The structure of thin microphase-separated polystyrene-block-polydimethylsiloxane (PS-PDMS) films has been studied using state-of-the-art top-down and cross-sectional electron microscopy. This is the first time that the profile of PS-PDMS films has been measured in situ and these measurements allowed us to image the shape of the PDMS domains within the film as well as examine the wetting behavior of the block copolymer film on a variety of substrates. It was found that for each polymer, substrate chemistry and annealing method combination examined, there was a small range of film thicknesses whereby the films exhibited the optimal characteristics of high levels of ordering without dewetting or multilayering. Specifically, the optimum thickness for films treated by thermal annealing was greater than that for the equivalent solvent annealed film; a change that was correlated with morphology variations related to solvent swelling of the solvent annealed films. The surface chemistry also induced changes in the optimum film thickness. Selective surfaces were shown to control whether a PDMS wetting layer was formed or not, leading to either thicker or thinner wetting optimum film thicknesses; while undulating morphologies were observed for less selective surfaces. Concomitant changes in the periodicity were then hypothesized to occur as a result of confinement effects and the selectivity of the surface.