Real-time intravital imaging of pH variation associated with osteoclast activity.
Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Hiroki
H
Maeda
WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.
Toshiyuki
T
Kowada
WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.|Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Junichi
J
Kikuta
Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Masayuki
M
Furuya
Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Mai
M
Shirazaki
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.|WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.
Shin
S
Mizukami
WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.|Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Masaru
M
Ishii
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.|WPI-Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.
Kazuya
K
Kikuchi
D000818:Animals; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D000069416:Intravital Microscopy / Q000379:methods*; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry; D010010:Osteoclasts / Q000378:metabolism*; D059008:Time-Lapse Imaging
D015335:Molecular Probes