Multicolor two-photon light-patterning microscope exploiting the spatio-temporal properties of a fiber bundle.

The development of efficient genetically encoded indicators and actuators has opened up the possibility of reading and manipulating neuronal activity in living tissues with light. To achieve precise and reconfigurable targeting of large numbers of neurons with single-cell resolution within arbitrary volumes, different groups have recently developed all-optical strategies based on two-photon excitation and spatio-temporal shaping of ultrashort laser pulses. However, such techniques are often complex to set up and typically operate at a single wavelength only. To address these issues, we have developed a novel optical approach that uses a fiber bundle and a spatial light modulator to achieve simple and dual-color two-photon light patterning in three dimensions. By leveraging the core-to-core temporal delay and the wavelength-independent divergence characteristics of fiber bundles, we have demonstrated the capacity to generate high-resolution excitation spots in a 3D region with two distinct laser wavelengths simultaneously, offering a suitable and simple alternative for precise multicolor cell targeting.

Descanned fast light targeting (deFLiT) two-photon optogenetics.

Two-photon light-targeting optogenetics allows controlling selected subsets of neurons with near single-cell resolution and high temporal precision. To push forward this approach, we recently proposed a fast light-targeting strategy (FLiT) to rapidly scan multiple holograms tiled on a spatial light modulator (SLM). This allowed generating sub-ms timely-controlled switch of light patterns enabling to reduce the power budget for multi-target excitation and increase the temporal precision for relative spike tuning in a circuit. Here, we modified the optical design of FLiT by including a de-scan unit (deFLiT) to keep the holographic illumination centered at the middle of the objective pupil independently of the position of the tiled hologram on the SLM. This enables enlarging the number of usable holograms and reaching extended on-axis excitation volumes, and therefore increasing even further the power gain and temporal precision of conventional FLiT.