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Characterization of Cellular Optoporation with Distance

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Characterization of Cellular Optoporation with Distance UC Irvine UC Irvine Previously Published Works Title Characterization of cellular optoporation with distance. Permalink https://escholarship.org/uc/item/41m9w7s0 Journal Analytical chemistry, 72(6) ISSN 0003-2700 Authors Soughayer, JS Krasieva, T Jacobson, SC et al. Publication Date 2000-03-01 DOI 10.1021/ac990982u License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Anal. Chem. 2000, 72, 1342-1347 Technical Notes Characterization of Cellular Optoporation with Distance Joseph S. Soughayer,² Tatiana Krasieva,³ Stephen C. Jacobson,§ J. Michael Ramsey,§ Bruce J. Tromberg,³ and Nancy L. Allbritton*,² Department of Physiology and Biophysics and Beckman Laser Institute, University of California, Irvine, California 92697-4560, and Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831 We have developed and characterized cellular optopora- ally, the speed at which cells can be loaded is very low and the tion with visible wavelengths of light using standard experimental geometry must be able to tolerate the introduction uncoated glass cover slips as the absorptive media. A of the large glass microinjection pipet. Lipid-based delivery frequency-doubled Nd:YAG laser pulse was focused at the systems (liposomes) often yield high survival rates, but the interface of the glass surface and aqueous buffer, creating percentage of cells loaded is frequently quite low and highly a stress wave and transiently permeabilizing nearby cells. variable among different cell types.1,2 Electroporation has been Following optoporation of adherent cells, three spatial shown to be an effective method for delivery of DNA and RNA zones were present which were distinguished by the into cells.1,3-6 However, the poor loading efficiency of electro- viability of the cells and the loading efficiency (or number poration has largely limited its use to genetic material, for which of extracellular molecules loaded). The loading efficiency the cell can synthesize much larger quantities of the protein also depended on the concentration of the extracellular product (compared to the introduced quantity of RNA/DNA). In molecules and the molecular weight of the molecules. In addition, the production of gas at the electrode surface can the zone farthest from the laser beam (>60 µm under complicate the use of electroporation. Laser-based methods have these conditions), nearly all cells were both successfully been shown to permeabilize cells to exogenous molecules but have loaded and viable. To illustrate the wider applicability of not become commonly used techniques.7-15 Potential reasons are this optoporation method, cells were loaded with a sub- the requirement for special absorptive coatings or membranes strate for protein kinase C and the cellular contents then with some of the laser-based methods, the frequent use of UV or analyzed by capillary electrophoresis. In contrast to pep- IR wavelengths, and the difficulty in combining the complicated tides loaded by microinjection, optoporated peptide showed illumination geometry with subsequent cellular analysis for some little proteolytic degradation, suggesting that the cells were of the strategies.7,8,10-12,15-17 The development and characterization minimally perturbed. Also demonstrating the potential for of laser-based methods utilizing visible wavelengths and simple future work, cells were optoporated and loaded with a optical geometries without the need for specialized coatings may fluorophore in the enclosed channels of microfluidic devices. (4) Potter, H. Anal. Biochem. 1988, 174, 361-373. (5) Tsong, T. Y. Biophys. J. 1991, 60, 297-306. (6) Ho, S. Y.; Mittal, G. S. Crit. Rev. Biotechnol. 1996, 16, 349-362. Many biologic and biochemical techniques require the intro- (7) Tsukakoshi, M.; Kurata, S.; Nomiya, Y.; Ikawa, Y.; Katsuya, Y. Appl. Phys. - duction of hydrophilic or membrane-impermeant molecules into B 1984, B35, 135 140. (8) Tao, W.; Wilkinson, J.; Stanbridge, E. J.; Berns, M. W. Proc. Natl. Acad. cells. A large number of methods have been developed, but Sci. U.S.A. 1987, 84, 4180-4184. typically the plasma membrane is transiently permeabilized and (9) Guo, Y.; Liang, H.; Berns, M. W. Physiol. Plant. 1995, 93,19-24. then the exogenous molecule is introduced into the cytoplasm. (10) Turovets, I.; Lewis, A.; Palanker, D.; Gilo, H.; Vilenz, A.; Broder, J. C.; Lewis, S. Biotechnol. 1993, 15, 1022-1027. One method is microinjection, which loads cells with relatively (11) Kurata, S.; Tsukakoshi, M.; Katsuya, Y.; Ikawa, Y. Exp. Cell Res. 1986, high concentrations of molecules. However, the extent of cellular 162, 372-378. damage can be unpredictable and is frequently high.1-3 Addition- (12) Palumbo, G.; Caruso, M.; Crescenzi, E.; Tecce, M. F.; Roberti, G.; Colasanti, A. J. Photochem. Photobiol., B 1996, 36,41-46. (13) Krasieva, T. B.; Chapman, C. F.; Lamorte, V. J.; Venugopalan, V.; Berns, * Corresponding author. Phone: 949-824-7784. Fax: 949-824-8540. E-mail: M. W.; Tromberg, B. J. Proc. SPIE-Int. Soc. Opt. Eng. 1998, 3260,38-44. [email protected]. (14) Doukas, A. P.; Flotte, T. J. Ultrasound Med. Biol. 1996, 22, 151-164. ² Department of Physiology and Biophysics, University of California. (15) Flotte, T. J.; Lee, S.; Zhang, H.; McAuliffe, D.; Douki, T.; Doukas, A. G. ³ Beckman Laser Institute, University of California. Proc. SPIE-Int. Soc. Opt. Eng. 1995, 2391, 202-207. § Oak Ridge National Laboratory. (16) Doukas, A. P.; McAuliffe, D. J.; Lee, S.; Venugopalan, V.; Flotte, T. J. (1) Hapala, I. Crit. Rev. Biotechnol. 1997, 17, 105-122. Ultrasound Med. Biol. 1995, 21, 961-967. (2) Celis, J. E. Biochem. J. 1984, 223, 281-291. (17) Lee, S.; Anderson, F.; Zhang, H.; Flotte, T. J.; Doukas, A. G. Ultrasound (3) McNeil, P. L. Methods Cell Biol. 1989, 29, 153-173. Med. Biol. 1996, 22, 1285-1293. 1342 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000 10.1021/ac990982u CCC: $19.00 © 2000 American Chemical Society Published on Web 02/17/2000 increase the utility of laser-based cell loading. Acid Facility at Stanford University. The C-terminus was amidated, Optoinjection and optoporation are laser-based methods ca- and the peptide was labeled on the N-terminus with fluorescein pable of loading exogenous molecules into selected cells.7-14 With (abbreviated F-PKC) as previously described.25 All other reagents both methods, a pulsed laser beam is used to transiently perme- were purchased from Fisher Scientific (Pittsburgh, PA). abilize cells. Mechanisms by which laser irradiation (photother- Conjugation of Texas Red to Glycine. Texas Red succin- molysis) and laser-generated stress transients perturb cell mem- imidyl ester was reacted overnight (21 °C) with a 20-fold molar branes are still under investigation.14,16-19 For optoinjection, a excess of glycine in an aqueous buffer containing HEPES (50 mM, single cell is directly irradiated by the focused laser beam and pH 8.0) and dimethyl formamide (15%). The reaction product was this cell alone is loaded with exogenous molecules.7-13,20 Under analyzed by HPLC on a reverse-phase C18 column (Altech, appropriate conditions, cells can be loaded with little visible Deerfield, IL) with a gradient of 0.1% trifluoroacetic acid (TFA) damage and with high survival rates.13,20 However, the throughput in water and 0.1% TFA in acetonitrile. No free Texas Red or Texas is low, the concentrations loaded are limited, and the cell must Red succinimidyl ester was observable. be directly aligned with the laser beam. For optoporation, the laser Optoporation of Cells. The microscopy system used for beam does not usually interact with the cell but rather interacts optoporating cells was similar to that described previously.13,22 with an absorptive medium, frequently polyimide.13-16,18 A me- Cells were cultured in chambers composed of a Teflon O-ring chanical transient or stress wave is then produced as a result of attached to a no. 1 glass cover slip. The cell chambers were optical breakdown, ablation, or rapid heating of the absorbing mounted on the stage of an inverted fluorescence microscope medium.14,21 This shock wave or mechanical transient interacts (Nikon). The cells were placed in buffer A (135 mM NaCl, 5 mM with nearby cells, producing temporary alterations in the plasma KCl, 10 mM HEPES, 2 mM MgCl2, 2 mM CaCl2, pH 7.4) membrane. The exact nature by which shock waves transiently containing Texas Red-dextran, Texas Red-glycine, Oregon permeabilize membranes remains unknown but is an active area Green, or F-PKC and maintained at 37 °C with an objective heater of investigation.14,16,18,21 Advantages of optoporation over opto- (Bioptechs, Butler, PA). A frequency-doubled Q-switched Nd:YAG injection are that more than one cell can be loaded simultaneously laser (New Wave, Sunnyvale, CA) was used to generate a single and that the cells and laser beam do not need to interact directly laser pulse (10 µJ, 5 ns pulse width, 532 nm) which was directed or be precisely coaligned. Recently Krasieva et al. reported the into the microscope. The pulse was focused (∼0.3-0.4 µmatits successful optoporation of cells with a visible wavelength of light waist) with a microscope objective (100×, 1.3 n.a., Olympus) and without specialized absorptive coatings.13 However, the within the cover slip adjacent to its interface with the buffer. After cellular mortality rate was as high as 50% making the technique delivery of the single laser pulse, cells were incubated for 3 min. of limited utility for many biologic and biochemical applications.13 The cells were washed by flowing buffer A containing 10 mM In this work, we characterize the parameters affecting the glucose (3-5 mL/min, 37 °C) over them for 20 min. Oregon cellular delivery of molecules by optoporation with a visible Green diacetate (10 µM) in buffer A was then added for 1 min to wavelength and without specialized absorbing surfaces.
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