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<div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"><a class="named-anchor" id="d18399804e171"><!-- named anchor --></a><h5 class="section-title" id="d18399804e172">Background:</h5><p dir="auto" id="d18399804e174">Bone marrow mesenchymal stem cell (BMSC) therapy holds great promise for regenerative medicine, but its clinical translation is hindered by the lack of non-invasive, real-time methods to track transplanted cell fate <i>in vivo</i>. Although photoacoustic (PA) imaging offers deep-tissue penetration and high sensitivity, existing cell-labeling strategies relying on endocytosis of contrast agents have drawbacks of prolonged incubation times and variable efficiency, thus potentially compromising cell viability and function.</p></div><div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"><a class="named-anchor" id="d18399804e179"><!-- named anchor --></a><h5 class="section-title" id="d18399804e180">Methods:</h5><p dir="auto" id="d18399804e182">To address these limitations, we developed a rapid, bioorthogonal pre-labeling strategy. Azide (N₃) groups were first metabolically engineered onto BMSC surfaces. Subsequently, dibenzocyclooctyne (DBCO)-functionalized gold nanostars (Au-star-DBCO) were conjugated to the cells via a highly efficient, copper-free click reaction, thereby enabling covalent membrane anchoring.</p></div><div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"><a class="named-anchor" id="d18399804e187"><!-- named anchor --></a><h5 class="section-title" id="d18399804e188">Results:</h5><p dir="auto" id="d18399804e190">This approach achieved rapid and specific stem cell labeling within 3 h, with an efficiency of 83.1% ± 0.67. The labeling process did not impair BMSC viability or multilineage differentiation potential. The Au-star-DBCO-labeled BMSCs generated strong, concentration-dependent PA signals both <i>in vitro</i> and in a rat subcutaneous model, and enabled dynamic monitoring for at least 72 h post-transplantation. The specificity of the bioorthogonal reaction provided a significantly higher signal-to-noise ratio than passive uptake methods.</p></div><div xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="section"><a class="named-anchor" id="d18399804e195"><!-- named anchor --></a><h5 class="section-title" id="d18399804e196">Conclusions:</h5><p dir="auto" id="d18399804e198">We successfully established a biocompatible and efficient platform for stem cell tracking by integrating metabolic glycoengineering, bioorthogonal chemistry, and nanostar-enhanced PA imaging. This strategy overcomes key limitations of traditional endocytosis-based labeling, and offers a rapid, specific, and functional cell-compatible tool for sensitive, longitudinal <i>in vivo</i> monitoring. This platform may advance precise assessment of cell therapies and facilitate their clinical application.</p></div>

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