Preprint: Review of Genetically Encoded Fluorescent Tags

I was recently asked to write a brief Technical Perspective on fluorescent tags for Molecular Biology of the Cell. These are meant to be introductions to a topic for novices in the field; I previously wrote one on light microscopy.

I’ve posted a preprint of the fluorescent tag review here; please send me any comments and I will incorporate them into the final version. I would have posted the preprint on BioRxiv, but it seems that they don’t host reviews.

Paper Roundup – November 2016

  • A detailed investigation of ER structure by multiple super-resolution methods [1]
  • Using deep convolutional neural networks to segment cells automatically with high accuracy [2]
  • A light sheet microscope that automatically adjusts the illumination plane to correct for sample-induced distortion [3]
  • Tools for scanning angle interference microscopy (SAIM) acquisition and analysis [4]
  • Super-resolution mapping of fluorophore orientation [5]
  • Isotropic point spread functions for fast cellular resolution 2-photon imaging [6]
  • CyRFP1, a long Stokes shift fluorescent protein co-excited with GFP but with separable emission [7]
  • mMaroon1, a new far-red fluorescent protein, and a four-color Fucci cell cycle sensor [8]
  • Multi-color electron microscopy [9]
  • A detailed review of fluorescent proteins [10]
  • A nice discussion of challenges in live cell time lapse imaging [11]
  • Ni2+ as a triplet state quencher for improved light output from Cy3 and Cy5 [12]
  • A python tool for image analysis [13]
  • Optimal reconstruction of 2D-SIM data [14]
  • A new bright monomeric red fluorescent protein, mScarlet [15]
  • A review of fluorescent tagging methods [16]
  • Adaptive SIM microscopy to reduce bleaching [17]
  • Tools for cluster analysis of single molecule localization microscopy methods [18]
  • A super-resolution microscope based on incoherent holography [19]


  1. J. Nixon-Abell, C.J. Obara, A.V. Weigel, D. Li, W.R. Legant, C.S. Xu, H.A. Pasolli, K. Harvey, H.F. Hess, E. Betzig, C. Blackstone, and J. Lippincott-Schwartz, "Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER", Science, vol. 354, pp. aaf3928-aaf3928, 2016.
  2. D.A. Van Valen, T. Kudo, K.M. Lane, D.N. Macklin, N.T. Quach, M.M. DeFelice, I. Maayan, Y. Tanouchi, E.A. Ashley, and M.W. Covert, "Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments", PLOS Computational Biology, vol. 12, pp. e1005177, 2016.
  3. L.A. Royer, W.C. Lemon, R.K. Chhetri, Y. Wan, M. Coleman, E.W. Myers, and P.J. Keller, "Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms", Nature Biotechnology, vol. 34, pp. 1267-1278, 2016.
  4. C.B. Carbone, R.D. Vale, and N. Stuurman, "An acquisition and analysis pipeline for scanning angle interference microscopy", Nature Methods, vol. 13, pp. 897-898, 2016.
  5. K. Zhanghao, L. Chen, X. Yang, M. Wang, Z. Jing, H. Han, M.Q. Zhang, D. Jin, J. Gao, and P. Xi, "Super-resolution dipole orientation mapping via polarization demodulation", Light: Science & Applications, vol. 5, pp. e16166, 2016.
  6. R. Prevedel, A.J. Verhoef, A.J. Pernía-Andrade, S. Weisenburger, B.S. Huang, T. Nöbauer, A. Fernández, J.E. Delcour, P. Golshani, A. Baltuska, and A. Vaziri, "Fast volumetric calcium imaging across multiple cortical layers using sculpted light", Nature Methods, vol. 13, pp. 1021-1028, 2016.
  7. T. Laviv, B.B. Kim, J. Chu, A.J. Lam, M.Z. Lin, and R. Yasuda, "Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins", Nature Methods, vol. 13, pp. 989-992, 2016.
  8. B.T. Bajar, A.J. Lam, R.K. Badiee, Y. Oh, J. Chu, X.X. Zhou, N. Kim, B.B. Kim, M. Chung, A.L. Yablonovitch, B.F. Cruz, K. Kulalert, J.J. Tao, T. Meyer, X. Su, and M.Z. Lin, "Fluorescent indicators for simultaneous reporting of all four cell cycle phases", Nature Methods, vol. 13, pp. 993-996, 2016.
  9. S. Adams, M. Mackey, R. Ramachandra, S. Palida Lemieux, P. Steinbach, E. Bushong, M. Butko, B. Giepmans, M. Ellisman, and R. Tsien, "Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species", Cell Chemical Biology, vol. 23, pp. 1417-1427, 2016.
  10. E.A. Rodriguez, R.E. Campbell, J.Y. Lin, M.Z. Lin, A. Miyawaki, A.E. Palmer, X. Shu, J. Zhang, and R.Y. Tsien, "The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins", Trends in Biochemical Sciences, vol. 42, pp. 111-129, 2017.
  11. S. Skylaki, O. Hilsenbeck, and T. Schroeder, "Challenges in long-term imaging and quantification of single-cell dynamics", Nature Biotechnology, vol. 34, pp. 1137-1144, 2016.
  12. V. Glembockyte, J. Lin, and G. Cosa, "Improving the Photostability of Red- and Green-Emissive Single-Molecule Fluorophores via Ni2+ Mediated Excited Triplet-State Quenching", The Journal of Physical Chemistry B, vol. 120, pp. 11923-11929, 2016.
  13. T.S.G. Olsson, and M. Hartley, "jicbioimage: a tool for automated and reproducible bioimage analysis", PeerJ, vol. 4, pp. e2674, 2016.
  14. V. Perez, B. Chang, and E.H.K. Stelzer, "Optimal 2D-SIM reconstruction by two filtering steps with Richardson-Lucy deconvolution", Scientific Reports, vol. 6, pp. 37149, 2016.
  15. D.S. Bindels, L. Haarbosch, L. van Weeren, M. Postma, K.E. Wiese, M. Mastop, S. Aumonier, G. Gotthard, A. Royant, M.A. Hink, and T.W.J. Gadella, "mScarlet: a bright monomeric red fluorescent protein for cellular imaging", Nature Methods, vol. 14, pp. 53-56, 2016.
  16. E.A. Specht, E. Braselmann, and A.E. Palmer, "A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging", Annual Review of Physiology, vol. 79, pp. 93-117, 2017.
  17. N. Chakrova, A.S. Canton, C. Danelon, S. Stallinga, and B. Rieger, "Adaptive illumination reduces photobleaching in structured illumination microscopy", Biomedical Optics Express, vol. 7, pp. 4263, 2016.
  18. J. Griffié, M. Shannon, C.L. Bromley, L. Boelen, G.L. Burn, D.J. Williamson, N.A. Heard, A.P. Cope, D.M. Owen, and P. Rubin-Delanchy, "A Bayesian cluster analysis method for single-molecule localization microscopy data", Nature Protocols, vol. 11, pp. 2499-2514, 2016.
  19. N. Siegel, V. Lupashin, B. Storrie, and G. Brooker, "High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers", Nature Photonics, vol. 10, pp. 802-808, 2016.

Access problems with https://

I’ve had a couple of reports of people unable read the posts by clicking on the front page links. At least some of this is due to the blog only being set up to work with HTTP.  If  you try and access pages through HTTPS, you’ll run into problems. The way to fix this seems to be to switch all links over to HTTPS.  I’ll do that in the next few weeks (unfortunately, it’s not totally straightforward), but in the meantime, if you can’t access a page, try accessing it via http:// instead of https://.

Testing a Point Grey Camera for Fluorescence Microscopy

About two years ago, I mentioned Point Grey cameras. These are cameras sold to the machine vision and industrial inspection market, and are much cheaper than typical microscopy cameras – most are <$1000. Point Grey puts out very nice spec sheets listing all of their cameras, and the specifications for some are pretty impressive – cameras with < 3e- read noise for ~$500. Nico Stuurman has recently written a Micro-manager driver for these cameras, and was kind enough to let me test one of these cameras. We mounted it opposite a Hamamatsu Flash4.0 (an older Flash4.0, with ~72% QE), and did a qualitative comparison by taking sequential images of the same test slide on both cameras.

The Point Grey camera we tested was a Chameleon3 CM3-U3-31S4M. This uses a Sony IMX265 sensor, which has 2048 x 1536 3.45 μm pixels, with 71% QE, <3e- read noise, and sells for ~$500. It can run at up to 55 fps. On paper, this camera should perform almost as well as the Flash 4.0. The images below are of a Texas red-phalloidin stained cell, captured with a 20x / 0.75 NA objective and a 10 ms exposure on both cameras. Click on the images to see the full size image.


The Flash 4.0 camera, 10 ms exposure. Click for full size.


The Point Grey camera, 10 ms exposure. Click for full size.

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New Nikon Stand

Nikon has just announced a new stand, the Ti2.  Some noteworthy features, including a 25mm camera port with an F-mount (with a new tube lens and larger filter cubes; it looks like the Plan Apo λ objectives are flat across this field), an LED brightfield illuminator with a fly-eye lens for uniform illumination, a motorized correction collar, an internal camera for back focal plane imaging, and encoding of all microscope components.