It is well known that excitations cause "bleaching" of GFP and virtually all fluorophores.

It is a very common issue in live cell microscopy or other applications that require multiple excitations/imagings/readings.

One key to minimise the bleaching is to minimise the excitation, both in intensity and time.

Also, if you could make the intervals of excitation longer than the protein turnover time, the bleaching may not be a problem.

Hi Shane,

Photobleahing of dyes are general problem when you are using them in live microscopy. Every dyes has a emission lifetime and with time the emission ability decreases.

At the time of live microscopy you have to reduced the excitation intensity (power) as well as the time of exposure.

Thanks both, that clears up the issues.

I was wondering as an alternative if I should;

1) Do a control plate and after 72 hours (duration of my experiment) take a reading of the RFU.

2) Use the data from above as a reference point with my actual working plates, then just take the end point reading; opposed to start and end / continuous?

Hi Shane,

A control plate isn't a bad idea, and I've done this for shorter exposure times on a different application. But there's two more problems. First, all GFP fluors are probably photobleached by 72 h. The second is more fundamental, and I'm stretching my experiences here (only worked with mammalian cells). Even with visible light, you're delivering a dose of photons (in J, you can calculate it if you have access to a light power sensor) that can cause damage to biologics. I've seen oxidative RNA damage in the 400-450 nm range with short (2 min) but high intensity, LED exposures. There may be an incredibly low absorbance cross section there, but throw enough photons at it...

Generally speaking, if you want to do extended fluorescence measurments over hours to days, take time lapse images where the cells are only exposed when imaged. If you don't have a system that can be computer controlled (e.g., via micromanager), then it's down to manual time points.

Hi Shane,

Yes, you can do this to minimise the error but whatever Matt is suggesting is quite significant. I never need such experiment for a long time period in my research work rather taking time lapse images. You can proceed whatever Matt has suggested..

Thanks again all, after reviewing + these comments; I'll be changing to manually counting each well in a 96 well plate to see if the cells have managed to replicate in each specific condition