Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy

Trafficking studies of plasma membrane-localized intracellular estrogen receptors have mainly relied on biochemical and histological techniques to locate the receptor before and after estradiol stimulation. More often than not these experiments were performed using postmortem, lysed, or fixed tissue samples, whose tissue or cellular structure is typically severely altered or at times completely lost, making the definitive localization of estrogen receptors difficult to ascertain. To overcome this limitation we began using total internal reflection fluorescence microscopy (TIRFM) to study the trafficking of plasma membrane estrogen receptors. This real-time imaging approach, described in this chapter, permits observation of live, intact cells while allowing visualization of the steps (in time and spatial distribution) involved in receptor activation by estradiol and movements on and near the membrane. TIRFM yields high-contrast real-time images of fluorescently labeled E6BSA molecules on and just below the cell surface and is ideal for studying estrogen receptor trafficking in living cells.