The temperature distribution within cells, especially the debate on "how hot are mitochondria?", has recently attracted widespread attention. Several studies have reported that mitochondrial temperature may be 10-15 degrees C higher than the ambient temperature, which poses a significant challenge to the understanding of cellular thermal signaling and metabolic regulation mechanisms. Yet, other studies have raised concerns regarding the origin of the measurement signal. To address this controversy, we evaluated the impact of the intracellular environment on mitochondrial temperature measurement and found that microenvironmental factors such as viscosity can significantly interfere with the accuracy of traditional one-dimensional response curves, leading to temperature reading deviations. Here, we propose a universal multidimensional calibration framework that eliminates confounding factors by leveraging an information-matrix-based correction, without any modification to the probe material, enabling high-precision temperature reconstruction across complex environments. We investigated the dynamic temperature changes in different organelles within the cell under stimulation and observed a distinct temperature gradient within the cell. In our experiments, no mitochondrial temperature exceeding 50 degrees C, and the upper range was approximately 42-43 degrees C, which is consistent with the inactivation temperature of enzymes. Our results help to reassess the underlying logic of metrology and promote a better understanding on cellular thermodynamics.
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