Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.
High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.
Eppell
S J
SJ
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106-7207.
Simmons
S R
SR
Albrecht
R M
RM
Marchant
R E
RE
D001792:Blood Platelets / Q000648:ultrastructure*; D002462:Cell Membrane / Q000648:ultrastructure; D005340:Fibrinogen / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D010980:Platelet Membrane Glycoproteins / Q000648:ultrastructure*
D010980:Platelet Membrane Glycoproteins; D005340:Fibrinogen