Bias in Intracellular Luminescence Thermometry: The Case of the Green Fluorescent Protein

Abstract

Measurement of intracellular temperature in a fast, accurate, reliable, and remote manner is crucial for the understanding of cellular processes. Nanothermometers based on the green fluorescence protein (GFP) are of special interest because intracellular temperature readouts can be obtained from the analysis of the polarization state of its luminescence. Despite the good results provided by GFP thermometers, the reliability of their intracellular thermal readouts is still a question of debate. Here, light is shed on this issue by introducing cell activity as a relevant bias mechanism that prevents the use of GFP for reliable intranuclear thermal measurements. Experimental evidence that this lack of reliability can affect not only GFP but also other widely used thermometers such as semiconductor nanocrystals is provided. It is discussed how differences observed between calibration curves obtained in presence and absence of cell activity can inform about the presence of bias. The presented results and discussion are aimed to warn the community working in intracellular thermometry and encourage authors to approach the issue in a conscious manner. The performance and reliability of the chosen intracellular thermometers must be judiciously assessed. This is the only way intracellular thermometry can progress and deliver indisputable results.

Spotlight on Luminescence Thermometry: Basics, Challenges, and Cutting‐Edge Applications

“…[55,321,322] One example is polarization-based intracellular thermometers in which the output signal has been recently discovered to depend on temperature as well as on the degree of cellular activity, precluding reliable thermal monitoring. [323] Yet, bias is not a complication exclusive to the field of biology. For example, luminescent thermometers have been also used to monitor thermal dynamics in micro and nanoelectronics, [108,191,324,325] whereby temperature changes are triggered by changes in the electric field.…”

“…[ 55,321,322 ] One example is polarization‐based intracellular thermometers in which the output signal has been recently discovered to depend on temperature as well as on the degree of cellular activity, precluding reliable thermal monitoring. [ 323 ]…”

Hybrid multifunctionalized mesostructured stellate silica nanoparticles loaded with β-diketonate Tb3+/Eu3+ complexes as efficient ratiometric emissive thermometers working in water

“…A. Thompson clearly demonstrated differences in the calibration curves obtained under different environmental conditions or in the absence and presence of cell activity for two well-established nanothermometers, such as GFP and QDs and encouraged the community working in thermometry to approach the issue in a conscious manner. 27 In different environments the correlation can follow a different parametrization. Absorption, autofluorescence, and diffusion among other properties specific to each system are indeed expected to affect the luminescent probe in a different manner.…”

“…Note also that an effort has been made for remote and local temperature measurements in living cells, [24][25][26] while the reliability of their intracellular thermal readout is still a question of debate. 27 Ln 3+ -based complexes have demonstrated their excellence as luminescence thermometry candidates. They exhibit unique photophysical properties, including long-lived emissions with lifetimes reaching the millisecond scale, specifically in the visible and/or infrared spectral regions.…”

“…Note also that an effort has been made for remote and local temperature measurements in living cells, 24–26 while the reliability of their intracellular thermal readout is still a question of debate. 27…”