Murine fibroblasts transfection by laser optoporation: preliminary notes on the use of a visible wavelength

Giuseppe Palumbo; Mario F. Tecce; Giuseppe Roberti; Alberto Colasanti

doi:10.1117/12.197507

22 December 1994 Murine fibroblasts transfection by laser optoporation: preliminary notes on the use of a visible wavelength

Giuseppe Palumbo, Mario F. Tecce, Giuseppe Roberti, Alberto Colasanti

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Proceedings Volume 2328, Biomedical Optoelectronic Devices and Systems II; (1994) https://doi.org/10.1117/12.197507
Event: International Symposium on Biomedical Optics Europe '94, 1994, Lille, France

Abstract

A novel approach to gene trasfection, i.e. the introduction of an extraneous gene into a host cell, by the 'non-contact forces' of a laser microbeam, is presented here. By means of a large magnification (100x) objective, the blue microbeam of an Argon laser (488 nm) has been focused on the cell membrane in culture in the presence of a pH indicator (namely the phenol-red), which is an usual component of culture media. Due to the local high light absorption of phenol red, which shows an absorption peak at 475 nm, at the site of the beam impact the cell membrane melts forming small circular holes. Throughout the holes, DNA purposely added to culture medium, may penetrate the cytoplasm. The wall damage, whose extension may be regulated by controlling the irradiation time, disappear spontaneously (membrane repair) within 1-2 minutes. By this technique, thereafter indicated as 'optoporation', we have successfully transfected into murine NIH3T3 fibroblasts (beta) -galactosidase and chloramphenicol-acetyltransferase bacterial genes. These conditions of transfection by means of 'non contact' forces, are very mild and do not require any addition of extraneous, potentially toxic chemicals. In addition, since the radiation used is in the visible region, where nucleic acids and most proteins do not absorb, no further deleterious effects for the cell are expected.

Citation Download Citation

Giuseppe Palumbo, Mario F. Tecce, Giuseppe Roberti, and Alberto Colasanti "Murine fibroblasts transfection by laser optoporation: preliminary notes on the use of a visible wavelength", Proc. SPIE 2328, Biomedical Optoelectronic Devices and Systems II, (22 December 1994); https://doi.org/10.1117/12.197507

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