Fast functional imaging of multiple brain regions in intact zebrafish larvae using Selective Plane Illumination Microscopy

Thomas Panier,Raphaël Candelier,Georges Debrégeas,Germán Sumbre,Raphaël Olive,Thomas Pietri,Sebastián A Romano

doi:10.3389/fncir.2013.00065

Abstract

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1–2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5–9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20–60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

Highlights

Cognitive processes generally implicate extended neural networks spanning several areas of the brain
We characterized the volume of the brain accessible to single-cell calcium imaging using Selective-plane Illumination Microscopy (SPIM)
We estimated the gain in the number of neurons that could be simultaneously sampled, and in the maximum accessible acquisition rate, that this imaging strategy provides compared to standard 2P-point-scanning microscopy (PSM) experiments

Summary

Introduction

Cognitive processes generally implicate extended neural networks spanning several areas of the brain. In order to shed light on the neural basis of these processes, it is necessary to simultaneously monitor the dynamics of multiple brain regions with single-cell resolution (Alivisatos et al, 2012). Siliconbased nanoprobes allow electrophysiologists to simultaneously record up to a few hundred neurons (Du et al, 2011; Stevenson and Kording, 2011). Their future development is limited by the intrinsically invasive nature of the technique. Further assets of neuro-imaging, with respect to electrophysiology, include the accurate localization of the monitored neurons, the possibility to distinguish cell identity using specific markers and the precise manipulation of neural activity using optogenetic methods (Wyart et al, 2009)

Methods

Results

Conclusion