Previous studies have shown that functionalized nanoparticles (NPs) topically applied on fresh tissues are able to rapidly target cell‐surface protein biomarkers of cancer. Furthermore, studies have shown that a paired‐agent approach, in which an untargeted NP is co‐administered with a panel of targeted NPs, controls for the nonspecific behavior of the NPs, enabling quantitative imaging of biomarker expression. However, given the complexities in nonspecific accumulation, diffusion, and chemical binding of targeted NPs in tissues, studies are needed to better understand these processes at the microscopic scale. Here, fresh tissues were stained with a paired‐agent approach, frozen, and sectioned to image the depth‐dependent accumulation of targeted and untargeted NPs. The ratio of targeted‐to‐untargeted NP concentrations—a parameter used to distinguish between tumor and benign tissues—was found to diminish with increasing NP diffusion depths due to nonspecific accumulation and poor washout. It was then hypothesized and experimentally demonstrated that larger NPs would exhibit less diffusion below tissue surfaces, enabling higher targeted‐to‐untargeted NP ratios. In summary, these methods and investigations have enabled the design of NP agents with improved sensitivity and contrast for rapid molecular imaging of fresh tissues. A microscopic investigation of a nanoparticle‐based molecular imaging strategy is presented, which is used to inform the design of larger nanoparticles that exhibit reduced diffusion and improved binding to cell‐surface biomarkers when topically applied onto the surfaces of fresh tissue specimens. Data and preliminary modeling suggest the presence of a binding‐site barrier that restricts the diffusion of biomarker‐targeted nanoparticles in comparison to untargeted control nanoparticles.