I recently came back from the east coast with an unwanted guest attached to me: a tick, probably a lone star tick, Amblyomma americanum. After removing it, I decided to have some fun with it – I dehydrated it in methanol, cleared it in methyl salicylate, and then imaged it on our spinning disk confocal. The movie below is stitched from four images, and is about 1.8 mm on a side and 1.2 mm thick. Total image size is ~2800 x 2800 x 306. The fluorescence is endogenous autofluorescence excited at 488 and 561 nm. This is probably a nymphal tick, and it looks like the mouthparts are missing.
I was recently contacted by a company that makes media designed to minimize phototoxicity to cells during fluorescence imaging. For those of you who are having problems with cell death or damage during extended fluorescence imaging, it may be worth trying. It’s called LiveLight, from Cell Guidance Systems, and they make a couple different formulations. This joins similar products from Evrogen and ThermoFisher (which may only reduce background, but we’ve had good luck with it). I haven’t tried the LiveLight media, so I would interested in hearing from anyone who has.
A number of our users have been testing out the low-fluorescence DMEM from Life Technologies, Fluorobrite DMEM, and many have found that it substantially improves the signal-to-background of their experiments, particularly for imaging dim samples. Recently we tried it when doing sptPALM of PA-GFP, and found that it made a substantial difference in image quality. Below are two movies, taken under identical conditions, and scaled identically, of single PA-GFP labeled receptors diffusing in the membrane of a cell. They are illuminated with modest 488 nm excitation and weak 405 nm excitation to switch PA-GFP on. The movies were acquired by TIRF, and are shown in real time. The top movie is of cells in conventional DMEM-based media; the bottom, cells in Fluorobrite DMEM. The Fluorobrite media substantially improves the image contrast; there’s also a noticeable reduction in background events outside of the cell.
If you’re doing low-signal imaging, particularly in the GFP channel, I think the Fluorobrite media is well worth trying out.
I mentioned the new CLARITY clearing method from Karl Deisseroth’s lab in one of my weekly paper roundups a while back and I’ve finally had a chance to read through it more carefully.
CLARITY cleared brain
Not only is it an impressive piece of work, but they have put together a nice website with detailed protocols and parts lists for building your own system. I’m considering putting one together at the NIC. I’m very curious to see what people are going to do with these techniques as they become available, but also a little terrified by the amount of microscopy data we’ll be able to generate with them.
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
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. 
- A set of fluorescent proteins for Chlamydomonas 
- A new super-bright green fluorescent protein, mNeonGreen 
- 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
- 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
- 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
This recent paper describes an improved dehydration and clearing method for clearing mouse brains and preserving GFP fluorescence. The use tetrahydrofuran, instead of ethanol, for dehydration, and dibenzyl ether for clearing, and show superior results to the use of BABB or methyl salicylate. They claim this works better than the Miyawaki lab Scale protocol in adult mouse brain.
This is figure 3 from their paper – the left side is conventionally cleared; the right side with their new method:
Reference: Chemical Clearing and Dehydration of GFP Expressing Mouse Brains
Becker K, Jährling N, Saghafi S, Weiler R, Dodt H-U (2012) Chemical Clearing and Dehydration of GFP Expressing Mouse Brains. PLoS ONE 7(3): e33916. doi:10.1371/journal.pone.0033916