Abstract
High resolution (≈1 nm lateral resolution) biological AFM imaging has been carried out almost exclusively using freshly cleaved mica as a specimen supporting surface, but mica suffers from a fundamental limitation that has hindered AFM's broader integration with many modern optical methods. Mica exhibits biaxial birefringence; indeed, this naturally occurring material is used commercially for constructing optical wave plates. In general, propagation through birefringent material alters the polarization state and bifurcates the propagation direction of light in a manner which varies with thickness. This makes it challenging to incorporate freshly cleaved mica substrates with modern optical methods, many of which employ highly focused and polarized laser beams passing through then specimen plane. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on common microscope cover glass following a straight-forward cleaning procedure. Direct comparison between data obtained on glass and on mica show no significant differences in AFM image fidelity. This work opens the door for combining high resolution biological AFM with powerful optical methods that require optically isotropic substrates such as ultra-stable1 and direct 3D AFM2. In turn, this capability should enable long timescale conformational dynamics measurements of membrane proteins in near-native conditions. 1.King, G.M., Carter, A.R., Churnside, A.B., Eberle, L.S. & Perkins, T.T. Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions. Nano Letters 9, 1451 (2009). 2.Sigdel, K.P., Grayer, J.S. & King, G.M. Three-dimensional atomic force microscopy: interaction force vector by direct observation of tip trajectory. Nano Lett 13, 5106-5111 (2013).
Highlights
854-Pos Board B634 Fibrinogenesis and Fibrinolysis Followed with Nano-Thrombelastography Tımea Feller, Miklos S.Z
Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on common microscope cover glass following a straight-forward cleaning procedure
Gold coated AFM tips were derivatized with long carbon chain thiols and thiols functionalized with carboxylic acid and amine head groups and used to measure the strength of interaction or adhesive force between the AFM tip and aggregate surface
Summary
854-Pos Board B634 Fibrinogenesis and Fibrinolysis Followed with Nano-Thrombelastography Tımea Feller, Miklos S.Z. 852-Pos Board B632 Glass: A Multi-Platform Specimen Supporting Substrate for Precision Single Molecule Studies of Membrane Proteins Nagaraju Chada, Krishna P. & King, G.M. Three-dimensional atomic force microscopy: interaction force vector by direct observation of tip trajectory.
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