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].

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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

Graphing Fluorescent Protein Properties

I spent the weekend learning d3.js, which is an javascript library for doing interactive graphics in the browser window. I used it to make a graph from my fluorescent protein list:

FPchartThe resulting graph plots excitation and emission wavelengths and brightness, and shows the identity of each protein on mouseover.  You can view it here.  I’m really only scratching the surface of what is possible with d3 (these examples show more of its power), and I hope to do more with it (and improve this graph) in the future.

 

New Fluorescent Proteins From DNA2.0

DNA2.0 has done something interesting: they’ve generated a bunch of new fluorescent proteins whose sequences are not covered by any existing patents. Specifically, they’ve taken a bunch of fluorescent protein sequences, shuffled them, synthesized the resulting sequences, and selected those that are fluorescent. These were then subjected to additional rounds of mutagenesis to produce their final set of fluorescent proteins. The entire process, and the resulting proteins, are described in this poster. They don’t describe the performance of these proteins compared to EGFP or other commonly used fluorescent proteins, so it’s hard to tell how good they are.  Nevertheless, these could be a useful resource for further development, unencumbered by IP restrictions.

Fluorescent Protein Aggregation

I’m currently at MBL, teaching microscopy in the Physiology course, hence the reduced rate of posting. Not surprisingly there’s been a lot of discussion about microscopy, including a great talk from Hari Shroff (about which more later). Today we had a talk from John Allen, standing in for Mike Davidson. John gave a very nice overview of the state of the art in the fluorescent protein field.  Most of what was covered is published and reasonably well known, but I did learn about a recent paper for a quantitative way of assessing fluorescent protein aggregation by fusing proteins to an ER membrane protein. Protein aggregation restructures the ER into smooth-ER like whorls that can be detected by imaging; the amount of such structures correlates with the aggregation of fluorescent proteins [1]. How well this correlates with protein function in other contexts remains to be seen, but a systematic assay for fluorescent protein aggregation potential is very welcome.

References

  1. L.M. Costantini, M. Fossati, M. Francolini, and E.L. Snapp, "Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.", Traffic (Copenhagen, Denmark), 2012. http://www.ncbi.nlm.nih.gov/pubmed/22289035

GFP photobleaching in live cells

One of the more surprising things (to me, anyway) that I’ve learned about GFP photobleaching in live cells is that it is strongly dependent on redox environment. It has been shown that GFP can undergo photochemical oxidation to a bleached form and then to a red form [1]. The electron acceptors in this oxidation can be various cellular components including flavins and flavoproteins and NAD+. This appears to be a major cause of GFP bleaching in vivo.  It can be greatly reduced by removing riboflavin or all vitamins from the culture medium DMEM [2]. More recent work showed that GFP bleaching due to riboflavin in the culture medium can be suppressed by adding rutin, a plant flavonol, 30 minutes prior to imaging [3].

If you’re concerned about GFP bleaching in your cells, it’s worth trying DMEM lacking all vitamins. It’s commercially available from Evrogen as DMEMgfp. It probably has lower fluorescent background as well. Rutin is commercially available as well, and easy to try if you don’t want to use DMEM without vitamins. There is a report in the literature that Trolox can reduce the bleaching of EBFP [4] but it is not clear if this is true for EGFP as well.

As an aside, the GFP oxidative reddening can be used for photoswitchable single molecule super-resolution imaging as well [5].

References

  1. A.M. Bogdanov, E.A. Bogdanova, D.M. Chudakov, T.V. Gorodnicheva, S. Lukyanov, and K.A. Lukyanov, "Cell culture medium affects GFP photostability: a solution", Nature Methods, vol. 6, pp. 859-860, 2009. http://dx.doi.org/10.1038/nmeth1209-859
  2. A.M. Bogdanov, E.I. Kudryavtseva, and K.A. Lukyanov, "Anti-Fading Media for Live Cell GFP Imaging", PLoS ONE, vol. 7, pp. e53004, 2012. http://dx.doi.org/10.1371/journal.pone.0053004
  3. A. Matsuda, L. Shao, J. Boulanger, C. Kervrann, P.M. Carlton, P. Kner, D. Agard, and J.W. Sedat, "Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones", PLoS ONE, vol. 5, pp. e12768, 2010. http://dx.doi.org/10.1371/journal.pone.0012768

Weekly paper roundup: Week of April 8th

I’ve decided to do a weekly roundup of interesting imaging papers I come across on a week by week basis.  Here’s what I’ve stumbled across this week:

  • From the Deisseroth lab – a new method of clearing tissues that permits amazingly deep microscopy. [1]
  • A set of fluorescent proteins for Chlamydomonas [2]
  • A new super-bright green fluorescent protein, mNeonGreen [3]

References

  1. K. Chung, J. Wallace, S. Kim, S. Kalyanasundaram, A.S. Andalman, T.J. Davidson, J.J. Mirzabekov, K.A. Zalocusky, J. Mattis, A.K. Denisin, S. Pak, H. Bernstein, C. Ramakrishnan, L. Grosenick, V. Gradinaru, and K. Deisseroth, "Structural and molecular interrogation of intact biological systems", Nature, vol. 497, pp. 332-337, 2013. http://dx.doi.org/10.1038/nature12107
  2. B.A. Rasala, D.J. Barrera, J. Ng, T.M. Plucinak, J.N. Rosenberg, D.P. Weeks, G.A. Oyler, T.C. Peterson, F. Haerizadeh, and S.P. Mayfield, "Expanding the spectral palette of fluorescent proteins for the green microalgaChlamydomonas reinhardtii", The Plant Journal, vol. 74, pp. 545-556, 2013. http://dx.doi.org/10.1111/tpj.12165
  3. N.C. Shaner, G.G. Lambert, A. Chammas, Y. Ni, P.J. Cranfill, M.A. Baird, B.R. Sell, J.R. Allen, R.N. Day, M. Israelsson, M.W. Davidson, and J. Wang, "A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum", Nature Methods, vol. 10, pp. 407-409, 2013. http://dx.doi.org/10.1038/nmeth.2413

Quantitative FRET microscopy

There’s a nice set of posts on the confocal listserv today about quantitative FRET microscopy using CFP and YFP.  A good cautionary introduction for those of you thinking about doing FRET microscopy.

As an aside, if you’re interested in biological light microscopy, you’d do well to join the confocal listserv – it covers a lot more than just confocal microscopy and has many knowledgeable and helpful readers.

New Addgene Plasmids

The Addgene newsletter turns out to be a surprisingly good source for news on new fluorescent reporters and optogenetic constructs.  In the March newsletter, there are plasmids for Loren Looger’s new glutamate reporter, iGluSnFr [1], a set of lentiviral vectors for RGB marking, which is like Brainbow for clonal analysis [2], and a new optogenetic channel [3]. December’s newsletter has the calcium reporter GCaMP6, a new split GFP, the Clover and mRuby2 FRET pair, and plasmids for RNA tagging using MS2 and a related system.

This seems like it’s worth keeping an eye on.

References

ABRF report, Day 2

The highlight of day two of ABRF, for me, was the talk by Robert Campbell on fluorescent reporters. He started off by saying that existing fluorescent proteins were quite good and unlikely to get dramatically better. He spent the rest of his talk discussing his labs work to improve fluorescent protein reporters, where there is a lot of work still to be done.  One of his guiding principles is “redder is better”.

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