Fluorescent Protein Photobleaching and Light Source

In the recent paper on the new Zoanthus derived fluorescent protein, mPapaya1 [1], I saw a figure in the supplementary material that gives a good illustration of the challenges comparing photobleaching rates between proteins  The figure is reproduced here:

pbleach

Photobleaching data on four different fluorescent proteins. A&B: excitation with a mercury arc lamp; 494/41 nm filter for mPapayas; 500/24 nm filter for mCitrine and mVenus. C&D: LED excitation; 525 nm LED with 494/41 nm filter for mPapayas; 460 nm LED for mCitrine and mVenus. E&F: Confocal laser scanning with a 515 nm laser. From [1].

It’s hard to compare between the mPapaya and mCitrine / mVenus proteins because different filters were used in each panel, but mCitrine / mVenus can be compared and mPapaya1 / 0.6 can be compared. mCitrine and mVenus behave the same under all illumination conditions, with mVenus slightly more photostable, but the photostability of the mPapayas vary depending under illumination conditions. Under mercury arc lamp illumination mPapaya0.6 and mPapaya1 are about equally photostable, but under LED or laser illumination mPapaya1 is substantially more photostable.

There are a number of reasons that there may be this difference in bleaching rates (excitation wavelength and peak intensity come immediately to mind) but what it does clearly show is the difficulty in coming up with a global ranking of photostability of proteins. If the relative photostability of proteins depends on the conditions they are measured under, then you need to specify the measurement conditions to know the relevant bleaching rate. If you’re trying to compile data for many proteins from many different papers (like we are), then saying something sensible about relative photostability becomes very challenging.

References

  1. H. Hoi, E. Howe, Y. Ding, W. Zhang, M. Baird, B. Sell, J. Allen, M. Davidson, and R. Campbell, "An Engineered Monomeric Zoanthus sp. Yellow Fluorescent Protein", Chemistry & Biology, vol. 20, pp. 1296-1304, 2013. http://dx.doi.org/10.1016/j.chembiol.2013.08.008

2 thoughts on “Fluorescent Protein Photobleaching and Light Source

  1. I’m a novice in this area. I would like to photobleach YFP of an aliquot of cells in suspension, which have a conformation-based CFP-YFP FRET reporter, so as to be able to measure the increased CFP emission that results from dequenching. The photobleaching needs to be relatively uniform, and I need to be able to recover enough cells so that they can be studied by flow cytometry, as compared to an aliquot of unbleached cells. Can anyone recommend a way to do this?

    • You could probably do this by focusing a filtered arc lamp into a cuvette of cells, but I suspect you would need to illuminate for a long time to get enough photobleaching to measure. You might be better off using a microscopy-based FRET assay, where the bleaching and the FRET assay can be done on a microscope, or not using photobleaching to assess FRET efficiency.

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