Abstract

Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free space bulk optical setup. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. Here we report the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining a microlens fiber based optical transfection system with a micro-capillary based microfluidic system. A fiber based illumination system is also incorporated in the system in order to achieve visual identification of the cell boundaries during transfection. A novel fabrication method is devised to obtain easy and inexpensive fabrication of microlensed fibers, which can be used for femtosecond optical transfection. This fabrication method offers the flexibility to fabricate a microlens which can focus ultra-short laser pulses at a near infrared wavelength to a small focal spot (~3 µm) whilst keeping a relatively large working distance (~20 µm). The transfection efficiency of the integrated system with localized plasmid DNA delivery, is approximately 50%, and is therefore comparable to that of a standard free space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of DNA for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, including the dosing of tissue slices, targeted drug delivery, and targeted gene therapy in vivo.

©2010 Optical Society of America

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  1. D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Transfection by optical injection,” in The Handbook of Photonics for Medical Science, T. V.V, ed. (CRC Press, Taylor & Francis Group, London, 2010), pp. 87–117.
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    [Crossref] [PubMed]
  6. 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(5), 3021–3031 (2008).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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2010 (1)

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010).
[Crossref] [PubMed]

2009 (2)

2008 (7)

H. Y. Choi, S. Y. Ryu, J. H. Na, B. H. Lee, I. B. Sohn, Y. C. Noh, and J. M. Lee, “Single-body lensed photonic crystal fibers as side-viewing probes for optical imaging systems,” Opt. Lett. 33(1), 34–36 (2008).
[Crossref] [PubMed]

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(5), 3021–3031 (2008).
[Crossref] [PubMed]

S. Y. Ryu, H. Y. Choi, J. Na, W. J. Choi, and B. H. Lee, “Lensed fiber probes designed as an alternative to bulk probes in optical coherence tomography,” Appl. Opt. 47(10), 1510–1516 (2008).
[Crossref] [PubMed]

A. Uchugonova, K. König, R. Bueckle, A. Isemann, and G. Tempea, “Targeted transfection of stem cells with sub-20 femtosecond laser pulses,” Opt. Express 16(13), 9357–9364 (2008).
[Crossref] [PubMed]

X. Tsampoula, K. Taguchi, T. Cizmár, V. Garces-Chavez, N. Ma, S. Mohanty, K. Mohanty, F. Gunn-Moore, and K. Dholakia, “Fibre based cellular transfection,” Opt. Express 16(21), 17007–17013 (2008).
[Crossref] [PubMed]

R. Le Harzic, M. Weinigel, I. Riemann, K. König, and B. Messerschmidt, “Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens,” Opt. Express 16(25), 20588–20596 (2008).
[Crossref] [PubMed]

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

2006 (2)

2004 (1)

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

2003 (1)

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

2002 (1)

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002).
[Crossref] [PubMed]

2001 (2)

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

A. Malki, R. Bachelot, and F. Van Lauwe, “Two-step process for micro-lens-fibre fabrication using a continuous CO2 laser source,” J. Opt. A, Pure Appl. Opt. 3(4), 291–295 (2001).
[Crossref]

2000 (1)

T. Held, S. Emonin, O. Marti, and O. Hollricher, “Method to produce high-resolution scanning near-field optical microscope probes by beveling optical fibers,” Rev. Sci. Instrum. 71(8), 3118–3122 (2000).
[Crossref]

1996 (1)

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(3), 135–140 (1984).
[Crossref]

Agate, B.

Aouani, H.

Bachelot, R.

X. H. Zeng, J. Plain, S. Jradi, P. R. Goud, R. Deturche, P. Royer, and R. Bachelot, “High speed sub-micrometric microscopy using optical polymer microlens,” Chin. Opt. Lett. 7(10), 901–903 (2009).
[Crossref]

A. Malki, R. Bachelot, and F. Van Lauwe, “Two-step process for micro-lens-fibre fabrication using a continuous CO2 laser source,” J. Opt. A, Pure Appl. Opt. 3(4), 291–295 (2001).
[Crossref]

Baumgart, J.

Bintig, W.

Brown, C. T. A.

Bueckle, R.

Campbell, P.

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010).
[Crossref] [PubMed]

Cao, C. L.

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

Cavallari, M.

Chang, S.

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

Cheng, W. H.

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

Choi, H. Y.

Choi, W. J.

Cizmár, T.

Deiss, F.

Deturche, R.

Dholakia, K.

Driscoll, T. J.

Emonin, S.

T. Held, S. Emonin, O. Marti, and O. Hollricher, “Method to produce high-resolution scanning near-field optical microscope probes by beveling optical fibers,” Rev. Sci. Instrum. 71(8), 3118–3122 (2000).
[Crossref]

Ertmer, W.

Esashi, M.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Ferrand, P.

Fischer, P.

Gale, G. M.

Garces-Chavez, V.

Goud, P. R.

Gunn-Moore, F.

Gunn-Moore, F. J.

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010).
[Crossref] [PubMed]

Guo, R.

Hache, F.

Haga, Y.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Han, M.

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

Hane, K.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Heisterkamp, A.

Held, T.

T. Held, S. Emonin, O. Marti, and O. Hollricher, “Method to produce high-resolution scanning near-field optical microscope probes by beveling optical fibers,” Rev. Sci. Instrum. 71(8), 3118–3122 (2000).
[Crossref]

Hollricher, O.

T. Held, S. Emonin, O. Marti, and O. Hollricher, “Method to produce high-resolution scanning near-field optical microscope probes by beveling optical fibers,” Rev. Sci. Instrum. 71(8), 3118–3122 (2000).
[Crossref]

Huang, W. H.

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(3), 135–140 (1984).
[Crossref]

Inoue, K.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Isemann, A.

Itagak, H.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

Jradi, S.

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(3), 135–140 (1984).
[Crossref]

Kim, J.

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

König, K.

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(3), 135–140 (1984).
[Crossref]

Le Harzic, R.

Lee, B. H.

Lee, J. M.

Lee, J. W.

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

Li, J. W.

Liu, Y. D.

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

Lu, Y. K.

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

Lubatschowski, H.

Ma, N.

Malki, A.

A. Malki, R. Bachelot, and F. Van Lauwe, “Two-step process for micro-lens-fibre fabrication using a continuous CO2 laser source,” J. Opt. A, Pure Appl. Opt. 3(4), 291–295 (2001).
[Crossref]

Marti, O.

T. Held, S. Emonin, O. Marti, and O. Hollricher, “Method to produce high-resolution scanning near-field optical microscope probes by beveling optical fibers,” Rev. Sci. Instrum. 71(8), 3118–3122 (2000).
[Crossref]

Messerschmidt, B.

Minh, P. N.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Mohanty, K.

Mohanty, S.

Murua Escobar, H.

Na, J.

Na, J. H.

Ngezahayo, A.

Noh, Y. C.

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(3), 135–140 (1984).
[Crossref]

Oh, K.

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

Ohkohchi, N.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

Ono, T.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Plain, J.

Riches, A.

Riemann, I.

Rigneault, H.

Royer, P.

Ryu, S. Y.

Sasaki, M.

P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

Satomi, S.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

Shirahata, Y.

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

Sibbett, W.

Sohn, I. B.

Sojic, N.

Stevenson, D.

Stevenson, D. J.

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010).
[Crossref] [PubMed]

Taguchi, K.

Tempea, G.

Tirlapur, U. K.

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002).
[Crossref] [PubMed]

Tsai, Y. C.

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

Tsampoula, X.

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(3), 135–140 (1984).
[Crossref]

Uchugonova, A.

Van Lauwe, F.

A. Malki, R. Bachelot, and F. Van Lauwe, “Two-step process for micro-lens-fibre fabrication using a continuous CO2 laser source,” J. Opt. A, Pure Appl. Opt. 3(4), 291–295 (2001).
[Crossref]

Weinigel, M.

Wenger, J.

Willenbrock, S.

Xia, A. D.

Xiao, S. Z.

Zeng, X. H.

Zhai, X. M.

Appl. Opt. (2)

Appl. Phys. B (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(3), 135–140 (1984).
[Crossref]

Chin. Opt. Lett. (1)

IEEE Photon. Technol. Lett. (1)

J. Kim, M. Han, S. Chang, J. W. Lee, and K. Oh, “Achievement of large spot size and long collimation length using UV curable self-assembled polymer lens on a beam expanding core-less silica fiber,” IEEE Photon. Technol. Lett. 16(11), 2499–2501 (2004).
[Crossref]

J. Investig. Med. (1)

Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, “New technique for gene transfection using laser irradiation,” J. Investig. Med. 49(02), 184–190 (2001).
[Crossref] [PubMed]

J. Micromech. Microeng. (1)

Y. C. Tsai, Y. D. Liu, C. L. Cao, Y. K. Lu, and W. H. Cheng, “A new scheme of fiber end-face fabrication employing a variable torque technique,” J. Micromech. Microeng. 18(5), 055003 (2008).
[Crossref]

J. Opt. A, Pure Appl. Opt. (1)

A. Malki, R. Bachelot, and F. Van Lauwe, “Two-step process for micro-lens-fibre fabrication using a continuous CO2 laser source,” J. Opt. A, Pure Appl. Opt. 3(4), 291–295 (2001).
[Crossref]

J. R. Soc. Interface (1)

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010).
[Crossref] [PubMed]

Nature (1)

U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002).
[Crossref] [PubMed]

Opt. Express (7)

A. Uchugonova, K. König, R. Bueckle, A. Isemann, and G. Tempea, “Targeted transfection of stem cells with sub-20 femtosecond laser pulses,” Opt. Express 16(13), 9357–9364 (2008).
[Crossref] [PubMed]

X. Tsampoula, K. Taguchi, T. Cizmár, V. Garces-Chavez, N. Ma, S. Mohanty, K. Mohanty, F. Gunn-Moore, and K. Dholakia, “Fibre based cellular transfection,” Opt. Express 16(21), 17007–17013 (2008).
[Crossref] [PubMed]

R. Le Harzic, M. Weinigel, I. Riemann, K. König, and B. Messerschmidt, “Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens,” Opt. Express 16(25), 20588–20596 (2008).
[Crossref] [PubMed]

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[Crossref] [PubMed]

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P. N. Minh, T. Ono, Y. Haga, K. Inoue, M. Sasaki, K. Hane, and M. Esashi, “Bach fabrication of microlens at the end of optical fiber using self-photolithgraphy and etching techniques,” Opt. Rev. 10(3), 150–154 (2003).
[Crossref]

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[Crossref]

Other (1)

D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Transfection by optical injection,” in The Handbook of Photonics for Medical Science, T. V.V, ed. (CRC Press, Taylor & Francis Group, London, 2010), pp. 87–117.

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Figures (10)

Fig. 1
Fig. 1 SEM image of the polymer microlens fabricated at the facet of an optical fiber. (inset): shows the apex of the microlens.

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Fig. 2
Fig. 2 Schematic of the experimental setup for fiber based optical transfection. A collimated laser beam generated by Ti: Sapphire laser is directed onto a half wave plate and an optical isolator. Lens L1 and L2 expands the incoming laser beam by 1.6 times and subsequently couples the beam into the 35 cm long optical fiber, through a fiber collimator. For axicon and microlens tipped fiber transfection, an LED light source is used for illumination. For integrated system, the illumination fiber is connected to a home built fiber illuminator and a micropipette is connected to liquid delivery tube. (a) SEM image of an axicon of 110.9° fabricated at the tip of an optical fiber (b) Side view of the tip of the integrated system.

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Fig. 3
Fig. 3 Illumination at the cell sample during optical transfection using (a) axicon tipped fiber; (b) microlens tipped fiber and (c) integrated system. It can be seen that, with the fiber based illumination, the cell boundaries are clearly visible during the transfection procedure, when transfected with the integrated system (c).

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Fig. 6
Fig. 6 Fluorescent microscope image of optically transfected CHO-K1 cells, incubated for 48 hours after transfection. The bright cells are transfected successfully resulting in the uptake of the plasmid and thereby expressing the mitochondrially targeted red fluorescent protein.

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Fig. 4
Fig. 4 [a] Design of the integrated system. A glass capillary tube with 580 μm inner diameter is connected to port 1 of a barbed T junction through a piece of plastic tube. One optical fiber for laser delivery and another multimode fiber for illumination are inserted into the glass capillary through port 2. A piece of plastic tube and a slide clamp are used to hold two fibers and seal the fiber inlet at the same time. Another piece of plastic tube for liquid delivery is connected to port 3 of T junction. [b] Photograph of the integrated system.

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Fig. 5
Fig. 5 The transfection results of CHO-K1 and HEK-293 cells using 3 different methods. The error bar shown is standard error of the mean.

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Fig. 7
Fig. 7 Microlens tipped fiber fabrication setup. A violet diode laser (405 nm) is coupled into a piece of single mode fiber (fiber 1) through a 10X objective lens to obtain an output beam with perfect Gaussian lateral distribution. The beam, directed by a 5 cm tube lens and a beam splitting cube is focused by a 60X objective at the cleaved facet of a fiber. The lens is placed at 6 cm away from the fiber to make the beam converging. The fiber on which microlens is to be fabricated (fiber 2) is mounted in the setup using an xyz stage with 1 µm resolution, after dipping it into the uncured UV curing adhesive which forms a drop of the adhesive at fiber tip. The adhesive at the tip of the fiber 2 is cured for 5 s with the laser power of 0.5 mW.

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Fig. 8
Fig. 8 Different steps involved in the fabrication of lens at the tip of the fiber.

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Fig. 9
Fig. 9 Different structures can be made by changing the curing beam distribution. Using curing beam (a), (b) and (c) with appropriate laser power and curing time structures like (d), (e) and (f) respectively were fabricated.

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Fig. 10
Fig. 10 Ray tracing model used to estimate the parameters of the fabricated microlens.

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Tables (1)

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Table 1 Transfection results

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