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The nematode  Caenorhabditis elegans  is an important model organism for biomedical research and genetic studies relevant to human biology and disease. Such studies are often based on high-resolution imaging of dynamic biological processes in the worm body tissues, requiring well-immobilized and physiologically active animals in order to avoid movement-related artifacts and to obtain meaningful biological information. However, existing immobilization methods employ the application of either anesthetics or servere physical constraints, by using glue or specific microfluidic on-chip mechanical structures, which in some cases may strongly affect physiological processes of the animals. Here, we immobilize  C .  elegans  nematodes by taking advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. We successfully demonstrated the applicability of this method in challenging worm imaging contexts, in particular by applying it for high-resolution confocal imaging of the mitochondrial morphology in worm body wall muscle cells and for the long-term quantification of number and size of specific protein aggregates in different  C .  elegans  neurodegenerative disease models. Our approach was also suitable for immobilizing other small organisms, such as the larvae of the fruit fly  Drosophila melanogaster  and the unicellular parasite  Trypanosoma brucei . We anticipate that this versatile technique will significantly simplify biological assay-based longitudinal studies and long-term observation of small model organisms.

Reversible and long-term immobilization in a hydrogel-microbead matrix for high-resolution imaging of Caenorhabditis elegans and other small organisms