More on CLARITY

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.

Weekly Paper Roundup: Week of April 22

  • A new quantum dot synthesis that gives smaller quantum dots with narrower linewidths and less bleaching [1].
  • A photoactivatible degron that allows protein degradation to be triggered by blue light.  Includes a cool photo taken with yeast. [2].

References

  1. O. Chen, J. Zhao, V.P. Chauhan, J. Cui, C. Wong, D.K. Harris, H. Wei, H. Han, D. Fukumura, R.K. Jain, and M.G. Bawendi, "Compact high-quality CdSe–CdS core–shell nanocrystals with narrow emission linewidths and suppressed blinking", Nature Materials, vol. 12, pp. 445-451, 2013. http://dx.doi.org/10.1038/nmat3539
  2. C. Renicke, D. Schuster, S. Usherenko, L. Essen, and C. Taxis, "A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function", Chemistry & Biology, vol. 20, pp. 619-626, 2013. http://dx.doi.org/10.1016/j.chembiol.2013.03.005

Weekly paper roundup: Week of April 15th

  • A new GPU-based deconvolution plugin for Fiji/ImageJ that promises Richardson-Lucy deconvolution in around a second. [1]
  • A careful comparison of localization between immunofluorescence and fluorescent protein tagging. Gratifyingly, the two methods agree well. [2]
  • A new approach to calculating localization for super-resolution microscopy approaches using high magnification on an EMCCD that claims a 2-fold improvement in accuracy [3]
  • Injectable, wireless, ultrasmall LEDs used for optogenetics in free-running mice [4]

References

  1. M.A. Bruce, and M.J. Butte, "Real-time GPU-based 3D Deconvolution", Optics Express, vol. 21, pp. 4766, 2013. http://dx.doi.org/10.1364/OE.21.004766
  2. C. Stadler, E. Rexhepaj, V.R. Singan, R.F. Murphy, R. Pepperkok, M. Uhlén, J.C. Simpson, and E. Lundberg, "Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells", Nature Methods, vol. 10, pp. 315-323, 2013. http://dx.doi.org/10.1038/nmeth.2377
  3. J. Chao, S. Ram, E.S. Ward, and R.J. Ober, "Ultrahigh accuracy imaging modality for super-localization microscopy", Nature Methods, vol. 10, pp. 335-338, 2013. http://dx.doi.org/10.1038/nmeth.2396
  4. T. Kim, J.G. McCall, Y.H. Jung, X. Huang, E.R. Siuda, Y. Li, J. Song, Y.M. Song, H.A. Pao, R. Kim, C. Lu, S.D. Lee, I. Song, G. Shin, R. Al-Hasani, S. Kim, M.P. Tan, Y. Huang, F.G. Omenetto, J.A. Rogers, and M.R. Bruchas, "Injectable, Cellular-Scale Optoelectronics with Applications for Wireless Optogenetics", Science, vol. 340, pp. 211-216, 2013. http://dx.doi.org/10.1126/science.1232437

Public Service Announcement: Fans

Don’t ever put any equipment with fans on your air table!

We just spent several days trying to track down the source of an image non-uniformity on our confocal. This was an intermittent problem that would manifest itself either as horizontal lines in the image of varying intensity, or sinusoidal image distortions. It occured after moving the microscope, and we first thought it was electrical interference. We spent an hour or two recabling the microscope without any luck.  Next we checked the laser launch and its table for vibration, made sure the air table was floated properly … no luck, again.  Finally, we realized that the arc lamp that used to be on its own shelf was now on the table with the confocal. Removing the arc lamp solved the problem – it turned out that vibration from the ventilation fan was coupled through the table to the confocal.

We’ve seen this problem with cameras as well, which is harder to solve since taking the camera off the microscope is not an option. Typically the solution is replacing the camera fan.

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 microalga Chlamydomonas 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

Choosing a PC for High Speed Imaging with the Andor Zyla

We’ve been fortunate enough to get our hands on a 10-tap Andor Zyla camera. This is a new sCMOS camera that is capable of reading out its full field of view (5.5 megapixels) at 100 frames per second. At two bytes per pixel, that’s 11 MB per image, for a total data rate of 1.1 gigabytes per second. It turns out that acquiring and storing 1.1 GB/s is not so easy. A solid state drive (SSD) can deliver sustained write speeds of 520 MB/s, about half of what we need. The solution is to use multiple solid state drives, in RAID 0, so that we can write to them in parallel. Both Hamamatsu (for their Flash4.0) and Andor recommend four SSDs in RAID 0.  In principle, with four SSDs at 520 MB/s, we should be able to get a total data transfer rate of 2.08 GB/s, assuming nothing else is limiting.

However, getting this data transfer rate in practice is not so easy. We just bought such a setup to go with our new Zyla. It has an Intel RAID card and four 240GB Intel SSDs. The host PC has two quad-core Xeons with a 1600 MHz bus clock. In principle it seems that it ought to run somewhere close to 2 GB/s. However, it turns out that getting these speeds in practice is not so easy. Out of the box, it ran at about 500 MB/s. The problem turned out to be that we had not enabled write caching on the device in Windows; once we did that the write speed jumped to about 950 MB/s.  This is still lower than it seems like we should be able to get and not quite the 1100 MB/s we would need to continually stream data from that camera.  Unfortunately, figuring out how to adjust the raid settings for optimal performance is not so easy (for example) and there is a lack of real-world documentation on how to optimize RAID settings for maximum write throughput.  Partly this is because sustained write performance is a little bit of an unusual requirement – most applications care a lot more about random read/write performance. So it seems like for now I’ll be adjusting settings on the RAID controller to see what combination of settings gives the highest write throughput. If anyone reading this knows how to set up our RAID for maximum speed with out me blindly trying settings, please let me know.

It turns out that the PC hardware isn’t the only limiting factor in handling this much data. The Micro-manager team has been busy rewriting the Micro-manager core to minimize overhead and handle these high data rates in Micro-manager.  Right now we are able to run continuously with 15 msec exposure times, or 66.7 frames per second. Hopefully we’ll be able to get to the full 100 fps rate with a little tweaking. Not surprisingly, you can fill up hard drives pretty fast this way – at 66.7 fps, we’ll fill our entire RAID 0 drive in about 20 minutes.

Assuming we do get our RAID up to speeds above 1.1GB/s, I’ll post how we did it.

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