FIB-SEM: An Additional Technique for Investigating Internal Structure of Pollen Walls

Abstract

Pollen grain morphology has been widely used in the classification of the Acanthaceae, where external pollen wall features have proved useful in determining relationships between taxa. Although detailed information has been accumulated using light microscopy, transmission electron microscopy and scanning electron microscopy (SEM) techniques, internal pollen wall features lack investigation and the techniques are cumbersome. A new technique involving precise cross sectioning or slicing of pollen grains at a selected position for examining wall ultrastructure, using a focused ion beam-scanning electron microscope (FIB-SEM), has been explored and promising results have been obtained. The FIB-SEM offers a good technique for reliable, high resolution, three-dimensional (3D) viewing of the internal structure of the pollen grain wall.

Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM

“…The FIB-SEM method was introduced in 2008 in the biological sciences for identifying synaptic contacts (Knott et al, 2008) and has been applied for 3-D analysis in animal tissues and cells (Ohta et al, 2012;Peddie and Collinson, 2014). Following its use in animal samples, FIB-SEM has also been used in plant tissues and cells (Lucas et al, 2012;House and Balkwill, 2013;Bhawana et al, 2014;Lütz-Meindl et al, 2016;Sviben et al, 2016); however, there are only a few reports showing 3-D reconstruction with segmentation: a root nodule of mung bean (Vigna radiata) (Lucas et al, 2012), green algae (Micrasterias and Nitella) (Lütz-Meindl et al, 2016) and coccolithophorid algae (Emiliana) (Sviben et al, 2016), and there has been no report for chlorenchyma in higher plants.…”

Exploring the interior of cuticles and compressions of fossil plants by FIB‐SEM milling and image microscopy

“…The FIB‐SEM technique has proven itself extremely useful in ultrastructural studies of various biological materials (Nalla et al ., ; House & Balkwill, and references therein), allowing for the in situ observation of previously unobserved features. Compared to other scanning electron or X‐ray volume imaging techniques, FIB‐SEM systems typically have very high imaging resolution and allow for direct observation of near‐subsurface structures.…”

“…The FIB-SEM technique has proven itself extremely useful in ultrastructural studies of various biological materials (Nalla et al, 2005;House & Balkwill, 2013 and references therein), Fig. 6.…”

“…FIB-SEM instruments were initially developed for the study of in situ nanostructures in industrial materials (Orloff et al, 2003). They eventually found applications in a great variety of research areas, such as lithography and nanodevice developments (Fujii & Kaito, 2005;Moberly et al, 2007;Volkert & Minor, 2007;Santschi et al, 2009;Javed et al, 2014), petrology and mineralogy (Heaney et al, 2001;Lee et al, 2003;Wirth, 2004;de Winter et al, 2009;Stokes & Hayles, 2009), biological materials (Drobne et al, 2005;Nalla et al, 2005;Orso, 2005;Milani et al, 2007;Moberly et al, 2007;de Winter et al, 2009;Marko, 2010;Grandfield & Engqvist, 2012;Rigort et al, 2012;Hoffmann et al, 2015;Wagenknecht et al, 2015), extant pollen grains and plant tissues (House & Balkwill, 2013;Bhawana et al, 2014), and the analysis of forensic data (Milani et al, 2012).…”

3D Plant cell architecture of Arabidopsis thaliana (Brassicaceae) using focused ion beam–scanning electron microscopy

“…FIB-SEM has been used on biological tissues in two ways: (1) a sample may be embedded, sectioned, and imaged using the FIB-SEM apparatus ( Heymann et al, 2006 ); or (2) in some cases, the FIB capability is used to produce high-quality sections for TEM imaging ( Wei et al, 2012 ). FIB-SEM has, to date, been used to produce three-dimensional renderings of animal cells ( Bushby et al, 2011 ), microorganisms ( Heymann et al, 2006 ;Wei et al, 2012 ), starch granules in plant cells ( Crumpton-Taylor et al, 2012 ), and pollen cells ( House and Balkwill, 2013 ).…”