Rowiak CS: Transfection of MTH53A cells by femtosecond laser opto-perforation

Transfection of MTH53A cells by femtosecond laser opto-perforationJ. Baumgart, W. Bintig, A. Ngezahayo, S. Willenbrock , H. Murua Escobar, W. Ertmer, H. Lubatschowski, A. Heisterkamp

J.Baumgart@lzh.dewww.biophotonics.uni-hannover.de

ImplementationThe 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!.

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 cal-

culated and measured to be about 0.4 times the

cell volume at an average membrane de-

polarization. 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.

References[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)

Fig. 7 MTH53A cells transfected with pEGFP-C1-HMGA2

vector by opto-perforation.

Fig. 2 Schematic setup of the opto-perforation

setup with integrated patch-clamp equipment.

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.

Fig. 6 (A) The viability of the cells and (B) the efficiency of the

uptake of propidium iodide into the cells, dependent on the pulse

energy and the irradiation time. (error bar: ± 10%)

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).

Fig. 5 A: Fluorescence image of granulosa cells during opto-perforation, the treated cells are high-

lighted by the dashed circles. 1.5 µM propidium iodide is solved in the media and the laser para-

meters 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 incu-

bation 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.

20 µm 20 µm

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.

IntroductionCompared to the conventional techniques femtosecond (fs) laser opto-

perforation 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 opto-

perforation 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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0.7 0.8 0.9 1.0 1.1 0.7 0.8 0.9 1.0 1.1pulse energy [nJ] pulse energy [nJ]

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time [ms] time [ms]

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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.

0 20 40 60 80 100 120 0 20 40 60 80 100 120

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CCD

laser attenuator

UV-lamp

amplifier

PC

objective

beam-

splitter

pipette

sample

patch-clamp pipette

and electrode

cells

glass bottom

petri dish

laser

objective

DNA solved in NaCl media

Transfection by fs laser irradiation

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.