All-optical fluorescence lifetime nanodiamond thermometry for intracellular photothermal hyperthermia

Nanodiamond-based thermometry provides a photostable and biocompatible platform for nanoscale temperature sensing in biological environments. Most nanodiamond thermometry approaches rely on continuous-wave optically detected magnetic resonance, which requires microwave delivery and specialized instrumentation. Here, we demonstrate an all-optical alternative based on the temperature dependence of the fluorescence lifetime of nitrogen-vacancy centers in 100 nm nanodiamonds. To evaluate the performance of fluorescence lifetime-based nanodiamond thermometry under biologically relevant conditions, we investigate intracellular temperature dynamics during near-infrared (808 nm) optical hyperthermia. Custom-fabricated gold nanorods encapsulated in mesoporous silica and functionalized with Rhodamine B serve as localized plasmonic heaters and reference thermometers. Fluorescence Lifetime Imaging Microscopy (FLIM) enables simultaneous, spatially resolved comparison between heater-coupled RhB fluorophores and freely distributed nanodiamond probes within the same cellular environment. Nanodiamonds measured intracellular temperature increases up to +24 °C, in close agreement with the +27 °C rise reported by RhB-based sensors. Real-time FLIM measurements further demonstrate the ability of nanodiamonds to track dynamic temperature evolution during prolonged irradiation. While absolute temperature values differ due to spatial separation between heater and probe, both sensors exhibit consistent relative trends. These results establish fluorescence lifetime-based nanodiamond thermometry as a practical, microwave-free approach for monitoring intracellular thermal processes, providing a complementary strategy for nanoscale temperature sensing during photothermal therapy.