Spatiotemporal activity patterns of proteases such as matrix metalloproteinases and cysteine proteases in organs have the potential to provide insight into how organized structural patterns arise during tissue morphogenesis and may suggest therapeutic strategies to repair diseased tissues. Toward imaging spatiotemporal activity patterns, recently increased emphasis has been placed on imaging activity patterns in three-dimensional culture models that resemble tissues in vivo. Here, we briefly review key methods, based on fluorogenic modifications either to the extracellular matrix or to the protease-of-interest, that have allowed for qualitative imaging of activity patterns in three-dimensional culture models. We highlight emerging plasmonic methods that address significant improvements in spatial and temporal resolution and have the potential to enable quantitative measurement of spatiotemporal activity patterns with single-molecule sensitivity.
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