Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes

Despite
the significant advantages of two-photon excitation microscopy
(TPEM) over traditional confocal fluorescence microscopy in live-cell
imaging applications, including reduced phototoxicity and photobleaching,
increased depth penetration, and minimized autofluorescence, only
a few metal ion-selective fluorescent probes have been designed and
optimized specifically for this technique. Building upon a donor–acceptor
fluorophore architecture, we developed a membrane-permeant, Zn­(II)-selective
fluorescent probe, chromis-1, that exhibits a balanced two-photon
cross section between its free and Zn­(II)-bound form and responds
with a large spectral shift suitable for emission-ratiometric imaging.
With a Kd of 1.5 nM and wide dynamic range,
the probe is well suited for visualizing temporal changes in buffered
Zn­(II) levels in live cells as demonstrated with mouse fibroblast
cell cultures. Moreover, given the importance of zinc in the physiology
and pathophysiology of the brain, we employed chromis-1 to monitor
cytoplasmic concentrations of labile Zn­(II) in oligodendrocytes, an
important cellular constituent of the brain, at different stages of
development in cell culture. These studies revealed a decrease in
probe saturation upon differentiation to mature oligodendrocytes,
implying significant changes to cellular zinc homeostasis during maturation
with an overall reduction in cellular zinc availability. Optimized
for TPEM, chromis-1 is especially well-suited for exploring the role
of labile zinc pools in live cells under a broad range of physiological
and pathological conditions.