Multi-dimensional decoding of the TSPO signal in animal models of neuroinflammation using fluorescence-conjugated DPA714

Neuroinflammation is a core pathological process in multiple central nervous system diseases. Although positron emission tomography (PET) targeting the translocator protein (TSPO) (e.g., ¹⁸F-DPA-714) is widely used for in vivo inflammatory imaging, the cellular heterogeneity and functional states underlying its imaging signals remain unclear, severely limiting its mechanistic research and clinical translational value. This study used the multifunctional fluorescent TSPO probe (Cy5-PEG3-DPA714) to establish a multidimensional decoding framework that enables systematic analysis of the TSPO signal in neuroinflammation across in vivo, tissue, and single-cell levels. Multiple animal models demonstrated the probe’s ability to dynamically monitor acute and chronic neuroinflammation noninvasively in vivo. Histopathology confirmed that the TSPO signal mainly comes from activated Iba1 + microglia. Using the probe’s fluorescence properties, we effectively isolated specific microglial subpopulations with distinct TSPO signaling profiles via flow cytometry. Single-cell analysis revealed that the TSPO signal is specifically enriched in a microglia subpopulation with TSPO probe high . Importantly, this subpopulation shares features with disease-associated microglia (DAM), exhibiting a robust pro-inflammatory phenotype and significant metabolic reprogramming. Furthermore, we found that TSPO probe high microglia are highly dependent on the Colony Stimulating Factor 1 Receptor (CSF1R) signal pathway, and selective elimination using the CSF1R inhibitor (PLX3397) reduces neuroinflammation. This study systematically decodes the TSPO signal in neuroinflammation through a multimodal approach, providing novel insights and tools for precision therapeutic strategies targeting specific pathological microglia subpopulations.