Indo-1 calcium imaging
Gayatri Venkiteswaran
Saturday, 01 March 2008 08:54 UTC
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?
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Replies
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Anonymous
Did you figure it out? I’m not sure, but the excitation spectrum of the molecule may change with pH. Also, if only 3 AM esters are cleaved, maybe it’s binding another metal ion, with different emission properties. Did you check your indo stock with a calibration series?
Good luck!
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Indo-1 is a dual excitation dual emission dye. Not only does Ca2+ binding change the peak emission wavelenght, but also the peak excitation wavelength.
See: Tsien RY (1999) Monitoring Cell Calcium. In: Calcium as a Cellular Regulator (Carafoli E, Klee C, eds), pp 28-54. Oxford: Oxford University Press
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