Ultra-structural imaging in cleared mouse cns using nanoparticles

Recent advances in tissue clearing methods such as CLARITY,
whereby whole organs become optically transparent and permeable to macromolecules, have allowed us to visualise structural and functional relationships at the cellular level using traditional fluorescent antibodies, without the necessity of multiple slicing, staining, imaging and subsequent 3D digital reconstruction. Although these methods have allowed us to visualise with higher resolution smaller, non-neuronal structures of the mouse brain, photo-bleaching and limited range of available emission wavelength of organic-dye fluorophores, has limited our ability to multiplex using traditional immuno-fluorescent techniques. Functionalized nanoparticles such as Nanodiamonds, Nanorubies and SuperDotsTM are specialised inorganic particles (with tunable monodisperse sizes, such as 10 nm, 15 nm, 20 nm, up to a few microns) coated with a bio-molecule such as protein, that can then bind to specific targets within tissue and be visualised using scanning microscopy or fibre optics. The benefit of these nanoparticles in a biological setting include; enhanced luminescence signal, reduced background and are more stable than traditional organicfluoro dyes. Furthermore, the broad emission spectra and lifetime barcodes of SuperDotsTM, which allows excitation of multiple SuperDotTM populations at a single wavelength at either 800 nm or 980 nm, far removed from their respective emissions, allows for multiplexing of up to 10 targets simultaneously. However, fluorescence labelling using nanoparticles has yet to be demonstrated in a biological system such as cleared mouse CNS tissue. We aim to develop a technique allowing high resolution fluorescence imaging of proteins in cleared mouse brain and spinal cord tissue, using bare and functionalized Nanodiamonds, Nanorubies and SuperDotsTM and visualised with specialised microscopy developed through the ARC Centre of Excellence for Nanoscale BioPhotonics.