Diffraction-unlimited imaging: from pretty pictures to hard numbers

Diffraction-unlimited fluorescence imaging allows the visualization of intact, strongly heterogeneous systems at unprecedented levels of detail. Beyond the acquisition of detailed pictures, increasing efforts are now being focused on deriving quantitative insights from these techniques. In this work, we review the recent developments on sub-diffraction quantization that have arisen for the various techniques currently in use. We pay particular attention to the information that can be obtained but also the practical problems that can be faced, and provide suggestions for solutions or workarounds. We also show that these quantitative metrics not only provide a way to turn raw data into hard statistics but also help to understand the features and pitfalls associated with sub-diffraction imaging. Ultimately, these developments will lead to a highly standardized and easily applicable toolbox of techniques, which will find widespread application in the scientific community.