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 long ipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external envi ronment. To preserve details of the highly hydrophilic ECM in the native st ate and to preserve, with a high degree of fidelity, the intracellular stru ctures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high-resolution visualization of su rfaces using cryo-field emission SEM (cryo-FESEM) and preservation for TEM observation of thin sections by high-pressure freezing (HPF) and freeze sub stitution (FS). The extracellular structures of diatoms plunge-frozen in li quid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those obse rved with light microscopy. Cryo-FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell-substratum and cel l-cell associations and revealed details of cell attachment processes not v isible using other SEM techniques or light microscopy. The layer of ECM coa ting the frustule and entirely encapsulating cells of A. longipes and C. ci stula was shown to have a significant role in initial cell adhesion and sub sequent interaction with the environment. Trails of raphe-associated ECM, g enerated 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 pres ervation of intra- and extracellular structures comparable to previous repo rts for animals and higher plants and revealed several organelles not descr ibed 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 th rough frustule pores during stalk production was closely related to the dia totepum. The stalks of A. longipes consist of highly organized, multilayere d, fine fibrillar materials with an electron-opaque layer organized as a sh eath at the stalk periphery.