Multiphoton microscopy is being used as a tool for tracking nanoparticles and assessing endogenous autofluorescent molecules. This technology is founded on femtosecond lasers that are capable of exciting unlabeled metal nanoparticles, quantum dots, and upconverting nanoparticles. The addition of time-correlated single-photon counting detectors enables fluorescence lifetime imaging. Fluorescence lifetime measurements result in high-resolution, quantitative data that can be used to distinguish nanoparticle signals from those of endogenous fluorophores. This application of multiphoton microscopy is capable of simultaneous nanoparticle and NAD(P)H imaging, resulting in the capacity for dye-free assessments of treatment-induced metabolic changes. The stage is set for advanced, clinical imaging focused nanoparticle trials that can directly address critical issues in nanomedicine and nanotoxicology.
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