Single cell optical transfection

Abstract

The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology.

Figure 1.

Patch clamping of a cell during irradiation with an 800 nm source allows the study of the timing of the photoporation event and the quantitative assessment of material exchange. (Reproduced with permission from Optical Society of America; Baumgart et al. (2008).)

Figure 2.

Concept diagram of Gaussian beam versus Bessel beam photoporation. The therapeutic central core of a Bessel beam used for optical injection extends over 100 µm, whereas the therapeutic volume of a traditional fs-pulsed Gaussian beam is of the order of 2–3 µm in height (Tsampoula et al. 2007; Niioka et al. 2008). Misaligned CW Gaussian beams also suffer greatly reduced effectiveness. Hence, an operator using a Bessel beam to optically inject a cell does not need to focus the plasma membrane carefully. CW, continuous wave; fs, femtosecond-pulsed. (Reproduced with permission from Brown et al. (2008). Copyright © Wiley-VCH Verlag GmbH & Co. KGaA.)

Figure 3.

Multiple Bessel beam arrays generated in real time by a spatial light modulator. Each beam is capable of transfecting a single cell, so multiple cells per field-of-view may be dosed. Scale bar, 10 µm. (Reproduced with permission from Optical Society of America; Čižmár et al. (2008).)

Figure 4.

(a) Optical injection of the membrane impermeable dye Lucifer Yellow. (b) Optical transfection of capped GFP mRNA into primary rat hippocampal neurons: (i) 0 min, (ii) 30 min, (iii) 120 min and (iv) 180 min. The injected neurons take up the yellow dye or express the GFP, while neighbouring neurons remain unaffected. Scale bars, 20 µm. (Reproduced with permission from Macmillan Publishers Ltd: Nature Methods; Barrett et al. (2006). Copyright © 2006.)