A Quick and Simple Technique for Orientating Diatoms for SEM and Light Microscopy Mark Webber1,

* and Elaine Humphrey2 1455-2555 Cook Rd., Galiano Island, British Columbia, Canada, V0N1P0 2Advanced Microscope Facility, University of Victoria, PO Box 1700 STN CSC, Victoria, British Columbia,

VSW 2Y2 Canada *

Abstract: A method was developed to expedite the imaging of diatoms by optimizing their orientation. This quick technique puts grooves into a SEM stub so that as the diatoms are laid down they either land on the flat surface or in a variety of positions in the grooves, thereby reducing time required to capture taxonomically critical features.

Keywords: Diatoms, imaging, orientation method, scanning electron microscopy, light microscopy

Introduction Diatoms are unicellular eukaryotic microalgae that play

important ecological roles on a global scale. Diatoms are responsible for 20% of global carbon fixation and 40% of marine primary productivity. Tus they are major contribu- tors to climate change processes and form a substantial basis of the marine food web [1]. Over the last few decades, the first author on this article, Mark Webber, has collected samples of fresh water and marine diatoms from all over the world for taxonomic and ecological research. To identify diatoms to the genera or species level has required the development of novel and quick methods for orientating specimens for scan- ning electron microscopy (SEM) imaging, especially when a limited number of specimens are available. Taxonomic and other critical information can only be derived from variable orientations. When preparing diatoms for micros-

copy they usually settle on slides, dishes, and stubs in either the valve or girdle views, which is seldom an optimal orientation for imaging ( Figure 1) or taxonomy. It can take considerable time to find cells in a favorable view for the requirements of the research. Tilting the scanning electron microscope (SEM) stage does not always provide a suf- ficiently steep angle for the optimal imag- ing of critical features. Similarly, a light microscope (LM) examination of a limited number of specimens can leave important features obscured from view. Occasionally, with enough cells, one is fortunate that a cell has settled in an advantageous incli- nation. To enhance the chances of optimal positioning occurring, we present a proce- dure of placing grooves on SEM stubs with


a diamond scribe at a steep enough angle for the viewing of diatoms. Tis oſten simplifies the SEM tilting and rotation procedure and minimizes the requirement to make time- consuming adjustments on the instrument. Te technique is also excellent for stereo microscopy. Tis method was inspired by a more elaborate method described by MacGilli- vary and Ehrman [2]. Tis method is fast, simple, and inex- pensive and can be used by almost any facility for randomly orienting diatoms. Adding diatoms to SEM stubs can follow a number of

procedures, most of which involve pipetting whole cells cleaned of organics onto glass coverslips, carbon stickies, filter papers, or membrane filters, which are then attached to the stub [3–8]. Alternatively, and more precisely, diatoms can be added directly to an aluminum stub [4]. For SEM examination of diatoms, prepared aluminum stubs are nor- mally sputter coated with a sufficient thickness (oſten 8–15 nm) of gold or gold/palladium (Au/Pd) alloy to increase the signal to noise ratio and to suppress charging.

Materials and Methods Making grooves in SEM stubs. Initially, a 12.7 mm alu- minum SEM stub was finely sanded of all roughness using

Figure 1: Images illustrating the limitations of examining diatoms lying flat on an un-grooved alu- minum stub. (a) A pennate freshwater diatom naturally oriented onto the valve face. The girdle mor- phology is not clearly visible even with fully tilting the SEM stage. (b) A colony of the marine diatom Skeletonema sp. in typical girdle facing orientation. Even with tilting of the stage, a clear view of the valve cannot be obtained. Bars in (a) = 5 μm, (b) = 10 μm.

doi:10.1017/S155192951900124X • 2020 January

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