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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - BrainBIT (All-optical brain-to-brain behaviour and information transfer)

Teaser

Summary

A more detailed information on neuronal encoding of information in the cortex is a crucial point in neuroscience and physiopathology studies. On one hand it is important for a deeper understanding of brain function and on the other hand to dissect the physiological mechanism that are altered in pathologies, diseases and injuries.
In this project, we setup and characterize new optical imaging and manipulation techniques to address brain functionality over the large scale.

Work performed

Two-photon microscopy (TPM) is a well-established technique for imaging even in highly scattering media. Being a point-scanning technique, however, TPM suffers from slow scan rates. To limit this issue we took advantage of multispot multiphoton microscopy (MMM), which increases the speed by multiplexing the excitation. In addition, to reduce the optical cross talk which prohibits the application of MMM deep within scattering samples, we capitalized on the advantages of confocal line detection by exploiting the rolling shutter of a fast sCMOS camera. This new combination of techniques will be useful to dissect fine structural features of neuronal architecture in mice brain, in both physiological and pathological conditions.
In addition, we characterized several red-shifted genetically engineered calcium indicators with large-scale cortical imaging in awake mice. To perform a full-optical imaging and transfer of neuronal activity, we chose these new calcium indicators with reduced optical cross-talk with the most commonly used neuronal actuators.

Final results

In Adam et al. (2018), a multispot two-photon excitation microscope with rolling shutter wide-field detection was developed. This method benefits from the increased penetration depth of two-photon excitation in scattering media and the increased imaging rates of multiplexed excitation while exploiting the rolling shutter of a fast sCMOS camera. Microscope performances were demonstrated in fixed semicleared brain slices by imaging dendritic spines up to 400-Î¼m deep.

In Montagni et al., 2018 we describe the evaluation of different indicators of neuronal activity to identify the most suited for the development of an all-optical system as stated in the WP2 T2.1 of the project. The problem addressed concern the identification of the best red-shifted indicator to reveal the cortical neuronal activity. To this aim, we tested different red-shifted genetically encoded calcium indicators. The overall objective is to identify a combination of indicator and actuator that could be successfully used to read and write from mouse cortex, towards the intra and inter subject transfer of neuronal functionality. In the paper by Montagni et al., we identified jRCaMP1a as the best red-shifted functional indicator that does not show photoswitch, resulting the best choice for the future development of an all-optical system for interrogation of neuronal circuits.

In Sancataldo et al. (Front. Neuroanat. 2019), we described the use of acousto-optic deflectors (AODs) in light-sheet microscopy to mitigate striping artefacts. This work paves the way to a more quantitative functional imaging in zebrafish larvae, which is a key aspect of the project, and which was also previously investigated by our group (Muellenbroich et al., Front. Cell Neurosci. 2018). Further, the use of AODs has been a technical training for the implementation of multiplexed ultrafast light-sheet microscopy, described previously in the report.