10:00 AM - 10:15 AM
▼ [20a-E214-4] Photoinjection of Fluorescent Nanoparticles into Plant Cells Using Femtosecond Laser Amplifier with Enzyme Treatment
Keywords:Photoporation, Laser Cell Manipulation, Single Plant Cell
Femtosecond laser photoinjection has become a popular method to deliver various kinds of molecules into mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery of objects with diameter larger than 5 nm. In this work, we demonstrated the photoinjection of large objects into cytoplasm of an intact single plant cell with femtosecond laser amplifier under moderate enzyme treatment conditions. A single 20 nJ pulse of femtosecond laser amplifier was focused through a 100x objective on a contact point of cell membrane and wall. The injection of green fluorescent nanoparticles with diameter of 80 nm was evaluated by comparing fluorescence images before and after the laser irradiation. To estimate level of injection, the difference of fluorescence intensities inside the cell between before and after photoinjection was calculated. Our results indicated that the enzyme treatment is needed to assist the injection of the nanoparticles by photoinjection. It contributes to increasement of the particle diffusion rate through the cell wall that permits injection of particles into the cytoplasm efficiently. With this finding, the application of the photoinjection using the amplified fs laser pulse has good potential for efficient injection of large objects to intact plant cells. It is expected to expand insights into plant systems being used in gene modifications.