Transfection of MTH53A cells by femtosecond laser opto-perforation J. Baumgart, W. Bintig, A. Ngezahayo, S. Willenbrock , H. Murua Escobar, W. Ertmer, H. Lubatschowski, A. Heisterkamp
Introduction Compared to the conventional techniques femtosecond (fs) laser optoperforation is a less invasive transfection method [1]. The fs laser is tightly focused into the cell membrane to induce transient pores in the order of magnitude of less than 1_µm. Due to the nonlinear cutting effects, the damage is limited to the focal volume of the laser beam. Thus, the cell is only affected in a small part of the membrane. The key factor of optoperforation by fs laser pulses is the exchange of intra and extracellular media so that the dye molecules or the DNA diffuse into the perforated cell. The measurement of this volume exchange was performed by the membrane potential measurement via patch-clamp technique [2] and a concentration determination.
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laser Fig. 1 Sketch of simultaneous patch-clamp and opto-perforation of a living cell. The induced pore allows the diffusion of ions through the membrane.
Implementation
Transfection by fs laser irradiation
The laser system is a tunable titanium sapphire laser (715_nm_–_955_nm) which generates ultrashort pulses of 140 fs at a repetition rate of 90 MHz. The laser beam is guided to the reflector revolver of the microscope. The beamsplitter reflects the beam into the high numerical aperture objective which focuses the laser into the cell membrane. Additionally, a patch-clamp setup was integrated to the microscope. The patch electrode with the pipette solution has a resistance of 10_M!.
Two different regimes of intracellular uptake of extracellular media could be found. In both cases, the potential depolarizes some mV. In the first regime, the potential then repolarizes or stays at the same level. Another behavior occurs, when a gas bubble is induced. Then the potential de-polarizes very fast about 10_mV to 20_mV (fig. 4). In both regimes, the irradiation time t is shorter than the depolarization time "t.
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Fig. 2 Schematic setup of the opto-perforation setup with integrated patch-clamp equipment.
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Fig. 5 A: Fluorescence image of granulosa cells during opto-perforation, the treated cells are highlighted by the dashed circles. 1.5 µM propidium iodide is solved in the media and the laser parameters were 0.9_nJ pulse energy and 40 ms irradiation time. All manipulated cells are fluorescence. B and D: Bright field image of the cells. C: Fluorescence image of the cells after 90 minutes incubation in PBS. The cells were re-stained with propidium iodide to verify the viability. The cell pointed out by the arrow is representative for a cell whose membrane is damaged and therefore permeable for the fluorophore.
The optimum parameters for high viability and high efficiency were found at 0.9_nJ pulse energy and 40 ms irradiation time (fig. 6). It was even to transfect MTH53A dog cells with pEGFP-C1-HMGA2 at the same parameters (fig. 7). A limiting factor was the bounding of the DNA to the glass bottom of the culture dishes. Thus, the dishes were coated by poly-L-lysine to prevent binding. At an extracellular concentration of 50 µg/µl DNA, about 10 fg of the molecules enter into the perforated cells during the manipulation at a cell diameter of 10_µm. [1] U.K. Tirlapur, K. König, Targeted transfection by femtosecond laser, Nature, 418, 290-291 (2002) [2] E. Neher, B. Sakmann, Single channel currents recorded from membrane of denervated frog muscle fibres, Nature, 260, 799-802 (1976) [3] D. Goldman, Potential, impedance, and rectification in membranes, Journal of General Physiology, 27, 37-60 (1943)
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Following the Nernst and Goldman equations [3], the volume exchange can be calculated assuming a constant cell volume during manipulation. The volume exchange was theoretically predicted to be 0.4 times the cell volume at a membrane potential de-polarization of 10 mV. The results of the opto-perforation experiments with propidium iodide verify this result very well (fig._5).
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Fig. 4 The membrane potential of a granulosa cell during fs laser perforation. The grey bar represents the laser irradiation time t for the opto-perforation. (A) No bubble formation during the treatment; (B) a small gas bubble was created during the treatment.
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Fig. 7 MTH53A cells transfected with pEGFP-C1-HMGA2 vector by opto-perforation.
Conclusion The measurements of the membrane potential combined with concentration determination give new insights about the exchanged cell volume and thus about the amount of uptaken molecules during opto-perforation. This volume was calculated and measured to be about 0.4 times the cell volume at an average membrane depolarization. Two different potential behaviors could be seen: with and without bubble formation. Only in the case of bubble formation, enough media is exchanged, so that the cell fluoresces after perforation. The optimum laser parameters for the opto-perforation were found at 0.9_nJ pulse energy and 40 ms irradiation time. Even the uptake of very big molecules as DNA strands was successfully realized.
J.Baumgart@lzh.de www.biophotonics.uni-hannover.de