Labeling for Quantitative Comparison of Imaging Measurements in Vitro and in Cells

Qualitative imaging of biomolecular localization and distribution inside cells has revolutionized cell biology. Most of these powerful techniques require modifications to the target biomolecule. Over the past 10 years, these techniques have been extended to quantitative measurements, from in-cell protein folding rates to complex dissociation constants to RNA lifetimes. Such measurements can be affected even when a target molecule is just mildly perturbed by its labels. Here, the impact of labeling on protein (and RNA) structure, stability, and function in cells is discussed via practical examples from the recent literature. General guidelines for selecting and validating modification sites are provided to bring the best from cell biology and imaging to quantitative biophysical experiments inside cells.