Labeling of single, intact plant root cells using laser-mediated optoperforation

The interface between physics and biology has resulted in advances in medical engineering and animal cell biology, but plant science has been slower to recognize this synergy. Current knowledge of plants is derived from tissue-level studies; while easy to analyze they do not provide insight into the cellular-level responses that are central to the function of the whole organism. Thus, single-cell research is needed to discover how cells influence everything from their immediate neighbors to distant tissues. Current methods to modify individual cells are cumbersome (i.e. microinjection), or imprecise and deleterious to the cell (i.e. biolistics). Recently, single animal and cell wall-free cultured plant cells were punctured using a laser-based method known as optoperforation. This method involves tightly focusing a pulsed laser onto a cell membrane. Within this small focal region, nonlinear optical processes occur, giving rise to multiphoton excitation of the water at the cell boundary. As a result of this interaction, transient cell membrane openings are created, allowing diffusion of surrounding particles into the cell. We have used this technique to show the first successful optoperforation of intact plant tissue via permeation of the cell wall and plasma membrane with fluorescent 70 kDa dextran molecules. Cell survival was monitored for 24 hours and the dextrans remained distributed throughout the cytoplasm while the nucleus stayed functional, as evidenced by vital staining. This research has applications in studying the metabolism of single cells such as cell-to-cell movement of large molecules and may replace difficult techniques for generating transgenic plants.