Abstract

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

© 2008 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
    [CrossRef] [PubMed]
  2. D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
    [CrossRef] [PubMed]
  3. E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
    [CrossRef] [PubMed]
  4. J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
    [CrossRef] [PubMed]
  5. W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
    [CrossRef] [PubMed]
  6. P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
    [CrossRef] [PubMed]
  7. U.K. Tirlapur and K. K¨onig, "Targeted transfection by femtosecond laser," Nature (London) 418, 290-291 (2002)
    [CrossRef] [PubMed]
  8. D. Stevenson, B. Agate, X. Tsampoula, P. Fischer, C. T. A. Brown,W. Sibbett, A. Riches, F. Gunn-Moore, and K. Dholakia, "Femtosecond optical transfection of cells: viability and efficiency," Opt. Express 14, 7125-7133 (2006)
    [CrossRef] [PubMed]
  9. V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
    [CrossRef]
  10. A. Vogel, J. Noack, G. Huettman, and G. Paltauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005)
    [CrossRef]
  11. A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
    [CrossRef]
  12. M. Bustin, "Regulation of DNA-Dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins," Mol. Cell. Biol. 19, 5237-5246 (1999)
    [PubMed]
  13. K. K¨onig, "Multophoton Microscopy in Life Sciences," J. Microscopy  2, 83-104 (2000)
    [CrossRef]
  14. O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
    [CrossRef] [PubMed]
  15. E. Neher and B. Sakmann, "Single-channel currents recorded from membrane of denervated frog muscle fibres," Nature (London) 260, 799-802 (1976)
    [CrossRef] [PubMed]
  16. M. Numberger and A. Draguhn, Patch-Clamp-Technik (Spektrum Akademischer Verlag, 1996)
  17. D. Goldman, "Potential, impedance, and rectification in membranes," J. Gen. Physiol. 27, 37-60 (1943)
    [CrossRef] [PubMed]

2006 (1)

2005 (3)

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
[CrossRef]

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

P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
[CrossRef] [PubMed]

2003 (2)

A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
[CrossRef]

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

2002 (1)

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

2000 (1)

K. K¨onig, "Multophoton Microscopy in Life Sciences," J. Microscopy  2, 83-104 (2000)
[CrossRef]

1999 (2)

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

M. Bustin, "Regulation of DNA-Dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins," Mol. Cell. Biol. 19, 5237-5246 (1999)
[PubMed]

1994 (1)

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

1992 (1)

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

1991 (1)

E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
[CrossRef] [PubMed]

1981 (1)

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

1976 (1)

E. Neher and B. Sakmann, "Single-channel currents recorded from membrane of denervated frog muscle fibres," Nature (London) 260, 799-802 (1976)
[CrossRef] [PubMed]

1943 (1)

D. Goldman, "Potential, impedance, and rectification in membranes," J. Gen. Physiol. 27, 37-60 (1943)
[CrossRef] [PubMed]

Acker, J. P.

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
[CrossRef]

Agate, B.

Altmann, B.

A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
[CrossRef]

Astumian, R. D.

E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
[CrossRef] [PubMed]

Bhatia, M.

P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
[CrossRef] [PubMed]

Brown, C. T. A.

Bustin, M.

M. Bustin, "Regulation of DNA-Dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins," Mol. Cell. Biol. 19, 5237-5246 (1999)
[PubMed]

Chalfie, M.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

Chock, P. B.

E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
[CrossRef] [PubMed]

Cormier, M. J.

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

Dholakia, K.

Doronin, K.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Eckenrode, V. K.

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

Elezzabi, A. Y.

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
[CrossRef]

Euskirchen, G.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

Fischer, P.

Goldman, D.

D. Goldman, "Potential, impedance, and rectification in membranes," J. Gen. Physiol. 27, 37-60 (1943)
[CrossRef] [PubMed]

Gunn-Moore, F.

Hamill, O. P.

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

Huettman, G.

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

K¨onig, K.

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

K. K¨onig, "Multophoton Microscopy in Life Sciences," J. Microscopy  2, 83-104 (2000)
[CrossRef]

Kohli, V.

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
[CrossRef]

Kolb, H.-A.

A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
[CrossRef]

Kuppuswamy, M.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Lichtenstein, D. L.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Marty, A.

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

McCarty, D. M.

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Menendez, P.

P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
[CrossRef] [PubMed]

Negishi, A.

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Neher, E.

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

E. Neher and B. Sakmann, "Single-channel currents recorded from membrane of denervated frog muscle fibres," Nature (London) 260, 799-802 (1976)
[CrossRef] [PubMed]

Ngezahayo, A.

A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
[CrossRef]

Noack, J.

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

Paltauf, G.

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

Prasher, D. C.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

Prendergast, F. G.

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

Riches, A.

Sakmann, B.

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

E. Neher and B. Sakmann, "Single-channel currents recorded from membrane of denervated frog muscle fibres," Nature (London) 260, 799-802 (1976)
[CrossRef] [PubMed]

Samulski, R. J.

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Sibbett, W.

Sigworth, F. J.

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

Stevenson, D.

Stilwell, J. L.

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Superfine, R.

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Tekle, E.

E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
[CrossRef] [PubMed]

Tirlapur, U. K.

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

Tollefson, A. E.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Toth, K.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Tsampoula, X.

Tu, Y.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

Vogel, A.

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

Wang, L.

P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
[CrossRef] [PubMed]

Ward, W. W.

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

Wold, W. S.

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Appl. Phys. B (1)

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

Biotechnol. Bioeng. (1)

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation," Biotechnol. Bioeng. 92, 7 (2005)
[CrossRef]

Curr. Gene Ther. (1)

P. Menendez, L. Wang, and M. Bhatia, "Genetic Manipulation of Human Embryonic stemm cells: A System to Study Early Human Development and Potential Therapeutic Applications," Curr. Gene Ther. 5, 375-385 (2005)
[CrossRef] [PubMed]

Curr. Opin. Immunol. (1)

W. S. Wold, K. Doronin, K. Toth, M. Kuppuswamy, D. L. Lichtenstein, and A. E. Tollefson, "Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic," Curr. Opin. Immunol. 11, 380-386 (1999)
[CrossRef] [PubMed]

Gene (1)

D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier, "Primary structure of the Aequorea Victoria green-fluorescent preotein," Gene 111, 229-233 (1992)
[CrossRef] [PubMed]

J. Gen. Physiol. (1)

D. Goldman, "Potential, impedance, and rectification in membranes," J. Gen. Physiol. 27, 37-60 (1943)
[CrossRef] [PubMed]

J. Membrane Biol. (1)

A. Ngezahayo, B. Altmann, and H.-A. Kolb, "Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells," J. Membrane Biol. 194, 165-176 (2003)
[CrossRef]

J. Microscopy (1)

K. K¨onig, "Multophoton Microscopy in Life Sciences," J. Microscopy  2, 83-104 (2000)
[CrossRef]

J. Virol. (1)

J. L. Stilwell, D. M. McCarty, A. Negishi, R. Superfine, and R. J. Samulski, "Development anf Characterization of Novel Empty Adenovirus Capsids and Teir Impact on Cellular Gene Expression," J. Virol.12881-12885 (2003)
[CrossRef] [PubMed]

Mol. Cell. Biol. (1)

M. Bustin, "Regulation of DNA-Dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins," Mol. Cell. Biol. 19, 5237-5246 (1999)
[PubMed]

Nature (London) (2)

E. Neher and B. Sakmann, "Single-channel currents recorded from membrane of denervated frog muscle fibres," Nature (London) 260, 799-802 (1976)
[CrossRef] [PubMed]

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

Opt. Express (1)

Pfl¨ugers Arch. (1)

O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pfl¨ugers Arch. 391, 85-100 (1981)
[CrossRef] [PubMed]

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

E. Tekle, R. D. Astumian, and P. B. Chock, "Electroporation by Using Bipolar Oscillating Electric Field: An Improved Method for DNA Transfection of NIH 3T3 Cells," Proc. Natl. Acad. Sci. USA 88, 4230-4234 (1991)
[CrossRef] [PubMed]

Science (1)

M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, "Green fluorescent protein as a marker for gene expression," Science 263, 802-805 (1994)
[CrossRef] [PubMed]

Other (1)

M. Numberger and A. Draguhn, Patch-Clamp-Technik (Spektrum Akademischer Verlag, 1996)

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (7)

Fig. 1.
Fig. 1.

(Color online) Schematic setup of the opto-perforation system.

Fig. 2.
Fig. 2.

(Color online) Sketch of simultaneous patch-clamp and opto-perforation of a living cell. The induced transient pore allows the diffusion of molecules through the membrane.

Fig. 3.
Fig. 3.

The membrane potential of a granulosa cell during fs laser perforation. The laser pulse energy was 0.9 nJ. The grey bar represents the laser irradiation time t for the optoperforation, Δt represents the maximum depolarization time. (A) There was no bubble formation during the treatment (n=7); (B) a small gas bubble was created during the treatment (n=4).

Fig. 4.
Fig. 4.

The fluorescence intensity of LY introduced in cells at different concentrations via the patch-clamp pipette. The data points represent average standard deviation for at least 5 different cells for each concentration. The linear fit (f(x)=0.46x+1.77) was used to estimate the concentration of LY in the cells after opto-perforation induced uptake of the chromophor dissolved in the extracellular solution. The area of interest for the used extracellular concentrations during opto-perforation is zoomed out. As an example, to the extracellular LY concentration ([LY]o) of 600 µM corresponds an intracellular concentration ([LY] i ) of 263 µM and a fluorescence intensity of 123 a.u. (table 3.2) represented by the grey triangle.

Fig. 5.
Fig. 5.

(Color online) Opto-perforated granulosa cells in presence of PI. (A) Fluorescence image of granulosa cells growing on a cover slit during opto-perforation, the treated cells are highlighted by the dashed circles. 1.5 µM PI is solved in the media and the laser parameters were 0.9 nJ and 40 ms. All manipulated cells are fluorescent. (B) Bright field image of the same cells. C: Fluorescence image of the cells after 90 minutes incubation in PBS. The cells were re-stained with PI to verify the viability. The cell pointed out by the arrow is representative for a cell whose membrane is damaged and therefore still permeable for the fluorophore. D: Bright field image after the incubation time. Scale bars: 30 µm.

Fig. 6.
Fig. 6.

(Color online) The viability of the cells (A) and the efficiency of the introduction of PI into the cells (B) dependent on the pulse energy and the irradiation time.

Fig. 7.
Fig. 7.

(Color online) MTH53a cells transfected with either pEGFP-C1 or pEGFP-C1-HMGB1 vectors. (A) Complete labelling of MTH53a cells by GFP. (B) Specific labelling of the MTH53a cell nucleus by pEGFP-C1-HMGB1 fusion proteins. The opto-perforation was performed at a wavelength of 800 nm, a pulse energy of 0.9 nJ and an irradiation time of 40 ms. The images were taken 48 hours after the treatment. Scale bars: 20 µm.

Tables (1)

Tables Icon

Table 1. Fluorescence intensity of LYmeasured in cells after application of opto-perforation in presence of different LY concentrations in the extracellular solution ([LY]o). The values were reported on the calibration line (Fig. 4) to estimate the intracellular LY concentration ([LY] i ). The measured relative volume exchanged is given as [LY] i /[LY] o . The expected intracellular concentrations were calculated by assuming α/V=0.4. These values were reported to the calibration curve to obtain the expected fluorescence intensities. All measured values include ± standard deviation.

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

Δ t > t
α V = ( 1 exp ( Δ U m F RT ) ( 1 exp ( U m F RT ) )

Metrics