Distribution and movement of membrane‐associated platelet glycoproteins: Use of colloidal gold with correlative video‐enhanced light microscopy, low‐voltage high‐resolution scanning electron microscopy, and high‐voltage transmission electron microscopy

Abstract

Scanning electron microscopy (SEM), especially low-voltage (1 KeV) high-resolution SEM, can be used in conjunction with stereo pair high-voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface-related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to "prepare" cells for electron microscopy precludes observation in the living state. The use of several types of video-enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy. We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold-labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD-containing adhesive proteins, with gold-fibrinogen or gold-anti-IIb/IIIa. The initial binding and subsequent movement of gold-fibrinogen-IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low-voltage (1 KeV) high-resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position-in-transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).

Diphosphoryl Lipid A from Rhodobacter sphaeroides Blocks the Binding and Internalization of Lipopolysaccharide in RAW 264.7 Cells

“…Additionally, the LPS conjugation sites for fluorescent probes are unknown, and contaminants may be labeled. Colloidal gold is a very common inert label that has been used extensively for labeling different classes of ligands and proteins to study their interaction and localization within the cell by electron microscopy (23)(24)(25). In nearly all instances, the conjugation of colloidal gold particles with Abs, ligands, or enzymes does not change their binding or biological activity (25).…”

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

“…The drudgery of early CM methods Despite the long history of CM as a concept, researchers had to wait until the second half of the 1980s to see the first practical applications of such methods to large sets of samples in order to address specific research questions about biological fine structure (e.g., Albrecht et al 1989;Donnell et al 1988;Goodman and Albrecht 1987;Rieder and Bowser 1985;Wynford-Thomas et al 1986). The CM approaches reported at that time were characterized mainly Fig.…”

Organization of von Willebrand factor on surface-activated platelets.

“…192024 - 27 However, the function of bound fibrinogen is to serve as a bridge between GPIIb-IIIa and subendothelial constituents or other platelets. The stable receptor-ligand complexes formed between surfaces are incapable of movement.…”