Preparation of an imaging chamber for visualizing single molecules in living Dictyostelium cells

Environmental changes result in signaling events at cell membranes. To develop the means to understand these events at the molecular level, it is essential to become familiar with the stochastic nature of signaling molecules in living cells. Using total internal reflection fluorescent microscopy (TIRFM), these signaling events can be directly observed at the single-molecule level. This protocol describes the preparation of an imaging chamber to visualize single molecules in living Dictyostelium cells, which are highly sensitive to chemoattractant stimulation. It also describes treatment of the cells to allow visualization. Chemotactically competent cells are treated with the desired chemical, and the cell suspension is delivered onto coverslips and then overlaid with a thin agarose sheet in preparation for imaging. The cells are typically treated with a fluorescently labeled stimulant or inhibitor. Alternatively, the cells can be stimulated with photoreactive chemicals by using caged compounds. A caged compound is photoactivatable by irradiation with ultraviolet (UV) light, which cleaves a linker conjugating a caging moiety and a chemical. Thus, at a desired moment during recording of the single-molecule images, the concentration of the chemicals can be increased by photolysis of the caged compounds included in the buffer.