Real-time intravital imaging of pH variation associated with osteoclast activity

Abstract

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.

Time-lapse reveals that osteoclasts can move across the bone surface while resorbing

“…a critical duration with respect with sealing zone turnover; see below) instead of every 21 min. Of note, our in vitro observations are in line with recent intravital time-lapse recordings visualizing the sub-osteoclastic resorption compartment itself (achieved with an acid-sensitive probe), and showing a crescent-shaped zone moving over the bone surface (Maeda et al, 2016). …”

In vivo multiphoton imaging of immune cell dynamics

“…Selective visualization of molecular events associated with endogenous target cells with small molecule fluorophores often requires ingenious in vivo labeling methods. For example, a recent study developed a pH-activatable small molecule probe that is fixed on the bone surface after the in vivo administration and reported intravital imaging of the bone resorption activity of osteoclasts [42]. …”

In vivovisualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption

“…Next, we examined the changes in acid production of mature osteoclasts using a pH-sensing chemical probe, pHocas-3, which we recently developed 29. This probe, which emits green fluorescence only in acidic environments, can detect local low pH in bone resorption areas in vivo .…”