Scaling exponents v, that describe the correlation between mean square end-to-end distances and contour lengths of macromolecules, were determined by statistical analysis of scanning force micrographs of single linear poly(2-vinylpyridine) and brush-like poly(butanoate-ethyl methacrylate)-graft-poly(n-butyl acrylate) macromolecules adsorbed on mica. Using an atmosphere-controlled scanning force microscope, single adsorbed molecules were collapsed and re-expanded upon being exposed to alcohol and water vapor, respectively. This manipulated collapse-unfolding was used to equilibrate the molecular structure/conformation. The in situ and real-time scanning force microscopy analysis allows the scientist to quantitatively characterize end-to-end distances and contour lengths of the molecules directly on the image and to observe differences in the spreading dynamics for the two types of macromolecules. A distinct difference has been observed between the expanded two-dimensional (2D) conformations of linear and brush-like polymer chains. Whereas a scaling exponent v of 0.73 was found for the expanded 2D conformation of the linear molecules, a v-value of 0.53 was determined for the expanded 2D conformation of the seemingly stiffer brush-like molecules. A theoretical explanation of the differences between the 2D conformations of brush-like and linear macromolecules is proposed here. (c) 2007 Wiley Periodicals, Inc.