 0.5’ diameter aluminium stubs with a plastic 1-mm pipette and air drying. After 20–30 min. the stubs were put into a JEOL JSM-7600F STEM at the Center for Research and Advanced Studies of the National Polytechnic Institute in Merida, Yucatan, Mexico. To take microphotographs in the low-voltage regime of a quality satisfactory for distinguishing particular morphological features allowing us to identify species correctly was the main purpose of this

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diminish specimen damage and charging, while keeping the electron probe diameter small to ensure high resolution. This mode provides high-resolution images whose quality is as high as those of higher accelerating voltages, even though the electron energies close to the sample are as low as 100 V to 3 kV. This way, the electron beam hits the sample without damaging the information from the specimen surface.26 After experimenting with HMDS and its application to marine dinoflagellates and their observation with the same SEM over the course of three months using 5 kV, we decided to change to a 1 kV regime. The selected options were as follows: voltage 1 kV (sometimes 1.2 kV), working distance about 20 mm (although sometimes we also


reduced it to 15 mm), gentle beam (GB), secondary electron detector (LEI, or lower secondary electron image), probe current 1–2 × 10−10 A, specimen tilt 0 to 20 .

3. RESULTS AND DISCUSSION The micrograph taken at 5 kV (Fig. 4(f)) clearly demonstrated a higher quality, especially in resolution. However, satisfactory resolution, contrast and magnification in the micrographs taken at 1.0–1.2 kV must be noted (Figs. 1–3, 4(a)–(e)). This implies a wide possibility for obtaining images of thecate dinoflagellates much less expensively and faster when it is necessary, due to a more rapid procedure that includes only washing and dehydration and does


Fig. 4. Scanning electron micrographs of Protoperidinium concinnum taken of non-metalized (a)–(e) and a metalized specimen (f) at different voltages, with gentle beam (GB) or without it (SEM), WD = 15 mm, magnification 1000×: (a)—1.0 kV, LEI, GB. (b)—1.5 kV, LEI, GB. (c)—2.0 kV, LEI, GB. (d)—2.0 kV, LEI, SEM. (e)—5.0 kV, LEI, SEM. (f)—5.0 kV, LEI, SEM. Scale bar: 10 m.

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4. CONCLUSIONS The Gentle Beam method in the low-voltage regime (< 5 kV) provides high-resolution images whose quality is as high as those of higher accelerating voltages. The described method applied to uncoated specimens can be a cheaper and more rapid option for routine studies of thecate dinoflagellates. A voltage of 1.2 kV or higher is recommended during observations with the JEOL JSM7600F. A comparatively longer working distance (15 to 20 mm) and LEI help the specimens to avoid charging. Acknowledgments: This study was partially supported by FOMIX-YUC No. 108160 (2008) and CONACYT LAB-2009-01 No. 123913 (2009) projects led by Patricia Quintana-Owen (CINVESTAV-IPN, Mérida, Mexico). We also appreciate the financial support of the Universidad Veracruzana and the Departamento de Recursos del Mar (CINVESTAV-IPN) given to YBO to perform a sabbatical year in CINVESTAV-IPN in Merida

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Received: 24 September 2012. Accepted: 24 October 2012.

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and the hospitality of the host scientist Jorge A. Herreranot include sputter coating with a metal. On the whole, Silveira and Fany del C. Merino-Virgilio in the Labothe general views of the cells looked rather good, as did ratory of Marine Plankton. María C. Ramírez-Jáuregui the close-ups of the APC/apex, the sulcal area or parts from ICMyL-UNAM, Mexico City, kindly helped us with of the theca at up to 11,000× (Figs. 1(f), 2(e), 3(b)). literature, and Marcia M. Gowing from Seattle improved Nevertheless, we noted a pronounced difference between the writing style. 1.0 kV and 1.2 kV shown by the more distinct trichocyst pores, so 1.2 kV or higher voltage is recommended during observations with the JEOL JSM-7600F (Figs. 1(b), (d), References and Notes 2(b), 3(a), (b), 4(b)). 1. K. A. Steidinger and E. R. Cox, Free-Living Dinoflagellates, edited The effect of the r-filter secondary-electron detection by E. R. Cox, Phytoflagellates, Elsevier/North Holland, New York system is to remove very low energy secondary-electrons. (1980), pp. 407–432. These electrons are the most affected by the charging of 2. A. R. Loeblich, III, Proceedings of the North American Paleontological Convention, Part G, Chicago, USA (1970), September 1969. the sample, and therefore removing them implies, in a way, 3. L. C. Morrill and A. R. Loeblich, III, Intern. Rev. Cytol. 82, 151 ignoring the charging of the samples due to the fact that (1983). they are non-conducting.27 4. J. P. Kokinos, T. I. Eglinton, M. Goni, J. J. Boon, P. A. Martoglio, It should be noted that in the approach proposed here and D. A. Anderson, Org. Geochem. 28, 265 (1998). 5. K. Bogus, G. Versteegh, I. Harding, U. Holzwarth, and K. Zonneveld, we used a long working distance (15–20 mm). These long Proceedings of the 44th Annual Meeting 2011, American Associdistances help the observation process by increasing the ation of Stratigraphic Palynologists, Southampton, UK, September depth of focus. The disadvantages usually related to long (2011). to: Delivered by Ingenta working distances (increased probe size—increased aber6. E. Balech, Neotrópica 21, 20 (1975). Vladimir Basiuk rations) are not large enough to cancel the better image 7. A. Boltovskoy, Limnobios 1, 1 (1976). IP : 132.248.29.219 8. J. D. Dodge and R. D. Saunders, Bot. Mar. 28, 99 (1985). quality obtained. Also, LEI was the preferred detector Fri, 11 Jan 2013 15:45:43 9. J. D. Donde and S. Toriumi, Bot. Mar. 36, 145 (1993). for these images because it is less susceptible to sample 10. A. Boltovskoy, Manual de Métodos Ficológicos, edited by K. Alveal, charging. During our SEM observations radiation damM. E. Ferrario, E. C. Oliveira, and E. Sar, Universidad de Concepage was not a problem, although charging sometimes was ción, Concepción, Chile (1995), pp. 119–138. 11. K. A. Steidinger, J. H. Landsberg, E. W. Truby, and B. A. Blakesley, (Figs. 4(b)–(d)).