Single-Cell Poration and Lysis by Microsecond-Laser-Pulse Induced Microbubbles

Abstract

In this dissertation, single-cell poration and single-cell lysis combined with single-cell manipulation via microsecond-laser-pulses induced microbubbles are integrated on a microfluidic chip for molecular biology applications. Molecular delivery is necessary in many clinical and fundamental biological research. Traditional cell poration techniques are well-suited to treating large groups of cells. However, the ability to transfer exogenous molecules into localized single cells is required in fields such as stem-cell research. Optoporation can achieve localized single-cell poration using continuous-wave lasers or nanosecond or femtosecond laser pulses, but with limitations for each method. In this dissertation, a new optoporation method, laser-induced microbubble poration (LMP), is demonstrated and characterized. LMP uses microsecond-long laser pulses to generate and control size-oscillating microbubbles in saline media. The bubble size oscillation induces microstreaming, and the associated shear stress, which causes small pores to open on the membrane of nearby cells. LMP method can achieve single-cell poration with a high efficiency of 95.2 ± 4.8%, and a cell viability as high as 97.6 ± 2.4%. The poration of cells above laser-induced microbubbles (PCALM) has been developed to further improve LMP, and provides higher throughput and the delivery of larger molecules. Single-cell lysis can release intracellular components which provide crucial information for biomedical research. In this report, localized single cells can be lysed precisely and selectively, using microbubbles induced by the microsecond laser pulses. The shear stress from the microstreaming of the laser induced microbubble and the direct contact with the expanding bubbles causes the rupture of the targeted cell membrane. It is also convenient to integrate the cell manipulation function into the cell lysis system, which can be used for cell-cell interaction studies. In the future, the parallel and automated control of microbubbles can be achieved by a laser scanning system or a spatial light modulator. This will enable the poration or lysis of multiple target cells at the same time, further improving the throughput.