Can anyone share their experience on the lifetime of different tracers or dyes? I am retrogradely labeling neurons with cholera toxin (CtB) 594 for chronic in-vivo imaging. I assume the lifetime of my label is limited by the fluorophore (e.g., degradation, excretion, or bleaching) and not what it is conjugated to (CtB). How many days out can I expect to still see a signal in my cells of interest?
Hi Steve J Sullivan I honestly think this question may have a broad answer. First of all it is important to know your system, your imaging conditions, dye concentration, microscope parameters/settings. Also note importantly how frequently you are imaging them. For example, if you do a long term imaging experiment, do you image once every 30 minutes or once every hour?. I do think if you don’t irradiate them so frequently like once every 2 minutes (for example) they should be able to survive imaging experiments, but questions can be raised about their phototoxicity and cytotoxicity and the stability during in vivo experiments. What are the dyes you are specifically using ?
Thanks for your input Chathura. My dye is Alexa 594 conjugated to the cholera toxin B subunit. Ideally I would image a few times a week, one scan per session, to identify the retrogradely labeled neurons. I realize there are probably a lot of variables to consider, but knowing how far out others have imaged in the past would help give me a ballpark figure—days, weeks, months?
Alexa 594 is a relatively photostable dye. Especially in your experiments, the number of scans you would use will be not much. So I hope this will be good for few weeks. Alexa 594 is a Rhodamine based dye. Another in addition to the possible photo damage to the dye with continuous irradiation, another concern is the stability of the dye. Since the dye is conjugated to the toxin, it may probably be an advantage.
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