Real‐Time and Site‐Specific Perturbation of Dynamic Subcellular Compartments Using Femtosecond Pulses

Understanding laser interactions with subcellular compartments is crucial for advancing optical microscopy, phototherapy, and optogenetics. While continuous-wave lasers rely on linear absorption, femtosecond (fs) lasers enable nonlinear multiphoton absorption confined to the laser focus, offering high axial precision. However, current fs laser delivery methods lack the ability to target dynamic molecular entities and automate target selection, making them incapable of performing real-time perturbation of mobile or complexly distributed biomolecules. Additionally, existing technologies separate fs pulse delivery and imaging, preventing simultaneous recording of cellular responses. To overcome these challenges, this study introduces fs real-time precision opto-control (fs-RPOC), which integrates a laser scanning microscope with a closed-loop feedback mechanism for automated, chemically selective subcellular perturbation. Fs-RPOC achieves superior spatial precision and fast response time, enabling single- and sub-organelle microsurgery of dynamic targets and localized molecular modulation. By applying a pulse-picking method, fs-RPOC independently controls laser average and peak power at any desired subcellular compartment. Targeting mitochondria, fs-RPOC reveals site-specific molecular responses resulting from fs-laser-induced reactive oxygen species formation, H₂O₂ diffusion, and low-density plasma generation. These findings offer new insights into fs laser interactions with subcellular compartments and demonstrate fs-RPOC's potential for precise molecular and organelle regulation.