Comb copolymers comprising a poly(methyl methacrylate) (PMMA) backbone and short, poly(ethylene oxide) (PEO) side chains, PMMA-g-PEO, have been proposed to self-organize at the polymer/water interface, resulting in the quasi-two-dimensional (2D) confinement of the backbone for chains at the immediate surface of PMMA-g-PEO films (Biomacromolecules 2001, 2, 85-94). To directly probe such 2D conformations, combs modified with maleimide groups on the PEO chain ends were blended at 0.5-10 wt % into unmodified PMMA-g-PEO (M-n 142 kg/mol, PDI 3.2, 32 wt % PEO) and cast into films similar to 35 nm thick. Films were immersed in aqueous solution to induce orientation of surface molecules, and maleimide-functionalized chains at the film/ water interface were labeled with 1.4 nm diameter Au nanoparticles. Transmission electron microscopy (TEM) was then used to trace the 2D trajectories of nanoparticle-decorated chains. The distribution of observed chain lengths was in good agreement with that from gel permeation chromatography. The 2D radius of gyration (R-g) calculated from the observed conformations scaled with the number of backbone segments (N) as R-g similar to N-0.69 +/- 0.02. Monte Carlo simulations of a 2D melt of comparable chain length distribution yielded a scaling exponent v = 0.67 +/- 0.03, suggesting that the deviation from classical 2D melt behavior arose from polydispersity.