Calcium Imaging

Calcium imaging and imaging of other ions

In this section we will provide an overview on different applications in ion fluorescence imaging.

There are a number of advantages of using fluorescence probes over other techniques. Depending on the experimental requirements different detector systems can be used to obtain high resolution information on the time course of events and/or on the spatial localisation of events.

High temporal resolution can be achieved using Photodiode based systems. Changes in Ca²⁺ concentration can be measured in the order of milliseconds using a photodiode-based Photometry setup.

High spatial resolution may be obtained using fast integrating CCD cameras which enable measurements over tens of milliseconds up to 200 Hz. This way additional spatial information of the intracellular environment can be obtained. Agilent can provide high quality imaging hardware and software packages in either of these fields.

Ion imaging is a technique which typically combines microscopy with the use of ion sensitive fluorescent dyes in order to measure and visualize intracellular ion concentrations.

Ion sensitive dyes are fluorescent molecules which reversibly bind to specific ions. These dyes are very sensitive to any change in ion concentration.

A measure for the affinity of ion binding to the dye is the dissociation constant (Kd) for that ion dye interaction. Different side groups at the dye molecule may influence the affinity of the dye to the specific ion (see figure 1).

Binding of the Ion causes conformational changes in the dye altering its fluorescence excitation and emission properties which can be used to report ion concentration.

Many different dyes are commercially available for measuring Ca²⁺ (or other ion) concentration and pH and it is important to select the most appropriate dye for your needs (http://probes.invitrogen.com)