Real-Time Background-Free Selective Imaging of Fluorescent Nanodiamonds in Vivo

Recent developments of imaging techniques have enabled
fluorescence
microscopy to investigate the localization and dynamics of intracellular
substances of interest even at the single-molecule level. However,
such sensitive detection is often hampered by autofluorescence arising
from endogenous molecules. Those unwanted signals are generally reduced
by utilizing differences in either wavelength or fluorescence lifetime;
nevertheless, extraction of the signal of interest is often insufficient,
particularly for in vivo imaging. Here, we describe a potential method
for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds.
This method is based on the property of NVCs that the fluorescence
intensity sensitively depends on the ground state spin configuration
which can be regulated by electron spin magnetic resonance. Because
the NVC fluorescence exhibits neither photobleaching nor photoblinking,
this protocol allowed us to conduct long-term tracking of a single
nanodiamond in both Caenorhabditis elegans and mice,
with excellent imaging contrast even in the presence of strong background
autofluorescence.