Microscopy101
Out of the Blue and into the Black: Preparation, Mounting, and Image Rendering of Complex, Chorate Dinoflagellate Cysts for Scanning Electron Microscopy Sandy M.S. McLachlan1
* and Elaine C. Humphrey2
1School of Earth and Ocean Sciences, University of Victoria, PO Box 1700, STN CSC, V8W 2Y2, Victoria, BC, Canada 2Advanced Microscopy Facility, Department of Mechanical Engineering, University of Victoria, PO Box 1700, STN CSC,
V8W 2Y2, Victoria, BC, Canada *
sandymcl@uvic.ca
Abstract: We describe an experimental approach for achieving an optimal black background for scanning electron photomicrographs of small samples with elaborate and intricate structures. Specimens of
the highly ornate,
background would otherwise pose a significant challenge for digital removal, makes them an ideal sample for this study.
66-million-year-old chorate dinoflagellate
cyst species Cannosphaeropsis franciscana were selected as the subject of this study. Photomicrographs collected following standard aluminum stub surface placement were compared to those taken of specimens mounted using a novel pin-and-pedestal method. This simplistic mounting technique minimizes the need for post-production image editing and extraneous background removal.
Keywords: dinoflagellate cysts, microfossils, SEM, specimen preparation, image post-production
Introduction “And blood-black nothingness began to spin. A system of cells interlinked, within cells
interlinked within one stem. And dreadfully distinct against the dark, a tall white fountain played.” —Vladimir Nabokov, Pale Fire [1] Clear presentation of data is the foundation of effective
science communication. A classic image is that represented by the remarkable grayscale resolution of photomicrographs rendered through scanning electron microscopy (SEM). No greater is the stark definition revealed by this practice than that which can be perceived by the contrast between the lighter foreground features of the subject and a darker background. Scanning electron image rendering has come a long way toward achieving a dark background since the use of film [2]. However, attempts to engineer a sample holder suitable for the generation of a uniform black background with a suitable gray scale for foreground features have been limited [3], with emphasis in recent years having shiſted to the now ubiquitous use of image editing soſtware. A pin-and-pedestal method is proposed as a means for collecting an immaculate dark background to minimize the need for post-production image editing. Specimens of Cannosphaeropsis franciscana, a fossil species of organic-walled chorate dinoflagellate cyst of Late Cretaceous– early Paleocene age recovered from the sedimentary rocks of the Oyster Bay Formation on Vancouver Island, British Columbia, Canada [4], were selected as the subjects for this study. Teir complex ornate structure, around which an undesirable bitmap
38 doi:10.1017/S1551929521001334
Materials and Methods Specimen recovery and preparation. Te dinoflagellate
cysts were prepared using a standardized extraction procedure [5]. Rock samples with a matrix of ∼2 cm3
were placed in 50ml
polypropylene test tubes and treated repeatedly with 10% room- temperature hydrochloric acid (HCl) to dissolve any carbonates. Following HCl
treatment, the samples were placed in 48% interlinked, within cells
room-temperature hydrofluoric acid (HF) where they were leſt to sit with daily stirring for an average of two weeks (Figure 1). Each sample then underwent one round of HCl and two rinses of reverse osmosis water to remove any HF residue before final sieving through 120μm and 15μm Nitex nylon mesh to remove remaining particles. Te samples were subjected to one minute of ultrasonic agitation to dislodge any minute sediment particles from the dinoflagellate cysts [6,7] and centrifuged at 3600rpm. Specimens prepared for SEM underwent up to an additional four minutes of ultrasonic agitation following sieving. A series of specimens were transferred to the surface of aluminum SEM stubs with a micropipette for standard presentation (Figure 2), while others were selected for mounting using the novel pin- and-pedestal technique described herein. Te creation of a pin-and-pedestal is fairly straightforward
and begins with the use of wire cutters to remove the ∼1mm end of an entomological pin, which is optimal for its narrow, machined point. Te pin tip is then gently touched to a carbon sticker to retain a minute bolus of sticker to hold the specimen in place. A small portion of carbon sticker is affixed to the stub surface into which the cut pin end is then embedded at a 90° angle and reinforced with Paraloid B-72 adhesive as shown in Figure 3A. A dry dinoflagellate cyst specimen is then touched with a pin and transferred from a slide on the stage of a transmitted light microscope to the pin tip mounted vertically on the stub with the aid of a reflected light microscope. Once mounting was completed, the SEM stubs were coated with either gold or a gold/palladium mix using either an Edwards S150B or Anatech Hummer VI sputter coater for a thickness of 10–50nm.
www.microscopy-today.com • 2021 November
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