Universal guidelines for the conversion of proteins and dyes into functional nanothermometers

Abstract

In the last decade, technological advances in chemistry and photonics have enabled real‐time measurement of temperature at the nanoscale. Nanothermometers, probes specifically designed to relay these nanoscale temperature changes, provide a high degree of temperature, temporal, and spatial resolution and precision. Several different approaches have been proposed, including microthermocouples, luminescence and fluorescence polarization anisotropy‐based nanothermometers. Anisotropy‐based nanothermometers excel in terms of biocompatibility because they can be built from endogenous proteins conjugated to dyes, minimizing any system perturbation. Moreover, the resulting fluorescent proteins can retain their native structure and activity while performing the temperature measurement, allowing precise temperature recordings from the native environment or during an enzymatic reaction in any given experimental system. To facilitate the future use of these nanothermometers in research, here we present a theoretical model that predicts the optimal sensitivity for anisotropy‐based thermometers starting with any protein or dye, based on protein size and dye fluorescence lifetime. Using this model, most proteins and dyes can be converted to nanothermometers. The utilization of these nanothermometers by a broad spectrum of disciplines within the scientific community will bring new knowledge and understanding that today remains unavailable with current techniques.

Real-Time Temperature Sensing Using a Ratiometric Dual Fluorescent Protein Biosensor

“…Theoretically, the uvGFP and mCherry coding sequences could easily be engineered into a synthetic operon or to produce a fusion protein in order to facilitate the topologically precise measurement of absolute temperature. The latter could enable the single wavelength excitation of both fluorophores as GFP and mCherry are a well characterized fluorescence lifetime imaging microscopy (FLIM) fluorescence resonance energy transfer (FRET) pair [ 34 , 35 ], and the fluorescence lifetime of uvGFP is also sufficiently high for use as a FLIM-FRET-based thermosensor [ 36 ]. While real-time thermocyclers are not capable of measuring FLIM-FRET, the conversion of the DFPTB into a fusion protein would simplify its expression and purification and enable new or improved applications.…”

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

“…However, the same thermal response is observed in all the cases: the FPA value decreases when the temperature increases, in agreement with previous studies. [42,[44][45][46]57] We calculated, for each nucleus, the slope of the evolution of FPA with tempera-…”

“…However, the same thermal response is observed in all the cases: the FPA value decreases when the temperature increases, in agreement with previous studies. [ 42,44–46,57 ] We calculated, for each nucleus, the slope of the evolution of FPA with temperature $\[\left( {\frac{{dFPA}}{{{\rm{d}}T}}} \right)\]$. Although the FPA value differs between cells, the mean FPA versus T slope is independent of the cell line (see the Supporting Information), with a value of −0.5 ± 0.1 × 10 −3 °C −1 .…”

Multichannel Fluorescence Microscopy: Advantages of Going beyond a Single Emission

“…[ 134 ] As for MRBFs (see Section 4.2), the fluorescent agent can be engineered to enhance its response to temperature and measure changes in this parameter through the variation in the sensor's mobility using FPA. [ 132 ] As Donner et al described, [ 130 ] the maximum sensitivity is found when the lifetime of the fluorescent probe is in the same range of its rotational lifetime. Utilizing the green fluorescent protein (GFP) as anisotropy‐based nanothermometers (ABNTs), they mapped the intracellular temperature of HeLa cancer cells.…”

“…It is directly related to the Brownian fluctuations and affects the viscosity. This can be exploited to develop FPA nanothermometers based on fluorescence molecules such as fluorescent proteins, [130,131] dyes, [132] proteins conjugated with dyes, [132,133] and DNA labeled with intercalated dyes. [134] As for MRBFs (see Section 4.2), the fluorescent agent can be engineered to enhance its response to temperature and measure changes in this parameter through the variation in the sensor's mobility using FPA.…”