Super resolution imaging of living intact cells and tissues is challenging if not impossible. Most of the studies at this spatial resolution, closer to the scale of macromolecule and small organelles inside the cell,
are conducted in fixed death cells. Typical imaging experiments such as recording pictures of a neuron and its compartment before and after treatment, such as long term potentiation or metabolic perturbation, is simply not possible with super resolution. This limitation makes difficult to study important compartment such as synapses while functioning, posing a limit of our ability to understand mechanisms underlining basic human function
such as learning, thinking and moving.
This proposal will open a completely new windows of observation for the life sciences. The MoNaLISA microscopy platform, proposed here, will enable accurate a sensitive investigation of protein machineries in intact neurons and neuronal tissues. The novel features of our microscope will make embryos and intact organisms accessible to super resolution fluorescence microscopy. In fact, MoNaLISA is a unique tool to image living sample at high spatial resolution and it has the potential to help solving basic questions in the life science as well as to future use for more sensitive tissues analysis of clinical relevance (tissue screening on the molecular level).
MoNaLISA will increase the applicability of the state of art of super resolution microscopy by adding two important features: decreasing photo-damage and imaging for longer time at the nanoscale. Essential advances to achieve intact live tissue imaging. This new technology will equip the life science community in Europe with a unique and very powerful methodological advance.
Particularly, it will significantly increase the sensitivity of the current state of art methods paving the road for innovative research with a potential of revealing fundamental biological processes at unprecedented level of details.
The proposal is structured in 4 Objectives, which consist of prototyping and building the microscope, developing new imaging schemes and probes, recording imaging data, developing new analysis tools and finally demonstrating its potential by performing live neuronal imaging with single synaptic vesicle spatial resolution.
The objectives have been developed step by step according to the 60 months time line and the results have been published in peer review journals such as Nature Communication (3 manuscripts), Nature Methods (1 manuscript), Journal of Applied Physics (2 manuscripts) and Biomedical optics (1 manuscript) for microscopy and probes methodological development and applications in journal such as EMBO (1 manuscript), PNAS (1 manuscript), and Cells (1 manuscript).
With MoNaLISA is now possible to record movies of trafficking proteins and organelles at the synapse.
