Ion Monitoring at Nanoscale Sites of Interorganelle Membrane Contact in Living Cells

Nanostructural contact sites formed by interorganelle membrane contacts, including mitochondria and lysosome contacts (MLC), facilitate the exchange of substances during various life processes. However, existing bioanalytical technologies have yet to provide accurate information on the exchange of substances, as these techniques exhibit limited spatial and temporal resolution. To address this limitation, a strategy is proposed that combines fluorescence resonance energy transfer (FRET) probes with high spatial resolution detection and super‐resolution microscopes (SRM) that feature time‐resolved imaging. Specifically, a proof‐of‐concept approach is presented for monitoring H+ fluctuations during MLC with a spatial H+ biosensor targeting lysosomes, BDP‐RhB. The biosensor comprises H+‐sensitive rhodamine B as a FRET acceptor connected by a flexible chain to a BODIPY derivative as a donor. The acidity of MLC sites may vary, influencing the spatial distance of the flexible chain and causing a fluorescence transition in BDP‐RhB. Consequently, the spatial distribution of H+ can be identified using SRM. Furthermore, an algorithm has been developed to screen and identify potential compounds that control substance exchange in the MLC. Collectively, this work presents the dynamic of H+ in lysosomes within living cells, which provides a drug screening tool for studying substance exchange through interorganelle membrane contacts.