We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation.
Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To address this issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-photon light-sheet microscopy the maximum achievable frame rate is limited by the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarisation, and we implemented remote scanning of the focal plane to record without disturbing the sample. Using this setup, as a preliminary result, we characterized the intensity spectra of neuronal calcium traces of 4 days post fertilisation larvae during the day/night phases. We aim to extend these results to multiple brain regions and frequency bands.
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.