All-optical intracellular thermal profiling using nanodiamond-based "thermal radar"

Abstract

The local thermal conductivity (\k{appa}) is a pivotal biophysical parameter, governing intracellular heat flux and underlying functional processes like metabolic regulation and stress response. However, label-free mapping with sub-micron resolution in living cells remains challenge. Here, we present frequency-domain fluorescence thermometry (FD-FTM), an all-optical method based on a hybrid nanodiamond-on-gold-membrane platform, which enables quantitative mapping of \k{appa} in biological systems. Fluorescence nanodiamonds (FNDs) are deposited on substrates coated with a 50 nm gold membrane, where FNDs function as nanoscale thermometers, and the gold membrane serves as a photothermal heat source. We validate FD-FTM across reference materials and biological media, with fitting uncertainties of ~10%. By varying the modulation frequency, we tune the thermal penetration depths, enabling controlled heat propagation from the substrate to the cell nucleus. The method delivers sensitivity sufficient to resolve changes in biofluid thermal conductivity on the order of 16% relative to water. Using these capabilities, we demonstrate non-invasive thermal profiling across scales: at the cellular level, nuclear chromatin packing yields \k{appa} higher by ~10% relative to the cytoplasm; at the organelle level, we resolve \k{appa} variations associated with protein aggregates formed during liquid-liquid phase separation in an amyotrophic lateral sclerosis disease model. Temporal measurements in living cells over 30 minutes further reveal spatially resolved intracellular responses to osmotic stress, linking nanoscale thermal dynamics to biomolecular condensates. These results establish FD-FTM as a label-free, robust, and quantitative platform for thermally decoding intracellular processes, opening avenues for studying metabolic heterogeneity, disease mechanisms, and therapeutic responses.