# Thermometric Characterization of Fluorescent Nanodiamonds Suitable for Biomedical Applications
## 2 Materials and Methods
### Fluorescent Thermometry  
The fluorescence spectra of 35 nm and 70 nm-sized nanodiamonds were recorded at different temperatures using a laser scanning confocal microscope Olympus BX41 (Japan) equipped an InGaAs detector using a 10x long working distance microscope objective with a NA 0.25 and an OBIS 532 nm laser with 20 mW of continuous power as the optical excitation. The laser was directed through a series of mirrors into the microscope system. Then, the laser was focused on the sample, the detector collected the signal, and then processed with Horiba Scientifics LabSpec software. Once these colour centres were well-localised and the optimal microscope parameters established for a maximised output signal, fluorescence spectra were recorded at different temperatures within the physiological range of 25 \({}^{\circ}\)C to 60 \({}^{\circ}\)C with the help of a heating plate in steps of 1 \({}^{\circ}\)C and 20 min of thermal equilibrium time. Fluorescence spectra were recorded using a detection range from 550 nm to 850 nm in steps of 1 nm with a 2 nm wide slit and 0.1 s of integration time.  
The fluorescence spectra of 100 nm-sized nanodiamonds were recorded at different temperatures using a Horiba Scientifics Fluorologo\({}^{\circledR}\)-3 FL3-22 (Japan) equipped with a photomultiplier tube Hamamatsu R928P and an OBIS 532 nm laser with 20 mW of continuous power as optical excitation. The temperature control was achieved with the F-3030 temperature bath and the FL-1027 Single-Position Thermostatted Cell Holder accessories from Horiba Scientifics. The software FluorEssense (Horiba Scientifics, Japan) was set for an emission scan from 25 \({}^{\circ}\)C to 60 \({}^{\circ}\)C in steps of 1 \({}^{\circ}\)C with a tolerance of 0.1 \({}^{\circ}\)C and 20 min of thermal equilibrium time. The fluorescence spectra were recorded using a detection range from 550 nm to 850 nm in steps of 1 nm with a 2 nm wide slit and 0.1 s of integration time.  
The software Origin (OriginLab, Northampton, MA, USA) was used to obtain peak information of the NV colour centres' zero phonon lines characteristics (NV\({}^{0}\), 575 nm and NV\({}^{-}\), 637 nm) in the form of intensity, full width at half maximum and position through a Gaussian fit. First, all the spectra were normalized from 0 to 1. Then, they had their baseline adjusted to include the phonon sidebands below ZPL, similarly to the background subtraction performed by [PERSON] et al. [44]. Finally, they had their NV centre peaks fit with a Gaussian curve. A linear fit was calculated for the thermal evolution of the NV centres' fluorescence, yielding very accurate results regarding their R\({}^{2}\) quality of fit value. Relative sensitivities were calculated according to the following equation:  
\[\text{S}_{\text{Q}}=(1/\text{Q})(\text{dQ}/\text{dT}) \tag{1}\]  
where S\({}_{\text{Q}}\) represents the sensibility, Q is the NV centre's fluorescence and T is the temperature.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Part Number** & **Concentration** & **NV Centres/Particle** & **Size** \\ \hline
900172-5 mL & 1 mg/mL & \(\leq\)4 & 35 nm \\
798169-5 mL & 1 mg/mL & \(>\)300 & 70 nm \\
900174-5 mL & 1 mg/mL & \(>\)900 & 100 nm \\ \hline \end{tabular}
\end{table}
Table 1: Sigma Aldrich’s nanodiamonds specifications.