Thrombosis is a frequent predisposing factor in high‐mortality cardiovascular diseases, which underscores the urgent need to precisely diagnose thrombosis formation at a less severe stage. However, it is still challenging to realize in vivo early thrombosis imaging due to lack of high sensitivity/‐specificity molecular imaging strategy. To address such problems, thrombin‐activatable scintillating nanoprobes are developed for background‐free X‐ray‐excited luminescence (XEL) imaging of in vivo early thrombosis. The nanoprobes are constructed by designing bright XEL‐emitting lanthanide‐doped scintillator nanocrystals (NCs) with dye labeled‐peptide modification. Such nanoprobes show XEL‐off originally and enable robust turn‐on XEL upon thrombin‐specific cleavage of the peptide, resulting from the rational manipulation of energy transfer between dye and NCs. Specially, due to high tissue penetration depth and background‐free attributes in XEL imaging, this strategy achieves high‐efficiency XEL imaging of the early thrombosis on the basis of in situ elevated thrombin levels. Moreover, XEL and magnetic resonance imaging capabilities can be integrated to further improve imaging accuracy and monitor thrombosis progression. As such, the exploited strategy demonstrates a novel paradigm for realizing timely thrombosis imaging and provides a new readily tailorable platform for sensitive in vivo deep‐tissue imaging.

Autofluorescence background in complex biological samples is a major challenge in achieving high sensitivity of fluorescence immunoassays (FIA). Here we report an X-ray luminescence-based immunoassay for high-sensitivity detection of biomarkers using X-ray scintillating nanotags. Due to the weak scattering and absorption of most biological chromophores by X-ray excitation, a low-dose X-ray source can be used to produce intense scintillating luminescence from the nanotags for autofluorescence-free biosensing. To demonstrate this concept, we designed and synthesized NaGdF:Tb@NaYF core/shell nanoparticles as kind of high-efficiency X-ray scintillating nanotags, which are able to convert high-energy X-ray photons to visible light without autofluorescence in biological samples. Notably, strong X-ray absorption and minimized surface quenching arising from the heavy Gd/Tb atoms and core/shell architecture of the nanoparticles were found to be critically important for high-efficiency X-ray excited luminescence for high-sensitivity biosensing. Our method allows for sensing alpha-fetoprotein (AFP) biomarkers with a detection limit down to 0.25 ng/mL. Moreover, the as-described X-ray luminescence immunoassay exhibited an excellent biological specificity, high stability, and sample recovery, implying an opportunity for applications in complex biological samples. Consequently, our method can be readily extended for multiplexing sensing and medical diagnosis.

Cell temperature monitoring is of great importance to uncover temperature-dependent intracellular events and regulate cellular functions. However, it remains a great challenge to precisely probe the localized temperature status in living cells. Herein, we report a strategy for in situ temperature mapping on an immune cell membrane for the first time, which was achieved by using the lanthanide-doped upconversion nanoparticles. The nanothermometer was designed to label the cell membrane by combining metabolic labeling and click chemistry and can leverage ratiometric upconversion luminescence signals to in situ sensitively monitor temperature variation (1.4% K–1). Moreover, a purpose-built upconversion hyperspectral microscope was utilized to synchronously map temperature changes on T cell membrane and visualize intracellular Ca2+ influx. This strategy was able to identify a suitable temperature status for facilitating thermally stimulated calcium influx in T cells, thus enabling high-efficiency activation of immune cells. Such findings might advance understandings on thermally dependent biological processes and their regulation methodology.