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Optofluidics, Microfluidics and Nanofluidics

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Laser switching contrast microscopy to monitor free and restricted diffusion inside the cell nucleus

Franz-Josef Schmitt
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Cornelia Junghans
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Matthias Sturm
  • Corresponding author
  • Technical University of Berlin, Institute of Optics and Atomic physics ER1-1, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Csongor Keuer
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Hans Joachim Eichler
  • Corresponding author
  • Technical University of Berlin, Institute of Optics and Atomic physics ER1-1, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Thomas Friedrich
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
Published Online: 2016-04-22 | DOI: https://doi.org/10.1515/optof-2016-0001

Abstract

A novel microscopic technique termed laser switching contrast microscopy (LSCM) allows for the imaging of the dynamics of optically switchable proteins in single cell compartments. We present an application for the monitoring of diffusive properties of single molecules of the photo-switchable fluorescent protein Dreiklang (DRK). LSCM in the cell nucleus of Chinese hamster ovary (CHO) cells cytoplasmically expressing DRK unravels quick diffusive equilibration of the DRK molecules inside the whole cytoplasm and inside the cell nucleus within seconds. The nuclear membrane is also highly permeable for DRK. Inside the nucleus entirely distinct regions are found that only partially enable diffusive protein redistribution with mean square displacement proportional to time while in other regions the mobility of the proteins seems to be restricted. After photo-switching string like patterns of light DRK molecules are observed in the cell nucleus. In addition a fraction of these DRK molecules appears immobile. The findings support recent theories of the cell interior described as a random obstacle model with an additional immobile fraction of DRK. Numerical simulations show that at different illumination intensity and different distance from the laser focus similar patterns for fluorescence recovery might be obtained in spite of strongly varying diffusion constants.

Keywords: Fluorescence microscopy; superresolution microscopy; Dreiklang; fluorescent protein; diffusion constant; photo-switchable molecules; random walk; nanofluidics

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About the article

Received: 2015-11-02

Accepted: 2016-01-08

Published Online: 2016-04-22



Citation Information: Optofluidics, Microfluidics and Nanofluidics, ISSN (Online) 2300-7435, DOI: https://doi.org/10.1515/optof-2016-0001. Export Citation

© 2016 Franz-Josef Schmitt et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0)

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