Paper Roundup – October 2016

  • A super-resolution reconstruction method applicable to both single-molecule data as well as denser data [1]
  • Multi-modal, multi-photon imaging for stain free histology [2]
  • uDISCO, an improved solvent-based clearing method compatible with fluorescent proteins [3]
  • Orientation measurement of single molecules in vivo [4]
  • Background estimation for single molecule microscopy [5]
  • A review of clearing methods and their methods of action [6]
  • A software tool for analyzing single molecule microscopy data [7]
  • Highly multiplexed STORM imaging using fluorescent nanodiamond fiducials and multiple rounds of antibody binding and elution [8]
  • A single-shot autofocusing method [9]
  • Assessing fluorescent protein aggregation by fusion to polyglutamine repeats [10]
  • Widefield epi-illumination for STORM using a custom illumination path to ensure uniform illumination [11]


  1. N. Gustafsson, S. Culley, G. Ashdown, D.M. Owen, P.M. Pereira, and R. Henriques, "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations", Nature Communications, vol. 7, 2016.
  2. H. Tu, Y. Liu, D. Turchinovich, M. Marjanovic, J.K. Lyngsø, J. Lægsgaard, E.J. Chaney, Y. Zhao, S. You, W.L. Wilson, B. Xu, M. Dantus, and S.A. Boppart, "Stain-free histopathology by programmable supercontinuum pulses", Nature Photonics, vol. 10, pp. 534-540, 2016.
  3. C. Pan, R. Cai, F.P. Quacquarelli, A. Ghasemigharagoz, A. Lourbopoulos, P. Matryba, N. Plesnila, M. Dichgans, F. Hellal, and A. Ertürk, "Shrinkage-mediated imaging of entire organs and organisms using uDISCO", Nature Methods, vol. 13, pp. 859-867, 2016.
  4. S.B. Mehta, M. McQuilken, P.J. La Riviere, P. Occhipinti, A. Verma, R. Oldenbourg, A.S. Gladfelter, and T. Tani, "Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells", Proceedings of the National Academy of Sciences, vol. 113, pp. E6352-E6361, 2016.
  5. S. Preus, L. Hildebrandt, and V. Birkedal, "Optimal Background Estimators in Single-Molecule FRET Microscopy", Biophysical Journal, vol. 111, pp. 1278-1286, 2016.
  6. K. Tainaka, A. Kuno, S.I. Kubota, T. Murakami, and H.R. Ueda, "Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling", Annual Review of Cell and Developmental Biology, vol. 32, pp. 713-741, 2016.
  7. S. Malkusch, and M. Heilemann, "Extracting quantitative information from single-molecule super-resolution imaging data with LAMA – LocAlization Microscopy Analyzer", Scientific Reports, vol. 6, 2016.
  8. J. Yi, A. Manna, V.A. Barr, J. Hong, K.C. Neuman, and L.E. Samelson, "madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy", Molecular Biology of the Cell, vol. 27, pp. 3591-3600, 2016.
  9. J. Liao, L. Bian, Z. Bian, Z. Zhang, C. Patel, K. Hoshino, Y.C. Eldar, and G. Zheng, "Single-frame rapid autofocusing for brightfield and fluorescence whole slide imaging", Biomedical Optics Express, vol. 7, pp. 4763, 2016.
  10. Y. Jiang, S.E. Di Gregorio, M.L. Duennwald, and P. Lajoie, "Polyglutamine toxicity in yeast uncovers phenotypic variations between different fluorescent protein fusions", Traffic, vol. 18, pp. 58-70, 2016.
  11. K.M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, "Super-resolution imaging of multiple cells by optimized flat-field epi-illumination", Nature Photonics, vol. 10, pp. 705-708, 2016.

Github pages

Github pages is awesome. I’ve been dimly aware of it for some time, but only just tried it. It’s really simple – if you have a Github repo that is a webpage, just tell Github that it should serve it as such, and it will become a live webpage. For instance, a few mouse clicks made my FPvisualization repository visible as a live webpage. Commits pushed to the repository automatically go live on the web.

Building a light sheet microscope around a Nikon AZ100, Part 1

A few years ago we got a Nikon AZ100 microscope on indefinite loan from a lab here that no longer was using. The AZ100 is an interesting microscope – it has low magnification objectives with relatively high numerical apertures (we have 1x / 0.1, 2x / 0.2, and 5x / 0.5 objectives) combined with a 1x – 8x optical zoom system to allow both large field-of-view imaging and high resolution imaging of the same sample. I initially set this up for routine fluorescence imaging, but it didn’t fill a useful niche and so largely went unused.

As groups on campus began testing various tissue clearing methods (CLARITY [1], PACT [2], iDISCO [3], …), I realized that this would make a good base for a simple “Ultramicroscope”-style [4] light sheet microscope. This is about the simplest kind of light sheet microscope you can build; you simply use a cylindrical lens to reshape an expanded laser beam to a sheet that propagates perpendicular to the optical axis of the microscope.  We had an old 561 nm Coherent Sapphire laser sitting around from a rebuild of the laser launch on our spinning disk confocal, so a few hundred dollars in Thorlabs parts sufficed to set up a demo system. The sample is placed in a cuvette on the microscope stage, illuminated with the light sheet from the side, and imaged with the objective from above.

The initial light sheet test system.

The initial light sheet test system. The laser is mounted on the black table; to the left you can see the mirrors used to direct the beam to propagate through the image plane, perpendicular to the optical axis. The cage system holds a Galilean beam expander and a slit; the cylindrical lens sits inside the dark enclosure. In the inset you can see the cylindrical lens and fluorescence excited in an agarose cylinder doped with fluorescent beads.

Continue reading


  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.
  2. B. Yang, J. Treweek, R. Kulkarni, B. Deverman, C. Chen, E. Lubeck, S. Shah, L. Cai, and V. Gradinaru, "Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing", Cell, vol. 158, pp. 945-958, 2014.
  3. N. Renier, Z. Wu, D. Simon, J. Yang, P. Ariel, and M. Tessier-Lavigne, "iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging", Cell, vol. 159, pp. 896-910, 2014.
  4. H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain", Nature Methods, vol. 4, pp. 331-336, 2007.