Abstract
Femtosecond laser photoporation has become a popular method to deliver various kinds of molecules such as genes, proteins, and fluorescent dyes into single mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery, especially for larger molecules than the mesh size of the cell wall. This work is the first demonstration of the efficient photoinjection of megadalton molecules into a cytoplasm of an intact single plant cell by employing a femtosecond laser amplifier under moderate enzyme treatment conditions. The intense femtosecond laser pulse effectively formed a pore on the cell wall and membrane of Tobacco BY-2, and 2 MDa dextran molecules were introduced through the pore. Along with the pore formation, induced mechanical tensile stresses on BY-2 cells were considered to increase permeability of the cell membrane and enhance the uptake of large molecules. Moreover, the moderate enzyme treatment partially degraded the cell wall thereby facilitating the increase of the molecular introduction efficiency.
Highlights
Introduction efficiencies under three conditionsThe level of introduced FITC molecules under the three conditions, untreated, mannitol treated and adjunctive enzyme treated, were quantified by differential fluorescence intensities measured in cytosol between before and after the photoporation (∆I)
In advance of the fs laser photoporation, we examined a morphological change of intracellular structures of tobacco BY-2 cell (TBY-2) cells (Fig. 1a) after the mannitol addition and enzyme treatments
When mannitol was added to the culture medium, the vacuoles shrunk, and the cell membrane was separated from the cell wall (Fig. 1b), which means that the turgor pressure decreases and the cell is plasmolyzed in the hypertonic mannitol solution
Summary
The level of introduced FITC molecules under the three conditions, untreated, mannitol treated and adjunctive enzyme treated, were quantified by differential fluorescence intensities measured in cytosol between before and after the photoporation (∆I). There was a small background fluorescence signal inside the cytoplasm that originated from cell’s auto fluorescence and/or scattered fluorescence by cells. The temporal fluctuations of the background fluorescence for 40 cells varied from -3.45 to 1.30 with a mean of -0.94. From the background temporal fluctuations estimation, we define ∆I as molecular introduction level and the ∆I value of 2 is called the molecular introduction threshold.
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