Hi,
Iam doing IF in HeLa cells and facing a weird problem. I use the standard protocol and when I expose the cell to laser light in the 488 channel, all the fluorescence fades the instant its exposed to light. Never faced this problem before. Used the same mounting medium and secondary antibody without any problem previously. Decreased the epifluorescence light even to 10%, but still the same thing happened thrice. No recovery of fluorescence later on. Gone once for all.
Has anyone faced this problem before. leads would be appreciated.
Thanks,
jaya
I need to know the full experiment details before i could give comment. But with whatever you have mentioned in your question, what i feel is could be the charging effect. kindly check the filters in the instrument. check with the instrument person of the right wavelength is hitting the sample.
This kind of charging effect is very common in electron microscopy if biological samples are used.
My answer still be vague unless you verify the instrument set up provided your samples preparation does not have a variation.
Thank you. Its normal IF taken in confocal 800 Airy scan. The light intensity for epifluorescence was 10% and during the experiment the laser light was 0.2%. No variation in the experiment. I faced the same when using the confocal 780 too.
you have mentioned that your samples are experiencing the same in two different confocal instruments. it means nothing could be from the instrument i suppose.
How about taking some other sample with which you it works. That way we can verify if your sample preparation has any problem
It is not phototoxicity if it does not kill or damage your cells. What you are decribing sounds like photobleaching (i.e. you only lose fluorescence, not so much damage the cells) that also happens always with all fluorophores given sufficient number of excitation events (required number depends on the fluorophore characteristics, its likelihood to undergo photochemical reactions instead of emitting a photon, and the number of excitation events within a given time depends also on the excitation intensity). What fluorophore are you using? Is it sensitive to photobleaching? Do you have something else in your system that increases the likelihood on photochemical reactions, e.g. a reactive chemical species, a chemical species that generates radicals when exposed to your excitation light or so? What is the laser power (in Watts, not percentages)?
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence