If you’ve spent any time talking to microscopists or other people doing a lot of digital image analysis, at some point you’ve undoubtedly heard a sternly worded warning against using lossy compression, because it changes your data. Xerox has run into this problem in a particularly nasty way on some of their copiers. As first reported here, certain Xerox copiers, when scanning to PDF, will randomly replace some numbers with others. For example, 66 turns into 86 when scanned. This happens because of a lossy compression algorithm, JBIG2. Continue reading
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 . 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 . 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 .
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  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 .
- 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
- 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
- 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
Apologies for the delay in blog posts – I’ve been co-directing the 2013 QB3/UCSF Course in Biological Light Microscopy for all of last week. We taught about 60 hours of microscopy over the course of 6 days, to 24 students. It was a lot of fun and I think everyone had a good time. I’ve posted some photos from it.
In addition to a number of excellent local speakers who gave didactic lectures on various aspects of microscopy, we also had Loren Looger from HHMI Janelia Farm and Mark Schnitzer from Stanford in to give scientific lectures. Loren gave a great talk on the various reporters his lab has been developing, which include not only the GCaMP calcium reporters and the iGluSnFR glutamate reporter, but also some new cool things like a photoconvertible calcium reporter where the photoconversion efficiency is calcium dependent. Mark Schnitzer talked about his amazing in vivo endoscopy imaging, which is now being commercialized by a new company, Inscopix.
If you’re interested in learning more microscopy, keep an eye out for the announcement for next years course.