Abstract

Photoporation is a rapidly expanding technique for the introduction of macromolecules into single cells. However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm. Photoporation of 4000 Chinese Hamster ovary cells was performed, representing the largest optical transfection study reported to date. We have investigated a range of laser fluences at the cell membrane and, at 1.2 μJ/cm2, have found an average transfection efficiency of 50 ± 10%. Contrary to recent literature, in which 100% efficiency is claimed, our measure of efficiency accounts for all irradiated cells, including those lost as a result of laser treatment, thereby providing a true biological measure of the technique.

© 2006 Optical Society of America

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  1. S. Mehier-Humbert and R. H. Guy, "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells," Adv. Drug Delivery Rev. 57,733-753 (2005).
    [CrossRef]
  2. H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
    [CrossRef] [PubMed]
  3. G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
    [CrossRef] [PubMed]
  4. L. Paterson, B. Agate, M. Comrie, R. Ferguson, T. K. Lake, J. E. Morris, A. E. Carruthers, C. T. A. Brown, W. Sibbett, P. E. Bryant, F. Gunn-Moore, A. C. Riches, and K. Dholakia, "Photoporation and cell transfection using a violet diode laser," Opt. Express 13, 595-600 (2005).
    [CrossRef] [PubMed]
  5. M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
    [CrossRef]
  6. Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
    [CrossRef]
  7. J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
    [CrossRef] [PubMed]
  8. S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
    [CrossRef] [PubMed]
  9. S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
    [CrossRef]
  10. U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
    [CrossRef] [PubMed]
  11. U. K. Tirlapur and K. Konig, "Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability," Plant J. 31, 365-374 (2002).
    [CrossRef] [PubMed]
  12. E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
    [CrossRef]
  13. A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
    [CrossRef]
  14. D. Stevenson, B. Agate, L. Paterson, T. K. Lake, M. Comrie, C. T. A. Brown, A. C. Riches, P. E. Bryant, W. Sibbett, F. Gunn-Moore, and K. Dholakia, "Optical transfection of mammalian cells," presented at SPIE Photonics Europe, Strasbourg, France, 3-7 April 2006.
  15. M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
    [CrossRef] [PubMed]
  16. A. Heisterkamp and H. Lubatschowski, "Subcellular photodisruption,"Femtosecond technology for technical and medical applications, Top. Appl. Phys. 96, 227-232 (2004).
    [CrossRef]
  17. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, "Pulse energy dependence of subcellular dissection by femtosecond laser pulses," Opt. Express 13,3690-3696 (2005).
    [CrossRef] [PubMed]
  18. U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
    [CrossRef] [PubMed]

2006 (1)

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

2005 (4)

2004 (1)

A. Heisterkamp and H. Lubatschowski, "Subcellular photodisruption,"Femtosecond technology for technical and medical applications, Top. Appl. Phys. 96, 227-232 (2004).
[CrossRef]

2003 (3)

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
[CrossRef]

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

2002 (3)

U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur and K. Konig, "Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability," Plant J. 31, 365-374 (2002).
[CrossRef] [PubMed]

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

2001 (2)

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

2000 (1)

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

1996 (1)

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

1984 (1)

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

Agate, B.

Alken, P.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Allbritton, N. L.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Brown, C. T. A.

Bryant, P. E.

Carruthers, A. E.

Caruso, M.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Colasanti, A.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Comrie, M.

Crescenzi, E.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Darash-Yahana, M.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Dholakia, K.

Ferguson, R.

Galun, E.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Gunn-Moore, F.

Gupta, P. K.

S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
[CrossRef]

Guy, R. H.

S. Mehier-Humbert and R. H. Guy, "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells," Adv. Drug Delivery Rev. 57,733-753 (2005).
[CrossRef]

Halbhuber, K. J.

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Heisterkamp, A.

A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, "Pulse energy dependence of subcellular dissection by femtosecond laser pulses," Opt. Express 13,3690-3696 (2005).
[CrossRef] [PubMed]

A. Heisterkamp and H. Lubatschowski, "Subcellular photodisruption,"Femtosecond technology for technical and medical applications, Top. Appl. Phys. 96, 227-232 (2004).
[CrossRef]

Hendinger, A.

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

Honigman, A.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Huttmann, G.

A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
[CrossRef]

Hyam, E.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Ikawa, Y.

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

Ingber, D. E.

Itagak, H.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

Jacobson, S. C.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Kasuya, T.

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

Khatchatouriants, A.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Knoll, T.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Konig, K.

U. K. Tirlapur and K. Konig, "Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability," Plant J. 31, 365-374 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Krasieva, T.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Krieg, R.

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Kumar, S.

Kurata, S.

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

Lake, T. K.

Lewis, A.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Lubatschowski, H.

A. Heisterkamp and H. Lubatschowski, "Subcellular photodisruption,"Femtosecond technology for technical and medical applications, Top. Appl. Phys. 96, 227-232 (2004).
[CrossRef]

Manevitch, A.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Maxwell, I. Z.

Mazur, E.

Mehier-Humbert, S.

S. Mehier-Humbert and R. H. Guy, "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells," Adv. Drug Delivery Rev. 57,733-753 (2005).
[CrossRef]

Michel, M. S.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Mohanty, S. K.

S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
[CrossRef]

Morris, J. E.

Nickerson, J. A.

Noack, J.

A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
[CrossRef]

Nomiya, Y.

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

O'Donnell, M.

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

Ohkohchi, N.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

Palumbo, G.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Pappo, O.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Paterson, L.

Paultauf, G.

A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
[CrossRef]

Peuckert, C.

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Ramsey, J. M.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Riches, A. C.

Roberti, G.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Sagi, S.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Sailer, R.

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

Satomi, S.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

Schaaf, A.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Schmidtt, M.

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

Schneckenburger, H.

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

Sharma, M.

S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
[CrossRef]

Shirahata, Y.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

Sibbett, W.

Soughayer, J. S.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Strauss, W. S. L.

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

Tavor, E.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Tecce, M. F.

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Tirlapur, U. K.

U. K. Tirlapur and K. Konig, "Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability," Plant J. 31, 365-374 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Trojan, L.

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Tromberg, B. J.

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Tse, C.

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

Tsukakoshi, M.

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

Underwood, J. M.

Vogel, A.

A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
[CrossRef]

Ye, J. Y.

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

Zeira, E.

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Zohdy, M. J.

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

Adv. Drug Delivery Rev. (1)

S. Mehier-Humbert and R. H. Guy, "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells," Adv. Drug Delivery Rev. 57,733-753 (2005).
[CrossRef]

Anal. Chem. (1)

J. S. Soughayer, T. Krasieva, S. C. Jacobson, J. M. Ramsey, B. J. Tromberg, and N. L. Allbritton, "Characterization of cellular optoporation with distance," Anal. Chem. 72, 1342-1347 (2000).
[CrossRef] [PubMed]

Appl. Phys. B (2)

M. Tsukakoshi, S. Kurata, Y. Nomiya, Y. Ikawa, and T. Kasuya, "A novel method of DNA transfection by laser microbeam cell surgery," Appl. Phys. B 35, 135-140 (1984).
[CrossRef]

A. Vogel, J. Noack, G. Huttmann, and G. Paultauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
[CrossRef]

Biotech. Lett. (1)

S. K. Mohanty, M. Sharma, and P. K. Gupta, "Laser-assisted microinjection into targeted animal cells," Biotech. Lett. 25, 895-899 (2003).
[CrossRef]

Exp. Cell Res. (1)

U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J. Halbhuber, "Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death," Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

IEEE Trans. Ultrason. Ferroelectr. Freq. Control (1)

M. J. Zohdy, C. Tse, J. Y. Ye, and M. O'Donnell, "Optical and acoustic detection of laser-generated microbubbles in single cells," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53, 117-125 (2006).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmidtt, "Laser-assisted optoporation of single cells," J. Biomed. Opt. 7,410-416 (2002).
[CrossRef] [PubMed]

J. Inv. Med. (1)

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, "New technique for gene transfection using laser irradiation," J. Inv. Med. 49, 184-190 (2001).
[CrossRef]

J. Photochem. Photobiol. B (1)

G. Palumbo, M. Caruso, E. Crescenzi, M. F. Tecce, G. Roberti, and A. Colasanti, "Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation," J. Photochem. Photobiol. B 36, 41-46 (1996).
[CrossRef] [PubMed]

Mol. Therapy (1)

E. Zeira, A. Manevitch, A. Khatchatouriants, O. Pappo, E. Hyam, M. Darash-Yahana, E. Tavor, A. Honigman, A. Lewis, and E. Galun, "Femtosecond infrared laser - an efficient and safe in vivo gene delivery system for prolonged expression," Mol. Therapy 8,342-350 (2003).
[CrossRef]

Nature (1)

U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

Opt. Express (2)

Plant J. (1)

U. K. Tirlapur and K. Konig, "Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability," Plant J. 31, 365-374 (2002).
[CrossRef] [PubMed]

Prostate Cancer Prostatic Dis. (1)

S. Sagi, T. Knoll, L. Trojan, A. Schaaf, P. Alken, and M. S. Michel, "Gene delivery into prostate cancer cells by holmium laser application," Prostate Cancer Prostatic Dis. 6, 127-130 (2003).
[CrossRef] [PubMed]

Top. Appl. Phys. (1)

A. Heisterkamp and H. Lubatschowski, "Subcellular photodisruption,"Femtosecond technology for technical and medical applications, Top. Appl. Phys. 96, 227-232 (2004).
[CrossRef]

Other (1)

D. Stevenson, B. Agate, L. Paterson, T. K. Lake, M. Comrie, C. T. A. Brown, A. C. Riches, P. E. Bryant, W. Sibbett, F. Gunn-Moore, and K. Dholakia, "Optical transfection of mammalian cells," presented at SPIE Photonics Europe, Strasbourg, France, 3-7 April 2006.

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Figures (7)

Fig. 1.
Fig. 1.

The photoporation/transfection apparatus. A femtosecond titanium sapphire laser was first passed though a variable neutral density filter and a beam-shutter in order to control the intensity and duration of the laser dose, and the beam was then expanded with a lens relay telescope to fill the back of a microscope objective (×60; N.A.= 0.85).

Fig. 2.
Fig. 2.

Detailed diagram of the “sample” from Fig. 1. The use of a floating coverslip allowed a hole to be generated by photoporation on the non-adhered (top) side of the cell of interest, allowing the surrounding pEGFP DNA solution to penetrate the cell. (Not to scale).

Fig. 3.
Fig. 3.

Representative images at 48h of selected CHO cells optically transfected with GFP, highlighting the specificity of the technique Cells are co-stained with the blue nuclear dye, DAPI (i.e. the blue nuclei surrounding the green targeted cells represent cells that have not been transfected). (a) ×10 and (b) ×60 magnification.

Fig. 4.
Fig. 4.

Typical reactions of adherent CHO cells exposed to 60 ms (A1–A3) and 250 ms (B1–B3) of femtosecond irradiation, 200 mW at focus, immediately before (A1, B1), during (A2, B2), and 1 minute after exposure (A3, B3). Scale bar = 10 μm.

Fig. 5.
Fig. 5.

(a) CHO cell (indicated) photoporated in the presence of the normally membrane impermeable dye, trypan blue; (b) 30 second later, photoporation has acted to permeabilise the cell membrane, allowing the external substance to enter by phototranslocation.

Fig. 6.
Fig. 6.

Transfection efficiency data for the photoporation of n = 4000 cells. Exposure times varied from 10 ms to 250 ms, and average laser powers at focus varied from 168 mW to 225 mW. Main graph: As a function of laser fluence, Φ, at the cell membrane: BOXES represent range of transfection efficiencies achieved; DATA POINTS represent the average transfection efficiency; BARS represent the standard error of the mean (calculated for laser fluences at which n > 400 cells were photoporated). Inset: Data set for photoporated cells at fluences up to 7 μJ/cm2. Each data point corresponds to n = 100 photoporated cells.

Fig. 7.
Fig. 7.

(Grayscale) Detail of a laser-micromachined region of interest pre-marked on a polystyrene culture dish by ablation, with CHO cells then added and grown to confluence. (a) Before laser treatment; (b) Following laser treatment and, after 2h, the subsequent addition of 0.1% trypan blue. Every cell within the upper region only was photoporated. Cells appearing darker had taken up the trypan blue dye and were assumed to be non-viable.

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