I want to use microspheres which have been dyed with a fluorescent dye for fluorescence microscopy in a solution that has a 1-5% concentration of hydrogen peroxide. Is this possible or will the hydrogen peroxide always bleach any particles I put in?


The Hydrogen peroxide will definitively accelerate bleaching of your fluorescent microspheres. In fact, we routinely add glucose oxidase, glucose and catalase to our single molecule fluorescence experiments in order to get rid of oxygen and hydrogen peroxide in our solution and thus reduce photobleaching. However, Fluorescence microspheres are very photostable and whether or not they work for your application depends on several factors:

  1. how long the microspheres have been in the solution before you start imaging (the sooner you start the better, because of less reaction time between dye and hydrogen peroxide).
  2. How long you need to image (the shorter the better, because of less photobleaching)
  3. How large the microspheres can be (the larger the better because large microsphere contain more dye that is less accessible to the hydrogen peroxide in solution).

Basically, I suggest you try (and let us know how it went :) )

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    $\begingroup$ It worked fine and the spheres I used did not get bleached by the hydrogen peroxide. I think they were dyed all through, rather than surface coated. I left them in the hydrogen peroxide solution for 4 hours and I could still see them just fine. $\endgroup$ – tannoreth Feb 12 '16 at 15:33

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