Any anti-bleaching strategies for cells mounted with PBS?

Probes & Prep
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1

Hi everyone,

I am now using water objective imaging samples in PBS, which provided no RI mismatch. But the sample in PBS was easier to bleach without any anti-bleaching agents added. I would like to change PBS to mounting media with RI 1.33 or add some chemicals to do the job.

Thus, first I found this Prolong Live RI 1.3,Links to an external site from therm-Fischer. which is suitable for live cells. Besides, I also found a chemical called Trolox (soluble vitamin E) that can be added directly to the PBS.

I would also need to image the PSF on a coverslip with Tetraspeck beads mounted and check the chromatic aberration. I also don’t want an RI mismatch when using the bead slides.

Would anyone have experience or recommendations for a good solution in this situation (either a suitable mounting medium close to RI 1.33, or additives that work well in PBS)?

Thank you so much for your attention!

2

You are imaging live cells? And you want them to stay alive and happy?

If this is not so important then one approach is to use an oxygen scavenging system like glucose oxidase / catalase (and glucose) along with trolox. You can add these to PBS without significantly changing the RI and (for most dyes) it is pretty effective at reducing photo-bleaching. However the cells won’t appreciate the resulting low oxygen conditions.

Also, what dye(s) are you imaging? Some are much more sensitive anti-bleaching chemicals than others.

3

Hi Hazen,

Thank you for your reply!

I am imaging fixed cells in a 96-well plate, so cell viability is not a concern in my case. I am using Alexa Fluor dyes (Alexa Fluor series 405，488，647 and mCherry protein).

Thanks again for your helpful suggestions!