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

Fast functional imaging of multiple brain regions in intact zebrafish larvae using Selective Plane Illumination Microscopy. - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

* Corresponding author 1 Laboratoire Jean PERRIN 2 ENS Paris - École normale supérieure - Paris 3 IBENS - Institut de biologie de l-Ecole Normale Supérieure 4 IBENS - Institut de biologie de l-école normale supérieure

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.

Keywords : Light-sheet imaging SPIM zebrafish brain

Autor: Thomas Panier - Sebastián A Romano - Raphaël Olive - Thomas Pietri - Germán Sumbre - Raphaël Candelier - Georges Debrégeas -



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