Common fluorescent proteins for single-molecule localization microscopy

Natalia V. Klementieva, Nina G. Bozhanova, Natalie M. Mishina, Elena V. Zagaynovaa, Konstantin A. Lukyanov, Alexander S. Mishin

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review


Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Singlemolecule 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, highresolution 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.

Original languageEnglish
Title of host publicationEuropean Conference on Biomedical Optics, ECBO 2015
PublisherOSA - The Optical Society
ISBN (Print)9781628417012
Publication statusPublished - 21 Jul 2014
Externally publishedYes
EventEuropean Conference on Biomedical Optics, ECBO 2015 - Munich, Germany
Duration: 21 Jun 201525 Jun 2015

Publication series

NameOptics InfoBase Conference Papers


ConferenceEuropean Conference on Biomedical Optics, ECBO 2015


  • Photobleaching
  • Red fluorescent proteins
  • Single-molecule localization microscopy


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