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BMC Biotechnol. 2010 Feb 2;10:6. doi: 10.1186/1472-6750-10-6.

A new approach to dual-color two-photon microscopy with fluorescent proteins.

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  • 1Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717, USA.



Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency.


Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein.


Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

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