Paper roundup: December 2013

  • A clever method for acquiring 0.5 gigapixel images by projecting a magnified image of the sample onto a modified flatbed scanner [1]
  • Electroporating small molecule fluorophore-labelled molecules into E. coli for long term imaging [2]
  • A Brainbow-like technique for labeling cells in Drosophila development [3]
  • A review of high speed nonlinear optical microscopy (two-photon, CARS, and similar techniques) [4]
  • Optogenetic control of Ras/Erk signaling [5]
  • Protocols for capillary mounting of spheroids [6] and cysts [7] for light sheet imaging.
  • Resolution doubling using a conventional spinning disk confocal microscope with stroboscopic illumination [8]
  • Using TALEs to target fluorescent proteins to specific genomic loci [9]
  • A review of fluorescent membrane probes [10]
  • A protocol for using spinach-based aptamers for imaging ADP concentration in bacteria [11]


  1. G. Zheng, X. Ou, and C. Yang, "05 gigapixel microscopy using a flatbed scanner", Biomedical Optics Express, vol. 5, pp. 1, 2013.
  2. R. Crawford, J. Torella, L. Aigrain, A. Plochowietz, K. Gryte, S. Uphoff, and A. Kapanidis, "Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules", Biophysical Journal, vol. 105, pp. 2439-2450, 2013.
  3. O. Kanca, E. Caussinus, A.S. Denes, A. Percival-Smith, and M. Affolter, "Raeppli: a whole-tissue labeling tool for live imaging of Drosophila development", Development, vol. 141, pp. 472-480, 2013.
  4. P. So, E. Yew, and C. Rowlands, "High-Throughput Nonlinear Optical Microscopy", Biophysical Journal, vol. 105, pp. 2641-2654, 2013.
  5. J. Toettcher, O. Weiner, and W. Lim, "Using Optogenetics to Interrogate the Dynamic Control of Signal Transmission by the Ras/Erk Module", Cell, vol. 155, pp. 1422-1434, 2013.
  6. J. Swoger, F. Pampaloni, and E.H.K. Stelzer, "Imaging Cellular Spheroids with a Single (Selective) Plane Illumination Microscope", Cold Spring Harbor Protocols, vol. 2014, pp. pdb.prot080176-pdb.prot080176, 2013.
  7. J. Swoger, F. Pampaloni, and E.H.K. Stelzer, "Imaging MDCK Cysts with a Single (Selective) Plane Illumination Microscope", Cold Spring Harbor Protocols, vol. 2014, pp. pdb.prot080184-pdb.prot080184, 2013.
  8. O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R.H. Kehlenbach, F.S. Wouters, J. Grosshans, G. Bunt, and J. Enderlein, "Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy", Proceedings of the National Academy of Sciences, vol. 110, pp. 21000-21005, 2013.
  9. H. Ma, P. Reyes-Gutierrez, and T. Pederson, "Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors", Proceedings of the National Academy of Sciences, vol. 110, pp. 21048-21053, 2013.
  10. A. Klymchenko, and R. Kreder, "Fluorescent Probes for Lipid Rafts: From Model Membranes to Living Cells", Chemistry & Biology, vol. 21, pp. 97-113, 2014.
  11. R.L. Strack, W. Song, and S.R. Jaffrey, "Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria", Nature Protocols, vol. 9, pp. 146-155, 2013.

ASCB roundup

I just got back from attending the 2013 ASCB meeting. I try to go every year as most of the microscopy vendors are there, so it’s a great place to see new products and network. I mostly didn’t go to the scientific talks, though there was one great talk by Eric Betzig on his latest developments on Bessel beam light sheet microscopy. He now has systems that can record a full volume in under a second and can image at high frame rates for hours with minimal phototoxicity. His results so outshone what you can do with commercial microscopes that I and several other microscopists found it thoroughly depressing.

On the vendor floor, there wasn’t any single new amazing product, but there were some nice developments.  There was also a lot of consolidation evident in the microscopy world: Andor, which recently purchased Spectral Applied Research and Apogee Imaging Systems, is itself being acquired by Oxford Instruments, and Prairie Technologies has been acquired by Bruker.  Here are some of the interesting things I saw:

  • Epi Technology – a startup making a reversed filter cube allowing you to image your epi-illumination source through the eyepieces. Useful for finding and removing dirt.
  • Datacolor is releasing a system for color calibration of transmitted light images. It uses a slide with known reference colors on it to allow putting your transmitted light images into a known color space using their own software. Unfortunately it doesn’t seem to plug in to any existing microscopy software.
  • Life Technologies has released a new DMEM formulation that reduces media fluorescence by about 9-fold.  It should be very nice for live cell imaging.
  • There were a number of companies showing off microfluidic systems for cell culture and imaging. Ebers, microfluidic ChipShop, and SynVivo were three that I talked to.
  • Nikon was showing off their new range of laser accessories, including a Mosaic-type micro-mirror system for patterned illumination, dual-arm TIRF, and a galvo scanning system for photobleaching.
  • Spectral Applied Research, recently acquired by Andor, which in turn is being acquired by Oxford Instruments, was showing off its all-in-one spinning disk, TIRF, and STORM system. It was quite nice looking but I can’t find any information about it on their website.
  • Bruker was showing off the swept-field confocal, formerly from Prairie technology. They now have it working for spectral detection, acquiring 16 wavelengths simultaneously at each point.

Installing a Filter in the Ti Eyepiece Path

We recently set up a Nikon Ti microscope with a photobleaching / photoactivation rig. This required installing a second filter turret in the microscope, which meant we could no longer put the Sutter filter wheel directly underneath the filter turret, and instead had to put the filter wheel on the camera port. This means that we can no longer use the eyepieces for fluorescence imaging. To allow us to once again do fluorescence imaging to the eyepieces, and to provide additional laser safety by preventing the lasers from reaching the eyepieces (they’re already interlocked), I wanted to install a filter in the eyepiece path.

If you take off the eyepieces, there is a nice hole in the stage up block that the eyepieces mount into. It’s just a bit larger than a 25 mm filter and there’s plenty of space around it for a filter to sit. We have a 3D printer in lab, so it was simple to draw up an adapter that sits in the hole in the stage up block and in turn holds a standard 25 mm filter.  You can see the results below.

TI-eyepieceeyepiece filter

The files for the part can be downloaded from the 3D parts page on the NIC wiki.

Paper Roundup: November 2013

  • A review on determining protein quaternary structure by FRET. I’m a little skeptical of their ability to extract as much information from (typically noisy) FRET data as they want to, but it’s an interesting review nonetheless. [1]
  • Using GFP-labeled TALEs to fluorescently label chromosomal regions [2]
  • Another compressed sensing algorithm for fast STORM/PALM imaging [3]
  • A review on Raman microscopy [4]
  • STED imaging of individual diamond nitrogen vacancies with ~10 nm resolution [5]
  • Ultra-high speed 3D imaging of second harmonic generation using holography [6]
  • Single molecule super-resolution imaging used to count molecules and measure the fraction of dimers [7]
  • High speed 3D holographic imaging of ciliary beating [8]
  • Temperature sensitive GFPs for measurement of the temperature inside cells [9]
  • An improved version of the spinach aptamer, spinach2, for fluorescent labeling of RNAs [10]
  • A new method for monovalent functionalization of quantum dots for single molecule labeling and imaging [11]
  • A new method for segmenting nuclei. It has a nice overview of current algorithms for nuclear segmentation in the introduction [12]
  • A microfluidic method for trapping cells in droplets and imaging them for long periods of time [13]
  • A review of superresolution localization methods, specifically on fitting algorithms [14]
  • An introduction to machine learning approaches for analyzing imaging data in cell biology [15]


  1. V. Raicu, and D. Singh, "FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells", Biophysical Journal, vol. 105, pp. 1937-1945, 2013.
  2. Y. Miyanari, C. Ziegler-Birling, and M. Torres-Padilla, "Live visualization of chromatin dynamics with fluorescent TALEs", Nature Structural & Molecular Biology, vol. 20, pp. 1321-1324, 2013.
  3. H.P. Babcock, J.R. Moffitt, Y. Cao, and X. Zhuang, "Fast compressed sensing analysis for super-resolution imaging using L1-homotopy", Optics Express, vol. 21, pp. 28583, 2013.
  4. K.A. Antonio, and Z.D. Schultz, "Advances in Biomedical Raman Microscopy", Analytical Chemistry, vol. 86, pp. 30-46, 2014.
  5. S. Arroyo-Camejo, M. Adam, M. Besbes, J. Hugonin, V. Jacques, J. Greffet, J. Roch, S.W. Hell, and F. Treussart, "Stimulated Emission Depletion Microscopy Resolves Individual Nitrogen Vacancy Centers in Diamond Nanocrystals", ACS Nano, vol. 7, pp. 10912-10919, 2013.
  6. D.R. Smith, D.G. Winters, and R.A. Bartels, "Submillisecond second harmonic holographic imaging of biological specimens in three dimensions", Proceedings of the National Academy of Sciences, vol. 110, pp. 18391-18396, 2013.
  7. X. Nan, E.A. Collisson, S. Lewis, J. Huang, T.M. Tamguney, J.T. Liphardt, F. McCormick, J.W. Gray, and S. Chu, "Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling", Proceedings of the National Academy of Sciences, vol. 110, pp. 18519-18524, 2013.
  8. L.G. Wilson, L.M. Carter, and S.E. Reece, "High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms", Proceedings of the National Academy of Sciences, vol. 110, pp. 18769-18774, 2013.
  9. S. Kiyonaka, T. Kajimoto, R. Sakaguchi, D. Shinmi, M. Omatsu-Kanbe, H. Matsuura, H. Imamura, T. Yoshizaki, I. Hamachi, T. Morii, and Y. Mori, "Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells", Nature Methods, vol. 10, pp. 1232-1238, 2013.
  10. R.L. Strack, M.D. Disney, and S.R. Jaffrey, "A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA", Nature Methods, vol. 10, pp. 1219-1224, 2013.
  11. J. Farlow, D. Seo, K.E. Broaders, M.J. Taylor, Z.J. Gartner, and Y. Jun, "Formation of targeted monovalent quantum dots by steric exclusion", Nature Methods, vol. 10, pp. 1203-1205, 2013.
  12. J. QI, "Dense nuclei segmentation based on graph cut and convexity-concavity analysis", Journal of Microscopy, vol. 253, pp. 42-53, 2013.
  13. M.A. Khorshidi, P.K.P. Rajeswari, C. Wählby, H.N. Joensson, and H. Andersson Svahn, "Automated analysis of dynamic behavior of single cells in picoliter droplets", Lab on a Chip, vol. 14, pp. 931, 2014.
  14. A.R. Small, and R. Parthasarathy, "Superresolution Localization Methods", Annual Review of Physical Chemistry, vol. 65, pp. 107-125, 2014.
  15. C. Sommer, and D.W. Gerlich, "Machine learning in cell biology – teaching computers to recognize phenotypes", Journal of Cell Science, vol. 126, pp. 5529-5539, 2013.


Some of you probably noticed that this blog was down for the last week. Unfortunately our server died right before Thanksgiving and I only now got it back up.  Please let me know if you encounter any problems with it.

To make up for the absence of the blog, I’ll share with you a cool movie we’ve just taken in the NIC. This is single particle tracking PALM or sptPALM [1] of the dopamine receptor labeled with mEos2.  Here, we’re imaging with pretty strong 561 nm illumination and very weak 405 nm illumination, so we’re continuously converting mEos2 molecules to the red state and imaging them until they bleach. Using this methodology we can track many different individual receptors over a long time and see how the population behavior changes when we stimulate the receptor.


  1. S. Manley, J.M. Gillette, G.H. Patterson, H. Shroff, H.F. Hess, E. Betzig, and J. Lippincott-Schwartz, "High-density mapping of single-molecule trajectories with photoactivated localization microscopy", Nature Methods, vol. 5, pp. 155-157, 2008.