Abstract

We report the transfection of human hepatocarcinoma (HepG2) cells by femtosecond (fs) laser pulses at 1554nm. It was found that HepG2 cells could be perforated transiently to admit propidium iodide by that laser. This photoporation was safe, as the membrane resealed itself within a short time and no mitochondrial depolarization was detected. The cells were next photoporated in the presence of DNA plasmids for the expression of green fluorescence protein, and about 80% of the exposed cells showed green fluorescence 24h later. Thus it could be concluded that it is safe and efficient to use fs laser at 1554nm to transfect foreign molecules into cells under a standard microscope.

© 2008 Optical Society of America

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2006 (1)

2005 (1)

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

2002 (2)

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur and K. Konig, Nature 418, 290 (2002).
[CrossRef] [PubMed]

2001 (1)

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

2000 (1)

S. R. Schwarze, K. A. Hruska, and S. F. Dowdy, Cell Biol. 10, 290 (2000).

1997 (1)

1996 (1)

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

1991 (2)

C. A. Sacchi, J. Opt. Soc. Am. B 8, 337 (1991).
[CrossRef]

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

1986 (1)

F. Docchio, C. A. Sacchi, and J. Marshall, Lasers Ophthalmol. 1, 83 (1986).

1980 (1)

M. R. Capecchi, Cell 22, 479 (1980).
[CrossRef] [PubMed]

Agate, B.

Atkin, A.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Brown, C. T. A.

Capecchi, M. R.

M. R. Capecchi, Cell 22, 479 (1980).
[CrossRef] [PubMed]

Chen, A.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Chen, L. B.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Dholakia, K.

Docchio, F.

F. Docchio, C. A. Sacchi, and J. Marshall, Lasers Ophthalmol. 1, 83 (1986).

Dowdy, S. F.

S. R. Schwarze, K. A. Hruska, and S. F. Dowdy, Cell Biol. 10, 290 (2000).

Fischer, P.

Gilbert, R.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Gratton, E.

Guerra, A.

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Gunn-Moore, F.

Halbhuber, K.-J.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

Heller, R.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Hruska, K. A.

S. R. Schwarze, K. A. Hruska, and S. F. Dowdy, Cell Biol. 10, 290 (2000).

Huttman, G.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Jaroszeski, M.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Konig, K.

U. K. Tirlapur and K. Konig, Nature 418, 290 (2002).
[CrossRef] [PubMed]

König, K.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

K. König, P. T. C. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef] [PubMed]

Krieg, R.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

Lin, M.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Mantulin, W. W.

Marshall, J.

F. Docchio, C. A. Sacchi, and J. Marshall, Lasers Ophthalmol. 1, 83 (1986).

Moradpour, D.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Mottola-Hartshorn, C.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Nahen, K.

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Nicolau, C.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Noack, J.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Paltauf, G.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Peuckert, C.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

Reers, M.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Riches, A.

Sacchi, C. A.

C. A. Sacchi, J. Opt. Soc. Am. B 8, 337 (1991).
[CrossRef]

F. Docchio, C. A. Sacchi, and J. Marshall, Lasers Ophthalmol. 1, 83 (1986).

Schwarze, S. R.

S. R. Schwarze, K. A. Hruska, and S. F. Dowdy, Cell Biol. 10, 290 (2000).

Sibbett, W.

Smiley, S. T.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Smith, T. W.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

So, P. T. C.

Steele, G. D.

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

Stevenson, D.

Tirlapur, U. K.

U. K. Tirlapur and K. Konig, Nature 418, 290 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

Tromberg, B. J.

Tsampoula, X.

Venugopalan, V.

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Vogel, A.

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Wands, J.

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

Appl. Phys. B (1)

A. Vogel, J. Noack, G. Huttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[CrossRef]

Cell (1)

M. R. Capecchi, Cell 22, 479 (1980).
[CrossRef] [PubMed]

Cell Biol. (1)

S. R. Schwarze, K. A. Hruska, and S. F. Dowdy, Cell Biol. 10, 290 (2000).

Exp. Cell Res. (1)

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88 (2001).
[CrossRef] [PubMed]

FEBS Lett. (1)

R. Heller, M. Jaroszeski, A. Atkin, D. Moradpour, R. Gilbert, J. Wands, and C. Nicolau, FEBS Lett. 389, 225 (1996).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

Lasers Ophthalmol. (1)

F. Docchio, C. A. Sacchi, and J. Marshall, Lasers Ophthalmol. 1, 83 (1986).

Nature (1)

U. K. Tirlapur and K. Konig, Nature 418, 290 (2002).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Phys. Rev. Lett. (1)

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA (1)

S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele, Jr., and L. B. Chen, Proc. Natl. Acad. Sci. USA 88, 3671 (1991).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

Effect of fs laser irradiation on a cell membrane. A healthy HepG2 cell was exposed to a fs laser for 5 s . (a) White light and (b) red PI fluorescence images were observed 2 h later. (c) A healthy cell without laser exposure and no PI fluorescence was found 4 h later.

Fig. 2
Fig. 2

Confirmation of membrane sealing after laser illumination: (a)–(d) images of a group A cell, (e)–(h) images for a group B cell. (a) Cell before laser exposure ( t = 0 ) ; (b) cell at 20 min after laser exposure ( t = 20 min ) ; (c) same cell after four more hours ( t = 4 h 20 min ); (d) absence of fluorescence from the same cell at t = 4 h 20 min ; (e) and (f) cell in group B before and after laser exposure ( t = 20 min ) ; (g) same cell at t = 4 h 20 min ; (h) fluorescence detected from the cell at t = 4 h 20 min .

Fig. 3
Fig. 3

Effect of fs 1554 nm laser exposure on the mitochondrial depolarization in HepG2 cells loaded with JC-1. Laser illumination times: (a) 0, (b) 5, (c) 10, and (d) 20 s . (e) Cell treated with valinomycin but no laser illumination. Images in (1)–(3) were acquired before, while those in (4)–(6) were acquired 1.5 h after laser exposure.

Fig. 4
Fig. 4

Typical GFP fluorescence from HepG2 cells with targeted transfection.

Tables (1)

Tables Icon

Table 1 Average R/G Ratio of Cells at Different Exposure Times

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