Calcium Imaging

Fluorescent dye parameters

Important dye parameters to consider are:

• specificity for the ion of interest
• dissociation constant Kd
• excitation and emission spectrum
• fluorescence intensity
• free Ca²⁺ range
• cell permeability (availability as a AM ester)
• efficiency of Photon absorption
• quantum yield (ratio of absorbed and emitted photons)
• sensitivity to photobleaching (chemical destruction of the dye during illumination)
• quenching (deactivation of excited dye molecules without the emission of any radiation)

For more information on fluorescent ion sensitive dyes, please refer to the Molecular Probes Handbook. There are three major classes of ion-sensitive dyes:

• single excitation dyes and
• dual excitation ratiometric dyes
• dual emission ratiometric dyes

Single excitation dyes

With single wavelength dyes the intensity of the fluorescence emission spectrum increases in proportion to free ion concentration. The problem with using a single wavelength dye is that it is difficult to distinguish between differences in ion concentration and variations in dye brightness caused by factors such as dye concentration, dye photobleaching and dye leakage from a cell. This makes absolute measurement of ion concentration with single wavelength dyes difficult. Examples are dyes of the Fluo- family, such as Fluo 4.

Dual excitation ratiometric dyes

These dyes may be excited successively at two different wavelengths. The emitted light increases with increasing [Ca]_i at one excitation wavelength, and decreases at a second wavelength (see figure 2). A prominent example is FURA 4. The following table summarizes some dual excitation ratiometric dyes:

For more information regarding different fluorescent ion sensitive dyes, please refer to the molecular probes homepage

Dual emission ratiometric dyes

These dyes are excited at one wavelength and fluorescence emission at two longer wavelengths. With indo-1 and cSNARF-1, the two emission wavelengths correspond to the ion binding and free forms of each dye. As the concentration of free Ca²⁺ or protons increases, there is a shift in the emission spectra of Indo-1 and cSNARF-1 respectively, to the shorter wavelengths of the ion binding forms of these dyes.

Please refer to the molecular probes homepage for more information on Indo-1 and cSNARF-1.