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


RESULTS
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.


CONCLUSIONS
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.
Tillo
Shane E
SE
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717, USA.
Hughes
Thomas E
TE
Makarov
Nikolay S
NS
Rebane
Aleks
A
Drobizhev
Mikhail
M
D005456:Fluorescent Dyes / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein