Closed imaging chamber. (A) A customized insert designed to hold 18-mm coverslips. The drawing shows a side view of the assembled insert. Note that the thickness of the Teflon ring is such that when the coverslips are in place, the assembly is flush with the top and bottom of the insert. To obtain an air-tight seal, a thin layer of silicon grease is applied to all areas of the ring in contact with the coverslips. When using the chamber in an open configuration, we use a thicker Teflon ring to compensate for the absence of the top coverslip. (B) The base and the top plate of the commercial platform (Series 20, Warner Instruments) are used to clamp the assembled insert in place. The base plate has a pair of resistive heater elements that are connected to a temperature controller. (C) The assembled chamber. For this illustration, the chamber volume was filled with phenol red-containing medium. For long-term imaging, we use medium lacking phenol red.