Laser-assisted Transfection, Optogenetic Stimulation And Its Detection In Mammalian Cells
Dhakal, Kamal Raj
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Stimulation of cells, especially neurons is of significant interest both for basic understanding of neuronal circuitry as well as clinical intervention. Existing electrode-based methods of stimulation is invasive and non-specific to single cells or cell type. Recently, optical stimulation of targeted neurons expressing light-sensitive proteins (opsins) has surfaced as an emerging and a powerful technique (Optogenetics) in neuroscience. Various transfection methods such as viral-based, lipofection, electroporation have been developed to express the opsin. However, these methods are not suitable for transfection of a single cell. In order to achieve single cell transfection, we have used femtosecond pulsed laser microbeam to make a transient hole in the cell membrane in order to deliver exogenous molecules such as plasmids:Channelrhodopsin-2 (ChR2), and red-activatable Channelrhodopsin (ReaChR) or cell impermeable dye (Rhodamine Phalloidin). This method allowed live cell imaging following injection of actin-staining dye Rhodamine Phalloidin. Plasmids encoding for light sensitive proteins ReaChR, ChR2 and white opsin were also transfected into the cells by laser-assisted optoporation. Using optogenetics tools as a minimally invasive technique for cells and tissues is limited to the study of superficial regions, since a significant amount of the visible light is lost due to absorption and scattering. We have used fiber optic two-photon optogenetic stimulation (FO-TPOS) in order to enhance the in-depth stimulation. Photothermal contributions which occur due to absorption of NIR light during optogenetic stimulation, has been modeled and discussed. Near-infrared TPOS will allow localized stimulation so as to enable probing and manipulation of neural circuitry with high spatial resolution. Several electrode-based methods have been developed in order to detect and measure the electrical activity of cells and tissues during stimulation such as patch-clamp and multi-electrode arrays(MEAs) recording. These methods are invasive, potentially non-sterile and require cumbersome instruments which often generate noise and artifacts. We have used calcium imaging to detect the activation of cells as a result of optogenetic stimulation.