Labeling of Cell Membranes and Compartments for Live Cell Fluorescence Microscopy

Abstract

Publisher Summary This chapter describes methods for fluorescently labeling the surface and organellar membranes, as well as the spaces they enclose. The fluorescence imaging of cytoskeletal elements is also discussed. The extracellular space of cells is easily labeled by fluorescent dextrans or calcein markers. The plasma membrane is the easiest cell membrane to label because it is very accessible. Fluorescent dyes and most commonly used dyes FM 1-43 family are used for labeling the plasma membrane. The cytosol is the intracellular space excluding the nucleus and the organelles. The cytosol is easy to label because aqueous fluorescence markers injected into eggs diffuse throughout the cytosol. In many cell types, mitochondria are easily labeled by water-soluble, lipophilic, positively charged fluorescent molecules, which cross the plasma membrane and become concentrated in mitochondria due to the large negative membrane potential of the mitochondria. Short-chain dicarbocyanine dyes—such as DiOC 6 (3)—are effective for staining endoplasmic reticulum (ER) in cultured fibroblasts. ER in echinoderm eggs are successfully stained by two other methods: (1) injecting an oil drop saturated with a long-chain dicarbocyanine dye, or (2) expressing a GFP chimera. Bodipy ceramide is a fluorescent marker used for labelling the Golgi apparatus. The most effective way to label the Golgi apparatus has been with GFP chimeras. The nuclear envelope is easily visible by transmitted light microscopy; however, its components (nuclear pores, lamina) are labeled by GFP chimeras to investigate how the nuclear envelope breaks down during meiosis or mitosis. The way in which the egg or embryo is kept on the microscope stage is critical for the success of a live cell experiment.