Measuring free Ca2+ concentration ([Ca2+]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca2+ sensors have led to the situation where cellular and subcellular [Ca2+] is examined by many non-specialists. In this chapter, we evaluate the most used Ca2+ indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca2+ and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca2+ measurement are discussed.
Keywords: Ca2+; Fluorescence; Laser confocal microscopy; Non-ratiometric; Ratiometric.