Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein Export

@inproceedings{citeulike:4779892, abstract = {Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.}, author = {Biteen, Julie S. and Thompson, Michael A. and Tselentis, Nicole K. and Shapiro, Lucy and Moerner, W. E.}, citeulike-article-id = {4779892}, citeulike-linkout-0 = {http://scitation.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=PSISDG00718500000171850I000001&idtype=cvips&gifs=yes}, citeulike-linkout-1 = {http://link.aip.org/link/?PSI/7185/71850I}, editor = {Enderlein, J\"{o}rg and Gryczynski, Zygmunt K. and Erdmann, Rainer}, journal = {Single Molecule Spectroscopy and Imaging II}, keywords = {biophysics, gfp, microscopy, super-resolution}, location = {San Jose, CA, USA}, number = {1}, pages = {71850I+}, posted-at = {2009-06-08 18:41:25}, priority = {2}, publisher = {SPIE}, title = {Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein}, url = {http://scitation.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=PSISDG00718500000171850I000001&idtype=cvips&gifs=yes}, volume = {7185}, year = {2009} }

TY - CONF ID - citeulike:4779892 L3 - citeulike-article-id:4779892 N2 - Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells. CY - San Jose, CA, USA IS - 1 JF - Single Molecule Spectroscopy and Imaging II TI - Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein PB - SPIE VL - 7185 SP - 71850I KW - biophysics KW - gfp KW - microscopy KW - super-resolution AU - Biteen, Julie AU - Thompson, Michael AU - Tselentis, Nicole AU - Shapiro, Lucy AU - Moerner, WE A2 - Enderlein, J\"{o}rg A2 - Gryczynski, Zygmunt A2 - Erdmann, Rainer PY - 2009/// UR - http://scitation.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=PSISDG00718500000171850I000001&idtype=cvips&gifs=yes ER -