Extracellular matrix assembly in diatoms (Bacillariophyceae).: IV.: Ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high-resolution visualization of surfaces using cryo-field emission SEM (cryo-FESEM) and preservation for TEM observation of thin sections by high-pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge-frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo-FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell-substratum and cell-cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe-associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo-FESEM revealed a sheet-like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra- and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron-opaque layer organized as a sheath at the stalk periphery.