**5. Conclusions**

Temperature is an important parameter influencing physical, chemical and biological processes: For this reason, the investigation of new materials, with enhanced performances, together with the definition of the more performing experimental protocol is a hot topic in the nanothermometry field. The experimental work reported in this article will contribute to the development of a new Raman based biocompatible nanothermometer, by investigating the optical performances of titanium dioxide, as anatase, with Raman technique.

The spectroscopic characterization of titanium dioxide has been carried out in the visible range, at 488.0, 514.5, 568.2 and 647.1 nm, and the Raman-active modes have been investigated to find the more performing one, as temperature sensor. Both Stokes and anti-Stokes spectra were collected at different temperature, input power and wavelengths, to investigate the temperature range, the temperature resolution, the eventual self-heating (due to the input laser power) of the sample and to identify the working range of the nanothermometer. A key point for the identification of the local temperature is the calibration of the experimental set-up, which allows defining the best experimental protocol. The calibration procedure has been conducted by controlling the sample temperature with a temperature-controlled stage and exploring the Raman signals in the temperature range of 283–323 K (with 5 K increment), as it is of interest for biological applications. The obtained values of the anti-Stokes/Stokes ratio allow the determination of the calibration constant, specified for all anatase Raman modes at each excitation wavelength. The calibration constant permits to determine the sample local temperature and to identify the power range where the local temperature is not affected by the laser power. Working with an incident laser power higher than 2 mW the sample experiences self-heating, while at lower power samples do not experience any self-heating. The validation of the proposed protocol has been finally achieved with the analysis of the Raman spectra of the *Test Sample*. Repeated anti-Stokes and Stokes measurements, have been performed on various positions of the sample at room temperature (~297 K), with an incident laser power of 1.5 mW. An excellent agreemen<sup>t</sup> between the temperature derived from the anti-Stokes/Stokes ratios and the expected temperature was found, with a standard deviation of repeated temperature measurements calculated to be in between 1 and 3 K, for the most intense peak, located at 143 cm<sup>−</sup>1, which has been demonstrated to be the most sensitive to temperature. This titanium dioxide mode seems to be an excellent candidate for the local temperature detection in the visible range from 488.0 to 647.1 nm, reaching the highest sensitivity in the red region.

**Author Contributions:** Conceptualization, D.P. and R.S.; Methodology, R.P., V.Z., D.P., R.S.; Software, V.Z., R.P.; Validation, V.Z., D.P., R.S. and R.P.; Formal analysis, V.Z.; Investigation, V.Z.; Resources, R.S. and R.P.; Data curation, V.Z.; Writing—original draft preparation, V.Z. and R.S.; Writing—review and editing, V.Z., D.P., R.P. and Signorini R; Supervision, R.S. and D.P.; Project administration, R.S.; Funding acquisition, R.S. and R.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Chemical Science Department of University od Padova, project P-DiSC#10BIRD2019-UNIPD.

**Conflicts of Interest:** The authors declare no conflict of interest.
