Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation.
Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.
Institute of Physics and Chemistry of Materials of Strasbourg, Organic Materials Group, CNRS and Université Louis Pasteur-Strasbourg I (UMR 7504), 23 rue du Loess, 67034 Strasbourg, France.
Ali
A
Hayek
Alexei
A
Grichine
Thomas
T
Huault
Clément
C
Ricard
Frédéric
F
Bolze
Boudewijn
B
Van Der Sanden
Jean-Claude
JC
Vial
Yves
Y
Mély
Alain
A
Duperray
Patrice Lilian
PL
Baldeck
Jean-François
JF
Nicoud
D000818:Animals; D001921:Brain / Q000166:cytology*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D036641:Microscopy, Fluorescence, Multiphoton; D017785:Photons
D005456:Fluorescent Dyes