Monthly Archives: July 2013

Graphing Fluorescent Protein Properties

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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

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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.

Speeding up the ASI Stage

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As I’ve discussed previously, I’ve been working on building a system for high speed image stitching. I’ve described previously a number of methods I’ve tried for image acquisition. To get high speed mosaic scanning, I’ve tried software and hardware control of the stage, as well as rapid stage scanning and strobe illumination. To date, however, I haven’t tried very hard to speed up the stage itself. I spent a few days last week trying to make the ASI stage move as fast as possible, and it turns out you can get more than a two-fold increase in speed just by configuring the stage correctly. Continue reading →

Problems and solutions with our SSD array

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I’ve talked about our high speed solid state drive array previously.  In general it’s been working well since we set it up.  However, a few weeks ago, after a fair amount of use, we discovered that its speed had dropped to 400 MB/sec from 2100 MB/sec when we set it up.  Since we want to be able to collect data at 1100 MB/sec, this was a problem.  I’m pretty sure that the problem is related to the way SSDs store data. Unlike magnetic hard drives, SSDs can’t just overwrite previously written areas – they must be erased first. This means that if you fill up an SSD, delete some files, and then want to write more files to the disk, the previously used areas must be erased first. However, erasing is slower than writing, so this erasing slows down performance of the drive.  For a good intro to SSDs and how they work, see this article.

Consistent with this, erasing our SSD RAID0 array through the RAID controller control panel and reformatting the disk (so that Windows could recognize it) restored it to its original performance. However, it would be nice if there was a way to deal with this that didn’t require spending an hour erasing the drive. Continue reading →

New Microscopes From the Shroff Lab

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One of the treats of being at this year’s running of the MBL Physiology course was getting to meet Hari Shroff, who’s at the course for the summer as an imaging scholar. He’s brought two students / postdocs with him and is building two novel microscopes: an all-optical structured illumination system (SIM) and a dual view SPIM / light-sheet system. I’ll discuss the all-optical SIM here and the SPIM in another post. Continue reading →

Paper Roundup: June 2013

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  • A systematic comparison of three flavin binding fluorescent proteins, including iLOV. [1]
  • A green fluorescent protein, named UnaG, has been discovered in the freshwater eel. It uses bilirubin as the chromophore, and is comparable in brightness to GFP. [2]
  • Lab on a Chip has a theme issue on optofluidics [3] which includes this interesting paper that uses GFP lasing as a readout for energy transfer from GFP to an acceptor molecule.  It claims to be much more sensitive than conventional FRET readouts [4].
  • Three STORM protocols have been published by the Zhuang lab: an introduction [5], a protocol for making dual-labeled antibodies [6], and a protocol for transfecting photoactivatible fluorescent proteins [7].  The two protocols are pretty short, but the introduction is detailed.
  • A nice paper looking at DNA looping by subresolution tracking of lacI and tetR bound to three repeats of their binding sites on the E. coli chromosome. [8]
  • A review of imaging in live animals [9].
  • A new clearing method for brain and other tissues.  It uses fructose and thioglycerol, clears a mouse brain in just a few days, and appears to work about as well as Scale. [10].
  • A review on super-resolution imaging of DNA [11].
  • A new set of infrared fluorescent proteins from the Verkushka lab.  Like the previous iRFP, these are dimeric, but they span the wavelength range from 670nm – 720nm and enable multicolor imaging in the near-IR. [12].
  • The latest issue of Nature Methods has two papers on open hardware SPIM setups: OpenSpinMicroscopy and OpenSPIM. Both use off-the-shelf hardware and open source software to run the system.  The OpenSpinMicroscopy page includes filter wheels and galvos controlled by an Arduino board. [13], [14]
  • An improved long Stokes shift protein, mBeRFP, that is supposed to be 3x brighter than mKeima and LSS-mKate2. [15].

References

  1. A. Mukherjee, J. Walker, K.B. Weyant, and C.M. Schroeder, "Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters", PLoS ONE, vol. 8, pp. e64753, 2013. http://dx.doi.org/10.1371/journal.pone.0064753
  2. A. Kumagai, R. Ando, H. Miyatake, P. Greimel, T. Kobayashi, Y. Hirabayashi, T. Shimogori, and A. Miyawaki, "A Bilirubin-Inducible Fluorescent Protein from Eel Muscle", Cell, vol. 153, pp. 1602-1611, 2013. http://dx.doi.org/10.1016/j.cell.2013.05.038
  3. A. Liu, and C. Yang, "Optofluidics 2013", Lab on a Chip, vol. 13, pp. 2673, 2013. http://dx.doi.org/10.1039/C3LC90054A
  4. Q. Chen, X. Zhang, Y. Sun, M. Ritt, S. Sivaramakrishnan, and X. Fan, "Highly sensitive fluorescent protein FRET detection using optofluidic lasers", Lab on a Chip, vol. 13, pp. 2679, 2013. http://dx.doi.org/10.1039/C3LC50207D
  5. M. Bates, S.A. Jones, and X. Zhuang, "Stochastic Optical Reconstruction Microscopy (STORM): A Method for Superresolution Fluorescence Imaging", Cold Spring Harbor Protocols, vol. 2013, pp. pdb.top075143, 2013. http://dx.doi.org/10.1101/pdb.top075143
  6. M. Bates, S.A. Jones, and X. Zhuang, "Preparation of Photoswitchable Labeled Antibodies for STORM Imaging", Cold Spring Harbor Protocols, vol. 2013, pp. pdb.prot075168, 2013. http://dx.doi.org/10.1101/pdb.prot075168
  7. M. Bates, S.A. Jones, and X. Zhuang, "Transfection of Genetically Encoded Photoswitchable Probes for STORM Imaging: Table 1.", Cold Spring Harbor Protocols, vol. 2013, pp. pdb.prot075150, 2013. http://dx.doi.org/10.1101/pdb.prot075150
  8. Z. Hensel, X. Weng, A.C. Lagda, and J. Xiao, "Transcription-Factor-Mediated DNA Looping Probed by High-Resolution, Single-Molecule Imaging in Live E. coli Cells", PLoS Biology, vol. 11, pp. e1001591, 2013. http://dx.doi.org/10.1371/journal.pbio.1001591
  9. R. Weigert, N. Porat-Shliom, and P. Amornphimoltham, "Imaging cell biology in live animals: Ready for prime time", Journal of Cell Biology, vol. 201, pp. 969-979, 2013. http://dx.doi.org/10.1083/jcb.201212130
  10. M. Ke, S. Fujimoto, and T. Imai, "SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction", Nature Neuroscience, vol. 16, pp. 1154-1161, 2013. http://dx.doi.org/10.1038/nn.3447
  11. C. FLORS, "Super-resolution fluorescence imaging of directly labelled DNA: from microscopy standards to living cells", Journal of Microscopy, vol. 251, pp. 1-4, 2013. http://dx.doi.org/10.1111/jmi.12054
  12. D.M. Shcherbakova, and V.V. Verkhusha, "Near-infrared fluorescent proteins for multicolor in vivo imaging", Nature Methods, vol. 10, pp. 751-754, 2013. http://dx.doi.org/10.1038/nMeth.2521
  13. E.J. Gualda, T. Vale, P. Almada, J.A. Feijó, G.G. Martins, and N. Moreno, "OpenSpinMicroscopy: an open-source integrated microscopy platform", Nature Methods, vol. 10, pp. 599-600, 2013. http://dx.doi.org/10.1038/nmeth.2508
  14. P.G. Pitrone, J. Schindelin, L. Stuyvenberg, S. Preibisch, M. Weber, K.W. Eliceiri, J. Huisken, and P. Tomancak, "OpenSPIM: an open-access light-sheet microscopy platform", Nature Methods, vol. 10, pp. 598-599, 2013. http://dx.doi.org/10.1038/nmeth.2507
  15. J. Yang, L. Wang, F. Yang, H. Luo, L. Xu, J. Lu, S. Zeng, and Z. Zhang, "mBeRFP, an Improved Large Stokes Shift Red Fluorescent Protein", PLoS ONE, vol. 8, pp. e64849, 2013. http://dx.doi.org/10.1371/journal.pone.0064849