Hi Elmira,

Do you have more details on this issue that you are observing? What type of setup are you using, and especially what is the magnification and NA of the objectives you used? What kind of sample are you measuring? Have you tried to use a lifetime standard sample (a fluorescent dye with known lifetime) to verify this issue?

What is the magnitude of change that you observe in the lifetime values measured at different magnifications?

Thanks,

Alessio

Phototoxicity is increased at higher magnification. Under some conditions, this can cause ROS generation that can cause problems in some tissue samples. Higher magnification results in higher fluence at the sample.

You have two issues:

1. Shot noise related fitting complications (Alessio suggests checking for this and other systematic error with a known standard - a good practice)

2. As Ron noted, excitation intensity dependent dynamics are real, and the presence of oxygen can be part of it. You must know your excitation intensities at every magnification you use!

A decrease in lifetime with increased intensity is usually caused by excited-state quenchers, like triplets or polarons/charges. If you tell me more about your results and your system of interest I may be able to speculate further.