CORDIS

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.

In the MoNaLISA proposal we planned to create a new cutting edge microscopy platform as described in Objective 1 and 2. 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.
I recruited the master students to work under my supervision on the establishment of the MoNALISA microscope. The students Andreas Boden and Jonatan Alvelid after completing their master continue as PhD students and under my supervision worked on Objective 1 d to extend the MoNaLISA imaging to different fluorescent proteins for multicolor experiments.
Based on this work we built and demonstrated the MoNaLISA microscope, which has been published in Nature Communication as open access. The new method has been used to image a plethora of cell types including brain cell in living and functional slices, human cancer cell and bacteria. The novelty in the MoNaLISA technology is the ability to record movies of trafficking organelle and proteins in living and functional cell without giving up the high spatial resolution.
Once the technology was built we proceed by developing new probes compatible with the MoNaLISA imaging scheme. The work published in Nature Methods consists of a new red shifted reversible fluorescent protein, which combined with a new MoNaLISA illumination also red-shifted allowed super resolution imaging at minimal illumination doses.
This innovation will help the imaging and life science research by providing way to minimally perturb the samples during sensitive bio-imaging recording. A second work on this topic of probe development involved another MoNaLISA team member, the PhD student Jonatan Alvelid, who published in Nature Communication as open source a new set of infrared probes compatible with super resolution imaging, the first in this part of the spectra which allowed multi-plex imaging of proteins.
Along with the recording of MoNaLISA imaging data we started developing objective 3, which focused on new software to control and automate the microscope (published in Journal of Physics and Nature Communication) as well as new analysis tools for quantitative imaging (published in Biomedical Optics). On this line, we made the Tempesta software which control the MoNaLISA system available in github testa lab, and we kept adding new feature for smart and automated recording.
The new theory for image formation and signal quantification provided the ground for new applications focusing on molecular counting during synaptic activity (on going collaborative work).

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.