Pre-bleaching of media?

Hi,

Is photobleaching culture media prior to live-cell time courses something people do?
The intention being to reduce the background caused by the media in widefield images.

For example below is the mean intensity over time of a region of the image that has no cells.
image
image
Since the media appears to photobleach the idea would be to photobleach it before imaging to reduce background.
(the media used above is FluoroBrite+FBS)

Thanks

Generally, I hope people aren’t purposefully exciting their culture media because media components (e.g. riboflavin) will lead to photodamage. (One source here)

Commercial background suppressors exist, but I don’t have any personal experience with any of them.

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I’ve experienced this so many times. My impression is that if/when you go to an area of no cells and bleach that due to diffusion, the molecules in the media that are responsible for the ‘background glow’ will simply diffuse back in (anecdote - the background glow does seem sometimes decrease somewhat over time in long timelapse runs, but this isn’t a good way to address the problem). A better solution is to buy some phenol red free medium, which is readily available for most standard formulations, this removes a lot of the glow. If you’re needing to also remove riboflavin and tryptophan (I think), you can get this medium, but it is traditionally a lot more expensive (I was once quoted $500/500 ml bottle). I’m not too familiar with the commercial background suppressors, but it sounds interesting. Depending on how long you plan to image, there are also products like “Live Cell Imaging Solution”, which is an optimized buffer where cells can be out for 4 hours or so, this has zero fluorescence. Good luck! Based on the image you provide, which look like nuclei at 10X in a wound assay (could be wrong), I’d say phenol-red free is the way to go.

Phenol red does not fluoresce but it quenches green fluorophores. If you want to get rid of green autofluorescence in culture medium, use a medium with low flavins and Vitamin B12 as these molecules fluoresce strongly in the green channel.

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“Of note, most tissue culture medium contains fluorescent compounds such as for example phenol red. In our hands, background fluorescence of regular DMEM or similar tissue culture media is negligible on our spinning disk confocal setup at 488 nm or 561 nm excitation with optimized filter sets. However, phenol red is highly fluorescent when excited at 440 nm, and phenol red free media has to be used for imaging of cyan FPs.”

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