In Situ Stable Generation of Reactive Intermediates by Open Microfluidic Probe for Subcellular Free Radical Attack and Membrane Labeling

Subcellular stimulation by free radicals is crucial for deeper insight of cell behaviors. However, it remains a tough challenge due to the high spatial precision requirement and short life of radicals. Herein, we report a versatile open microfluidic probe for stable generation of free radical and subcellular stimulation. By optimizing parameters, the chemical reaction can be confined in a microregion with a diameter of several mu m, and the real-time produced reactive radicals can attack the desired subcellular region of a single cell. In order to reveal the attacked region, fluorescent cyanine 3 labeled tyramide free radicals are synthesized, and the target microregion on a single cell is successfully stained by the covalent linking reaction between radicals and membrane proteins, which proves the feasibility of our method. We believe this method will open new avenues for short-lived reactive intermediates stimulation at the single-cell/sub-cell level and selective membrane labeling.

Automated summary

A versatile open microfluidic probe has been developed for stable generation of free radicals and subcellular stimulation. By utilizing fluorescent labeled radicals, the method successfully targets specific subcellular regions in single cells, proving its feasibility in real-time imaging and manipulation.