Abstract
Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).
Highlights
Recent scientific advances, both in microscopy technology [1,2,3,4,5] and in fluorescent sensors of neuronal activity [6,7], have led to a profound revolution in the field of brain functional imaging, making it pivotal for understanding brain functions
We show that the presented 2P Light-sheet fluorescence microscopy (LSFM) design enables high spatio-temporal resolution mapping of seizure dynamics, allowing the emergence of peculiar postero-anterior propagation patterns to be revealed for the first time
We devised a 2P LSFM setup to perform fast whole-brain imaging in zebrafish larvae
Summary
Both in microscopy technology [1,2,3,4,5] and in fluorescent sensors of neuronal activity [6,7], have led to a profound revolution in the field of brain functional imaging, making it pivotal for understanding brain functions. Owing to its unique architecture, which couples optical sectioning ability with parallelization of the acquisition process within each frame, LSFM has allowed for the first time the high-speed volumetric imaging of a vertebrate brain in its entirety, employing the intrinsically transparent zebrafish larva as a sample [9]. This technique has enabled the simultaneous functional investigation of large populations of neurons throughout the entire zebrafish brain expressing fluorescent calcium indicators, leading to novel insights into circuit dynamics [9,10,11,12,13,14]. We describe a 2P LSFM setup achieving fast whole-brain imaging (5 volumes per second) in zebrafish larvae [29,30] through SNR boosting, while keeping low the incident power on the sample, a crucial aspect for in-vivo imaging [23,31]
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