Agar pads prevent both motility of live cells and Brownian motion of fixed cells. This makes them the method of choice for live imaging of multiple model organisms: bacteria, yeast, worm embryos and adult worms, plant, zebrafish embryos. They can be coupled to additional methods to further enhance immobilization: for instance, the use of anesthetics for C. elegans.

What is an agar pad? Although they are commonly referred as such, they are commonly made of agarose. Agar is a polysaccharide obtained from red algae, while agarose is the main component of agar, obtained after agar purification. Therefore, they are thin flat layers of an agarose matrix (see image). As a polymer, agarose creates a porous matrix where cells (or molecules for molecular biology applications) are trapped. However, nutrient and gas exchange occurs through the matrix making this method compatible with live imaging. No toxicity for the cells due to growth in agar has been described.

Agarose percentage to make pads can vary. Very standard percentages are from 0.5% to 2%. Higher percentages of agarose (up to 10%) have been used to immobilize adult worms, with the advantage of the lack of toxicity compared to other methods. Adult worms in agarose pads can be imaged for hours if the pad is properly sealed (avoiding desiccation) [3]. Replacement of agarose by other polysaccharides is as well possible. For instance, while 2% agar is the standard pad used for C. elegans embryo imaging, this can be replaced for 4% sucrose 0.1M NaCl for delicate early embryos or embryos sensitive to osmotic stress (Hyman Lab protocol, [2])

Melting and gelling temperatures depend on the type of agarose. Standard agarose is liquid after dissolution in water at high temperatures and solidifies around 35-45°C (depending on the concentration). Low melting agaroses remain fluid at much low temperatures (30°C).