Femtosecond cellular transfection using novel laser beam geometries

Lasers in cytology;Beam optics;Femtosecond lasers

Tsampoula, Xanthi ; Dholakia, Kishan ; Dholakia, Kishan

University:University of St Andrews

Department:Physics & Astronomy (School of)

关键词: Lasers in cytology;    Beam optics;    Femtosecond lasers;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/909/XanthiTsampoulaPhDThesis.pdf?sequence=3&isAllowed=y

来源: DR-NTU

【 摘 要 】

In this thesis, femtosecond (fs) cellular transfection of Chinese Hamster Ovary (CHO)cells was performed using a tightly focused Gaussian beam. The beam focus waspositioned on the cell membrane and three laser doses, each of 40 ms duration, weredelivered allowing for the formation of a highly localized pore on the cell membrane. Themembrane pore, induced as a result of a multiphoton process known as photoporation,permitted the surrounding DNA to diffuse into the cell cytoplasm. 48 hours after laserirradiation, the viable photoporated cells expressed a red fluorescent protein. Thetopography of a photoporated cell, targeted with tightly focused fs pulses, was alsomonitored as a function of the input power using Atomic Force Microscopy. Followingthis, I generated and implemented a “non-diffracting” quasi-Bessel beam (BB) by meansof a conical shaped lens, the axicon, which successfully provided an alternative route forphotoporation to the highly divergent Gaussian beam. A comparison was given betweenthe two beam approaches for photoporation. The “non-diffracting” character of the BBresulted in the first successful attempt towards automating optical transfection. This wasachieved by using an axicon and a spatial light modulator (SLM) to provide phasemodulation on the annular spatial spectrum field of the BB. This approach providedcontrol over the lateral and axial position of the beam with respect to the cell membrane,allowing for point and click photoporation. Successful photoporation of CHO cells wasalso demonstrated using for the first time an axicon tipped optical fibre. The applicabilityprospects of this method are significant, ranging from potential endoscopic embodimentsof the technique to advanced studies of tissue properties in vitro and in vivo.