Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms.

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans.

Microfluidic devices offer several advantages for C. elegans research, particularly for presenting precise physical and chemical environments, immobilizing animals during imaging, quantifying behavior, and automating screens. However, challenges to their widespread adoption in the field include increased complexity over conventional methods, operational problems (such as clogging, leaks, and bubbles), difficulty in obtaining or fabricating devices, and the need to characterize biological results obtained from new assay formats. Here we describe the preparation and operation of simple, reusable microfluidic devices for quantifying behavioral responses to chemical patterns, and single-use devices to arrange animals for time-lapse microscopy and to measure neuronal activity. We focus on details that eliminate or reduce the frustrations commonly experienced by new users of microfluidic devices.

A polymer index-matched to water enables diverse applications in fluorescence microscopy.

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.