The endoplasmic reticulum (ER) is a complex and highly dynamic three-dimensional intracellular membranous system, which acts as a dynamic calcium store in the majority of eukaryotic cells. The special arrangement of intra-ER Ca2+ buffers, characterized by low affinity for Ca2+, in combination with SERCA pump activity keeps intraluminal Ca2+ ([Ca2+]L) at ∼0.1–0.8 mM (Cell Calcium 38:303–310, 2005), thus creating a steep electrochemical gradient aimed at the cytosol. Activation of ER Ca2+ channels results in Ca2+ release, which contributes to [Ca2+]i elevation, whereas SERCA-dependent Ca2+ uptake assists termination of cytosolic Ca2+ signals. In addition, the continuous luminal space can act as a travelling route for free Ca2+ ions (“Ca2+ tunnels”), thus bypassing cytosolic Ca2+ buffers and preventing mitochondrial Ca2+ uptake or loss of Ca2+ over the plasma membrane. Furthermore, changes in [Ca2+]L regulate ER-resident chaperones, responsible for postranslational protein processing. Thus, [Ca2+]L integrates various signalling events and establishes a link between fast signalling, associated with the ER Ca2+release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. This paper overviews modern techniques for the imaging of [Ca2+]L using synthetic fluorescent Ca2+ dyes. The methods for ER dye loading, with a particular emphasis on employment of ER targeted esterases (the Targeted-Esterase induced Dye loading, TED) to increase specific accumulation of the probes within the ER lumen are described in detail.