Hi there,

Just a couple of caveats of the protocols you have used so far:

TdTomato has a pKa=4.5-4.7, so at pH6.5, TdTomato shouldn't be far away from its max. fluorescence (it is not going to be too sensitive to this pH)

https://www.fpbase.org/protein/tdtomato/

Article Improved monomeric red, orange and yellow fluorescent protei...

https://pubmed.ncbi.nlm.nih.gov/27240257/

https://www.nature.com/articles/s41598-019-38913-z

TdTomato has a t1/2 for photobleaching of approx 30" but at 561nm and 80uW laser illumination (really close to its excitation peak that is 555nm).

https://pubmed.ncbi.nlm.nih.gov/27240257/

Table S1

Have you considered using this wavelength (561 laser) instead of the UV light? Or at least a TRITC filter in a fluorescence microscope. Maybe it's better to achieve complete bleaching of your sample (TdTomato).

However, in your case (and if your protocol allows you to do so), I think you should definitely give a try to Methanol/Acetone (1:1) fixation. It's usually stated everywhere that you should never use it with fluorescent proteins because they will lose the fluorescence. Be careful with methanol if you need to perform IHF afterwards, it could damage your epitope of interest.

But you will need to try. Getting rid of all the fluorescence from TdTomato could be really difficult (is one of the brightest fluorescent proteins and is really photostable),

Hope this helps,

Cheers,

J

Hi J. Ramirez-Franco , thanks for the answers! I actually had no clue what pKa of a fluor meant! I primarily used pH6.5 (and also 3), because of this paper:

Effect of pH on thermal- and chemical-induced denaturation of GFP https://pubmed.ncbi.nlm.nih.gov/16118468/

The published pKa of eGFP is 6, and it photobleaches best at 6.5in some papers, and in my own hands. The paper claims it's some sort of structural change, alternatively, it's more easily photobleached because it's at it's max? In which case maybe it would be faster/easier to photobleach tdtomato at pH 4.5.

Yeah, I've tried using a tritc filter to photobleach tdtomato, and in acidic PBST, with H2O2, tdtomato does beautifully bleach, over the course of 5-10 min. *However* quite astonishingly, to my at least, when I replace it back into normal PBS, it quite quickly recovers. So for some reason this photobleaching isn't irreversible, which was quite frustrating, and kind of confuses my understanding of the mechanism of photobleaching.

I have tried methanol, and that didn't kill TdTomato, also interesting is that normal GFP is sensitive to methanol, but eGFP is not. I hadn't thought of using acetone, or doing methanol->acetone, or acetone->methanol....I'll have to try that out and see how it goes. Thanks for the suggestion!

Hello again,

Check the links below.

https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/fluorescence/fluorescenceintro/

https://www.leica-microsystems.com/science-lab/basic-principles-of-luminescence/

Quenching is reversible, bleaching is not. Most likely you have quenching (or a mix of quenching and bleaching) and not complete bleaching when you put TdTomato in acidic PBS with H2O2, that's why the reaction reverses. Even though they both lead to a decrease in fluorescence they're different processes.

Moreover, you can't bleach something that's quenched (because fluorophores are in the ground state), so, under this conditions, all the fluorescence that's lost because of quenching processes will not be affected and will not be bleached, and thus, will be recovered when you put the sample back in a non-quenching solution. This applies for both, pH changes and oxygen, they're quenchers, and fluorescence will recover when they disappear.

So try to photobleach (even if it takes much longer) in normal PBS, that way you won't see a recovery of the fluorescence. If you have access to a confocal, you can try it with a 561nm laser, it will be more effective.

Cheers,

J

Hmm, interesting. Thanks for this discussion J!

I tried out the acetone, as well as methanol/acetone mixture, and amazingly, Tdtomato is still as bright as it was after 10 minutes of submersion in this. When it didn't work at -20, I even did RT incubation, no effect. Shocking, and kind of amazing hahaha.

I have yet to try just photobleaching in PBS, I would like to to do in on epi, instead of a confocal because I'm doing this on tissue, not sections, and it would take forever to photobleach the entire thing by confocal. In any case, I shall report back if anything works :D