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Proceedings Paper

Common fluorescent proteins for single-molecule localization microscopy
Author(s): Natalia V. Klementieva; Nina G. Bozhanova; Natalie M. Mishina; Elena V. Zagaynova; Konstantin A. Lukyanov; Alexander S. Mishin
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Paper Abstract

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Paper Details

Date Published: 14 July 2015
PDF: 6 pages
Proc. SPIE 9536, Advanced Microscopy Techniques IV; and Neurophotonics II, 953609 (14 July 2015); doi: 10.1117/12.2184924
Show Author Affiliations
Natalia V. Klementieva, Nizhny Novgorod State Medical Academy (Russian Federation)
Nina G. Bozhanova, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation)
Natalie M. Mishina, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation)
Elena V. Zagaynova, Nizhny Novgorod State Medical Academy (Russian Federation)
Konstantin A. Lukyanov, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation)
Alexander S. Mishin, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation)


Published in SPIE Proceedings Vol. 9536:
Advanced Microscopy Techniques IV; and Neurophotonics II
Emmanuel Beaurepaire; Peter T. C. So; Francesco Pavone; Elizabeth M. Hillman, Editor(s)

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