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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - MoNaLISA (Long-term molecular nanoscale imaging of neuronal function)

Teaser

Summary

This proposal will open a completely new windows of observation for the life sciences. The MoNaLISA novel microscope will enable accurate a sensitive investigation of protein machineries in intact neurons and neuronal tissues. The novel features of our scopes will leverage super resolution imaging with light microscopy to embryos and intact organisms. 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.

Work performed

In the MoNaLISA proposal we planned to dedicate the first two years to the creation of a new cutting edge microscopy platform as described in Objective 1 and 2:
1) To build a stable microscopy platform that will extend switching mechanisms to parallelized images acquisition in order to enable rapid 3D imaging of large fields of view.
2) To adapt the platform to single molecule imaging, 3D localization, and molecule counting.
To pursue Objectives 1-2 my effort focused on three main activities, first to find and purchase the initial required equipment, second to recruit and coach the personnel with the right expertise and third to design and to perform the planned experiments.
According to the Objective 1a of the proposal, I organized the hardware dedicated to the microscope such as optics, illumination sources and sensitive detectors.
I recruited the master students Luciano Masullo and Andreas Boden to work under my supervision on the establishment of the MoNALISA microscope. Most of their works focused on selecting the right optics, designing part of the set-up and assembling it in different modules. The master student Aurelien Barbotin dedicated his master thesis work to develop part of the MoNaLISA illumination modules with spatial light modulator. All the students worked on the development of specific modules of the microscope, hardware and software. According to Objective 1b the microscope has been further developed in order to perform parallelized recording with periodic light patterns in order to speed up the acquisition process and to achieve 3D ability. We implemented orthogonally and incoherently crossed standing waves to parallelize the scanning. To address Objective 1c we developed the MoNaLISA imaging ability at minimal power. In fact we applied the parallelized illumination scheme to the fluorescent proteins rsEGFP2 and rsEGFP205S fused with the protein Vimentine and Map2. This imaging modality works in three steps: switching the protein ON with single or multi-photon absorption, switching the protein OFF in the periphery of the focal spot, and recording the fluorescence of the remaining proteins. These steps required an extensive photo-physical characterization of the fluorescent molecules, as an example we showed the ON-OFF kinetics of rsEGFPN205S measured with MoNaLISA in the last months. The student Andreas Boden under my supervision is currently working on Objective 1 d to extend the MoNaLISA imaging to different fluorescent proteins for multicolor experiments.
At the moment we are finalizing the data recording and analysis, which will lead to a first manuscript for publication in a high impact peer review journal in the next three months.

Final results

World leading institutes in the field of bio-imaging are currently investing immense efforts to attract experts from the field of super resolution microscopy with the ultimate goal of resolving the dynamics of the cell in physiological relevant systems at high spatial resolution. This is because some of the central biological problems such as live following of neuronal proteins during plasticity-induced treatments or imaging intermediate time-point during vesicle budding within cells, are simply not possible to tackle with available conventional methods. The next generation super resolution nanoscope proposed in this project has the potential to look at macromolecular complexes in their native functional states in intact tissues.
The equipment and personnel required in this proposal are intended to reach this goal. However, it will not only boost my effort in creating a highly competitive super resolution bio-imaging unit, capable of creating functionally annotated movies with molecular resolution, but will also encourage the application of the presented innovative technology to a wide range of biological problems investigated in Europe. The financial support therefore is expected to have high impact on the quality of science produced in my group in the very near future.