Cellular laserfection.

Many studies in modern biology often rely on the introduction of a foreign molecule (i.e., transfection), be it DNA plasmids, siRNA molecules, protein biosensors, labeled tracers, and so on, into cells in order to answer the important questions of today's science. Many different methods have been developed over time to facilitate cellular transfection, but most of these methods were developed to work with a specific type of molecule (usually DNA plasmids) and none work well enough with difficult, sensitive, or primary cells to meet the needs of current life science researchers. A novel procedure that uses laser light to gently permeabilize large number of cells in a very short time has been developed and is described in detail in this chapter. This method allows difficult cells to be efficiently transfected in a high-throughput manner, with a wide variety of molecules, with extremely low toxicity.

Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types.

Efficient delivery of compounds and macromolecules into living cells is essential in many fields including basic research, applied drug discovery, and clinical gene therapy. Unfortunately, current delivery methods, such as cationic lipids and electroporation, are limited by the types of macromolecules and cells that can be employed, poor efficiency, and/or cell toxicity. To address these issues, novel methods were developed based on laser-mediated delivery of macromolecules into cells through optoinjection. An automated high-throughput instrument, the laser-enabled analysis and processing (LEAP) system, was utilized to elucidate and optimize several parameters that influence optoinjection efficiency and toxicity. Techniques employing direct cell irradiation (i.e., targeted to specific cell coordinates) and grid-based irradiation (i.e., without locating individual cells) were both successfully developed. With both techniques, it was determined that multiple, sequential low radiant exposures produced more favorable results than a single high radiant exposure. Various substances were efficiently optoinjected--including ions, small molecules, dextrans, siRNAs (small interfering RNAs), plasmids, proteins, and semiconductor nanocrystals--into numerous cell types. Notably, cells refractory to traditional delivery methods were efficiently optoinjected with lower toxicity. We establish the broad utility of optoinjection, and furthermore, are the first to demonstrate its implementation in an automated, high-throughput manner.