A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.

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We describe an electrofusion-based technique for combinatorial synthesis of individual liposomes. A prototype device with containers for liposomes of different compositions and a fusion container was constructed. The sample containers had fluid contact with the fusion container through microchannels. Optical trapping was used to transport individual liposomes and cells through the microchannels into the fusion container. In the fusion container, selected pairs of liposomes were fused together using microelectrodes. A large number of combinatorially synthesized liposomes with complex compositions and reaction systems can be obtained from small sets of precursor liposomes. The order of different reaction steps can be specified and defined by the fusion sequence. This device could also facilitate single cell-cell electrofusions (hybridoma production). This is exemplified by fusion of transported red blood cells.

This paper describes the use of focused electric fields and focused optical fields for the highresolution manipulation of single cells. A focused electric field, obtained with the use of ultramicroelectrodes (tip diameter ~ 5 µm), is used to electroporate and electrofuse individual cells selectively and with high spatial resolution. A focused optical field, in the form of an optical tweezer, is used to isolate single organelles from a cell as well as to position liposomes incorporated with receptors and transporters along the cell for the high-resolution sampling and probing of the cellular microenvironment.