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Transfection of MTH53A cells by femtosecond laser opto-perforation
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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
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.
irrad
iatio
n tim
e [m
s]
irrad
iatio
n tim
e [m
s]
viability efficiency
A B
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]
60
50
40
30
60
50
40
30
95%
85%
75%
65%
55%
45%65%
55%45%
35%
25%15%
A
30 µm
B
30 µm
C
30 µm
D
30 µm
time [ms] time [ms]
no
rma
lize
dp
ote
ntia
l [m
V]
no
rma
lize
dp
ote
ntia
l [m
V]A B
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
1816141210
86420
18
16
14
12
10
8
6
4
2
0
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.