Fluorescence Imaging for Life Sciences forum: topic
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calcium imaging with fluorescent dyes
Gayatri Venkiteswaran
Saturday, 01 March 2008 08:48 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 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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when you say you checked the optics on your microscope, what do you mean? because this sounds like bleed through. are your filters old?
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Hey Jennifer, thank you for the response.My filters are not old, though i too think bleed through could be a possibility .Even though my emission filters are narrow, the dichroic for the 485 nm chromophore emission is a 400 nm LP, and since the emission profile of 405nm chromophore is quite significant even at 470nm, bleed through can happen.
I was thinking of determining the percentage bleed through using a calcium saturated solution with the dye, where the emission at 485nm should be negligible but i am not sure how would i determine what part of the fluorescence at 485nm is inherent to the fluorophore and what part is the bleed through. Any inputs on this? -
After looking at the emission spectrum, it is almost certainly bleed-through. I think that you need a red-shifted dichroic. According to Chroma, you would need a 440 LP and very tight filters in order to be sure that you have minimal bleedthrough. Check here for more information. Good luck!
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