Intracellular bottom-up generation of targeted nanosensors for single-molecule imaging

Organic dyes are useful tools for sensing cellular activities but unfavorable in single-molecule imaging, whereas quantum dots (QDs) are widely applied in single-molecule imaging but with few sensing applications. Here, to visualize cellular activities by monitoring the response of a single probe in living cells, we propose a bottom-up approach to generate nanoprobes where four organic dyes are conjugated to tetravalent single-chain avidin (scAVD) proteins via an intracellular click reaction. We demonstrate that the nanoprobes, exhibiting increased brightness and enhanced photostability, were detectable as single dots in living cells. The ease of intracellular targeting allowed the tracking of endoplasmic reticulum (ER) remodeling with nanometer spatial resolution. Conjugating thermosensitive dyes generated temperature-sensitive nanoprobes on ER membranes that successfully monitored local temperature changes in response to external heat pulses. Our approach is potentially a suitable tool for visualizing localized cellular activities with single probe sensitivity in living cells.