TY - GEN
T1 - Tag-free labeling of tubulin in live cells with fluorescent organic dyes
AU - Schvartz, Tomer
AU - Alush, Noa
AU - Nachmias, Dikla
AU - Arbely, Eyal
AU - Elia, Natalie
PY - 2016/12/20
Y1 - 2016/12/20
N2 - High-resolution fluorescence imaging, combined with the latest labeling techniques available today have yet been able to provide the necessary spatiotemporal resolution to record all cellular processes in live cells. Substituting the bulky fluorescent protein tags (such as GFP) currently used in live-cell applications with much smaller fluorescent dyes that possess superior photophysical characteristics will markedly improve these advanced imaging techniques. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non-invasive way to specifically and directly attach such fluorescent dyes to proteins in live cells. Here we employ this strategy to directly tag α-tubulin in live mammalian cells. By screening different conditions we have optimized the system for quantitative high-resolution recording of microtubules in live cells. We will present data demonstrating the feasibility and efficiency of the approach and will discuss the advantages and limitations of using genetic code expansion for quantitative high-resolution microscopy.
AB - High-resolution fluorescence imaging, combined with the latest labeling techniques available today have yet been able to provide the necessary spatiotemporal resolution to record all cellular processes in live cells. Substituting the bulky fluorescent protein tags (such as GFP) currently used in live-cell applications with much smaller fluorescent dyes that possess superior photophysical characteristics will markedly improve these advanced imaging techniques. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non-invasive way to specifically and directly attach such fluorescent dyes to proteins in live cells. Here we employ this strategy to directly tag α-tubulin in live mammalian cells. By screening different conditions we have optimized the system for quantitative high-resolution recording of microtubules in live cells. We will present data demonstrating the feasibility and efficiency of the approach and will discuss the advantages and limitations of using genetic code expansion for quantitative high-resolution microscopy.
UR - https://www.mendeley.com/catalogue/0dcb3222-310b-3bfe-81ee-92bb31452029/
U2 - 10.1002/9783527808465.emc2016.6296
DO - 10.1002/9783527808465.emc2016.6296
M3 - Conference contribution
T3 - European Microscopy Congress 2016: Proceedings
SP - 45
EP - 46
BT - European Microscopy Congress 2016: Proceedings
PB - Wiley - VCH Verlag GmbH & CO. KGaA
ER -