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.

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.

High‐throughput pharmacology will play an increasingly important role in drug development as companies strive to limit the likelihood that a compound will be removed from the market or fail phase II and III clinical trials because of unexpected side effects or toxicity. Identification of the correct compounds to advance from hit to lead‐compound candidate requires the determination of efficacy and evaluation of potential compound liabilities (solubility, adsorption, toxicity) and specificity of action. We propose that the characterization of the response of hit compounds against a broad panel of natural human cells will be very useful in making the correct selection. Because genes are not overexpressed in these cells, the signaling pathways are not altered by changes in protein stoichiometry. In addition, our cell bank has been genetically indexed and characterized for responses to known pharmacological agents and stored in a database. Thus, the pattern of functional responses (such as membrane potential, changes in intracellular calcium, cAMP etc.) obtained with test compounds can be compared with the known responses and gene expression, allowing for the ready identification of compounds with unexpected toxicities and effects. In addition, in vitro assays for evaluation of the adsorption and metabolism properties of compounds can be used to screen for compounds with drug‐like properties. These systems and methods provide high definition, content‐rich information about cellular responses and have sufficient throughput for use in screening hit compounds.