A SIMPLE METHOD FOR HIGH TEMPORAL RESOLUTION CALCIUM IMAGING WITH DUAL EXCITATION DYES

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cel ls. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a p otential drawback of ratiometry is that it requires equally fast switc hing of the excitation light between two wavelengths. To circumvent co ntinuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined w ith the acquisition of a single ratiometric pair of fluorescence image s at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change durin g the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs a cquired at regular intervals during the recording period, that further more includes a correction for the changing dye concentration in the c alculation of [Ca2+].