Nanostructure of the Diatom Frustule as Revealed by Atomic Force and Scanning Electron Microscopy

Abstract

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of crosslinking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in crosssection revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 Ϯ 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 Ϯ 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 Ϯ 1.4 nm and that silica particles from the girdle bands were 38.1 Ϯ 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.

Application of AFM in understanding biomineral formation in diatoms

“…Images from height data indicated that the silica was loosely packed, suggesting the material was porous. The average diameter of silica particles was consistent in several areas of the valve and in the girdle bands at 45 and 40 nm, respectively [8]. In cross-sections of H. amphioxys, similar particulate silica was seen, with average diameter of 37 or 38 nm in the valves and girdle bands, respectively.…”

“…Scanning of the AFM tip across the surface enabled removal of the mucilage, revealing silica surface features. The surface was quite smooth, although high resolution revealed some small-scale granularity, and other larger-scale features were superimposed on the basic structure [8]. Force curves of the surface showed that the material was noncompressible.…”

“…The first AFM examinations of diatom silica were by Almqvist et al [1] on the pennate diatom, Navicula pelliculosa, and Crawford, et al [8] on the pennates, Pinnularia viridis and Hantzschia amphioxys. These are all freshwater diatom species; N. pelliculosa is small (valve is 3×7 μm), whereas P. viridis and H. amphioxys are large (valves are about 100× 25 and 75×16 μm, respectively).…”

“…Examination of P. viridis focused on imaging and mechanical property measurements of extracellular mucilage covering the silica and the silica itself [8]. Scanning of the AFM tip across the surface enabled removal of the mucilage, revealing silica surface features.…”

“…There are two general morphological types of diatoms based on their valve structures: the centrics, which have rotational symmetry, and the pennates, which have bilateral symmetry [39]. Diatom silica is made from the soluble precursor form of silicic acid, which is transported into the cell [19,20] and into a specialized membranebound compartment called the silica deposition vesicle [8,11,36,44], or SDV, where it is polymerized to form the mineral. Once the mineral structure of the valve or girdle band is completed, it is moved in its entirety across the plasma membrane (exocytosed), where it becomes an exoskeleton surrounding the cell.…”