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Visualising individual green fluorescent proteins with a near field optical microscope
M F Garcia-Parajo1, J A Veerman, G M Segers-Nolten
1Department of Applied Physics & MESA Research Institute, University of Twente, Enschede, The Netherlands. m.f.garciaparajo@tn.utwente.nl
Cytometry
|July 15, 1999
Summary
Individual green fluorescence protein (GFP) photodynamics are more complex than previously thought. Even apparent bleaching is reversible over extended observation times, revealing new insights into GFP behavior.
Area of Science:
- Molecular Biology
- Biophysics
- Optical Microscopy
Background:
- Green fluorescence protein (GFP) is crucial for molecular biology applications.
- Understanding GFP's photophysical and photodynamical properties is essential for its use as a marker.
- Ensemble-averaged measurements may not fully capture individual protein behavior.
Purpose of the Study:
- To investigate the photophysical and photodynamical properties of individual S65T mutants of GFP.
- To correlate topographic and optical features of GFP.
- To understand the time-resolved behavior of single GFP molecules.
Main Methods:
- Near-field scanning optical microscopy (NSOM) with dual polarization detection channels.
- Simultaneous shear-force and near-field fluorescence imaging.
- Real-time monitoring of S65T-GFP mutants over extended periods (2+ hours) with high temporal resolution (0.5 ms).
Main Results:
- The emission dipole moment of individual GFPs is fixed within the protein structure and gel matrix.
- Observed reversible on-off fluorescence behavior on timescales from 10^-4 to 10^3 seconds.
- Apparent "bleaching" of GFP was found to be reversible with sufficient observation time.
Conclusions:
- Individual GFP molecules exhibit complex photodynamical behavior not evident in ensemble studies.
- Reversible photodynamics challenge conventional understanding of GFP photobleaching.
- Detailed single-molecule studies are vital for accurate characterization of fluorescent proteins.