Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

Each one of us in his life has, at least once, smelled the scent of roses, read one canto of Dante’s Commedia or listened to the sound of the sea from a shell. All of this is possible thanks to the astonishing capabilities of an organ, such as the brain, that allows us to collect and organize perceptions coming from sensory organs and to produce behavioural responses accordingly. Studying an operating brain in a non-invasive way is extremely difficult in mammals, and particularly in humans. In the last decade, a small teleost fish, zebrafish (Danio rerio), has been making its way into the field of neurosciences. The brain of a larval zebrafish is made up of 'only' 100000 neurons and it’s completely transparent, making it possible to optically access it. Here, taking advantage of the best of currently available technology, we devised optical solutions to investigate the dynamics of neuronal activity throughout the entire brain of zebrafish larvae

Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

Whole-brain functional imaging to highlight differences between the diurnal and nocturnal neuronal activity in zebrafish larvae

Most living organisms show highly conserved physiological changes following a 24-hour cycle which goes by the name of circadian rhythm. Among experimental models, the effects of light-dark cycle have been recently investigated in the larval zebrafish. Owing to its small size and transparency, this vertebrate enables optical access to the entire brain. Indeed, the combination of this organism with light-sheet imaging grants high spatio-temporal resolution volumetric recording of neuronal activity. This imaging technique, in its multiphoton variant, allows functional investigations without unwanted visual stimulation. Here, we employed a custom two-photon light-sheet microscope to study whole-brain differences in neuronal activity between diurnal and nocturnal periods in larval zebrafish. We describe for the first time an activity increase in the low frequency domain of the pretectum and a frequency-localised activity decrease of the anterior rhombencephalic turning region during the nocturnal period. Moreover, our data confirm a nocturnal reduction in habenular activity. Furthermore, whole-brain detrended fluctuation analysis revealed a nocturnal decrease in the self-affinity of the neuronal signals in parts of the dorsal thalamus and the medulla oblongata. Our data show that whole-brain nonlinear light-sheet imaging represents a useful tool to investigate circadian rhythm effects on neuronal activity.Comment: 18 pages, 6 figure

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

Light-sheet microscopy (LSM) is a powerful imaging technique that uses a planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM is a variant of LSM that exploits the 2P absorption effect for sample excitation. The light polarization state plays a significant, and often overlooked, role in 2P absorption processes. The scope of this work is to test whether using different polarization states for excitation light can affect the detected signal levels in 2P LSM imaging of typical biological samples with a spatially unordered dye population. Supported by a theoretical model, we compared the fluorescence signals obtained using different polarization states with various fluorophores (fluorescein, EGFP and GCaMP6s) and different samples (liquid solution and fixed or living zebrafish larvae). In all conditions, in agreement with our theoretical expectations, linear polarization oriented parallel to the detection plane provided the largest signal levels, while perpendicularly-oriented polarization gave low fluorescence signal with the biological samples, but a large signal for the fluorescein solution. Finally, circular polarization generally provided lower signal levels. These results highlight the importance of controlling the light polarization state in 2P LSM of biological samples. Furthermore, this characterization represents a useful guide to choose the best light polarization state when maximization of signal levels is needed, e.g. in high-speed 2P LSM.Comment: 16 pages, 4 figures. Version of the manuscript accepted for publication on Biomedical Optics Expres

Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts

The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD), to overcome this obstacle. We report the advantages provided by flexible and fast AODs in generating simultaneous angled multiple beams from a single laser beam and in fast light sheet pivoting and we demonstrate the suppression of illumination artifacts

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

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).Comment: Replacement: accepted version of the manuscript, to be published in Biomedical Optics Express. 36 pages, 15 figure

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast