NONCONTACT VERSUS CONTACT IMAGING - AN ATOMIC-FORCE MICROSCOPIC STUDYON HEPATIC ENDOTHELIAL-CELLS IN-VITRO

Liver sinusoidal endothelial cells (LEG) contain fenestrae, which cont rol the exchange of fluids, solutes, and particles between the sinusoi dal blood and the microvillous surface of the parenchymal cells. The s urface of LEC can be imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM images of LEC can be used t o study dynamic changes in fenestrae by comparing specimens fixed afte r different experimental treatments. In this article, we report the di fferent results obtained when contact (using a constant force) or nonc ontact (amplitude detection) imaging on the same cells was applied. Sp ecial attention was paid on the optimalization of the image acquisitio n of fenestrae, because quality SEM examinations of fenestrae have alr eady extensively been described. The following advances and conclusion s are presented here: 1) High-resolution imaging of slightly fixed LEC in fluid can be performed in noncontact AFM; 2) correct acquisition o f images of fenestrae with regard to their size (phi, +/- 200 nm) and shape (oval, without deformation) under liquid was possible with nonco ntact AFM, which was hitherto only feasible with fixed, dried, and coa ted LEC in contact AFM or SEM; 3) this mode of operation is more gentl e to cells than contact mode; 4) images of LEC obtained in noncontact mode are of higher quality and are devoid of smearing artefacts, promi nently present in contact-mode images; 5) it is of great importance to optimize feedback and scan parameters to obtain correct surface infor mation; and 6) LEC isolated and cultured by our method are physiologic ally responsive and represent an ideal object for AFM studies, because the cells are thin, smooth, and well attached to the culture substrat e and show dynamic fine structural details, such as fenestrae and coat ed pits, which cannot be seen by light microscopy. (C) 1997 John Wiley & Sons, Inc.