Abstract

Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

© 2008 Optical Society of America

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  1. 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]
  2. U. K. Tirlapur and K. Konig, "Targeted transfection by femtosecond laser," Nature 418, 290-291 (2002).
    [CrossRef] [PubMed]
  3. A. Vogel, J. Noack, G. Huttman, and G. Paltauf, "Mechanisms of femtosecond laser nanosurgery of cells and tissues," Appl. Phys. B 81, 1015-1047 (2005).
    [CrossRef]
  4. 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]
  5. J. Baumgart, W. Bintig, A. Ngezahayo, S. Willenbrock, H. Murua Escobar, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, "Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells," Opt. Express 16, 3021-3031 (2008).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  7. V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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  13. F. Hache, T. J. Driscoll, M. Cavallari, and G. M. Gale, "Measurement of ultrashort pulse durations by interferometric autocorrelation: Influence of various parameters," Appl. Opt. 35, 3230-3236 (1996).
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2008 (1)

2007 (2)

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

2006 (3)

2005 (3)

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]

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

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation," Biotechnol. Bioeng. 92, 889-899 (2005).
[CrossRef] [PubMed]

2003 (1)

S. K. Eah, W. Jhe, and Y. Arakawa, "Nearly diffraction-limited focusing of a fiber axicon microlens," Rev. Sci. Instrum. 74, 4969-4971 (2003).
[CrossRef]

2002 (1)

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

1996 (1)

Acker, J. P.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation," Biotechnol. Bioeng. 92, 889-899 (2005).
[CrossRef] [PubMed]

Agate, B.

Arakawa, Y.

S. K. Eah, W. Jhe, and Y. Arakawa, "Nearly diffraction-limited focusing of a fiber axicon microlens," Rev. Sci. Instrum. 74, 4969-4971 (2003).
[CrossRef]

Baumgart, J.

Bintig, W.

Brown, C. T. A.

Bryant, P. E.

Cancela, M. L.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

Carruthers, A. E.

Cavallari, M.

Comrie, M.

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

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]

Dholakia, K.

Driscoll, T. J.

Eah, S. K.

S. K. Eah, W. Jhe, and Y. Arakawa, "Nearly diffraction-limited focusing of a fiber axicon microlens," Rev. Sci. Instrum. 74, 4969-4971 (2003).
[CrossRef]

Elezzabi, A. Y.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation," Biotechnol. Bioeng. 92, 889-899 (2005).
[CrossRef] [PubMed]

Ertmer, W.

Ferguson, R.

Fischer, P.

Gale, G. M.

Garces-Chavez, V.

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

Gunn-Moore, F.

Guo, C. K.

Hache, F.

Heckenberg, N. R.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Heisterkamp, A.

Huttman, G.

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

Jhe, W.

S. K. Eah, W. Jhe, and Y. Arakawa, "Nearly diffraction-limited focusing of a fiber axicon microlens," Rev. Sci. Instrum. 74, 4969-4971 (2003).
[CrossRef]

Knoner, G.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Kohli, V.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation," Biotechnol. Bioeng. 92, 889-899 (2005).
[CrossRef] [PubMed]

Konig, K.

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

Lake, T. K.

Liu, Z. H.

Lubatschowski, H.

Morris, J. E.

Murua Escobar, H.

Ngezahayo, A.

Nieminen, T. A.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Noack, J.

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

Paterson, L.

Ratnapala, A.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Riches, A.

Riches, A. C.

Robles, V.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

Rubinsztein-Dunlop, H.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Sibbett, W.

Stevenson, D.

Stevenson, D. J.

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

Tirlapur, U. K.

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

Tsampoula, X.

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

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]

Vale, C. J.

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Vogel, A.

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

Waskiewicz, A. J.

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

Willenbrock, S.

Yang, J.

Yuan, L. B.

Appl. Opt. (1)

Appl. Phys. B (1)

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

Appl. Phys. Lett. (1)

X. Tsampoula, V. Garces-Chavez, M. Comrie, D. J. Stevenson, B. Agate, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, "Femtosecond cellular transfection using a nondiffracting light beam," Appl. Phys. Lett. 91, 053902 (2007).
[CrossRef]

Biotechnol. Bioeng. (2)

V. Kohli, J. P. Acker, and A. Y. Elezzabi, "Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation," Biotechnol. Bioeng. 92, 889-899 (2005).
[CrossRef] [PubMed]

V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, "An alternative method for delivering exogenous material into developing zebrafish embryos," Biotechnol. Bioeng. 98, 1230-1241 (2007).
[CrossRef] [PubMed]

Lab on a Chip (1)

G. Knoner, A. Ratnapala, T. A. Nieminen, C. J. Vale, N. R. Heckenberg, and H. Rubinsztein-Dunlop, "Optical force field mapping in microdevices," Lab on a Chip 6, 1545-1547 (2006).
[CrossRef]

Nature (1)

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

Opt. Express (4)

Rev. Sci. Instrum. (1)

S. K. Eah, W. Jhe, and Y. Arakawa, "Nearly diffraction-limited focusing of a fiber axicon microlens," Rev. Sci. Instrum. 74, 4969-4971 (2003).
[CrossRef]

Other (1)

T. Cizmar, Optical traps generated by non-traditional beams, (Masaryk University, Brno, 2006), pp. 127, Phd thesis.

Supplementary Material (1)

» Media 1: MPG (2916 KB)     

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Figures (4)

Fig. 1.
Fig. 1.

The simulated and measured axial intensity profiles of the fibre output beam (a) in air and (b) water media. ρ and z are the cylindrical coordinates with the z-axis pointing in the direction of the beam propagation. Experimental data are obtained from the azimuthally averaged beam profiles of the manufactured fibre output taken by CCD camera. The units I0 used in the simulations correspond to on-axis intensity of the fibre mode.

Fig. 2.
Fig. 2.

(a). Optimal angle of the axicon tip as a function of the working distance (b) the maximum beam intensity obtained at each working distance using the optimal fibre. I0 is the on-axis intensity of the fibre mode (c) comparison of the on-axis intensity of the fabricated fibre with the maximal achievable intensity for the optimal fibre (Fig. 2(b)) and the bare-tipped fibre.

Fig. 3.
Fig. 3.

(a). The microscope based photoporation apparatus using an axicon tipped fibre. The laser beam generated by the mode locked Ti: Sapphire laser was sent through a x1.6 demagnifing telescope and was subsequently coupled into the axicon tipped fibre which was rigidly mounted on a xyz translation stage. Each irradiated cell experienced three laser doses, each of 80 ms duration. (b). The distance of the fibre with respect to the cell membrane was the minimum possible distance, approximately 13 µm, as dictated by the geometry of the setup. The fibre is optimally positioned at 70 degrees with respect to the cell monolayer regardless of the cell shape. The working distance is determined to be the distance between the axicon tip and the cell membrane.

Fig. 4.
Fig. 4.

CHO cells during and after laser irradiation (a) The axicon tipped fibre is inserted inside the CHO sample and irradiates the cell from a distance equal to 13 µm. Each irradiated cell experiences three doses of femtosecond pulses, each of 80 ms duration. During photoporation no visual response was observed (Media 1). (b) Upon successful photoporation the cells uptake the plasmid and express the mitochondrially targeted red fluorescent protein.

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