Hi, I am a Phd. student and my work is mostly calcium imaging in primary neuronal cultures. Lately, I have been trying to standardise imaging with Indo-1 AM.FYI, Indo-1 is a ratiometric single excitation, dual emission dye. So, exciting at 338 nm should give emission at 405nm ( which is the fluorescence of the Ca2+ bound form) and 485 nm (Ca2+ free form). Increasing cytosolic calcium would therefore cause an increase in the 405nm emission with a concomitant drop in the 485nm emission. However, increasing the calcium inside with ionomycin, i not only see an increase in the emission at 405 nm but also a small increase in 485 nm.i have checked the optics on my microscope and everything appears fine. Does any body have any suggestions?