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Manipulation of cellular light from green fluorescent protein by a femtosecond laser

Abstract

Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry and cell biology1. It emits fluorescence following optical excitation, which is usually provided by a laser2,3. Here, we report that fluorescence from enhanced GFP can be ‘turned off’ by exposing cells to laser light. A short flash of femtosecond laser light is shown to deplete calcium in the endoplasmic reticulum of cells. Calcium-release-activated calcium channels are then activated by stromal interaction molecule 1 (STIM1). The rise in intracellular Ca2+ depolarizes mitochondria and increases the leakage of reactive oxygen species, which then permanently bleach the GFP. This controllable optical scheme for reactive oxygen species generation can also be used to modulate the photoconversion4 of GFP fluorescence from green to red emission and provide a mechanism for influencing cellular molecular dynamics.

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Figure 1: Experimental set-up.
Figure 2: GFP bleaching following exposure to a flash of light from a femtosecond laser.
Figure 3: Calcium in the endoplasmic reticulum is depleted by femtosecond laser exposure and is critical to GFP bleaching.
Figure 4: Femtosecond exposure can decrease MMP and increase ROS by releasing Ca2+.
Figure 5: Proposed mechanism for the femtosecond laser light turning off fluorescence from GFP.

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Acknowledgements

The authors thank W. Wang for effective discussions. S. He and J. Song are thanked for assistance with illustrations and X. Tang is thanked for supplying the STIM1–YFP plasmid. This work was supported by grants from the National Basic Research Program of China (grants 2011CB808101 and 2010CB327604), the National Natural Science Foundation of China (NSFC; grants 61108080, 60838004 and 81171556) and the Ministry of Science and Technology of China (2012CB917204).

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Contributions

H.H. developed the concept. H.H. and S.W. performed the optical experiments. S.L. conceived all biological experiments and materials. H.H., S.L. and M.H. analysed the data. H.H., S.L., M.H. and Y.C. wrote and revised the manuscript. Y.C. and C.W. supervised this research. All authors contributed to discussions.

Corresponding authors

Correspondence to Hao He or Chingyue Wang.

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The authors declare no competing financial interests.

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He, H., Li, S., Wang, S. et al. Manipulation of cellular light from green fluorescent protein by a femtosecond laser. Nature Photon 6, 651–656 (2012). https://doi.org/10.1038/nphoton.2012.207

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