Quantitative Analysis of Structure and Dynamics in AFM Images of Lipid Membranes

Abstract

AFM is now established as a powerful and direct technique for studying lipid membranes, and is highly complementary with other techniques. It is the only method for direct imaging and mechanical probing of lipid phase structure in a liquid environment down to the nanometer level. In order to understand the structure, function, and interactions of membranes at this level, we must be able to reliably and quantitatively measure the AFM images. Here we describe the methods used to process and analyze AFM images of phase-separated supported lipid bilayers . This initially takes a static approach, where we simply quantify the % of domain area, number of domains, and morphology, and quantify how many images must be taken to obtain reliable statistics. We then look at dynamics, describing the methods we use to study the nanometer scale motion of the domain perimeter as observed using Fast Scan AFM, and hence extract a quantitative line tension.

Appendix 1: ImageJ Macros

domain center macro:  {setSlice(1); for (i=1; i<=nSlices; i++) {      setSlice(i);   run("Measure");}} updateResults() X Coordinate Macro: {setSlice(1); for (i=1; i<=nSlices; i++) {      setSlice(i); doWand(200, 200);    getSelectionCoordinates(x, y);     for (j=0; j<x.length; j++){ setResult(getSliceNumber(), j, x[j]);}}}  updateResults() Y Coordinate Macro: {setSlice(1); for (i=1; i<=nSlices; i++) {      setSlice(i); doWand(200, 200);    getSelectionCoordinates(x, y);     for (j=0; j<y.length; j++){ setResult(getSliceNumber(), j, y[j]);}}}  updateResults()