Visualizing Calcium Flux in Freely Moving Nematode Embryos

Abstract

The lack of physiological recordings from Caenorhabditis elegans embryos stands in stark contrast to the comprehensive anatomical and gene expression datasets already available. Using light-sheet fluorescence microscopy to address the challenges associated with functional imaging at this developmental stage, we recorded calcium dynamics in muscles and neurons and developed analysis strategies to relate activity and movement. In muscles, we found that the initiation of twitching was associated with a spreading calcium wave in a dorsal muscle bundle. Correlated activity in muscle bundles was linked with early twitching and eventual coordinated movement. To identify neuronal correlates of behavior, we monitored brainwide activity with subcellular resolution and identified a particularly active cell associated with muscle contractions. Finally, imaging neurons of a well-defined adult motor circuit, we found that reversals in the eggshell correlated with calcium transients in AVA interneurons.

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

“…We used our original fiber-coupled diSPIM system 44 in addition to another, recently described fibercoupled diSPIM system 45 to acquire volumetric time lapse datasets of zebrafish embryo lateral line and nematode embryo neurodevelopment, respectively. Data were acquired in light-sheet scan mode (scanning the light sheet through the stationary sample) with the ASI diSPIM Micromanager 46,47 (http://dispim.org/software/micro-manager) plugin instead of the LabVIEW control software used previously 44 . For zebrafish data, the XY stage was manually moved periodically in order to ensure that the growing tip of the lateral line did not exit the field of view.…”

“…Data were acquired by moving the stage in a raster pattern with aid of the ASI diSPIM Micromanager 46 plugin (http://dispim.org/software/micro-manager, ref. 47 ). The number of imaging tiles/rows as well as other acquisition parameters of interest are reported in Supplementary Table 3.…”

Advances in Imaging Plant Cell Dynamics

“…LSFM also can track physiological processes such as calcium fluxes by using ratiometric calcium indicators in the intact and freely moving nematode Caenorhabditis elegans (Ardiel et al, 2017) or during the neuronal activity of zebrafish larvae (Barykina et al, 2017). In the first case, fast exposure (2.5-4.5 ms) was translated to a 5-Hz volumetric acquisition frame rate, allowing imaging of the entire animal.…”

Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours

“…LSFM has enabled monitoring of embryo development over several hours in liquid-filled open-top chambers 29–31 . However, diffraction-limited optical imaging occurs only when both the excitation light sheet and the emitted fluorescence pass through refractive index-matched materials, making LSFM challenging to use with standard methods for mounting larval and adult animals 31, 32 , but convenient to use with hydrogels that are nearly index-matched with water.…”