Methods to Detect Ca²⁺ in Living Cells

Abstract

Measurements of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) or free Ca²⁺ concentration in cellular organelles have become more routine. The primary reason for this is the availability of membrane permeant forms of Ca²⁺ indicators that can easily enter cells. In this chapter, the properties required of an ideal Ca²⁺ indicator are identified and the advantages and disadvantages of available Ca²⁺ indicators are pointed out. The pitfalls associated with usage of Ca²⁺ indicators together with the clear advantages of ratiometric over non-ratiometric indicators are discussed. The excitation of Ca²⁺ indicators and detection of the emitted fluorescence light require dedicated equipment; epifluorescence or confocal microscopes are most frequently used for this purpose and the advantages and disadvantages of these are discussed. Calibration experiments are required to translate changes in the fluorescence of Ca²⁺ indicators into real [Ca²⁺]_i changes, but this procedure is non-trivial and potential sources of error are identified. Future developments in the field of Ca²⁺ detection are discussed.