Single-cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites, and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single-cell activity. To obtain quantitative information (e.g., molecular quantity, kinetics, and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single-cell studies, both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live-cell analysis. Although a considerable proportion of single-cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single-cell analysis.