Paper Roundup: February 2016

  • Modeling the performance of light sheet microscopes in highly scattering tissues [1]
  • A review  of clearing techniques [2]
  • Combining multifocal microscopy and a cylindrical lens for 3D single-molecule localization over very large Z ranges [3]
  • Endogenous fluorescence tagging using CRISPR/Cas9 [4]
  • Improved versions of Clover and mRuby2 [5]
  • An improved multifocal microscope [6]
  • In situ hybridization in CLARITY-cleared tissues [7]
  • An inverted light-sheet microscope for imaging mouse embryo development [8]
  • Two papers describing imaging neuronal activity in free moving C. elegans [9] [10]
  • The 2016 single molecule localization microscopy challenge (see [11]


  1. A.K. Glaser, Y. Wang, and J.T. Liu, "Assessing the imaging performance of light sheet microscopies in highly scattering tissues", Biomedical Optics Express, vol. 7, pp. 454, 2016.
  2. E. Susaki, and H. Ueda, "Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals", Cell Chemical Biology, vol. 23, pp. 137-157, 2016.
  3. B. Hajj, M. El Beheiry, and M. Dahan, "PSF engineering in multifocus microscopy for increased depth volumetric imaging", Biomedical Optics Express, vol. 7, pp. 726, 2016.
  4. J. Stewart-Ornstein, and G. Lahav, "Dynamics of CDKN1A in Single Cells Defined by an Endogenous Fluorescent Tagging Toolkit", Cell Reports, vol. 14, pp. 1800-1811, 2016.
  5. B.T. Bajar, E.S. Wang, A.J. Lam, B.B. Kim, C.L. Jacobs, E.S. Howe, M.W. Davidson, M.Z. Lin, and J. Chu, "Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting", Scientific Reports, vol. 6, 2016.
  6. S. Abrahamsson, R. Ilic, J. Wisniewski, B. Mehl, L. Yu, L. Chen, M. Davanco, L. Oudjedi, J. Fiche, B. Hajj, X. Jin, J. Pulupa, C. Cho, M. Mir, M. El Beheiry, X. Darzacq, M. Nollmann, M. Dahan, C. Wu, T. Lionnet, J.A. Liddle, and C.I. Bargmann, "Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging", Biomedical Optics Express, vol. 7, pp. 855, 2016.
  7. E. Sylwestrak, P. Rajasethupathy, M. Wright, A. Jaffe, and K. Deisseroth, "Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution", Cell, vol. 164, pp. 792-804, 2016.
  8. P. Strnad, S. Gunther, J. Reichmann, U. Krzic, B. Balazs, G. de Medeiros, N. Norlin, T. Hiiragi, L. Hufnagel, and J. Ellenberg, "Inverted light-sheet microscope for imaging mouse pre-implantation development", Nature Methods, vol. 13, pp. 139-142, 2015.
  9. V. Venkatachalam, N. Ji, X. Wang, C. Clark, J.K. Mitchell, M. Klein, C.J. Tabone, J. Florman, H. Ji, J. Greenwood, A.D. Chisholm, J. Srinivasan, M. Alkema, M. Zhen, and A.D.T. Samuel, "Pan-neuronal imaging in roaming Caenorhabditis elegans", Proceedings of the National Academy of Sciences, vol. 113, pp. E1082-E1088, 2015.
  10. J.P. Nguyen, F.B. Shipley, A.N. Linder, G.S. Plummer, M. Liu, S.U. Setru, J.W. Shaevitz, and A.M. Leifer, "Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans", Proceedings of the National Academy of Sciences, vol. 113, pp. E1074-E1081, 2015.
  11. S. Holden, and D. Sage, "Super-resolution fight club", Nature Photonics, vol. 10, pp. 152-153, 2016.

Interlock distribution board

I assembled the interlock distribution box I mentioned previously. It was pretty straightforward to solder up three relays on a piece of perfboard. There is a single BNC input for the interlock loop, and BNC and phono jack outputs for our laser interlocks. Power is drawn from a 5V wall transformer. Pretty straightforward, and it works when installed on the microscope. The only surprising thing I learned is that the CSU-W1 interlock doesn’t close until the shutter on the CSU-W1 is open, so that shutter needs to be open for any lasers to operate.


XKCD on étendue

Today’s xkcd what if is about one of my favorite topics, étendue.  I discussed it briefly a little while ago, but briefly it says that the area of a light source times its solid angle as seen by the optical system must be a constant when propagated through that optical system. It’s a topic that can be counter-intuitive and takes some time to understand, and the XKCD explanation is actually quite good and relatively easy to follow – I would definitely recommend it as an introduction to the topic.

Interlocking multiple devices on a microscope

Over the last few years, we have been building out a progressively more complex microscope. It started life as a high speed widefield microscope (posted about here and here), was later upgraded to include a photoactivation and photobleaching system (see this post), and now has had a CSU-W1 spinning disk confocal added to it, courtesy of an S10 we were awarded.


A sketch of the overall microscope. The CSU-W1 has two camera ports, one with an Zyla 4.2 sCMOS camera and one with an EMCCD. The laser light for the CSU-W1 is delivered by fiber from the ILE laser launch. The box labeled Rapp is the photobleaching scanner, which has fiber connections to the 405 and 473 nm lasers. The Lumencor Spectra-X provides brightfield illumination, and the Zyla 5.5 is used for widefield imaging.

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Paper Roundup: January 2016

  • Microscopy Image Browser: a new open source tool for image analysis and visualization, written in Matlab [1]
  • A microscopy system for imaging neuronal activity in freely moving C. elegans [2]
  • A fluorescent protein tag that features rapid reversible binding of the chromophore [3]
  • Protocols for STORM imaging in tissue sections [4]
  • Fluorescent proteins for three and four color imaging in yeast [5]
  • Selecting protein markers for optimal phenotypic screening of drugs [6]
  • A set of refined protocols for CLARITY clearing, refractive index matching, and antibody penetration called ACT-PRESTO [7]
  • Simple chemical methods for making dye-photostabilizer conjugates resulting in dyes with reduced photobleaching rates [8]
  • Proximal cyclooctatetraene is a general dye photostabilizer [9]
  • Combining Fourier ptychography, pupil function estimation, and deconvolution [10]
  • Using a spherically aberrated point spread function to improve imaging depth in light-sheet microscopy [11]
  • Fluorescent speckle microscopy by speckled photoswitching [12]
  • Whole brain two-photon imaging with clearing and integrated sectioning [13]
  • Combining STED and RESOLFT to improve contrast and photostability [14]
  • Measuring resolution in coherent microscopy (technical, but has a nice set of references for resolution limits in microscopy) [15]
  • Photoacoustic tomography with a reversibly photoswitchable probe to improve contrast [16]
  • Incorporating probes into live cells by squeezing them through a aperture [17]


  1. I. Belevich, M. Joensuu, D. Kumar, H. Vihinen, and E. Jokitalo, "Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets", PLOS Biology, vol. 14, pp. e1002340, 2016.
  2. J.P. Nguyen, F.B. Shipley, A.N. Linder, G.S. Plummer, M. Liu, S.U. Setru, J.W. Shaevitz, and A.M. Leifer, "Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans", Proceedings of the National Academy of Sciences, vol. 113, pp. E1074-E1081, 2015.
  3. M. Plamont, E. Billon-Denis, S. Maurin, C. Gauron, F.M. Pimenta, C.G. Specht, J. Shi, J. Quérard, B. Pan, J. Rossignol, K. Moncoq, N. Morellet, M. Volovitch, E. Lescop, Y. Chen, A. Triller, S. Vriz, T. Le Saux, L. Jullien, and A. Gautier, "Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo", Proceedings of the National Academy of Sciences, vol. 113, pp. 497-502, 2015.
  4. L. Barna, B. Dudok, V. Miczán, A. Horváth, Z.I. László, and I. Katona, "Correlated confocal and super-resolution imaging by VividSTORM", Nature Protocols, vol. 11, pp. 163-183, 2015.
  5. R. Higuchi-Sanabria, E.J. Garcia, D. Tomoiaga, E.L. Munteanu, P. Feinstein, and L.A. Pon, "Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae", PLOS ONE, vol. 11, pp. e0146120, 2016.
  6. J. Kang, C. Hsu, Q. Wu, S. Liu, A.D. Coster, B.A. Posner, S.J. Altschuler, and L.F. Wu, "Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines", Nature Biotechnology, vol. 34, pp. 70-77, 2016.
  7. E. Lee, J. Choi, Y. Jo, J.Y. Kim, Y.J. Jang, H.M. Lee, S.Y. Kim, H. Lee, K. Cho, N. Jung, E.M. Hur, S.J. Jeong, C. Moon, Y. Choe, I.J. Rhyu, H. Kim, and W. Sun, "ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging", Scientific Reports, vol. 6, 2016.
  8. J.H.M. van der Velde, J. Oelerich, J. Huang, J.H. Smit, A. Aminian Jazi, S. Galiani, K. Kolmakov, G. Gouridis, C. Eggeling, A. Herrmann, G. Roelfes, and T. Cordes, "A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization", Nature Communications, vol. 7, 2016.
  9. Q. Zheng, S. Jockusch, G.G. Rodríguez-Calero, Z. Zhou, H. Zhao, R.B. Altman, H.D. Abruña, and S.C. Blanchard, "Intra-molecular triplet energy transfer is a general approach to improve organic fluorophore photostability", Photochemical & Photobiological Sciences, vol. 15, pp. 196-203, 2016.
  10. J. Chung, J. Kim, X. Ou, R. Horstmeyer, and C. Yang, "Wide field-of-view fluorescence image deconvolution with aberration-estimation from Fourier ptychography", Biomedical Optics Express, vol. 7, pp. 352, 2016.
  11. R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, "SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function", Cell, vol. 163, pp. 1796-1806, 2015.
  12. A.J. Pereira, P. Aguiar, M. Belsley, and H. Maiato, "Inducible fluorescent speckle microscopy", Journal of Cell Biology, vol. 212, pp. 245-255, 2016.
  13. M.N. Economo, N.G. Clack, L.D. Lavis, C.R. Gerfen, K. Svoboda, E.W. Myers, and J. Chandrashekar, "A platform for brain-wide imaging and reconstruction of individual neurons", eLife, vol. 5, 2016.
  14. J.G. Danzl, S.C. Sidenstein, C. Gregor, N.T. Urban, P. Ilgen, S. Jakobs, and S.W. Hell, "Coordinate-targeted fluorescence nanoscopy with multiple off states", Nature Photonics, vol. 10, pp. 122-128, 2016.
  15. R. Horstmeyer, R. Heintzmann, G. Popescu, L. Waller, and C. Yang, "Standardizing the resolution claims for coherent microscopy", Nature Photonics, vol. 10, pp. 68-71, 2016.
  16. J. Yao, A.A. Kaberniuk, L. Li, D.M. Shcherbakova, R. Zhang, L. Wang, G. Li, V.V. Verkhusha, and L.V. Wang, "Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe", Nature Methods, vol. 13, pp. 67-73, 2015.
  17. A. Kollmannsperger, A. Sharei, A. Raulf, M. Heilemann, R. Langer, K.F. Jensen, R. Wieneke, and R. Tampé, "Live-cell protein labelling with nanometre precision by cell squeezing", Nature Communications, vol. 7, 2016.