I'm planning to characterize my liposome vesicles using Atomic Force Microscopy (AFM). Most research papers recommend using freshly cleaved mica discs due to their atomically flat and clean surfaces, which seem ideal for imaging soft nanoscale structures like liposomes.
However, in my lab we currently have only glass discs available. What are the key differences between mica and glass discs in terms of AFM imaging performance? Could using glass significantly affect the resolution or stability of liposome observation?
If anyone has experience using glass substrates for liposome imaging in AFM, I’d really appreciate your insights or suggestions.
In deciding whether glass can replace mica for AFM characterization of liposomes, the choice depends on weighing resolution needs, sample characteristics, and experimental convenience. Mica is the gold standard because it has an atomically flat surface (≤0.1 nm roughness), which is essential for resolving nanoscale features such as small liposomes (200 nm) if well prepared. Both substrates are negatively charged at neutral pH, but adhesion can be increased for liposomes by functionalizing glass with APTES (to generate a positively charged amine surface) or poly-L-lysine, as in treatments for mica with divalent cations. Tapping mode imaging is necessary in glass to reduce lateral forces due to its rough topography, and cleanliness should be emphasized in sample preparation (e.g., piranha solution, plasma treatment) to avoid contaminants. Although mica allows greater resolution, glass is possible with modifications such as longer incubation periods, greater concentrations of liposomes, and liquid imaging to retain structural stability. For initial studies or applications where ultra-high resolution is non-essential, glass provides a working alternative, particularly if combined with surface treatments and stringent protocols. Researchers with goals towards fine nanoscale morphology or smaller liposomes must still prefer mica or consider intermediate alternatives such as silicon wafers with oxide layers. Finally, the selection depends on experimental objectives, liposome size, and availability of resources, with glass functioning as a viable alternative under optimal conditions.
I do not know enough abput your question/proposed method to give a full authoritative answer. However many years ago (~1976 ) while I was on sabbatical in the, then, Department of Ultrastructure Research at the (previous) John Innes Institute (Norwich). Dr Robert Horne showed me a method used to arrange ordered displays of tobacco mosaic and other viruses on the surface of freshly cleaved mica. I was seeking to negatively-stain filaments (of 'P-protein'?) that I found in phloem exudate from the cut ends of petioles of tobacco plants.
Viruse can arrange thenselves in orderly arrays on the new suraces of freshly cleaved MICA. I understand that they do so because the newly-cleaved surface developes an electric charge, or charges. Presumably, glass would not do that. Those charges on the mica might be essential to your method? Glass surfaces might not provide them? Apologies for lack of futher detail/references!
Best wishes, Richard P.C. Johnson (via Researchgate) 09/05/ 2025
Thank you very much Richard P. C. Johnson for sharing your experience!, your point helped deepen my understanding of surface functionality, and I truly appreciate your support!
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