Fluorescence nanothermometers—widely used from microelectronics to cell biology—face a critical yet often overlooked challenge: both their calibration and actual temperature readout can be significantly distorted by laser-induced heating on substrates with poor thermal conductivity or surface contamination. Here, we report a systematic investigation of how substrate thermal conductivity and interfacial polymer layers influence laser-induced heating of silicon-vacancy (SiV−) diamonds used as fluorescence-based secondary thermometers. Using substrates spanning several orders of magnitude in thermal conductivity and different thickness polymer layers between the sensor and the surface, we quantify the temperature rise of the diamonds relative to room temperature. Results reveal that under identical excitation conditions, bulk diamond—the highest-conductivity substrate exhibits only a negligible temperature rise (ΔT ≈ 0.95 °C), whereas thin amorphous holey carbon—the lowest-conductivity substrate studied induces an extreme increase of ΔT ∼530 °C. Furthermore, the presence of an interfacial polymer layer leads to a substantial temperature rise of ∼60 °C, in stark contrast to the near-zero heating observed on clean substrates (ΔT ≈ 0.02 °C). Experimental findings are further validated using COMSOL Multiphysics simulations with a steady-state 3D heat transfer model. Our work provides practical guidelines for substrate selection, surface preparation, and calibration methodology for nanoscale thermometry.
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