Hydrogels are commonly used in DNA and protein analysis, and as biomedical scaffolds for an array of applications. Determining the pore size distribution of these materials is critical in order to tune the transport properties of biomolecules through the porous network. While there are many indirect approaches to estimating a mean pore size, there are few, if any, studies published in which several direct pore size measurement techniques are compared side-by-side. In this study, Dr Imanda Jayawardene and colleagues compared electron microscopy, atomic force microscopy, and super-resolution fluorescence microscopy, to determine the pore size ditributions in agarose gel as a function of agarose concentration. One of the key findings was the the commonly employed approach of fluorescence microscopy simply doesn't have the resolution required for the task, and that SEM combined with high pressure freezing to maintain native structure was still the most reliable approach.