I have a material containing coumarin in it. When my collaborator takes fluorescence microscopic images of the material using DAPI filter, he mentioned me that the sample is photobleaching. As a result he couldn't take good fluorescence microscopic images. I didn't understand this completely.
When a fluorophore photobleaches, it means that it is 'destroyed', in the sense that it doesn't fluoresce anymore. Exciting your fluorophores is what is causing photobleaching, so imaging your sample using a fluorescence microscope will slowly reduce the amount of fluorescence that you see (because of photobleaching). One way to prevent this is to use a lower excitation power, although this will probably also reduce your sensitivity (except if you are using a very high laser power). Another solution would be to change the fluorescent dye you are using.
Hi Laurent and Bryan,
Thank you so much for the information. I appreciate your time.
You can also change the mounting medium, if you want to save your experiment and if you didn't yet use a polymerising one. Commercial mounting media that prevent photobleaching and are working well in my hands are vectashield (vector) and/or Prolong (Invitrogen). If it's too expansive for you, you can also add N propyl galate (0.1-0,2 M) to PBS-Glycerol (Science (New York, N.Y.) [1982, 217(4566):1252-1255]).
If the bleaching is really due to a photoinduced oxidation I suggest to bubble your cuvette or setup with nitrogen out of a simple gas bottle. For example the photoinduced bleaching of a polymer (P3HT) is strongly reduced under nitrogen (see my publications).
Hi Murthy,
It is simple. Your collaborator is using DAPI filter set to image means he is using 360nm excitation. Since coumarin molecule can absord at this wavelength what he is noticing is the dimerization of coumarin rather than photobleaching. Since you say you have polymeric material you may have enough local concentration in film or solution for dimerization. If he sees pure photobleaching he may have to check the laser power settings as coumarin generally has low quantum yield for bleaching. As Laurent suggested you may also try adding propyl gallate (a good singlet oxygen scavenger) and see if the photobleaching reduces. This will prove that the mechanism indeed is photobleaching rather than dimerization.
If you need a somewhat simpler reaction, eventually your fluorescence stain will lose the ability to fluoresce. This will happen if your light is very bright or if you expose it to light for too long. This is a particular problem in the DAPI end of the spectrum because these are high energy/short wavelength forms of light, and they tend to exhaust or burn out much faster. Others suggested reducing the light intensity (you can use a neutral density filter), or exposure time, and both of those will help. An electronic shutter on the microscope can help limit the exposure to the light and that will help reduce photobleaching, because you will only have the light on your sample for a few milliseconds while you take the picture.
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