Functional imaging of zebrafish neuronal activity by Bessel beam light-sheet microscopy

Abstract

One of the most exciting challenges of neurosciences in the last few years is the real-time recording of neuronal activity with single cell resolution across the entire brain. Thanks to the use of optical methods, together with animal models in which the whole encephalon is optically accessible, this goal is getting within reach. In this work, we use a transgenic zebrafish line expressing the genetically encoded calcium indicator GCaMP6s in which binding of calcium ions leads to an increase in the emitted fluorescence of the reporter. GCaMP6s is the most sensitive calcium reporter within the genetically encoded calcium indicator family and allows us to record zebrafish larva neuronal activity with a high signal-to-noise ratio and single neuron resolution. To record the fluorescence emitted by the GCaMP6s reporter we use a custom-made confocal light-sheet microscope (LSM), in which the sample is illuminated with a thin sheet of light and the detection optical axis is perpendicular to the illumination axis. Owing to its intrinsic optical sectioning, this technique provides cellular resolution with high frame-rates and low photobleaching, allowing us to record the neuronal activity of zebrafish larvae with high spatio-temporal resolution. Conventional one-sided illumination LSM can suffer from limitations arising from even low levels of pigmentation in the sample or the presence of other obstructions reducing the quality of the incoming excitation light sheet. This can lead to shadowing in the image and possibly dynamic artefacts when the components responsible for the optical perturbations move. We have therefore implemented an illumination system based on a Bessel beam to overcome these limitations. Due to their nondiffractive and “self-healing” properties Bessel beams improve the quality of the images obtained from zebrafish larvae, reducing shadowing effects and increasing image homogeneity.