In vivo multicolor imaging is important for monitoring multiple biomolecular or cellular processes in biology. 19 F magnetic resonance imaging (MRI) is an emerging in vivo imaging technique because it can non‐invasively visualize 19 F nuclei without endogenous background signals. Therefore, 19 F MRI probes capable of multicolor imaging are in high demand. Herein, we report five types of perfluorocarbon‐encapsulated silica nanoparticles that show 19 F NMR peaks with different chemical shifts. Three of the nanoprobes, which show spectrally distinct 19 F NMR peaks with sufficient sensitivity, were selected for in vivo multicolor 19 F MRI. The nanoprobes exhibited 19 F MRI signals with three colors in a living mouse. Our in vivo multicolor system could be utilized for evaluating the effect of surface functional groups on the hepatic uptake in a mouse. This novel multicolor imaging technology will be a practical tool for elucidating in vivo biomolecular networks by 19 F MRI.

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsiveF MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged byF MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Tandemly-repeated peptide design enhanced enzyme accessibility and cleavage efficiency of substrates on the nanoparticle surface, resulting in high MRI signal amplification.