In this Feature Application, we showcase the enormous potential that Nanolive live cell imaging holds for single cell characterization. We begin, by analyzing micro-heterogeneity at the population and the temporal level in unperturbed cells (case study one). We then extend our analysis to include a quantitative assessment of lipid droplet dynamics (case study two), before investigating how intracellular trafficking (case study three) and respiratory perturbation (case study four) impacts microheterogeneity in cell morphology.