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

BackgroundTwo-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.ResultsHere 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.ConclusionOur 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.

[1]  Atsushi Miyawaki,et al.  A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy , 2006, Nature Biotechnology.

[2]  Yi Luo,et al.  Predictions of novel two-photon absorption bands in fluorescent proteins. , 2007, The journal of physical chemistry. B.

[3]  Thomas Schmidt,et al.  Two-photon excitation action cross-sections of the autofluorescent proteins , 2001 .

[4]  M. Drobizhev,et al.  Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. , 2009, The journal of physical chemistry. B.

[5]  Robert E Campbell,et al.  Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging , 2008 .

[6]  A. Miyawaki,et al.  Hidden electronic excited state of enhanced green fluorescent protein. , 2008, Journal of Physical Chemistry B.

[7]  Robert E Campbell,et al.  Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. , 2006, The Biochemical journal.

[8]  Joachim Goedhart,et al.  Bright monomeric red fluorescent protein with an extended fluorescence lifetime , 2007, Nature Methods.

[9]  Robert E Campbell,et al.  Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. , 2007, Biochemistry.

[10]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[11]  A. Miyawaki,et al.  Two-photon dual-color imaging using fluorescent proteins , 2008, Nature Methods.

[12]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[13]  Michael Kasha,et al.  Characterization of electronic transitions in complex molecules , 1950 .

[14]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[15]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.

[16]  M. Drobizhev,et al.  Resonance enhancement of two-photon absorption in fluorescent proteins. , 2007, The journal of physical chemistry. B.

[17]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[18]  Robert E Campbell,et al.  Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors , 2008, Nature Methods.