Characterizing System Performance in Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141–145, 1981; Gingell et al., J Cell Biol 100:1334–1338, 1985; Patel et al., J Cell Sci 121:1159–1164, 2008), actin (Bretschneider et al., Curr Biol 14:1–10, 2004; Gerisch, Biophys J 87:3493–3503, 2004; Merrifield et al., Nat Cell Biol 4:691–698, 2002), and membrane dynamics (Oheim et al., Eur Biophys J 27:83–98, 1998; Steyer et al., Nature 388:474–478, 1997; Weisswange et al., J Cell Sci 118:4375–4380, 2005). In TIRF-M, dim fluorescence from cortical structures can be imaged with high contrast despite large cytoplasmic background from the bulk of the cell body. With any imaging method, standard samples are required to ensure correct alignment and monitor system performance over time. Here, we describe procedures for the production and use of a test sample to characterise and optimize TIRF system performance.