Monomeric red fluorescent proteins with a large Stokes shift.
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 mum the breast cancer cells show significant polarization towards vessels in living mice.
Piatkevich
Kiryl D
KD
Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Hulit
James
J
Subach
Oksana M
OM
Wu
Bin
B
Abdulla
Arian
A
Segall
Jeffrey E
JE
Verkhusha
Vladislav V
VV
D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply / Q000235:genetics / Q000378:metabolism; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton; D016296:Mutagenesis; D009368:Neoplasm Transplantation; D055668:Photochemical Processes; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014183:Transplantation, Heterologous; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein