Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light

Abstract

One of the fundamental goals of neuroscience is to understand how external sensory inputs and internal states are represented and processed by neuronal circuits in the nervous system to generate behavior. While in both simple and more complex organisms there is evidence of the existence of discrete anatomically pathways that compute sensory input and generate motor output, there is an increasing understanding that, most sensory functions and behavioral states are represented in a flexible and distributed fashion across large neuronal networks 1, 2 elegans nervous system, such as correlated activity among groups of neurons and their responses to sensory stimuli. This paves the way for future investigations on how sensory information is processed at the level of the whole brain, and for establishing a functional map of C. elegans' nervous system and potentially also in other model organisms. RESULTS Volumetric imaging using WF-TEFOOur approach for volumetric imaging is based on temporal focusing 15-18, 20, 21 .This two-photon excitation scheme allows to independently control the axial and lateral confinement of the excitation area while taking advantage of the high depth penetration and low scattering properties 22, 23 of two-photon excitation. In temporal focusing, the spectrum of a femtosecond laser pulse is spatially dispersed by a grating. The illuminated spot on the grating is imaged via a telescope, consisting of a relay lens and the microscope objective, inside the sample (Fig. 1A). Thereby, the spectral components of the laser pulse overlap in time and space only in the focus region where near-diffraction limited axial confinement of the excitation is achieved leading to high twophoton excitation probability, while wide-field excitation is maintained (see Online Methods for details). Outside of the focal region the pulse is chirped which reduces the two-photon excitation probability. Temporal focusing is compatible with conventional inverted or upright microscopes (Fig. 1A). Using this technique a wide-field two-photon excitation area of ~ 60 × 60 μm with an axial confinement of ~ 1.9 μm is generated (Online Methods) ( Fig. 1B-C).Furthermore, we have implemented a fast detection scheme based on an sCMOS camera coupled to a high-gain image intensifier. This eliminates the common trade-off between acquisition speed and sensitivity when comparing sCMOS and EMCCD cameras. Thereby we could retain the low noise floor of the sCMOS camera while benefiting from the signal enhancement of the intensifier ( Fig. 1D-F).Volumetric imaging is thus performed by scanning in the axial direction only, speeding up the volume acquisition time. For the lateral size described above and an axial range of over 30 μm, our microscope can achieve a volume acquisition rate of ~13Hz. However, due to limitations set by baseline fluorescence and signal to noise ratio of the Ca 2+ -sensor, as well as tolerable expression levels in C. elegans (see Online Methods for details), we performed the imaging at volumetric acquisition speeds of ...

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