Periodic Reporting for period 1 - NanoHeadTail (Head to tail imaging of fluorescent carbon nanotubes for the characterization of the brain extracellular space)
Período documentado: 2023-09-01 hasta 2025-08-31
In the "NanoHeadTail" project, we developed a new class of nanoprobes and combined it with state-of-the-art fluorescence microscopy to study the morphological and rheological properties of the brain extracellular space (ECS) at the nanoscale. Studying the ECS is critical as it is linked to a number of brain mechanisms, including some pathologies such as Parkinson’s disease. Previous studies have provided insights on the characteristics of the ECS but measured 2D trajectories and focused on the analysis of the translational diffusion properties of the probes. To overcome these limitations, we proposed to investigate the ECS using a new asymmetric nanoprobe based on the bi-functionalization of SWCNTs (bf-SWCNTs) with two fluorescent color centers (CCs) located at each end of the SWCNT (the “head” and the “tail”) and emitting at two distinct wavelengths. A custom-made microscope was envisioned to track the diffusion of individual bf-SWCNTs in 3D, thus allowing to to determine the characteristics of the ECS volume locally.
To this end, the project followed two main scientific directions:
1) The functionalization of SWCNTs with CC and the characterization of the functionalized SWCNTs.
The functionalization of carbon nanotubes with two different CCs necessitated a precise control of both the density of CCs and on the locations where CC form on the nanotube to avoid the creation of too many unwanted byproducts. Because, the CC functionalization reaction was poorly understood at the single nanotube level in the field and mostly viewed as stochastic, we dedicated the first part of this project to study the formation of CCs on individual SWCNTs. For that, an experiment has been designed to perform the functionalization reaction in situ while simultaneously resolving both the nanotube structure and the position of each CC forming in real time. This work lays the foundation of many future avenues for the functionalization of SWCNTs with CC, such as the bi-functionalization of SWCNTs with two CC types.
2) The analysis of SWCNT diffusion in 3D and in biological tissues.
A custom super resolution microscopy setup was built for the tracking of the functionalized SWCNTs in 3D using a double helix phase mask. The tracking was performed on nanotubes functionalised with either aryl and oxygen CCs demonstrating that bi-functionalization is in principle possible. To improve even further the diffusion of SWCNTs in tissues, we tracked ultrashort functionalized nanotubes exhibiting lengths smaller by one order of magnitude compared to conventional nanotube probes. We performed the first demonstration of 3D single particle tracking of these ultrashort SWCNTs in brain organotypic slice.
To this end, the project followed two main scientific directions:
1) The functionalization of SWCNTs with CC and the characterization of the functionalized SWCNTs.
The functionalization of carbon nanotubes with two different CCs necessitated a precise control of both the density of CCs and on the locations where CC form on the nanotube to avoid the creation of too many unwanted byproducts. Because, the CC functionalization reaction was poorly understood at the single nanotube level in the field and mostly viewed as stochastic, we dedicated the first part of this project to study the formation of CCs on individual SWCNTs. For that, an experiment has been designed to perform the functionalization reaction in situ while simultaneously resolving both the nanotube structure and the position of each CC forming in real time. This work lays the foundation of many future avenues for the functionalization of SWCNTs with CC, such as the bi-functionalization of SWCNTs with two CC types.
2) The analysis of SWCNT diffusion in 3D and in biological tissues.
A custom super resolution microscopy setup was built for the tracking of the functionalized SWCNTs in 3D using a double helix phase mask. The tracking was performed on nanotubes functionalised with either aryl and oxygen CCs demonstrating that bi-functionalization is in principle possible. To improve even further the diffusion of SWCNTs in tissues, we tracked ultrashort functionalized nanotubes exhibiting lengths smaller by one order of magnitude compared to conventional nanotube probes. We performed the first demonstration of 3D single particle tracking of these ultrashort SWCNTs in brain organotypic slice.
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).
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).
The outputs of the “NanoHeadTail” action are expected to have a strong scientific impact. The work carried out on the super resolution of the SWCNT structure, on the fluorescence properties of ultrashort nanotubes functionalized with CCs, and more importantly on the understanding of the CC functionalization reaction are particularly hot topics in the carbon nanotube community. The tools developed for the super resolution of SWCNTs can be used to resolve other types of systems at the nanoscale and could therefore set a new standard for the characterization of nanoparticles thanks to the improved throughput. Moreover, our understanding on how shortening functionalized SWCNTs can help reaching unrivalled QY values and challenge state-of-the-art probes such as QDs overcomes a bottleneck that was limiting the applications of carbon nanotubes as biological nanoprobes. Furthermore, being able to control the way SWCNTs are functionalized with CCs can also have important implications in material sciences to enable the creation of quantum light sources at the telecom wavelengths for quantum communications, which is currently a challenge. In addition, the results obtained on the 3D single particle tracking of functionalized ultrashort SWCNTs in the brain ECS are envisioned to have an important influence on the way biological systems can be characterized. Indeed, we showed the possibility to image and measure the rheological properties of the biological environment in 3D and with nanometric precision at depths. This could allow a better understanding on how these environments are organized, how they may change when affected by a pathology and how the diffusion of a drug to the regions of interest would be affected. While mostly fundamental, the advances made throughout the project have the potential for industrial applications (e.g. in material science) which can thus lead to a significant societal impact, both within and outside of Europe. The research carried out during the fellowship can also help improving the visibility of nanotechnologies and more particularly carbon nanotubes to the broad audience and to show the benefits these can bring to society. Finally, the resources shared throughout the project, analysis workflows and experimental methodologies, already start to be used by other laboratories and are therefore expected to have a significant impact in academia.