Ultra-short carbon nanotubes: near-infrared nanolabels for single molecule tracking in live brain tissues – UshortNT

Carbon nanotubes have unique optical properties for single molecule microscopy. We propose to develop preparation and bio-functionalization strategies of ultrashort carbon nanotubes, and to develop near infrared microscopy techniques for studying the spatio-temporal organization of neurotransmitter receptors in neurons.

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We studied the luminescence properties of empty and water filled carbon nanotubes at the single tube level and showed that the water filling substantially affects their luminescence properties (published in ACS Nano in 2012).

The ultimate sensitivity in imaging biological phenomenon in live cells is achieved by single-molecule detection, generally based on luminescence. Single-molecule methods have changed the way we think and carry out experiments on complex molecular systems by providing access to distributions of molecular properties which is of importance in biological systems that display static or time-dependent heterogeneity. Single-molecule detection also provides the possibility to determine molecule positions with nanometer accuracies, far below the optical resolution. The current luminescence-based single molecule techniques however suffer major limitations: short live times of probes (fluorescent molecules), large probe size (nanoparticles) but also the difficulty to detect the 3D trajectories of single molecules as well as the inability to detect single molecules in thick living samples where the interference from cellular autofluorescence is severe.
In this context, the need for small nano-labels with red-shifted optical responses appeared crucial for further applications single molecule methods to tissues.
A class of nanomaterials displays strong optical resonances in the nearIR: single wall carbon nanotubes (SWNTs). SWNTs are characterized by a high ratio of length (hundreds of nanometers up to millimeters) over diameter (~1nm). The discovery of their strong fluorescence in the nearIR opened the possibility to directly detect them at the single molecule level. They also bear remarkable absorption properties and could be detected at the single molecule level by a photothermal method that we recently developed. Their cell penetrating properties raised recently great hopes for applications in drug delivery. To serve as nanoprobes, standard SWNTs are nevertheless not readily applicable due to their long 1D dimension (>100nm) despite their unique optical properties. Interestingly, ultrashort (<10nm) SWNTs (UshortNTs) have been recently processed and their ensemble luminescence in the nearIR has been observed. We strongly believe that UshortNTs constitute promising nearIR nanolabels for 3D single molecule detection in tissues providing that specific microscopy methods and nanotube chemistry are now developed.
In this project, we propose to develop new 3D single molecule tracking schemes of UshortNTs (which will be based on luminescence and photothermal detection), to bio-functionalize them and to apply them to study the spatio-temporal organization of neurotransmitters receptors in brain slices.
Indeed, an important application of single molecule microscopy concerns the study of neurotransmitter receptors trafficking between sub-cellular compartments which play a key role in neuronal physiology, e.g. during synaptic plasticity. Over the last years, we have demonstrated the invaluable power of single molecule detection for understanding the complexity and key role of receptor diffusion processes in and out of the synapses of neurons. However, since “standard” single molecule methods still suffer from the previously mentioned limitations, current approaches are inappropriate for recording accurately the full 3D movements of a single receptor, especially in brain slices. This is a strong limitation because fluxes of receptors in and out synapses not only involve lateral diffusion in the plane of the membrane, but also endo/exocytosis and intracellular trafficking. As well, little is known about receptor dynamics in intact neuronal networks constituted e.g. by brain slices. The main biological outcome of this project will thus to perform such studies. More generally, this will constitute the first single molecule study in thick living tissues and this new approach should be transposable to many important biological questions. The cytotoxicity of the UshortNTs will also be studied in this project.