TIRF: using evanescent waves for high resolution membrane fluorescent imaging

Changes in the intracellular Ca2+ concentration ([Ca2+]i) accompany many diverse biological phenomena from neuronal excitability and muscle contraction to activation of transcription and cell death. Thanks to the introduction of fluorescent intracellular Ca2+ dyes our understanding of Ca2+ signalling has greatly advanced; it is established that depending on the signalling molecule and cell type, the global increase in cytoplasmic Ca2+ is mediated by the mobilisation of Ca2+ from intracellular stores and by the opening channels on the plasma membrane, or by a combination of both mechanisms. In this application note, we use TIRF to monitor intracellular free calcium ion concentration [Ca2+]i on or very close to the plasma membrane of muscle cells including human myotube