High‐Efficiency Targeted Gene Transfection of Cells Using Temporal and Spatial Shaping Femtosecond Laser Irradiation

Laser-irradiation-assisted cell gene transfection is sterile and nontoxic, but the low transfection efficiency cannot meet the application requirements. To improve the efficiency, a temporal and spatial shaping method of a femtosecond laser is proposed. Using the time shaping method, we can segment the pulse into subpulses of varying energies and with a defined delay, thereby influencing the interaction between electrons and photons, ultimately enhancing transfection efficiency. The transfection efficiency is further improved by spatially shaping the laser pulse to extend the focusing beam's working distance and reduce the cell's sensitivity to the focal position. Through the characterization of the viability and transfection efficiency of HEK-293T cells, the method achieved efficient and active transfection, with a maximum transfection efficiency of 45.1% and a cell survival rate of 93.6%. This method provides key technical support for femtosecond laser transfection and promotes its further application in clinical practice.