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.

近期成像技术的进步使得荧光显微镜能够探究感兴趣细胞内物质的定位和动态，甚至达到单分子水平。然而，这种高灵敏度的检测常常受到内源性分子产生的自发光的干扰。这些不必要的信号通常通过利用波长或荧光寿命的差异来降低；然而，对于体内成像而言，感兴趣信号的提取往往不足。在本研究中，我们描述了一种针对纳米钻石中氮空位中心（NVCs）选择性成像的潜在方法。该方法基于NVCs的属性，即其荧光强度对基态自旋配置的依赖性，该配置可通过电子自旋磁共振进行调节。由于NVC荧光既不发生光漂白也不发生光闪烁，该协议使我们能够在存在强烈背景自发光的情况下，对秀丽隐杆线虫和老鼠中的单个纳米钻石进行长期跟踪，并实现优异的成像对比度。