title: Targeted microinjection into cells and retina using optoporation.
abstract: The laser microbeam has enabled highly precise noncontact delivery of exogenous materials into targeted cells without compromising cell viability, which has been a highly challenging task for traditional methods. Here, we report targeted delivery of impermeable substances into mammalian cells and goldfish retinal explants subsequent to ultrafast laser microbeam assisted injection. Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time constant for injection. Using optimized laser intensity, exposure, and a spatial irradiation pattern, desired spatial transfection patterns in goldfish retina explants were achieved as confirmed by the expression of injected plasmids encoded for light-activable channel rhodopsin-2 ion-channel, tagged with fluorescent protein. Laser assisted delivery of exogenous material into a specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the functioning of neuronal circuitry of normal and degenerated retina.
authors.affiliation: University of Texas at Arlington, Department of Physics, Biophysics and Physiology Lab, Arlington, Texas 76019, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Gu
authors.forename: Samarendra K
authors.initials: SK
authors.lastname: Mohanty
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002466:Cell Nucleolus; D003584:Cytological Techniques / Q000295:instrumentation*; D004867:Equipment Design; D004912:Erythrocytes / Q000528:radiation effects; D006054:Goldfish; D006801:Humans; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections / Q000295:instrumentation*; D055095:Optics and Photonics / Q000295:instrumentation*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D016062:Porosity; D011419:Propidium; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012160:Retina; D059008:Time-Lapse Imaging; D014162:Transfection / Q000295:instrumentation* / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C523009:channelopsin-2, Chlamydomonas reinhardtii; C054562:yellow fluorescent protein, Bacteria; D011419:Propidium