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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 13, Iss. 2 — Jan. 24, 2005
  • pp: 595–600
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Photoporation and cell transfection using a violet diode laser

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  »View Author Affiliations


Optics Express, Vol. 13, Issue 2, pp. 595-600 (2005)
http://dx.doi.org/10.1364/OPEX.13.000595


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Abstract

The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

© 2005 Optical Society of America

1. Introduction

The introduction of foreign DNA into cells (transfection) is an essential procedure in genetic manipulation of cells and recombinant protein experiments. Genetic material is introduced to the cells in the form of plasmid expression vectors, which are circular, double-stranded units of DNA and contain genes of interest that can be replicated and expressed within a cell. Various methods for puncturing the cell plasma membrane, while minimising collateral damage, have been investigated and optimised for the many different cell types under investigation. Such methods include chemical transfection [1

1. F.L. Graham and A.J. Van der Eb, “A new technique for the assay of infectivity of human adenovirus 5 DNA,” Virology 52, 456–467 (1973). [CrossRef] [PubMed]

], lipid-based techniques [2

2. R. Fraley, S. Subramani, P. Berg, and D. Papahadjopolous, “Introduction of liposome-encapsulated SV40 DNA into cells,” J. Biol. Chem. 255, 10431–10435 (1980). [PubMed]

], the use of viral vectors [3

3. D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M.T. McManus, F.B. Gertler, M.L. Scott, and L. Van Parijs, “A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,” Nature Genet. 33, 401–406 (2003). [CrossRef] [PubMed]

], electroporation [4

4. T.K. Wong and E. Neumann, “Electric-field mediated gene-transfer,” Biochem. and Biophys. Res. Commun. 107, 584–587 (1982). [CrossRef]

] and laser-assisted photoporation [5

5. H. Schneckenburger, A. Hendinger, R. Sailer, W.S.L. Strauss, and M. Schmitt, “Laser-assisted optoporation of single cells,” J. Biomed. Opt. 7, 410–416 (2002). [CrossRef] [PubMed]

12

12. 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. Therapy 8, 342–350 (2003). [CrossRef]

].

Recently, laser-assisted photoporation has been demonstrated using sources such as continuous-wave argon-ion lasers operating at 488 nm [5

5. H. Schneckenburger, A. Hendinger, R. Sailer, W.S.L. Strauss, and M. Schmitt, “Laser-assisted optoporation of single cells,” J. Biomed. Opt. 7, 410–416 (2002). [CrossRef] [PubMed]

, 6

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-Biol. 36, 41–46 (1996). [CrossRef]

], pulsed and frequencyupconverted Nd:YAG lasers operating at 355 nm [7

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

], 532 nm [8

8. J.S. Soughayer, T. Krasieva, S.C. Jacobson, J.M. Ramsey, B.J. Tromberg, and N.L. Allbritton, “Characterization of cellular optoporation with distance,” Anal. Chem. 72, 1342–1347 (2000). [CrossRef] [PubMed]

] and 1064 nm [9

9. S.K. Mohanty, M. Sharma, and P.K. Gupta, “Laser-assisted microinjection into targeted animal cells,” Biotechnol. Lett. 25, 895–899 (2003). [CrossRef] [PubMed]

], and more recently with femtosecond titanium-sapphire lasers [10

10. U.K. Tirlapur and K. Konig, “Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability,” Plant J. 31, 365–374 (2002). [CrossRef] [PubMed]

12

12. 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. Therapy 8, 342–350 (2003). [CrossRef]

]. By using a tightly focused laser beam (with a localised focal volume of ~10-19 m3), the plasma membrane of cells can be perforated, allowing foreign molecules to enter the cell. Membrane fluidity facilitates subsequent closure of the perforation. Whilst such photoporation has proven successful, the laser systems employed are relatively cumbersome and typically involve two-photon processes which require extremely precise positioning of the laser focus on the cell plasma membrane. Such techniques relying on two-photon processes have proven relatively difficult to achieve. As an alternative, more direct one-photon processes have received less attention in the literature due the potentially detrimental effects on the targeted cell. By using sufficient care and control of the incident laser beam, we demonstrate a key advance in this field by employing a highly compact, inexpensive laser system. We have porated cells effectively via a single-photon process while avoiding any noticeable damage to the cell. Individual cells successfully transfected using single-photon laser-assisted poration were expanded into clonal groups which were then used to create stable cell lines.

In the work reported here, a commercial violet diode laser has been employed to photoporate Chinese hamster ovary (CHO-K1) cells and transfect them with a plasmid expression vector containing an antibiotic resistance gene (Geneticin/G418 resistance) and the green fluorescent protein (GFP) gene (pEGFP-N3, BD Biosciences, Oxford, U.K.). Geneticin/G418 resistance is used so that successfully transfected individual cells will survive subsequent drug treatment and divide to form colonies that can be selected, grown and analysed [15

15. P. Southern and P. Berg, “Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter,” J. Mol. Appl. Genet. 1, 327–341 (1982). [PubMed]

]. Such cells will also exhibit green fluorescence as a result of the presence of the GFP gene. The use of a violet diode laser allows an eminently practical and efficient means of photoporation in comparison with other laser sources used in similar experiments, and is readily implemented by directing the beam from this portable, miniature source into a simple home-built microscope setup.

2. Experiment

A strongly focused and attenuated beam from a violet diode laser yielding a power density of 1200 MWm-2 is sufficient to optically porate a cell membrane. Due to the strong cell-absorption characteristics of violet light, less than a milliwatt of laser power subjected to the cell plasma membrane for several tens of milliseconds can be sufficient for successful photoporation with no apparent cell damage. For our investigation, we made use of an inverted optical microscope configuration, incorporating the violet diode laser (Toptica Photonics CVLS-LH050-2V1, 405 nm, 40 mW output) which can be seen in Fig. 1. The violet diode laser provided a high-quality circularized beam (dx=dy) with an associated Msquared parameter of 1.2 in both transverse planes, with a beam diameter, d, of around 2 mm. This laser output was first passed though a variable neutral density (ND) filter to reduce the beam-focus power to the few milliwatts required for photoporation. The beam was then expanded with a simple telescope to fill the back of the ×100 oil-immersion microscope objective (N.A.=1.25). A CCD camera positioned below the sample cell allowed direct observation and selection of individual cells for targeted transfection. A simple beam-shutter unit taken from a single lens reflex (SLR) camera was used to provide the short exposure times required. Cells were positioned by manipulating the xyz translation stage such that the beam spot, when exposed, would be focused on the cell membrane, away from the nucleus. The overall footprint of our optical setup (Fig. 1), which can also be used either as an optical tweezer or confocal microscope, was 65×20×60 cm.

Fig. 1. Photoporation apparatus. A violet diode laser (Toptica Photonics, 405 nm, 40 mW output power) is directed towards the sample through an inverted optical microscope setup.

CHO-K1 cells were grown to sub-confluence in a 30 mm diameter Petri dish (with a glass bottom approximately 0.1 mm thick) at a concentration of approximately 105 cells per dish. The plasmid expression vector containing the antibiotic resistance gene and the GFP gene was added to the surrounding medium prior to photoporation.

The parameters for photoporation were determined empirically by firstly exposing cells to the maximum laser power (4 mW at focus, calculated using the measured transmission of the optical components) for several seconds. Severe, irreversible damage to the membrane was observed after exposure at this power for only three seconds. At a power of 0.3 mW at the focal spot, similar damage occurred after one minute of exposure. A shutter time of 40 ms (with a rise/fall time of ~1 ms) was then chosen in combination with a laser power of 0.3 mW. Using these parameters we expected the laser to transiently porate the cell plasma membrane, whilst avoiding irreparable cell damage. This was verified to be the case experimentally. To the best of our knowledge, the exact mechanism for cell photoporation is yet to be determined, but possibilities include localized heating of the lipid bilayer membrane leading to a phase transition from gel to a liquid crystalline state [16

16. Y Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68, 2137–2144 (1995). [CrossRef] [PubMed]

]; chromophore absorption; or widening of pre-existing channels. In using a single-photon process to achieve such laser-assisted poration, we have found it relatively straightforward to position the beam focus at the cell membrane. Such positioning of the beam focus is significantly more difficult when exploiting a two-photon process due to the greater precision required as a result of the highly localised nature of two-photon excitation.

Fig. 2. Captured image of a cell being exposed to the focused violet diode laser beam (0.3 mW average power for 40 milliseconds).

Twenty cells per plate were irradiated in the presence of 3 µg of the plasmid expression vector. Figure 2 shows the membrane of a CHO-K1 cell exposed to the 405 nm laser diode beam at a power of 0.3 mW for 40 ms. It can be seen clearly that the cell is irradiated away from the nucleus, thus avoiding any potential DNA damage at this wavelength. After photoporation, cells were incubated under normal conditions (37°C/5% CO2). Two days after photoporation, the surrounding medium was changed and 0.5 mg/ml of the antibiotic (Geneticin/G418) was added. The cells were then incubated for a further 10 days, during which time two further changes of media were made, and fresh antibiotic added, until cell colonies had formed.

3. Results

Antibiotic-resistant cell colonies were detected in the irradiated dishes containing the plasmid expression vector twelve days after photoporation, ten days after the antibiotic was introduced. No live cells were detected in either the non-photoporated control, nor in the GFP-free photoporated control, thus confirming that antibiotic resistance had been transferred to the cells that had been photoporated in the presence of the plasmid expression vector. Colonies were picked twelve days after photoporation and sub-cultured in a fresh dish in the presence of the antibiotic (Geneticin/G418). Cells were viewed three weeks after photoporation under fluorescence, and exhibited strong GFP expression as can be seen from Fig. 3. Cells continued to grow in the presence of the antibiotic, thereby indicating that the cells had been stably transfected with the plasmid expression vector containing the antibiotic resistance gene as well as the GFP gene.

Fig. 3. Fluorescence images of cells transfected with an antibiotic-resistance gene and the GFP gene, taken several weeks after photoporation (images taken using FITC filter on a Zeiss Axioplan 2 imaging microscope). Cells are those that were subcultured from the antibiotic resistant colonies. GFP expression throughout the cells is clear. (a) Live cells (×20); (b) Fixed cells in Vectashield (×100).

In a separate experiment, a second plasmid expression vector was also used, containing the antibiotic resistance gene and the DsRed-Mito gene, which expresses the red fluorescent protein (RFP) within the mitochondria of a cell. We again achieved successful transfection of CHO-K1 cells as can be seen from Fig. 4. This red fluorescent protein targets the cytoplasm-based mitochondria and as such no red fluorescence is observed in each cell nucleus.

Fig. 4. Fluorescence images of live cells transfected with a plasmid containing an antibiotic-resistance gene and a gene encoding a red fluorescent protein (pDsRed-Mito, BD Biosciences, Oxford, U.K.), taken several weeks after photoporation. Expression of the red fluorescent protein in the cell mitochondria is clear. (a) ×20 magnification; (b) ×100 magnification

The diode laser operating at 405 nm can porate cells at sub-milliwatt powers due to the high absorption by cells in this spectral region. Femtosecond lasers operating in the near-infrared porate cells due to a two-photon effect, explaining why such high peak intensities are required (TWcm-2). By contrast, this method is a single-photon effect that requires very low power. Cells are porated and subsequently transfected by exposing each cell, only once, to sub-milliwatt powers for tens of milliseconds. These parameters do not induce any visible changes in the cell (nor do they impede the formation of clonal groups) and therefore cell integrity is maintained.

4. Conclusions and discussion

Transfection of cells by photoporation with a violet diode laser is easy to implement, particularly if an existing optical microscope setup exists, as the laser source is inexpensive, compact and portable in comparison to previously used sources for laser cell poration. Cells can be selected visually for targeted photoporation, and the low power and short exposure time used in this experiment does not appear to cause irreversible cell membrane damage. While various successful transfection procedures using laser poration have resulted in stable cell lines [7

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

], this is the first time to our knowledge that it has been achieved with such low powers from a simple commercial diode laser. Interestingly, with the low power employed we can introduce the option of multiplexing the light source using a spatial light modulator to simultaneously porate many tens of cells [17

17. D. McGloin and K. Dholakia, private communication

] (Holoeye LC-R 2500). Future developments include incorporating this method of cell poration with a confocal microscope utilising a single violet diode laser for both these modalities.

We observe no adverse effects from using a single-photon process for laser-based transfection. Individual cells successfully transfected using laser-assisted poration expanded into clonal groups which were used to create stable cell lines. While other work in the literature reports that a two-photon process should be used in order to prevent cellular damage, we have found that the single-photon approach can also achieve transfection of a mammalian cell-line. This potentially offers an important step in simplification for laser assisted poration that in turn may lead to widespread use of this technique.

Acknowledgments

The authors wish to acknowledge the support of the UK Engineering and Physical Sciences Research Council, and the Scottish Higher Education Funding Council for funding through a Strategic Research Development Grant award.

References and links

1.

F.L. Graham and A.J. Van der Eb, “A new technique for the assay of infectivity of human adenovirus 5 DNA,” Virology 52, 456–467 (1973). [CrossRef] [PubMed]

2.

R. Fraley, S. Subramani, P. Berg, and D. Papahadjopolous, “Introduction of liposome-encapsulated SV40 DNA into cells,” J. Biol. Chem. 255, 10431–10435 (1980). [PubMed]

3.

D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M.T. McManus, F.B. Gertler, M.L. Scott, and L. Van Parijs, “A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,” Nature Genet. 33, 401–406 (2003). [CrossRef] [PubMed]

4.

T.K. Wong and E. Neumann, “Electric-field mediated gene-transfer,” Biochem. and Biophys. Res. Commun. 107, 584–587 (1982). [CrossRef]

5.

H. Schneckenburger, A. Hendinger, R. Sailer, W.S.L. Strauss, and M. Schmitt, “Laser-assisted optoporation of single cells,” J. Biomed. Opt. 7, 410–416 (2002). [CrossRef] [PubMed]

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-Biol. 36, 41–46 (1996). [CrossRef]

7.

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

8.

J.S. Soughayer, T. Krasieva, S.C. Jacobson, J.M. Ramsey, B.J. Tromberg, and N.L. Allbritton, “Characterization of cellular optoporation with distance,” Anal. Chem. 72, 1342–1347 (2000). [CrossRef] [PubMed]

9.

S.K. Mohanty, M. Sharma, and P.K. Gupta, “Laser-assisted microinjection into targeted animal cells,” Biotechnol. Lett. 25, 895–899 (2003). [CrossRef] [PubMed]

10.

U.K. Tirlapur and K. Konig, “Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability,” Plant J. 31, 365–374 (2002). [CrossRef] [PubMed]

11.

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

12.

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. Therapy 8, 342–350 (2003). [CrossRef]

13.

J.M. Girkin and A.I. Ferguson, “Confocal microscopy using an InGaN violet laser diode at 406nm,” Opt. Express 7, 336–341 (2000). http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336 [CrossRef] [PubMed]

14.

T.K. Lake, A.E. Carruthers, L. Paterson, M. Taylor, F. Gunn-Moore, J.W. Allen, W. Sibbett, and K. Dholakia, “Optical trapping and fluorescence excitation with violet diode lasers and extended cavity surface emitting lasers,” Opt. Express 12, 670–678 (2004). http://www.opticsexpress.org/abstract.cfm?URI=OPEX-12-4-670 [CrossRef] [PubMed]

15.

P. Southern and P. Berg, “Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter,” J. Mol. Appl. Genet. 1, 327–341 (1982). [PubMed]

16.

Y Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68, 2137–2144 (1995). [CrossRef] [PubMed]

17.

D. McGloin and K. Dholakia, private communication

OCIS Codes
(140.2020) Lasers and laser optics : Diode lasers
(140.7240) Lasers and laser optics : UV, EUV, and X-ray lasers
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.1420) Medical optics and biotechnology : Biology
(170.1530) Medical optics and biotechnology : Cell analysis

ToC Category:
Research Papers

History
Original Manuscript: December 21, 2004
Revised Manuscript: January 13, 2005
Published: January 24, 2005

Citation
L. Paterson, B. Agate, M. Comrie, R. Ferguson, T. Lake, J. Morris, A. Carruthers, C. T. Brown, W. Sibbett, P. Bryant, F. Gunn-Moore, A. Riches, and Kishan Dholakia, "Photoporation and cell transfection using a violet diode laser," Opt. Express 13, 595-600 (2005)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-13-2-595


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References

  1. F.L. Graham and A.J. Van der Eb, �??A new technique for the assay of infectivity of human adenovirus 5 DNA,�?? Virology 52, 456-467 (1973). [CrossRef] [PubMed]
  2. R. Fraley, S. Subramani, P. Berg, and D. Papahadjopolous, �??Introduction of liposome-encapsulated SV40 DNA into cells,�?? J. Biol. Chem. 255, 10431-10435 (1980). [PubMed]
  3. D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M.T. McManus, F.B. Gertler, M.L. Scott, and L. Van Parijs, �??A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,�?? Nature Genet. 33, 401-406 (2003). [CrossRef] [PubMed]
  4. T.K. Wong and E. Neumann, �??Electric-field mediated gene-transfer,�?? Biochem. and Biophys. Res. Commun. 107, 584-587 (1982). [CrossRef]
  5. H. Schneckenburger, A. Hendinger, R. Sailer, W.S.L. Strauss, and M. Schmitt, �??Laser-assisted optoporation of single cells,�?? J. Biomed. Opt. 7, 410-416 (2002). [CrossRef] [PubMed]
  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-Biol. 36, 41-46 (1996). [CrossRef]
  7. Y. Shirahata, N. Ohkohchi, H. Itagak, and S. Satomi, �??New technique for gene transfection using laser irradiation,�?? J. Invest. Med. 49, 184-190 (2001). [CrossRef]
  8. J.S. Soughayer, T. Krasieva, S.C. Jacobson, J.M. Ramsey, B.J. Tromberg, and N.L. Allbritton, �??Characterization of cellular optoporation with distance,�?? Anal. Chem. 72, 1342-1347 (2000). [CrossRef] [PubMed]
  9. S.K. Mohanty, M. Sharma, and P.K. Gupta, �??Laser-assisted microinjection into targeted animal cells,�?? Biotechnol. Lett. 25, 895-899 (2003). [CrossRef] [PubMed]
  10. U.K. Tirlapur and K. Konig, �??Femtosecond near-infrared laser pulses as a versatile non- invasive tool for intra-tissue nanoprocessing in plants without compromising viability,�?? Plant J. 31, 365-374 (2002). [CrossRef] [PubMed]
  11. U.K. Tirlapur and K. Konig, �??Targeted transfection by femtosecond laser,�?? Nature 418, 290-291 (2002). [CrossRef] [PubMed]
  12. 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. Therapy 8, 342-350 (2003). [CrossRef]
  13. J.M. Girkin and A.I. Ferguson, �??Confocal microscopy using an InGaN violet laser diode at 406nm,�?? Opt. Express 7, 336-341 (2000). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336</a> [CrossRef] [PubMed]
  14. T.K. Lake, A.E. Carruthers, L. Paterson, M. Taylor, F. Gunn-Moore, J.W. Allen, W. Sibbett, and K. Dholakia, �??Optical trapping and fluorescence excitation with violet diode lasers and extended cavity surface emitting lasers,�?? Opt. Express 12, 670-678 (2004). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-12-4-670">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-12-4-670</a> [CrossRef] [PubMed]
  15. P. Southern and P. Berg, �??Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter,�?? J. Mol. Appl. Genet. 1, 327-341 (1982). [PubMed]
  16. Y Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman and B J. Tromberg, �??Evidence for localized cell heating induced by infrared optical tweezers,�?? Biophys. J. 68, 2137-2144 (1995). [CrossRef] [PubMed]
  17. D. McGloin and K. Dholakia, private communication

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