Fura-2 is a ratiometric dye which changes its excitation spectrum depending on the level of calcium ions. Its emission spectrum, on the other hand, changes very little. Usually what one does is to alternate between two (UV) excitation wavelengths using a controllable monochromator. Alternatively, you will have a broad spectrum light source and switches the excitation wavelength using different excitation filters.

In your case, you are using a confocal laser scanning microscope (CLSM), which uses laser excitation. Did you also change the excitation wavelength of the laser (i.e. switch the laser)? I am a bit confused when you say you used "Fura (free) and Fura (saturated) filters", because usually on a CLSM this is for the emission/detection side. Could that be that your Fura (saturated) filter is blocking the excitation laser, leading to no fluorescence?

It would be very helpful to know the spectra of your filters and which laser you used for excitation for troubleshooting the problem.

Aaron Benson Wong Thank you for your valuable suggestion. I need a clarification in usage of Fura 2- AM. when we are analyzing the dye under multispec reader, we use two excitation wavelengths and one emission wavelength and we perform the calculations later to identify the calcium signalling. So, my question is that whether we need dual filters to analyse Fura under microscope or using only Fura-free filater is sufficient to analyse calcium flux..!

Kindly provide your valuable suggestions.

Thank you once again.