Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

Various small molecules have been used as functional probes for tis-sue imaging in medical diagnosis and pharmaceutical drugs for dis-ease treatment. The spatial distribution, target selectivity, and diffu-sion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for pre-cisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed "fixation-driven chemical cross-linking of exogenous ligands (FixEL),"which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribu-tion of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffu-sion/retention kinetics of MOIs such as the scaffold of PET tracer de-rivatives or drug-like small molecules.