Understanding the intricacies of gene expression is important for unraveling the mechanisms of growth, development, and maintenance of normal cell metabolism. Recently developed techniques such as cDNA-microarray hybridization or high-throughput RNA sequencing provide overall snapshots of the entire transcriptome of a given sample and have greatly shaped the development of biomedical research since the late 1990s. With the availability of next-generation sequencing technologies, whole transcriptome analysis is quickly becoming affordable and common practice in biological research. Quantification of gene expression is critical to understand gene regulatory networks, interpret epigenetic gene regulation, identify noncoding regulatory RNAs, and pinpoint genes involved in disease states or disorders. However, understanding the variability of gene expression from cell-to-cell demands a set of complementary tools that can be used to characterize gene expression networks in single cells. In this chapter, we review the recent advances in probe design chemistry, developments in imaging, and the need for spatial and temporal single-molecule transcript quantification. Additionally, we discuss the applicability of novel nanoparticle-based approaches for imaging RNA dynamics and quantification in medicine and molecular biology.