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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 1, Iss. 2 — Sep. 1, 2010
  • pp: 527–536

Optical injection of mammalian cells using a microfluidic platform

Robert F. Marchington, Yoshihiko Arita, Xanthi Tsampoula, Frank J. Gunn-Moore, and Kishan Dholakia  »View Author Affiliations

Biomedical Optics Express, Vol. 1, Issue 2, pp. 527-536 (2010)

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The use of a focused laser beam to create a sub-micron hole in the plasma membrane of a cell (photoporation), for the selective introduction of membrane impermeable substances (optical injection) including nucleic acids (optical transfection), is a powerful technique most commonly applied to treat single cells. However, particularly for femtosecond photoporation, these studies have been limited to low throughput, small-scale studies, because they require sequential dosing of individual cells. Herein, we describe a microfluidic photoporation system for increased throughput and automated optical injection of cells. Hydrodynamic focusing is employed to direct a flow of single-file cells through a focused femtosecond laser beam for photoporation. Upon traversing the beam, a number of transient pores potentially open across the extracellular membrane, which allows the uptake of the surrounding fluid media into the cytoplasm, also containing the chosen injection agent. The process is entirely automated and a rate of 1 cell/sec could readily be obtained, enabling several thousand cells to be injected per hour using this system. The efficiency of optically injecting propidium iodide into HEK293 mammalian cells was found to be 42 ± 8%, or 28 ± 4% taking into account the requirement of post-injection viability, as tested using Calcein AM. This work now opens the way for combining photoporation with microfluidic analyses, sorting, purification or on-chip cell culture studies.

© 2010 OSA

OCIS Codes
(000.1430) General : Biology and medicine
(020.4180) Atomic and molecular physics : Multiphoton processes
(140.7090) Lasers and laser optics : Ultrafast lasers
(170.3890) Medical optics and biotechnology : Medical optics instrumentation
(170.7160) Medical optics and biotechnology : Ultrafast technology
(140.3538) Lasers and laser optics : Lasers, pulsed

ToC Category:

Original Manuscript: June 1, 2010
Revised Manuscript: July 19, 2010
Manuscript Accepted: August 2, 2010
Published: August 9, 2010

Virtual Issues
Advances in Optical Coherence Tomography, Photoacoustic Imaging, and Microscopy (2010) Biomedical Optics Express

Robert F. Marchington, Yoshihiko Arita, Xanthi Tsampoula, Frank J. Gunn-Moore, and Kishan Dholakia, "Optical injection of mammalian cells using a microfluidic platform," Biomed. Opt. Express 1, 527-536 (2010)

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  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).
  2. 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(2), 595–600 (2005).
  3. M. L. Torres-Mapa, L. Angus, M. Ploschner, K. Dholakia, and F. J. Gunn-Moore, “Transient transfection of mammalian cells using a violet diode laser,” J. Biomed. Opt. 15(4), 041506 (2010).
  4. H. Schneckenburger, A. Hendinger, R. Sailer, W. S. L. Strauss, and M. Schmitt, “Laser-assisted optoporation of single cells,” J. Biomed. Opt. 7(3), 410–416 (2002).
  5. A. V. Nikolskaya, V. P. Nikolski, and I. R. Efimov, “Gene printer: laser-scanning targeted transfection of cultured cardiac neonatal rat cells,” Cell Commun. Adhes. 13(4), 217–222 (2006).
  6. 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(1), 41–46 (1996).
  7. I. B. Clark, E. G. Hanania, J. Stevens, M. Gallina, A. Fieck, R. Brandes, B. O. Palsson, and M. R. Koller, “Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types,” J. Biomed. Opt. 11(1), 014034 (2006).
  8. T. Knoll, L. Trojan, S. Langbein, S. Sagi, P. Alken, and M. S. Michel, “Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma,” Lasers Med. Sci. 19(1), 33–36 (2004).
  9. H. Schinkel, P. Jacobs, S. Schillberg, and M. Wehner, “Infrared picosecond laser for perforation of single plant cells,” Biotechnol. Bioeng. 99(1), 244–248 (2008).
  10. C. T. A. Brown, D. J. Stevenson, X. Tsampoula, C. McDougall, A. A. Lagatsky, W. Sibbett, F. J. Gunn-Moore, and K. Dholakia, “Enhanced operation of femtosecond lasers and applications in cell transfection,” J Biophotonics 1(3), 183–199 (2008).
  11. L. E. Barrett, J. Y. Sul, H. Takano, E. J. Van Bockstaele, P. G. Haydon, and J. H. Eberwine, “Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor,” Nat. Methods 3(6), 455–460 (2006).
  12. M. Lei, H. Xu, H. Yang, and B. Yao, “Femtosecond laser-assisted microinjection into living neurons,” J. Neurosci. Methods 174(2), 215–218 (2008).
  13. P. Mthunzi, K. Dholakia, and F. Gunn-Moore, “Photo-transfection of mammalian cells using femtosecond laser pulses: optimisation and applicability to stem cell differentiation,” J. Biomed. Opt. 15(4), 041507 (2010).
  14. C. Peng, R. E. Palazzo, and I. Wilke, “Laser intensity dependence of femtosecond near-infrared optoinjection,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 75(4 Pt 1), 041903 (2007).
  15. 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(16), 7125–7133 (2006).
  16. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002).
  17. 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(5), 053902–053903 (2007).
  18. 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).
  19. A. Yamaguchi, Y. Hosokawa, G. Louit, T. Asahi, C. Shukunami, Y. Hiraki, and H. Masuhara, “Nanoparticle injection to single animal cells using femtosecond laser-induced impulsive force,” Adv. Mater. (Deerfield Beach Fla.) 93, 39–43 (2008).
  20. 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. Ther. 8(2), 342–350 (2003).
  21. C. McDougall, D. J. Stevenson, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, “Targeted optical injection of gold nanoparticles into single mammalian cells,” J Biophotonics 2(12), 736–743 (2009).
  22. 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).
  23. 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).
  24. 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(2), 127–130 (2003).
  25. D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “The Handbook of Photonics for Medical Science,” 1 ed., V. V. Tuchin, ed. (CRC Press, USA, 2010), pp. 87–117.
  26. A. Vogel, N. Linz, S. Freidank, and G. U. Paltauf, “Femtosecond-laser-induced nanocavitation in water: implications for optical breakdown threshold and cell surgery,” Phys. Rev. Lett. 100(3), 038102 (2008).
  27. T. Cizmár, V. Kollárová, X. Tsampoula, F. Gunn-Moore, W. Sibbett, Z. Bouchal, and K. Dholakia, “Generation of multiple Bessel beams for a biophotonics workstation,” Opt. Express 16(18), 14024–14035 (2008).
  28. A. Noori, P. R. Selvaganapathy, and J. Wilson, “Microinjection in a microfluidic format using flexible and compliant channels and electroosmotic dosage control,” Lab Chip 9(22), 3202–3211 (2009).
  29. A. Adamo and K. F. Jensen, “Microfluidic based single cell microinjection,” Lab Chip 8(8), 1258–1261 (2008).
  30. N. Bao, Y. Zhan, and C. Lu, “Microfluidic electroporative flow cytometry for studying single-cell biomechanics,” Anal. Chem. 80(20), 7714–7719 (2008).
  31. J. Wang, M. J. Stine, and C. Lu, “Microfluidic cell electroporation using a mechanical valve,” Anal. Chem. 79(24), 9584–9587 (2007).
  32. Y. Zhan, J. Wang, N. Bao, and C. Lu, “Electroporation of cells in microfluidic droplets,” Anal. Chem. 81(5), 2027–2031 (2009).
  33. T. Zhu, C. Luo, J. Huang, C. Xiong, Q. Ouyang, and J. Fang, “Electroporation based on hydrodynamic focusing of microfluidics with low dc voltage,” Biomed. Microdevices 12(1), 35–40 (2010).
  34. R. Ziv, Y. Steinhardt, G. Pelled, D. Gazit, and B. Rubinsky, “Micro-electroporation of mesenchymal stem cells with alternating electrical current pulses,” Biomed. Microdevices 11(1), 95–101 (2009).
  35. E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider, “Gene transfer into mouse lyoma cells by electroporation in high electric fields,” EMBO J. 1(7), 841–845 (1982).
  36. J. A. Wolff, R. W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, and P. L. Felgner, “Direct gene transfer into mouse muscle in vivo,” Science 247(4949 Pt 1), 1465–1468 (1990).
  37. N. S. Yang, J. Burkholder, B. Roberts, B. Martinell, and D. McCabe, “In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment,” Proc. Natl. Acad. Sci. U.S.A. 87(24), 9568–9572 (1990).
  38. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, “Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure,” Proc. Natl. Acad. Sci. U.S.A. 84(21), 7413–7417 (1987).
  39. K. L. Douglas, “Toward development of artificial viruses for gene therapy: a comparative evaluation of viral and non-viral transfection,” Biotechnol. Prog. 24(4), 871–883 (2008).
  40. M. M. Wang, E. Tu, D. E. Raymond, J. M. Yang, H. Zhang, N. Hagen, B. Dees, E. M. Mercer, A. H. Forster, I. Kariv, P. J. Marchand, and W. F. Butler, “Microfluidic sorting of mammalian cells by optical force switching,” Nat. Biotechnol. 23(1), 83–87 (2005).
  41. A. Y. Lau, L. P. Lee, and J. W. Chan, “An integrated optofluidic platform for Raman-activated cell sorting,” Lab Chip 8(7), 1116–1120 (2008).
  42. R. F. Marchington, M. Mazilu, S. Kuriakose, V. Garcés-Chávez, P. J. Reece, T. F. Krauss, M. Gu, and K. Dholakia, “Optical deflection and sorting of microparticles in a near-field optical geometry,” Opt. Express 16(6), 3712–3726 (2008).
  43. K. Dholakia, W. M. Lee, L. Paterson, M. P. Macdonald, R. Mcdonald, I. Andreev, P. Mthunzi, C. T. A. Brown, R. F. Marchington, and A. C. Riches, “Optical separation of cells on potential energy landscapes: enhancement with dielectric tagging,” IEEE J. Sel. Top. Quantum Electron. 13(6), 1646–1654 (2007).
  44. J. C. McDonald and G. M. Whitesides, “Poly(dimethylsiloxane) as a material for fabricating microfluidic devices,” Acc. Chem. Res. 35(7), 491–499 (2002).
  45. K. Haubert, T. Drier, and D. Beebe, “PDMS bonding by means of a portable, low-cost corona system,” Lab Chip 6(12), 1548–1549 (2006).
  46. G.-B. Lee, C.-C. Chang, S.-B. Huang, and R.-J. Yang, “The hydrodynamic focusing effect inside rectangular microchannels,” J. Micromech. Microeng. 16(5), 1024–1032 (2006).
  47. C. M. Pitsillides, E. K. Joe, X. Wei, R. R. Anderson, and C. P. Lin, “Selective cell targeting with light-absorbing microparticles and nanoparticles,” Biophys. J. 84(6), 4023–4032 (2003).
  48. W. M. Lee, P. J. Reece, R. F. Marchington, N. K. Metzger, and K. Dholakia, “Construction and calibration of an optical trap on a fluorescence optical microscope,” Nat. Protoc. 2(12), 3226–3238 (2007).
  49. P. B. Howell, J. P. Golden, L. R. Hilliard, J. S. Erickson, D. R. Mott, and F. S. Ligler, “Two simple and rugged designs for creating microfluidic sheath flow,” Lab Chip 8(7), 1097–1103 (2008).

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