[18a-A25-1~11] 3.6 Laser processing

11:45 AM - 12:00 PM JST(2:45 AM - 3:00 AM UTC)English Presentation

[18a-A25-11] Photoinjection of Fluorescent Molecules into Tobacco BY-2 Cells Adapted with a Microchip Laser

〇(D)Muhammad Ridho Jatmiko¹, Naomi Tanga^1,4, Koichiro Kishima², Arvydas Kausas³, Yuji Sano³, Takunori Taira³, Yoichiroh Hosokawa^1,4,5 (1.Div. Mat. Sci., NAIST, 2.Pinpoint Photonics, Inc.,, 3.Div. Rsc. Innov, and Collabo., IMS, 4.CDG NAIST, 5.MLC NAIST)

Keywords:

Microchip laser,Laser ablation,Tobacco BY-2 cells

Photoinjection is a method that utilizes laser irradiation to create pores in cell membranes. This method introduces external molecules, such as proteins, nanoparticles, or plasmid DNA into cells. Previously, we successfully demonstrated that plasmid DNA is injected into softened cell wall Tobacco BY-2 cells using a femtosecond laser. Now we consider some advantages of a pulsed microchip laser, which is compact, maintenance-free, and low-cost compared with the femtosecond laser. The problem of the microchip laser is that the picosecond laser pulse requires higher power energy for ablation, which induces bigger scale of ablation area and larger size of cavitation bubble. Namely, it is difficult to optimize the laser irradiation condition. As demonstrated by Hausladen et al.1, using animal cells as a sample, molecules can be introduced into cells by randomly ablating, not targeting each cell individually. Their results suggest that cells in the laser focus area were destroyed or blasted, but some of the surrounding cells were successfully injected. In this work, we applied this method to a plant cell, Tobacco BY-2 cells. A fluorescent molecule, FITC-dextran (2 MDa) was used as the external molecules to check the injection capability. Initially, the photoinjection was performed in the same way as that by a femtosecond laser2. In addition, we used poly-D-lysine to fix the cells on a glass substrate to reduce the impact of ablation on cells. This procedure assists fine observation of the photoinjection for optimizing the laser irradiation condition. Unfortunately, we did not find the certain successful cells, although there were many cells broken by the laser irradiation. The possibility of microchip laser-induced photoinjection for plant cells is confirmed by conducting this experiment with adjusting the laser irradiation and cell preparation conditions.