sample dish
3.3 Conclusion
In this chapter, I detailed photoporation and optical transfection of CHO cells using a tightly focused Gaussian beam. Femtosecond photoporation is a powerful technique for modifying the genetic profile of cells at will. This is due to its non-invasive, highly
selective character that allows us to target a single or a group of cells randomly located within a sea of other cells. Due to the short duration of fs pulses, photoporation offers ultra-precise energy deposition at the target area, resulting in ultra-fine pore formation on the cellular membrane without the occurrence of collateral damage.
I have presented an extensive study of fs photoporation and transfection of CHO cells whereby the transfection rate of CHO cells was investigated as a function of the position of the cells along the axial direction of the beam propagation. When the cell membrane was positioned at the laser focus, one out of two cells were successfully transfected whereas when the cells were axially displaced from the focus the transfection rate significantly dropped and became negligible at axial displacements beyond the beam’s Rayleigh range. From these results, it became evident that Gaussian transfection introduces strict requirements in the positioning of the laser focus on the cell membrane due to the multiphoton nature of the pore creation. The highly divergent nature of a Gaussian beam requires demanding alignment when moving manually from cell to cell during photoporation. This makes the technique tedious to perform and be adopted by life-scientists who more often than not have a preference towards automated, alignment free optical setups and microscopes.
For the purposes of these studies, plasmid DNA encoding for a red fluorescent protein, the DS-Red, was introduced in the solution surrounding the cell monolayer during laser irradiation. Successful internalization of the DS-Red plasmid was evident 48h upon laser treatment and identified by means of a fluorescent microscope. The transfected and
have normal and healthy morphology. Precursor photoporation studies were also performed, in order to ascertain the correct laser parameters (i.e. laser dose duration, power level at laser focus, number of laser doses, site of irradiation) leading to successful internalization of the plasmid DNA. For the purposes of these initial studies, the membrane impermeable stain Trypan Blue was implemented. Successful internalization of Trypan Blue, results in local blue staining within the cytoplasm, around the area of irradiation. The blue staining occurs within several seconds upon cell irradiation ultimately leading to cell necrosis due to its toxicity. Trypan Blue photoporation studies might have appeared helpful in early experimental stages; however they did not offer conclusive results as to the optimum laser parameters for photoporation. Alternative stains such as Propidium Iodine and Calcein AM will be used in the future for performing transfection efficiency and viability studies.
In order to elucidate the effect of the fs laser pulses on the cell membrane, the topography of the photoporated cells with respect to the laser power was also studied and presented using AFM. Upon laser irradiation, the cells were fixed in formaldehyde solution and were subsequently scanned using the AFM. At low power at the laser focus, close to the threshold for safe photoporation, an ultra-fine pore was formed on the membrane which appeared to be of circular shape with diameter just below 1 μm. This manifested the multiphoton character of the interaction, which resulted in a selective and ultra-confined energy deposition on the targeted area, significantly decreasing the possibility of collateral damage. However, when the power was increased above the threshold of safe membrane perforation, thermal effects in conjuction with multiphoton chemical effects came to play that resulted in greater pore diameters. At sufficiently high powers, the
membrane morphology showed strong signs of collateral damage ultimately leading to cell necrosis.
Femtosecond optical transfection of cells is a powerful technique that surpasses in various aspects many of the currently employed transfection techniques. The limitations introduced by the highly divergent nature of the Gaussian laser beam make the technique less popular due to the demanding alignment when moving from cell to cell within a cell sample. As detailed in the chapter that follows, such limitations can and have been overcome by means of a “non-diffracting” BB. As shall be explained, such laser beam possess an elongated depth of focus which permits longer axial mismatching in positioning of the laser focus on the cell membrane. This significantly improves the way the photoporation process is performed in terms of alignment and paves the way towards complete automation of the optical transfection.
References
[1] B. Saleh and M. Teich, "Fundamentals of Photonics",2nd ed. (2001), John Wiley & Sons,
[2] A. Siegman, "Chapter 17: Physical properties of Gaussian beams", inLasers,
(1986), University Science Books, California, USA, p. 663. [3] E. Hecht, "Optics",4th ed. (2002), Addison Wesley, New York
[4] V.N. Mahajan, "Uniform versus Gaussian beams-A comparison of the effects of diffraction, obscuration, and aberrations,"Journal of the Optical Society of
[5] A. Diaspro, "Confocal and Two-Photon Microscopy: Foundations, Applications and Advances",1st ed. (2002), John Wiley and Sons, New York
[6] H.A. Haus, et al., "Structures for additive pulse mode locking,"Journal of Optical Society of America B8(10), p. 2068, 1991.
[7] I. Cormack, "PhD thesis: Rapid techniques for ultrashort optical pulse characterisation", Supervisor: W. Sibbett, (2001), School of Physics & Astronomy, University of St Andrews
[8] A. Miller and D.T. Reid, "Measuring ultrafast laser pulses", inUltrafast Photonics, (2004), Institute of Physics publishing, London, p. 59.
[9] D. Stevenson, et al., "Femtosecond optical transfection of cells: viability and efficiency,"Optics Express14(16), p. 7125, 2006.
[10] D. Lechardeur and G. Lukacs, "Intracellular barriers to non-viral gene transfer,"
Current Gene Therapy2(2), p. 183, 2002.
[11] G.L. Lukacs, et al., "Size-dependent DNA mobility in cytoplasm and nucleus,"
Journal of Biological Chemistry275(3), p. 1625, 2000.
[12] S. Kurata, et al., "The Laser Method for Efficient Introduction of Foreign DNA into Cultured-Cells,"Experimental Cell Research162(2), p. 372, 1986.
[13] V. Kohli, et al., "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation,"Biotechnology and Bioengineering 92(7), p. 889, 2005.
[14] S. Sagi, et al., "Gene delivery into prostate cancer cells by holmium laser application,"Prostate Cancer and Prostatic Diseases6, p. 127, 2003.
[15] H. He, et al., "Targeted photoporation and transfection in human HepG2 cells by a fiber femtosecond laser at 1554 nm,"Optics Letters33(24), p. 2961, 2008.
[16] U.K. Tirlapur and K. Konig, "Cell biology - Targeted transfection by femtosecond laser,"Nature418(6895), p. 290, 2002.
[17] A. Vogel, et al., "Mechanisms of femtosecond laser nanosurgery of cells and tissues,"Applied Physics B-Lasers and Optics81(8), p. 1015, 2005.