Practical considerations for feature assignment in high-speed AFM of live cell membranes

Abstract

High speed atomic force microscopy (HS-AFM) is, in principle, capable of yielding nanometer level detail about the surface of static structures. However, for highly dynamic samples HS-AFM may struggle with the correct feature assignment both within and between frames. Feature assignment in HS-AFM is dependent on (i) the intrinsic sampling rate, and (ii) the rate of internal redistribution of the sample. Whilst the first quantity (the sampling rate) is defined by the device parameters, the second quantity is frequently unknown, and is often the desired target of the measurement. This work examines how, even in the absence of gross cell morphological change, the rapid dynamics of living cell membranes, may impose an upper spatial limit to the frame-to-frame assignment of cell micro-topography and other related properties (such as local elasticity) whose motion may be described stochastically. Such a practical maximum may prove useful in the setup of HS-AFM experiments involving dynamic surfaces thereby facilitating selection of the most parsimonious relationship between observation size, image pixilation and sampling rates. To assist with performing the described calculations a graphical user interface-based software package called HS-AFM UGOKU is made freely available.

Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action

“…It is important to consider that the data for all pixels in an image are not acquired at the same time, but at different times during the lateral scanning of the sample stage; this is called data asynchronicity. Accordingly, one can easily imagine that a slow scan of a fast-moving object would result in a blurred image of the object, resulting in low spatial resolution [ 128 , 129 ]. Biological molecules exhibit translational diffusion, rotational diffusion and conformational changes related to their functions, all of which affect the appearance of observed objects ( Fig.…”