Femtosecond cellular transfection using novel laser beam geometries
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Date
30/11/2009Author
Supervisor
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Abstract
In this thesis, femtosecond (fs) cellular transfection of Chinese Hamster Ovary (CHO)
cells was performed using a tightly focused Gaussian beam. The beam focus was
positioned on the cell membrane and three laser doses, each of 40 ms duration, were
delivered allowing for the formation of a highly localized pore on the cell membrane. The
membrane 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 laser
irradiation, the viable photoporated cells expressed a red fluorescent protein. The
topography of a photoporated cell, targeted with tightly focused fs pulses, was also
monitored as a function of the input power using Atomic Force Microscopy. Following
this, I generated and implemented a “non-diffracting” quasi-Bessel beam (BB) by means
of a conical shaped lens, the axicon, which successfully provided an alternative route for
photoporation to the highly divergent Gaussian beam. A comparison was given between
the two beam approaches for photoporation. The “non-diffracting” character of the BB
resulted in the first successful attempt towards automating optical transfection. This was
achieved by using an axicon and a spatial light modulator (SLM) to provide phase
modulation on the annular spatial spectrum field of the BB. This approach provided
control 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 was
also demonstrated using for the first time an axicon tipped optical fibre. The applicability
prospects of this method are significant, ranging from potential endoscopic embodiments
of the technique to advanced studies of tissue properties in vitro and in vivo.
Type
Thesis, PhD Doctor of Philosophy
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