The scientific work carried out during the "NanoHeadTail" action can be divided into two main parts:
1) The functionalization of SWCNTs with CC and the characterization of the functionalized SWCNTs.
The initial phase of the project was focused on mastering the functionalization reaction of SWCNTs with CCs. To this end, SWCNTs were functionalized with p-nitro aryl groups (-C6H5NO2) in aqueous environment and using light as a reaction catalyst. Extensive work was carried out to optimize both the reaction and optical imaging conditions by adapting various parameters such as light excitation wavelength, nanotube and surfactant coating type. In our experiments, we used (6,5) purified SWCNTs coated with sodium dodecylbenzenesulfonate (SDBS). We then designed an experiment to study the functionalization reaction in situ on each individual SWCNT. For that, the SWCNTs were immobilized on a glass surface using poly-l-lysine and along with gold nanoparticles aggregates which act as fiducials necessary to correct drift correction during the measurement. A new microscope was built able to record simultaneously the SWCNT and CC emission using a two cameras system. To localize where the CCs form on the SWCNTs, it was first necessary to resolve the nanotube structure. To this end, we compared the performances of 3 analytical super resolution algorithms for the accurate determination of the SWCNT structure by comparing the results obtained along with the real SWCNT length, structure and orientation obtained from AFM and SWCNT emission polarization. We found that the deblurring by pixel reassignment (DPR) algorithm was the most accurate for that purpose. The localization of the CCs was determined by analyzing the variations in intensity as a function of time in the region centered around the SWCNT. The localizations of the CCs were then reconstructed altogether and analyzed with respect to the SWCNT structure. This work resulted in several breakthroughs. First, the analysis workflow we developed to compare the performances of analytical super resolution algorithms can be used in combination with the images resolved by DPR to readily determine the length distribution in a SWCNT sample with similar accuracy as AFM but with much higher throughput. These findings led to the publication of an article in a high-impact journal. In addition, this project is also the first visualization of the CC formation in situ on carbon nanotubes and at the single molecule level. Preliminary results indicate a stronger than expected inhomogeneity in the CC functionalization. These advances will soon result in the submission of another article. The results associated to both aforementioned subjects have also been respectively presented in two oral talks at the 2024 ECS conference (San Francisco, CA, US).
2) The analysis of SWCNT diffusion in 3D and in biological tissues.
One advantage of the CC functionalization is the possibility to trap excitons where CCs are located, thereby making it possible to shorten SWCNTs to lengths below 100 nm, resulting in the creation of luminescent ultrashort nanotubes which could gain access to more restrained parts of the biological tissue. CCs can also be used as an efficient way to brighten SWCNT emission by trapping excitons which would otherwise diffuse to quenching sites on the nanotube surface. In this work package, we compared the performances of ultrashort SWCNTs functionalized with two different CC types, aryl and oxygen groups, for single particle tracking in brain tissue. We observed that the SWCNTs functionalized with oxygen CCs were brighter than their aryl counterparts, both when considering ultrashort and longer nanotubes. Interestingly, we also observed that the uCCNTs were considered too bright compared to what was expected, regardless of the CC type. The intensity of SWCNT, and therefore CC, emission is proportional to the nanotube length. However, in our experiments, even though the uCCNTs were on average 10 times shorter than their longer counterparts, they were only 1.5 times dimmer. We explained this discrepancy by the presence of bright and dim parts along the functionalized SWCNTs, which were confirmed by super resolution of the CC localizations. These dim parts therefore limit the QY of the functionalized SWCNTs to values close to 2%. Upon cutting, the bright and dim parts are separated into different ultrashort fragments. The bright fragments, the only ones detected in the uCCNT samples, are thus devoid of any quenched sections and therefore exhibit much higher QYs up to 20% for the ox-uCCNTs. Because of this high brightness, the NIR emission of uCCNTs could be detected up to 100 um deep through brain tissue while maintaining sufficient photon count to allow super localization with precisions reaching ca. 30 nm. In contrast, using the same configuration, quantum dots (QDs) considered as the state-of-the-art for single particle tracking applications could not be detected at such depths due to their emission in the visible range. Finally, we showcased the capabilities of the ox-uCCNTs by performing 3D single particle tracking in brain tissue (organotypic slice). To this end, we built a microscopy setup capable of tracking the diffusion of CC-functionalized SWCNTs in 3D using double helix point spread function (PSF) engineering. The trajectories of the uCCNTs revealed the presence of hollow cylindrical structures in the brain ECS, which could not be detected in the previous single particle tracking work performed in 2D. These achievements resulted in one scientific publication and were presented in an oral talk at the 2024 ECS conference (San Francisco, CA, US).
