Single molecule wide-field polarization fluorescence imaging is an experimental method to determine the self-assembly of molecules dispersed in a thin film. Through a combination of wide-field imaging and confocal spectroscopy, the effect of synthetic structure of the oligomeric PPV-based materials was investigated to understand the effect of conjugation length, role of hydrogen bonding side chains, and influence of regioregularity on controlling chromophore folding. By studying alkoxy-linked and alkyl-linked bis(2-ethylhexyl)-p-phenylene vinylene (BEH-PPV) units of varying lengths (three, five, or seven), it was determined that conjugation length controlled the extent of molecular ordering and emission properties. Comparison of the experimental results to molecular dynamics simulations performed by collaborators confirmed that the materials became increasingly ordered as conjugation length increased. Further regulation of the assembly can be obtained through inclusion of hydrogen bonding side chains as seen in the altered amine and carboxylic acid alkoxy-linked trimer BEH-PPV in contrast to the bulky side chain tert-butyl trimer. The study of regio-regular (RR) and regio-random (RRa) alkoxy-linked pentamer poly(2-methoxy-5-(2’-ethylhexyloxy)-p-phenylenevinylene) (MEH-PPV) illustrates the limited effect of regioregularity of the side chains on self-assembly. Through synthetic structure, it is possible to design highly ordered materials through control of conjugation length and selection of side chains. / text