Microscopy 101

within 10–15 min. Centrifuging quickens the process. A more detailed review of cleaning methods is in preparation. Te cells were then re-suspended in

Figure 4: (a) The centric diatom Biddulphia antediluviana, oriented in both valve and girdle views (stereo micrograph). (b) SEM of B. antediluviana in a groove, girdle view. Bars in (a) = 75 μm, in (b) = 10 μm.

either DDW or an ethanol/DDW mix- ture (50–80% ethanol) and micro- pipetted directly onto the aluminum stubs. Te advantage of using the ethanol mixture was rapid evaporation to reduce drying time of the samples before sputter coating, and movement of the cells as the ethanol dried allowed for a greater chance of ran- dom orientation and less clumping and pil- ing of cells, one on top of another. It also effectively reduces drying time of the stub before SEM observation, oſten eliminating the use of an oven. Aſter air-drying (either overnight or for 10–15 min.

in a 60°C

Figure 5: (a–b) The marine diatom Skeletonema sp. in a groove in a valve-up orientation, also showing the valve view. Black arrow showing the width of the groove is 176 μm. Bars in (a) = 20 μm, in (b) = 10 μm.

oven), the cell density and distribution was checked with a stereo microscope to sys- tematically observe the number of cells and to make sure the necessary orientations obtained for SEM imaging were available. Tere needs to be enough cells as a single layer but not overlapping each other. Once accepted, samples were sputter coated with 8–15 nm Au/Pd for viewing on the SEM. Imaging. All SEM images were taken with the Hitachi S-4800 FESEM at


Advanced Microscope Facility, Univer- sity of Victoria, BC Canada, except Figure 1a, which was imaged on a JEOL 6700F FESEM, Microscopy Otago, University of Otago, Dunedin, New Zealand. LM images were taken with a Leica S8APO Stereo- microscope with a Canon Rebel EOS T1i digital camera with a Martin Microscopes MM SLR adaptor.

Figure 6: (a) The diatom Cocconeis sp. laying on a groove, valve face up. (b) Internal view of a Cocconeis sp. valve. Minimal tilting of the SEM stage is required. Bars = 20 μm.

Results Producing a gridded pattern on

the stub surface increased the number of grooves per stub, thereby increasing favorable orientations available for imag- ing (Figures 3–7). Tis method allowed for many diatoms to randomly orient so surfaces and features, especially internal structures that are normally obscured using a flat surface, were advantageously viewed by SEM and LM. Less


time was required to find cells in differ- ent orientations, and less time was spent rotating and tilting the SEM stage, which oſten meant

less need to adjust beam alignment if the stage had been tilted.

Figure 7: (a) A centric diatom, Thalassiosira sp., oriented so the girdle is in view: a non-typical orientation. (b) The marine diatom Paralia sulcata, lying in a groove (double arrow), both in girdle and valve orientations. Both are images of cleaned diatoms, taken with a Leica S8APO stereo microscope using side lighting. Bar = 100 μm.


Conclusions Creating grooves in a grid pattern on an SEM stub in this manner is an effective, • 2020 January

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