Ultrasensitive triple-mode Nile red-L64 niosome nanothermometers for real-time cellular thermogenesis mapping

Temperature dynamics in living cells provide critical insights into metabolic processes and cellular energy pathways. However, precise intracellular thermometry to measure minute temperature fluctuations, typically 0.1-0.2 °C changes within the human physiological temperature range of 37-39 °C, remains challenging due to interference from cellular components. We report a triple-mode fluorescence imaging nanothermometer, using Nile red (NR)-loaded thermoresponsive L64 niosomes (NR-L64), enabling ultrasensitive, intracellular thermal mapping via measuring the single- and two-photon fluorescence intensities and lifetime of NR-L64. The thermoresponsive niosomes undergo reversible phase transitions that modulate NR's emission, achieving an exceptional relative sensitivity of 107 °C^-1 with astounding temperature resolution of less than 0.001 °C, under two-photon fluorescence imaging. NR-L64 demonstrates remarkable photostability for over an hour and maintains a linear thermal response in the range of 37-39 °C. The nanothermometers are successful in monitoring real-time glucose-induced thermogenesis in live FaDu cells, by detecting a sequential temperature increase of 0.2 °C with subcellular resolution using confocal and two-photon fluorescence microscopy. Cross-validation between intensity and lifetime imaging confirms reliable thermal quantification across the linear range. These findings establish NR-L64 niosomes as versatile, minimally invasive tools for real-time cellular thermometry with broad applications in metabolic studies and precision therapeutic monitoring.