TY - JOUR
T1 - Monomeric red fluorescent proteins with a large Stokes shift
AU - Piatkevich, Kiryl D.
AU - Hulit, James
AU - Subach, Oksana M.
AU - Wu, Bin
AU - Abdulla, Arian
AU - Segall, Jeffrey E.
AU - Verkhusha, Vladislav V.
PY - 2010/3/23
Y1 - 2010/3/23
N2 - Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.
AB - Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.
KW - Cell polarity
KW - Intravital imaging
KW - Keima
KW - Two-photon microscopy
KW - mKate
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U2 - 10.1073/pnas.0914365107
DO - 10.1073/pnas.0914365107
M3 - Article
C2 - 20212155
AN - SCOPUS:77950400530
SN - 0027-8424
VL - 107
SP - 5369
EP - 5374
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 12
ER -