Ultrasensitive Triple-Mode Nile Red-L64 Niosome Nanothermometers for Real-Time Cellular Thermogenesis Mapping

Abstract

Temperature dynamics in living cells provide critical insights into metabolic processes and cellular energy pathways. However, precise intracellular thermometry to measure minimal temperature fluctuations, typically 0.1°C -0.2°C change in the human physiological temperature range of 37°C -39°C, remains challenging due to the 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 single and two-photon fluorescence intensities and lifetime of NR-L64. The thermoresponsive niosomes undergo reversible phase transitions that modulate NR's emission, achieving exceptional relative sensitivity of 107 °C⁻¹ with astounding temperature resolution below 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°C -39°C. The nanothermometers are successful to monitor the real-time glucose-induced thermogenesis in live FaDu cells, by detecting 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.