Light interaction with neurons offers a sensitive non-invasive approach of probing and mimicking brain activity. Recent advances in what is now called neurophotonics have opened the route for simultaneously recording information from cell populations, with pure optical methods: light is used to target neurons that have been previously genetically modified to express fl uorescent sensors indicating their activity (via calcium or voltage imaging), or light-sensitive ion channels or pumps that control their activity (via optogenetics).

Here, I will present optical techniques developed in our group that optimize the excitation volume inside the sample for effi cient multi-neuronal photoactivation and functional imaging. Specifically, I will present methods for three-dimensional light patterning of the excitation laser beam, based on the modulation of beam’s optical wavefront through liquid crystal spatial light modulators [1]. The methods presented can be used for one- and multi-photon excitation. Particularly for the non-linear regime, controlling the volume of the excitation patterns in the axial direction can be achieved by simultaneous temporal focusing of the laser pulses [2,3]. Applications of these methods to functional imaging with voltage sensors [4], and neuronal optogenetic activation [5,6] will be shown.

References
1) Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).
2) Papagiakoumou, E., de Sars, V., Oron, D. & Emiliani, V. Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses. Opt. Express 16, 22039 22047 (2008).
3) Papagiakoumou, E., Ronzitti, E. & Emiliani, V. Scanless two-photon excitation with temporal focusing. Nat. Methods 17, 571–581 (2020).
4) Sims, R. R. et al. Scanless two-photon voltage imaging. Research Square Under revision, (2023).
5) Chen, I.-W. et al. In Vivo Submillisecond Two-Photon Optogenetics with Temporally Focused Patterned Light. Journal of Neuroscience 39, 3484–3497 (2019).
6) Spampinato, G. L. B. et al. All-optical inter-layers functional connectivity investigation in the mouse retina. Cell Reports Methods 2, 100268 (2022).