Paper Roundup – October 2014

  • PSFj is a new tool to automatically measure point spread functions and report the performance of your microscope across the field of view. It looks promising, although I would love a similar tool that not only measured PSF size but also could fit a set of Zernike polynomials to evaluate astigmatishm, spherical aberration, etc. [1]
  • FEBS Letters has a special issue on single molecule approaches, including in vivo single molecule imaging.
  • There is a nice introduction to fluorescence and confocal microscopy in CSH Protocols [2]
  • An improved method for wavefront correction in the two-photon microscope for correcting sample-induced aberrations in deep imaging [3]
  • Single molecule imaging in living cells using 24 repeats of a peptide sequence fused to the protein of interest that is then bound by a coexpressed scFv [4]
  • Combinations of fluorescent proteins for 2-photon imaging [5]
  • Generation of long light sheets by scanning the focus of a gaussian beam using a acoustically tunable lens [6]
  • An improved system for localization microscopy using both astigmatism and biplane imaging [7]
  • The Betzig group has put together another technical tour-de-force – a lattice light sheet microscope capable of high resolution 3D imaging over long periods of time [8]
  • Plans for an open-hardware two-photo system using Thorlabs parts for the optomechanics [9]
  • Computational evolution of a resorufin ligase to covalently attach resorufin to a small, 13-aa tag [10]
  • A protocol describing assembly and alignment of the diSPIM system [11]
  • A high speed 3D imaging system using remote focusing and structured illumination [12]
  • A brighter green fluorescent RNA aptamer that binds DHFBI, Broccoli [13]
  • Using a fluorogen-activating protein for single particle tracking where particle density can be titrated by controlling the level of the fluorogen [14]


  1. P. Theer, C. Mongis, and M. Knop, "PSFj: know your fluorescence microscope", Nature Methods, vol. 11, pp. 981-982, 2014.
  2. M.J. Sanderson, I. Smith, I. Parker, and M.D. Bootman, "Fluorescence Microscopy", Cold Spring Harbor Protocols, vol. 2014, pp. pdb.top071795-pdb.top071795, 2014.
  3. C. Wang, R. Liu, D.E. Milkie, W. Sun, Z. Tan, A. Kerlin, T. Chen, D.S. Kim, and N. Ji, "Multiplexed aberration measurement for deep tissue imaging in vivo", Nature Methods, vol. 11, pp. 1037-1040, 2014.
  4. M. Tanenbaum, L. Gilbert, L. Qi, J. Weissman, and R. Vale, "A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging", Cell, vol. 159, pp. 635-646, 2014.
  5. S. Gossa, D. Nayak, B.H. Zinselmeyer, and D.B. McGavern, "Development of an Immunologically Tolerated Combination of Fluorescent Proteins for In vivo Two-photon Imaging", Scientific Reports, vol. 4, 2014.
  6. K.M. Dean, and R. Fiolka, "Uniform and scalable light-sheets generated by extended focusing", Optics Express, vol. 22, pp. 26141, 2014.
  7. J. Min, S.J. Holden, L. Carlini, M. Unser, S. Manley, and J.C. Ye, "3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction", Biomedical Optics Express, vol. 5, pp. 3935, 2014.
  8. B. Chen, W.R. Legant, K. Wang, L. Shao, D.E. Milkie, M.W. Davidson, C. Janetopoulos, X.S. Wu, J.A. Hammer, Z. Liu, B.P. English, Y. Mimori-Kiyosue, D.P. Romero, A.T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R.D. Mullins, D.M. Mitchell, J.N. Bembenek, A. Reymann, R. Böhme, S.W. Grill, J.T. Wang, G. Seydoux, U.S. Tulu, D.P. Kiehart, and E. Betzig, "Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution", Science, vol. 346, pp. 1257998, 2014.
  9. D.G. Rosenegger, C.H.T. Tran, J. LeDue, N. Zhou, and G.R. Gordon, "A High Performance, Cost-Effective, Open-Source Microscope for Scanning Two-Photon Microscopy that Is Modular and Readily Adaptable", PLoS ONE, vol. 9, pp. e110475, 2014.
  10. D.S. Liu, L.G. Nivon, F. Richter, P.J. Goldman, T.J. Deerinck, J.Z. Yao, D. Richardson, W.S. Phipps, A.Z. Ye, M.H. Ellisman, C.L. Drennan, D. Baker, and A.Y. Ting, "Computational design of a red fluorophore ligase for site-specific protein labeling in living cells", Proceedings of the National Academy of Sciences, vol. 111, pp. E4551-E4559, 2014.
  11. A. Kumar, Y. Wu, R. Christensen, P. Chandris, W. Gandler, E. McCreedy, A. Bokinsky, D.A. Colón-Ramos, Z. Bao, M. McAuliffe, G. Rondeau, and H. Shroff, "Dual-view plane illumination microscopy for rapid and spatially isotropic imaging", Nature Protocols, vol. 9, pp. 2555-2573, 2014.
  12. H. Choi, D.N. Wadduwage, T.Y. Tu, P. Matsudaira, and P.T.C. So, "Three-dimensional image cytometer based on widefield structured light microscopy and high-speed remote depth scanning", Cytometry Part A, vol. 87, pp. 49-60, 2014.
  13. G.S. Filonov, J.D. Moon, N. Svensen, and S.R. Jaffrey, "Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution", Journal of the American Chemical Society, vol. 136, pp. 16299-16308, 2014.
  14. S.L. Schwartz, Q. Yan, C.A. Telmer, K.A. Lidke, M.P. Bruchez, and D.S. Lidke, "Fluorogen-Activating Proteins Provide Tunable Labeling Densities for Tracking FcεRI Independent of IgE", ACS Chemical Biology, vol. 10, pp. 539-546, 2014.

Building a diSPIM

Hari Shroff and Abhishek Kumar have a fun post and video up describing building a diSPIM system at the Bangalore Microscopy Course. The video in particular is entertaining – it’s a time lapse showing the system assembly from start to finish. The 2015 course is now being organized – the web page isn’t up yet, but I encourage you to keep an eye out for it if you’re looking for a microscopy course to attend – the faculty are very good and the course is terrific (and a lot of fun!)

LEDs for Deep UV Imaging

As a result of our work with lanthanide imaging, I’ve acquired an interest in excitation sources in the deep UV (DUV; around 280 nm). When we started a few years ago, the only options were arc lamps.  Now there are a number of companies that produce DUV LEDs with decent emission powers. I haven’t yet tried any of these, though I plan to soon; I’m posting this list in case someone else is looking for suppliers.

Thorlabs makes a mounted LED that provides 25 mW at 280 nm.  Other suppliers of component LEDs (i.e. you supply the power source and mounting) are:

Firstcut CNC Machining

A colleague recently introduced me to Firstcut CNC Machining – a branch of ProtoLabs that does custom 3-axis CNC machining. They’re very easy to use – you upload them a 3D drawing (I use Autocad Inventor, because it’s free for academic use) and they will generate a quote for you.  I uploaded the part below, which is a prototype of a LED ring light that sits on top of an objective, and got a quote back for $240 to machine it out of aluminum, although they can’t machine the angled holes in the cone perfectly, since they are doing 3-axis milling.  Nevertheless, this was cheaper than I expected for such a complicated part, and their website is very easy to use. They also quote a 3 day turnaround, which is pretty nice (you can do one day turnaround, but it doubles the price).4x LED ring light 2

Paper Roundup – September 2014

  • A simple Airy beam light sheet microscope based on a tilted cylindrical lens. [1]
  • Simple illumination for phase contrast microscopy based on a ring of LEDs. [2]
  • A protocol for constructing a TIRF microscope that uses micromirrors placed under the objective for positioning the TIRF bea.m [3]
  • Use of small molecule dye binding tags (CLIP, SNAP, etc.) for rapid labeling (30 min) of whole mount Drosophila brains and mouse brain sections. [4]
  • An optimized Dronpa variant for two-color PALM imaging with PAmCherry1. [5]
  • A set of new red-shifted fluorescent proteins for detection under 633 nm excitation; however they are not that bright. [6]
  • An interesting paper studying the effect of weak dimerization between fluorescent proteins on the dynamic range of FRET probes [7]
  • A photoswitchable long Stokes shift protein. It is initially excited at 445 nm and emits at 621 nm; photoswitching converts it to a red form that is excited at 573 nm and emits at 621 nm. It is not very bright, but is sufficiently different from existing photoswitchable FPs that it may be of interest. [8]
  • A structure of the fluorescent RNA tag, Spinach, along with a smaller version, Baby Spinach [9]
  • Another paper on imaging in the IR with carbon nanotubes [10]
  • The Histochemical Society’s standards of practice for vaildating immunohistochemical data [11]


  1. Z. Yang, M. Prokopas, J. Nylk, C. Coll-Lladó, F.J. Gunn-Moore, D.E.K. Ferrier, T. Vettenburg, and K. Dholakia, "A compact Airy beam light sheet microscope with a tilted cylindrical lens", Biomedical Optics Express, vol. 5, pp. 3434, 2014.
  2. K.F. WEBB, "Condenser‐free contrast methods for transmitted‐light microscopy", Journal of Microscopy, vol. 257, pp. 8-22, 2014.
  3. J. Larson, M. Kirk, E.A. Drier, W. O'Brien, J.F. MacKay, L.J. Friedman, and A.A. Hoskins, "Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope", Nature Protocols, vol. 9, pp. 2317-2328, 2014.
  4. J. Kohl, J. Ng, S. Cachero, E. Ciabatti, M. Dolan, B. Sutcliffe, A. Tozer, S. Ruehle, D. Krueger, S. Frechter, T. Branco, M. Tripodi, and G.S.X.E. Jefferis, "Ultrafast tissue staining with chemical tags", Proceedings of the National Academy of Sciences, vol. 111, pp. E3805-E3814, 2014.
  5. A.B. Rosenbloom, S. Lee, M. To, A. Lee, J.Y. Shin, and C. Bustamante, "Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant", Proceedings of the National Academy of Sciences, vol. 111, pp. 13093-13098, 2014.
  6. U. Schoetz, N.C. Deliolanis, D. Ng, J. Pauli, U. Resch-Genger, E. Kühn, S. Heuer, W. Beisker, R.W. Köster, H. Zitzelsberger, and R.B. Caldwell, "Usefulness of a Darwinian System in a Biotechnological Application: Evolution of Optical Window Fluorescent Protein Variants under Selective Pressure", PLoS ONE, vol. 9, pp. e107069, 2014.
  7. L.H. Lindenburg, M. Malisauskas, T. Sips, L. van Oppen, S.P.W. Wijnands, S.F.J. van de Graaf, and M. Merkx, "Quantifying Stickiness: Thermodynamic Characterization of Intramolecular Domain Interactions To Guide the Design of Förster Resonance Energy Transfer Sensors", Biochemistry, vol. 53, pp. 6370-6381, 2014.
  8. K. Piatkevich, B. English, V. Malashkevich, H. Xiao, S. Almo, R. Singer, and V. Verkhusha, "Photoswitchable Red Fluorescent Protein with a Large Stokes Shift", Chemistry & Biology, vol. 21, pp. 1402-1414, 2014.
  9. K.D. Warner, M.C. Chen, W. Song, R.L. Strack, A. Thorn, S.R. Jaffrey, and A.R. Ferré-D'Amaré, "Structural basis for activity of highly efficient RNA mimics of green fluorescent protein", Nature Structural & Molecular Biology, vol. 21, pp. 658-663, 2014.
  10. D. Ghosh, A.F. Bagley, Y.J. Na, M.J. Birrer, S.N. Bhatia, and A.M. Belcher, "Deep, noninvasive imaging and surgical guidance of submillimeter tumors using targeted M13-stabilized single-walled carbon nanotubes", Proceedings of the National Academy of Sciences, vol. 111, pp. 13948-13953, 2014.
  11. S.M. Hewitt, D.G. Baskin, C.W. Frevert, W.L. Stahl, and E. Rosa-Molinar, "Controls for Immunohistochemistry", Journal of Histochemistry & Cytochemistry, vol. 62, pp. 693-697, 2014.