Two-photon light-sheet microscopy for high-speed whole-brain functional imaging of zebrafish neuronal physiology and pathology
Giuseppe de Vito, Lapo Turrini, Chiara Fornetto, Pietro Ricci, Marie Caroline Müllenbroich, Giuseppe Sancataldo, Elena Trabalzini, Giacomo Mazzamuto, Natascia Tiso, Leonardo Sacconi, Duccio Fanelli, Ludovico Silvestri, Francesco Vanzi, Francesco S. Pavone
Abstract
We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation.