Ca2+ Imaging of Intracellular Organelles: Mitochondria

Calcium handling by mitochondria is important both because mitochondria can shape the cytosolic Ca²⁺ signals and because changes in mitochondrial Ca²⁺ concentration ([Ca²⁺ ]_M ) are important for controlling physiological functions such as respiration or programmed cell death. Accurate measurements of [Ca²⁺ ]_M require selective location of the Ca²⁺ probe inside mitochondria and this is best achieved by targeting protein probes to the mitochondrial matrix. Aequorins are very adequate as Ca²⁺ probes because: (1) they allow molecular engineering for targeting or for changing the Ca²⁺ affinity; (2) do not require irradiation for measurements; (3) Ca²⁺ buffering is small; (4) have a very steep Ca²⁺ -dependence and a very wide dynamic range, which makes them ideal for detecting and quantifying Ca²⁺ microdomains. Consumption and low light output are some of its drawbacks that make calcium imaging a hard task. Here, we describe a procedure that overcomes these disadvantages by combining herpes simplex virus type 1(HSV-1)-based expression of targeted aequorins with photon-counting imaging. This methodology allows real-time resolution of changes of [Ca²⁺ ]_M by photon counting imaging at the single-cell level. Since HSV virus is neurotrophic, the method is adequate for measuring [Ca²⁺ ]_M in living neurons.