I'm using the fluorophore Nile Red to image what happens to lipid samples over time in solution. Everywhere you look people talk about photobleaching, but in my case the sample gets brighter over time (e.g. after 1 hour of non-stop imaging).
The same thing happens with FRAP analysis; the bleached spot brightens after recovery.
Can anybody tell me what might be happening?
Dye and lipids are simply mixed together, before being spincoated onto a microscope glass.
Peter, the lipids are dissolved in toluene. A tiny amount of Nile Red dissolved in methanol is added to the lipid solution. This is my stock.
The lipids are different triglycerides. I don't know if they should degrade and change emission, but I'm not excluding the possibility.
A few drops are then spincoated onto a glass surface and imaged.
Johannes, as Peter mentioned , please give sufficient time for incorporation of NR in hydrophobic lipid domain and then performed the experiment.
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