Dankovich TM, Rizzoli SO
iScience. 2021 Feb 3;24(3):102134.
Optical super-resolution microscopy (SRM) has enabled biologists to visualize cellular structures with near-molecular resolution, giving unprecedented access to details about the amounts, sizes and spatial distributions of macromolecules in the cell. Precisely quantifying these molecular details requires large datasets of high-quality, reproducible SRM images. In this review, we discuss the unique set of challenges facing quantitative SRM, giving particular attention to the shortcomings of conventional specimen preparation techniques and the necessity for optimal labeling of molecular targets. We further discuss the obstacles to scaling SRM methods, such as lengthy image acquisition and complex SRM data analysis. For each of these challenges, we review the recent advances in the field that circumvent these pitfalls and provide practical advice to biologists for optimizing SRM experiments.