Hydrogel encapsulation of living organisms for long-term microscopy

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging, across development or behavioral states, places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple and inexpensive method for rapid encapsulation of small animals of any developmental stage within a photocrosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintained a normal, uncompressed morphology in a hydrated environment and could be exposed to different aqueous chemicals. We focus in particular on the nematode C. elegans , an organism that is typically viewed with paralyzing reagents, nanobeads, adhesives, or microfluidic traps. The hydrogel is optically clear, non-autofluorescent, and nearly index-matched with water for use with light-sheet microscopy. We captured volumetric images of optogenetically-stimulated responses in multiple sensory neurons over 14 hours using a diSPIM light-sheet microscope, and immobilized worms were recoverable and viable after 24 hours encapsulation. We further imaged living pygmy squid hatchlings to demonstrate size scalability, characterized immobilization quality for various crosslinking parameters and identified paralytic-free conditions suitable for high-resolution single cell imaging. PEG hydrogel encapsulation enables continuous observation for hours of small living organisms, from yeast to zebrafish, and is compatible with multiple microscope mounting geometries.