Success of optogenetic intervention of neural activity requires optimization of delivery of genes encoding light sensitive proteins (opsins) to specific cells, and to record the changes in cells and tissue during optogenetic stimulation. The most-commonly used method for delivering opsin(s) is use of viral vector, which is prone to cause unexpected inflammatory responses, immunological reactions, and improper gene integration. Further, the viral methods limit the size of plasmid that can be packaged and delivered and therefore cannot carry multiple opsin-encoding genes or large promoters. Further, in several cases of human diseases such as retinitis pigmentosa (RP) where progressive loss of photoreceptors happens in peripheral retina, it will be useful to localize the expression of the opsins not only in specific cell types, but in a restricted spatial region (peripheral in RP).
The first aim of the proposal is to optimize a non-viral delivery method to mitigate the challenges posed by viral delivery. We have recently used focused near-IR ultrafast laser beam method to deliver opsins (ChR2) into spatially-patterned regions of neural tissue (retina). However, this technique needs to be optimized so as to minimize the deleterious effects. Further, it will prove useful to develop a label-free optical technique (in contrast to electrophysiology) to non-invasively evaluate functional activation of optogenetically-sensitized neurons with high spatial resolution and large throughput. Recently, we demonstrated use of Phase-Sensitive Frequency Domain Optical Coherence Tomography (PSFD-OCT) for detection of fluctuations in optogenetically-stimulated cells. PSFD-OCT is a novel technique based on the principles of low-coherence interferometry that can detect displacements of the order of tens of picometers. Because of the low-coherence length of the light, the detected signal has to be within the coherence length of the light source (~10?m). This enables PSFD-OCT to investigate sub- nanometer changes within a very small region of the tissue volume and is suitable for highly localized detection. The overall aim of this study is to optimize optical delivery of gene encoding opsins, and develop label-free non-invasive optical readout method based on PSFD-OCT to monitor the changes in cortical neurons and tissue resulting from the activation. 1
Using optogenetic stimulation, chemically identical neurons can be activated by light with high temporal and spatial resolution, after introduction of genes encoding light activated molecular channels. The most-commonly used method for gene delivery such as viral vectors, are prone to cause unexpected inflammatory responses, immunological reactions, and improper gene integration. The overall aim of this study is to develop and optimize optical delivery of gene encoding for light-sensitive proteins, and develop label-free non-invasive optical readout method based on phase-sensitive optical coherence tomography to monitor the changes in the optically- stimulated cells. 1
|Dhakal, Kamal; Black, Bryan; Mohanty, Samarendra (2014) Introduction of impermeable actin-staining molecules to mammalian cells by optoporation. Sci Rep 4:6553|
|Mondal, Argha; Black, Bryan; Kim, Young-tae et al. (2014) Loop formation and self-fasciculation of cortical axon using photonic guidance at long working distance. Sci Rep 4:6902|
|Gu, Ling; Koymen, Ali R; Mohanty, Samarendra K (2014) Crystalline magnetic carbon nanoparticle assisted photothermal delivery into cells using CW near-infrared laser beam. Sci Rep 4:5106|
|Dhakal, Kamal R; Gu, Ling; Shivalingaiah, Shivaranjani et al. (2014) Non-scanning fiber-optic near-infrared beam led to two-photon optogenetic stimulation in-vivo. PLoS One 9:e111488|