In the context of this project we developed multiple optical setups to help us achieve the stated goals:
- We developed an optical setup that helps us evolve GEVIs by imaging a large number of cells, which each express a mutated version of a GEVI. We can then select the cells that show a good response to our stimulus, and sequence the plasmid in it, to find the mutations that provided better functionality. A version of this setup was also used to identify aggressive cancer cells (L. You et al; Nature Biomedical Engineering, 6, 667–675 (2022) ). This setup is part of a licencing deal being set up with a commercial drug screening party.
- We developed a setup for nonlinear screening and in vivo imaging of GEVIs, including all the hardware and software needed for the evolution and nonlinear optogenetic experiments (X. Meng et al; Journal of Optics, 24, 054004 (2022)
- We created a setup for pump-probe experiments on GEVIS in cellular environments, to understand their response to nonlinear excitation under different voltages. We found that we could influence the photocycle of the GEVIs and with it their response to nonlinear excitation by making a hybrid version of the GEVI coupled to a plasmonic antenna. this influening of the photocycle makes the GEVI respond with kinetics that we otherwise do not see using nonlinear excitation and makes it possible to use them for nonlinear excitation under circumstances where they otherwise would not (Locarno et al, Adv. Materials, revision, 2025)
- Covid delayed the progress on some aspects of the project. To catch up, we created a separate in vivo imaging setup, which aims for more superficial voltage imaging in vivo, which can already start working on the proof-of-principle learning task while the other parts of the project develop further. We have taken this part of the project in two directions: we use pulsed excitation of GEVIs to perform FLIM imaging in zebrafish, invetigating their development (Wu, Silva et al, under review; Wu et al in prep); and we used voltage imaging in the cerebellum to investigate motor memory formation (Silva et al, in prep). we find correlations in complex spike occurance between different parts of the olivocerebellar loop using this voltage imaging technique.
Apart from the optical work:
- We set up a number of cell lines that are helpful to us in our GEVI evolution, characterization of GEVI dynamics and chanracterization of neural dynamics (used in a.o. Q. Li et al; Optics Express, 29, 21, 34097-34108 (2021); Flamourakis et al, Adv Func. Mat 35 (5), 2409451 (2025))
- We set up a theoretical framework for the analysis and manipulation of GEVI photocycles (Meng et al, ACS Phys Chem Au 3 (4) 320 (2023);
- We evaluated different GEVI families for dynamics under nonlinear excitations, and screened them for evolved brightness and functionality. We developed a novel GEVI with improved NIR fluorescence (Ganapathy et al, J. Bio. Chem. 299 (6) 2023). our screening setup has been augmented with AI based computational screening and we are no also sitting on a database of voltage sensitive rhodopins that we are characterizing (work in progress).
