Optical transfection

Optical transfection is the process of introducing nucleic acids into cells using light. Typically, a laser is focussed to a diffraction limited spot (~1 µm diameter) using a high numerical aperture microscope objective. The plasma membrane of a cell is then exposed to this highly focussed light for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane. The generation of a photopore allows exogenous plasmid DNA, RNA, organic fluorophores, or larger objects such as semiconductor quantum nanodots to enter the cell. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.

To put the above simply, cells do not usually allow certain types of substances into their interior space. Lasers can be used to burn a tiny hole on the cell surface, allowing those substances to enter. This is tremendously useful to biologists who are studying disease, as a common experimental requirement is to put things (such as DNA) into cells.

This technique was first demonstrated in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection of normal rat kidney cells.^[1] Since this time, the optical transfection of a host of mammalian cell types has been demonstrated using a variety of laser sources, including the 405 nm continuous wave (cw),^[2] 488 nm cw,^[3] or pulsed sources such as the 800 nm femtosecond pulsed Ti:Sapphire^{[4][5][6][7][8][9][10][11][12][13]} or 1064 nm nanosecod pulsed Nd:YAG.^[14][15]

Terminology

The meaning of the term transfection has evolved.^[16] The original meaning of transfection was "infection by transformation", i.e. introduction of DNA (or RNA) from a eukaryote-infecting virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA (or other nucleic acid species such as RNA or SiRNA).

Because of this strict definition of transfection, optical transfection also refers only to the introduction of nucleic acid species. The introduction of other impermeable compounds into a cell, such as organic fluorophores or semiconductor quantum nanodots is not strictly speaking "transfection," and is therefore referred to as "optical injection" or one of the other many terms now outlined.

The lack of a unified name for this technology makes reviewing the literature on the subject very difficult.^[17] Optical injection has been described using over a dozen different names or phrases (see bulleted lists below). Some trends in the literature are clear. The first term of the technique is invariably a derivation of word laser, optical, or photo, and the second term is usually in reference to injection, transfection, poration, perforation or puncture. Like many cellular perturbations, when a single cell or group of cells is treated with a laser, three things can happen: the cell dies (overdose), the cell membrane is permeabilised, substances enter, and the cell recovers (therapeutic dose), or nothing happens (underdose). There have been suggestions in the literature to reserve the term optoinjection for when a therapeutic dose is delivered upon a single cell,^[18][19][20] and the term optoporation for when a laser generated shockwave treats a cluster of many (10s to 100s) cells.^{[21][22][23][24]} The first definition of optoinjection is uncontroversial. The definition of optoporation, however, has failed to be adopted, with a similar number of references using the term to denote the dosing of single cells^{[25][26][27][28]} as those using the term to denote the simultaneous dosing of clusters of many cells ^{[29][30][31][32]}

As the field stands, it is the opinion of the authors of a review article on the subject^[33] that the term optoinjection always be included as a keyword in future publications, regardless of their own naming preferences.

Terms agreed by consensus

• Optoinjection (or any derivations of laser injection, optical injection, photoinjection): The transfer of any membrane impermeable substance into a cell using light. A general term that also encompasses optical transfection.
• Optical transfection (or any derivations of laser transfection, optotransfection, phototransfection): A specific type of optical transfection - the transfer of nucleic acids into a cell using light for the purposes of eliciting protein translation from those acids. To be in line with the current definition of transfection in the biological community, non-nucleic acids (such as fluorophores) cannot, by definition, be optically transfected (only optically injected).
• Photoporation (or any derivations of [laser-] or [optical-] or [opto-] or [photo-] AND [ poration] or [-permeabilisation] or [-puncture] or [-perforation]): The generation of a transient hole or holes on the plasma membrane (or cell wall) of a cell usually for the purpose of optical injection. See possible exception: Optoporation
• -surgery (such as cell nanosurgery, laser nanosurgery, laser surgery): A general term that incorporates all of the above definitions, but also includes the concepts of the ablation or optical manipulation of cell material for other purposes besides pore generation. Examples include selective cell ablation to purify cell populations, chromosome dissection, cytoskeleton disruption, organelle ablation, axotomy,^[34] or the optical tweezing or isolation of intracellular material.

Terms under deliberation

• Optoporation: Has been suggested to mean the dosing of a cluster of cells with a shockwave mediated mechanism, which usually results in a doughnut shaped therapeutic zone.^{[35][36][37][38]} On the contrary, has also been synonymously used with the term photoporation.^{[39][40][41][42]}
• Laserfection: Has been suggested to mean the dosing of a cluster of cells with a circularly shaped therapeutic zone. Term reserved for Cyntellect’s laser-enabled analysis and processing (LEAP) system.
• Light-induced convective transmembrane transport: A newly coined term for optoinjection.^[43]

Some of the above was reproduced with permission from.^[44]

Methods

A typical optical transfection protocol is as follows:^[45] 1) Build an optical tweezers system with a high NA objective 2) Culture cells to 50-60% confluency 3) Expose cells to at least 10 µg/ml of plasmid DNA 4) Dose the plasma membrane of each cell with 10-40 ms of focussed laser, at a power of <100 mW at focus 5) Observe transient transfection 24-96h later 6) Add selective medium if the generation of stable colonies is desired

See also

• Transfection
• Transformation
• Transduction
• Cationic liposome
• Nucleofection
• Magnet assisted transfection

References

1. M. Tsukakoshi, S. Kurata, Y. Nomiya, et al., "A Novel Method of DNA Transfection by Laser Microbeam Cell Surgery". Applied Physics B-Photophysics and Laser Chemistry. 35(3): 135-140 (1984)
2. Photoporation and cell transfection using a violet diode laser, Paterson, L., Agate, B., Comrie, M., Ferguson, R., Lake, T. K., Morris, J. E., Carruthers, A. E., Brown, C. T. A., Sibbett, W., Bryant, P. E., Gunn-Moore, F., Riches, A. C., and Dholakia, K. Optics Express 13(2) 595-600 (2005)
3. Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation, Palumbo, Caruso, Crescenzi, Tecce, Roberti, and Colasanti. Journal of Photochemistry and Photobiology B-Biology 36(1) 41-46 (1996)
4. Fibre based cellular transfection, Tsampoula, X., Taguchi, K., Cizmar, T., Garces-Chavez, V., Ma, N., Mohanty, S., Mohanty, K., Gunn-Moore, F., and Dholakia, K. Opt Express 16(21) 17007-17013 (2008)
5. Targeted transfection of stem cells with sub-20 femtosecond laser pulses, Uchugonova, A., Konig, K., Bueckle, R., Isemann, A., and Tempea, G. Opt Express 16(13) 9357-9364 (2008)
6. Enhanced operation of femtosecond lasers and applications in cell transfection, Brown, C. T. A., Stevenson, D. J., Tsampoula, X., McDougall, C., Lagatsky, A. A., Sibbett, W., Gunn-Moore, F., and Dholakia, K. Journal of Biophotonics 1(2) 183-199 (2008)
7. Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells, Baumgart, J., Bintig, W., Ngezahayo, A., Willenbrock, S., Murua Escobar, H., Ertmer, W., Lubatschowski, H., and Heisterkamp, A. Opt Express 16(5) 3021-3031 (2008)
8. Femtosecond laser-assisted microinjection into living neurons, Lei, M., Xu, H., Yang, H., and Yao, B. J Neurosci Methods 174(2) 215-218 (2008)
9. Femtosecond cellular transfection using a nondiffracting light beam, Tsampoula, X., Garces-Chavez, V., Comrie, M., Stevenson, D. J., Agate, B., Brown, C. T. A., Gunn-Moore, F., and Dholakia, K. Appl. Phys. Lett. 91(5) 053902-053903 (2007)
10. Laser intensity dependence of femtosecond near-infrared optoinjection, Peng, C., Palazzo, R. E., and Wilke, I. Phys Rev E Stat Nonlin Soft Matter Phys 75(4 Pt 1) 041903 (2007)
11. Femtosecond optical transfection of cells: viability and efficiency, Stevenson, D., Agate, B., Tsampoula, X., Fischer, P., Brown, C. T. A., Sibbett, W., Riches, A., Gunn-Moore, F., and Dholakia, K. Optics Express 14(16) 7125-7133 (2006)
12. Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor, Barrett, L. E., Sul, J. Y., Takano, H., Van Bockstaele, E. J., Haydon, P. G., and Eberwine, J. H. Nature methods 3(6) 455-460 (2006)
13. Targeted transfection by femtosecond laser, Tirlapur, and Konig. Nature 418(6895) 290-291 (2002)
14. Characterization of cellular optoporation with distance, Soughayer, Krasieva, Jacobson, Ramsey, Tromberg, and Allbritton. Analytical Chemistry 72(6) 1342-1347 (2000)
15. Mohanty, Sharma, and Gupta. (2003) Laser-assisted microinjection into targeted animal cells, in Biotechnol. Lett., pp 895-899
16. "Transfection" at Dorland's Medical Dictionary
17. Single cell optical transfection, Stevenson DJ, Gunn-Moore FJ, Campbell P, Dholakia K, Journal of The Royal Society Interface 7: 863-871, (2010)
18. Cell permeabilization and molecular transport by laser microirradiation, Krasieva TB, Chapman CF, LaMorte VJ, Venugopalan V, Berns MW, Tromberg BJ, Proc SPIE 3260: 38-44, (1998)
19. Cellular Laserfection, Rhodes K, Clark I, Zatcoff M, Eustaquio T, Hoyte KL, Koller MR, Michael WB, Greulich KO, Methods in Cell Biology 82: 309-333, (2007)
20. Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation, Venugopalan V, Guerra A, 3rd, Nahen K, Vogel A, Physical Review Letters 88: 078103, (2002)
21. Cell permeabilization and molecular transport by laser microirradiation, Krasieva TB, Chapman CF, LaMorte VJ, Venugopalan V, Berns MW, Tromberg BJ, Proc SPIE 3260: 38-44, (1998)
22. Cellular Laserfection, Rhodes K, Clark I, Zatcoff M, Eustaquio T, Hoyte KL, Koller MR, Michael WB, Greulich KO, Methods in Cell Biology 82: 309-333, (2007)
23. Characterization of cellular optoporation with distance, Soughayer JS, Krasieva T, Jacobson SC, Ramsey JM, Tromberg BJ, Allbritton NL, Analytical Chemistry 72: 1342-1347, (2000)
24. Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation, Venugopalan V, Guerra A, 3rd, Nahen K, Vogel A, Physical Review Letters 88: 078103, (2002)
25. Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation, Palumbo G, Caruso M, Crescenzi E, Tecce MF, Roberti G, Colasanti A, Journal of Photochemistry and Photobiology B-Biology 36: 41-46, (1996)
26. Targeted transfection of stem cells with sub-20 femtosecond laser pulses, Uchugonova A, Konig K, Bueckle R, Isemann A, Tempea G, Optics Express 16: 9357-9364, (2008)
27. Laser-assisted microinjection into targeted animal cells, Mohanty SK, Sharma M, Gupta PK, Biotechnology Letters 25: 895-899, (2003)
28. Laser-assisted optoporation of single cells, Schneckenburger H, Hendinger A, Sailer R, Strauss WSL, Schmidtt M, Journal of Biomedical Optics 7: 410-416, (2002)
29. Cell permeabilization and molecular transport by laser microirradiation, Krasieva TB, Chapman CF, LaMorte VJ, Venugopalan V, Berns MW, Tromberg BJ, Proc SPIE 3260: 38-44, (1998)
30. Cellular Laserfection, Rhodes K, Clark I, Zatcoff M, Eustaquio T, Hoyte KL, Koller MR, Michael WB, Greulich KO, Methods in Cell Biology 82: 309-333, (2007)
31. Characterization of cellular optoporation with distance, Soughayer JS, Krasieva T, Jacobson SC, Ramsey JM, Tromberg BJ, Allbritton NL, Analytical Chemistry 72: 1342-1347, (2000)
32. Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation, Venugopalan V, Guerra A, 3rd, Nahen K, Vogel A, Physical Review Letters 88: 078103, (2002)
33. Single cell optical transfection, Stevenson DJ, Gunn-Moore FJ, Campbell P, Dholakia K, Journal of The Royal Society Interface 7: 863-871, (2010)
34. Reversible permeabilization using high-intensity femtosecond laser pulses: Applications to biopreservation, Kohli V, Acker JP, Elezzabi AY, Biotechnology and Bioengineering 92: 889-899, (2005)
35. Cell permeabilization and molecular transport by laser microirradiation, Krasieva TB, Chapman CF, LaMorte VJ, Venugopalan V, Berns MW, Tromberg BJ, Proc SPIE 3260: 38-44, (1998)
36. Cellular Laserfection, Rhodes K, Clark I, Zatcoff M, Eustaquio T, Hoyte KL, Koller MR, Michael WB, Greulich KO, Methods in Cell Biology 82: 309-333, (2007)
37. Characterization of cellular optoporation with distance, Soughayer JS, Krasieva T, Jacobson SC, Ramsey JM, Tromberg BJ, Allbritton NL, Analytical Chemistry 72: 1342-1347, (2000)
38. Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation, Venugopalan V, Guerra A, 3rd, Nahen K, Vogel A, Physical Review Letters 88: 078103, (2002)
39. Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation, Palumbo G, Caruso M, Crescenzi E, Tecce MF, Roberti G, Colasanti A, Journal of Photochemistry and Photobiology B-Biology 36: 41-46, (1996)
40. Targeted transfection of stem cells with sub-20 femtosecond laser pulses, Uchugonova A, Konig K, Bueckle R, Isemann A, Tempea G, Optics Express 16: 9357-9364, (2008)
41. Laser-assisted microinjection into targeted animal cells, Mohanty SK, Sharma M, Gupta PK, Biotechnology Letters 25: 895-899, (2003)
42. Laser-assisted optoporation of single cells, Schneckenburger H, Hendinger A, Sailer R, Strauss WSL, Schmidtt M, Journal of Biomedical Optics 7: 410-416, (2002)
43. Laser modulated transmembrane convection: Implementation in cancer chemotherapy, Sommer AP, Zhu D, Scharnweber T, Journal of Controlled Release 148: 131-134, (2010)
44. Single cell optical transfection, Stevenson DJ, Gunn-Moore FJ, Campbell P, Dholakia K, Journal of The Royal Society Interface 7: 863-871, (2010)
45. Femtosecond optical transfection of cells: viability and efficiency, Stevenson, D., Agate, B., Tsampoula, X., Fischer, P., Brown, C. T. A., Sibbett, W., Riches, A., Gunn-Moore, F., and Dholakia, K. Optics Express 14(16) 7125-7133 (2006)

External links

• Research in optical transfection at the University of St Andrews
• Transfection at the US National Library of Medicine Medical Subject Headings (MeSH)
• Overview of transfection methods in Nature Methods 2, 875 - 883 (2005)