This study addresses the effect of sample preparation conditions on the structural integrity and composition of heavy hydrocarbon mixtures imaged by noncontact atomic force microscopy (nc-AFM). We designed and prepared a set of organic molecules mimicking well-accepted key characteristics of heavy oil asphaltenes including molecular architecture, molecular weight, boiling point, atomic H/C ratio, and bond strength. We deliberately focused on multicore molecule structures with long aliphatic linkers as this architecture was largely absent in previous nc-AFM studies of petroleum samples. The results confirm that all these molecules can be successfully imaged and remain intact under the same preparation conditions. Moreover, comparison with ultrahigh resolution FT ICR-MS of a steam-cracked tar asphaltene sample suggests that the single molecules identified by nc-AFM span the entire molecule spectrum of the bulk sample. Overall, these results suggest that petroleum molecules within the scope of chosen molecules studied herein can be prepared intact and without bias and the imaged data can be representative.