Biotechnology Letters 25: 895–899, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 895 Laser-assisted microinjection into targeted animal cells Samarendra K. Mohanty, Mrinalini Sharma & Pradeep K. Gupta ∗ Biomedical Applications Section Center for Advanced Technology, Indore-452013, India ∗ Author for correspondence (Fax: +91-731-2488430; E-mail: pkgupta@cat.ernet.in) Received 5 February 2003; Revisions requested 6 March 2003; Revisions received 31 March 2003; Accepted 4 April 2003 Key words: microinjection, optoporation, photodynamic treatment, transfection Abstract A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer. Introduction Microinjection of exogenous genes, fluorochromes, antibodies and photoactivable compounds into cells is required for a variety of applications in genetics, cell biology and biotechnology (Verma & Somia 1997, Kaplan & Somlyo 1989, Greulich 1999). Several tech- niques are being employed for this purpose. These include chemical methods, electroporation, direct mi- croinjection into cells through mechanical means, and liposome- and recombinant viral vector-mediated transfer. However, all these techniques have some major drawback. These techniques are often tedious, require considerable skill and the efficacy of transfer are highly variable especially with small animal cells. This has motivated exploration of the use of laser- assisted microinjection. This approach offers two im- portant advantages. First, it can be applied to all types of cells and secondly, it can be used on cells in sus- pension as well as attached cells. Lasers in the UV spectral range were the first to be investigated for op- toporation (Tao et al. 1987, Shirahata et al. 2001) due to strong absorption by the constituents of the mem- brane in this spectral region. However, the use of UV light raises concern about the damage to membranes, or cells or even exogenous DNA being transferred into the cell (Tao et al. 1987, Palumbo et al. 1996). The use of visible laser irradiation at 488 nm for op- toporation has also been demonstrated exploiting the fact that the indicator dye, Phenol Red, present in the culture medium has strong absorption at this wave- length. Laser absorption-induced rise in temperature may lead to changes in the membrane permeability (Palumbo et al. 1996, Schneckenburger et al. 2002). However, since several cellular components have sig- nificant absorption at this wavelength (488 nm), the possibility of deleterious effects even at this wave- length cannot be ruled out. Indeed, reports exist on argon laser-induced cytogenetic damage such as sis- ter chromatid exchange, chromatid and chromosome aberrations (Nakajima et al. 1983). Use of lasers with wavelength in near infrared region (700–1000 nm) would be more desirable for optoporation because ab- sorption by cellular components in this wavelength range is significantly lower than that beyond this spec- tral range. Recently, efficient gene transfection has indeed been reported using a near-infrared (800 nm) femtosecond laser (Tirlapur & Konig 2002). However, to the best of our knowledge use of nanosecond in- frared lasers for transfection has not been reported. We have investigated the use of a pulsed nanosecond 1064 nm Nd:YAG laser for microinjection of imper- meable fluorochromes such as propidium iodide and merocyanine 540 as well as transfection of green flu- orescent protein encoding plasmid into MCF-7 cells. Further, we have also investigated the possibility of en- hancement in the efficiency of laser-assisted microin-