Each cell has distinct gene-expression patterns even when sharing morphological similarities. Because the functions of a cell are determined by its location and time, single-cell resolution of gene expression is important to elucidate gene function. To achieve single-cell resolution of gene function, reliable single-cell transfection methods are needed. Some physical transfection methods have been applied to single-cell transfection with good results. Examples are:

All methods are performed under a microscope so that transfected cells can be trailed in real time. Micro-injection is straightforward and efficient but all the types of injectors actually perforate cell membranes resulting in physical damage to the cells. Single-cell electroporation efficiently delivers nucleic acids into single cells and can easily be applied in vivo. Single-cell electroporation of enhanced green fluorescence protein (EGFP) plasmid has shown the morphology and growth characteristics of a single neuron in vivo [38]. Phototransfection is the most accurate means of delivering nucleic acids (Fig. 3). Because the numbers and sizes of holes on the cell membrane can be adjusted, this method is the most suitable way of delivering population mRNAs. The additional advantage of phototransfection is that we can dictate subcellular location through which nucleic acids pass (e.g. axon or dendrite on neuron), which is not possible by electroporation. Introducing nucleic acids into a subcellular location is important for study of single polarized cells in which different cellular domains perform distinct activities. Neurons, especially, have soma, dendrites, and axons, each with a different function and localized gene expression. For example, transfection of E-26-like protein 1 (Elk-1) mRNA into dendrites of intact primary rat neurons induced cell death but introduction of Elk-1 mRNA in cell body did not cause cell death [20]. This experiment proved that localization of specific mRNA significantly altered the function of the mRNA, which was impossible to do using traditional transfection methods. The experiment could not be performed without a combination of mRNA transfection and subcellular locational transfection. Therefore, the combination of mRNA transfection and phototransfection is a powerful tool for study of gene function in single cells by virtue of point-directed delivery and immediate action of mRNA.

An illustration of phototransfection. Laser beams (green flashes) create holes at specific regions of single cell (subcellular locations) and nucleic acids (red dots) are delivered into the local areas