Introduction of impermeable actin-staining molecules to mammalian cells by optoporation

Abstract

The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as viral transfection and lipofection, are not realizable with a high degree of spatial selectivity. Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons). Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed. Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

Optical delivery of multiple opsin-encoding genes leads to targeted expression and white-light activation

“…Targeted laser-assisted transfection of single opsins To achieve the targeted transfection of cells with opsin-encoding plasmids (ChR2 and ReaChR), we used optimized laser parameters 23 for optoporation into HEK293 cells. Before optoporation, the extracellular medium was exchanged with new DMEM medium-containing DNA plasmids (ReaChR or ChR2) with a final concentration of 1 mg mL 21 and incubated for 30 min.…”

“…Recently, there has been considerable interest in the optical transfection of cells using ultrafast pulsed light [15][16][17][18][19][20][21][22][23] because this approach offers selective targeting and higher efficiency and viability (.90% reported in vitro 22 ) than other methods. Furthermore, femtosecond (fs) near-infrared (NIR) laser-based transfection has been shown to be safe, resulting in high efficiency and high survival rates (93%) of optoporated embryos during development 20 , as well as suitable for in vivo gene delivery 24 .…”

“…Although several non-viral approaches, such as electroporation and lipofection, exist, these methods suffer from low transfection efficiency, loss of cell viability, and non-targeted transfection, similar to viral methods. Ultrafast NIR laser-mediated optoporation 23,25 can address these issues and holds promise for the delivery of large multi-opsin constructs via targeted transfection with single-cell resolution and high efficiency.…”

Biodegradable microsphere-mediated cell perforation in microfluidic channel using femtosecond laser

“…[1][2][3][4][5] Cell membrane perforation by focused femtosecond laser pulses provides an exclusive technology to introduce external molecules into a single cell, i.e., optoinjection, without significant cell damage because of a nonlinear interaction in a tiny focused spot. [6][7][8][9] Recently, the use of nanoparticles or microparticles has been accelerating attractive achievements to expand the capability of ultrashort laser optoinjection technology toward simultaneous treatment of multiple cells. Chakravarty et al 10 reported the delivery of DNA, protein, and fluorescence molecules into cells in a cuvette by using carbon black nanoparticles and a femtosecond laser.…”

Laser-assisted photoporation: fundamentals, technological advances and applications

“…In recent years, cellular imaging by direct laser photoporation for delivery of dyes into live cells is being explored as a new and promising application. In particular, fs laser-assisted direct photoporation was used to deliver actin-staining fluorophores into rat cortical neurons for visualizing the cytoskeleton of dendrites [159].…”