Paper Roundup: August 2013

  • A temporally encoded method for imaging multiple focal points simultaneously in two-photon microscopy. [1]
  • A review of “Ultramicroscopy”, a variant of light sheet or SPIM microscopy for imaging cleared embryos. [2]
  • A nice review on the chemistry behind single molecule switching for super-resolution imaging.  It discusses both how to stabilize molecules to prevent them from blinking and how to promote photoswitching for STORM-type approaches. [3]
  • A review of two novel methods for labeling proteins with fluorophores, one based on β-lactamase; the other based on photoactive yellow protein.[4] A review of fluorogenic labeling probes in general, by the same authors [5]
  • A review of photoswitchable fluorescent proteins [6]
  • Using heavy water (D2O) can improve your single molecule super-resolution imaging. [7]
  • A method for determining the heights of objects in an image to better than the diffraction limit by measuring the center of mass of the point spread function. [8]
  • A fluorescent indicator of caspase activity that switches from non-fluorescent to fluorescent when cleaved by caspase. [9]
  • A method for improving red/orange FRET pairs by inducing weak dimerization between the donor and acceptor that locks them into a favorable conformation for energy transfer. [10]
  • Two photon activation rates for PAmCherry, PAmKate, and PAGFP. [11]
  • There is a good review on biological applications of FRET microscopy by the Periasamy group. [12]
  • A very high speed focusing system for a SPIM microscope using a lens whose focal length is electrically tunable. Using this system they were able to record 17-slice volumes at 30 Hz. [13]


  1. M. Ducros, Y.G. Houssen, J. Bradley, V. de Sars, and S. Charpak, "Encoded multisite two-photon microscopy", Proceedings of the National Academy of Sciences, vol. 110, pp. 13138-13143, 2013.
  2. K. Becker, N. Jahrling, S. Saghafi, and H. Dodt, "Ultramicroscopy: Light-Sheet-Based Microscopy for Imaging Centimeter-Sized Objects with Micrometer Resolution", Cold Spring Harbor Protocols, vol. 2013, pp. pdb.top076539-pdb.top076539, 2013.
  3. S. van de Linde, and M. Sauer, "How to switch a fluorophore: from undesired blinking to controlled photoswitching", Chem. Soc. Rev., vol. 43, pp. 1076-1087, 2014.
  4. S. Mizukami, Y. Hori, and K. Kikuchi, "Small-Molecule-Based Protein-Labeling Technology in Live Cell Studies: Probe-Design Concepts and Applications", Accounts of Chemical Research, vol. 47, pp. 247-256, 2014.
  5. Y. Hori, and K. Kikuchi, "Protein labeling with fluorogenic probes for no-wash live-cell imaging of proteins", Current Opinion in Chemical Biology, vol. 17, pp. 644-650, 2013.
  6. X.X. Zhou, and M.Z. Lin, "Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications", Current Opinion in Chemical Biology, vol. 17, pp. 682-690, 2013.
  7. S.F. Lee, Q. Vérolet, and A. Fürstenberg, "Improved Super-Resolution Microscopy with Oxazine Fluorophores in Heavy Water", Angewandte Chemie International Edition, vol. 52, pp. 8948-8951, 2013.
  8. C. Chiu, and E. Gratton, "Axial super resolution topography of focal adhesion by confocal microscopy", Microscopy Research and Technique, vol. 76, pp. 1070-1078, 2013.
  9. J. Zhang, X. Wang, W. Cui, W. Wang, H. Zhang, L. Liu, Z. Zhang, Z. Li, G. Ying, N. Zhang, and B. Li, "Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage", Nature Communications, vol. 4, 2013.
  10. L.H. Lindenburg, A.M. Hessels, E.H.T.M. Ebberink, R. Arts, and M. Merkx, "Robust Red FRET Sensors Using Self-Associating Fluorescent Domains", ACS Chemical Biology, vol. 8, pp. 2133-2139, 2013.
  11. T.M.P. Hartwich, F.V. Subach, L. Cooley, V.V. Verkhusha, and J. Bewersdorf, "Determination of two-photon photoactivation rates of fluorescent proteins", Physical Chemistry Chemical Physics, vol. 15, pp. 14868, 2013.
  12. Y. Sun, C. Rombola, V. Jyothikumar, and A. Periasamy, "Förster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells", Cytometry Part A, vol. 83, pp. 780-793, 2013.
  13. F.O. Fahrbach, F.F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, "Rapid 3D light-sheet microscopy with a tunable lens", Optics Express, vol. 21, pp. 21010, 2013.

More on High Speed Streaming to Solid State Drives

I’ve previously discussed our PC for acquiring images at 1.1 GB/s, and some of the problems we’ve had with that SSD array slowing down over time.  I just got a second PC for high speed streaming acquisition, this one based on a Core i7 platform, rather than a Xeon platform.  In addition to being about 60% of the cost of the Xeon system, I am also hoping that the TRIM support in the Intel RST drivers will prevent the slowing down of the array we’ve seen before.

The system has an Asus Maximus VI EXTREME Intel Z87 motherboard, a Core i7-4770K processor, and four 256 GB Samsung 840 Pro SSDs in RAID 0.  The system was built for us by Central Computers.  As measured by Crystal Disk Mark, the sequential write speed for the RAID array is about 1115 MB/s.  This is about half the speed we see for our Xeon system with the Intel RAID card (2050 MB/s) and indicates that there is some bottleneck when using the Intel motherboard RAID support. However, the write speeds should be just fast enough to cope with the 1.1GB/s produced by an Andor Zyla operating at top speed, and is easily fast enough to handle the full data rate of 800 MB/s produced by a Hamamatsu Orca-Flash4.0.

To test TRIM support and slowing down of both RAID arrays, I then wrote 900GB of data to the drive, measured the speed, then erased this data and repeated this test twice. At the end of this test we’ve written about 2.8x more data than capacity of the drive, which I think means that it should need to erase pages on the drive.  However, I don’t see a significant slowdown on either system – the sequential write speeds are essentially unchanged at the end of this test.

Clearly, there is something I don’t understand about where the slowing of write speeds over time is coming from. I’m going to continue monitoring the performance of both systems as we use them for data acquisition. However, for many high speed streaming applications, a Core i7-based system is likely to be fast enough, and save the expense of a RAID card.

Color Image Stitching


I’ve now gotten color image acquisition with the ScopeLED working with tiled image acquisition and image stitching, resulting in the following 2.26 gigapixel image (image links to Gigapan):

SitchedSpleenThis is stitched from 725 images, which took about ten minutes to acquire. These were overlaid, flat-fielded, and white balanced in Matlab, which took about another ten minutes, and stitched and exported in Microsoft ICE, which took about an hour. I don’t yet understand why there is a color gradient across the image. I’ll have to figure out where that comes from and fix it.

Color imaging with RGB LEDs and a grayscale camera

As readers of this blog will know, we’ve been putting a lot of work into doing stitching of large numbers of brightfield images to acquire large fields of view. You will also have noticed that all the images I’ve shown so far were grayscale. That’s because we have a grayscale camera. However, many biological samples that people want to image are color. Rather than buy a color camera (and because color camera support in Micro-Manager is poor right now) we’ve instead tried using a red/green/blue (RGB) LED source from ScopeLED to generate color images. It works pretty well:ScopeLED_color

Continue reading

When Lossy Compression Goes Bad

If you’ve spent any time talking to microscopists or other people doing a lot of digital image analysis, at some point you’ve undoubtedly heard a sternly worded warning against using lossy compression, because it changes your data. Xerox has run into this problem in a particularly nasty way on some of their copiers. As first reported here, certain Xerox copiers, when scanning to PDF, will randomly replace some numbers with others. For example, 66 turns into 86 when scanned. This happens because of a lossy compression algorithm, JBIG2. Continue reading

Paper roundup: July 2013

  • Multiplexed immunoassay using the new GE technology to bind antibody, image, then destroy fluorophore. Uses 20 different antibodies on tissue microarrays. [1]. The technology is described in more detail here. [2]
  • A fluorescent activity probe for ammonia transporters. These are insertions of a circularly permuted GFP into an ammonia transporter such that the fluorescence of the GFP reports on the activity of the transporter. [3].
  • A new set of yeast fluorescent tagging proteins (I helped develop these and will post more about them later) [4].
  • A review on measuring membrane potential with fluorescent proteins [5].
  • A nice review on imaging in neurobiology, focusing on multi-photon and light sheet microscopy [6].
  • A parallel RESOLFT microscope capable of ~80 nm (FWHM) resolution and acquiring a 100 μm field of view in < 1 sec. [7]
  • A description of a set of open hardware microscopes for wide-field, confocal, FLIM imaging.  More details are available at their website.[8]
  • Super-resolution imaging of the nuclear pore complex using single color STORM-type imaging with Alexa 647. [9].
  • New photo-activatible infrared fluorescent proteins using biliverdin as a cofactor. [10]
  • A new calcium sensor, GCaMP6, from Janelia Farm, that is more sensitive than previous versions. [11]
  • Ultrahigh resolution dual-color single molecule switching imaging from Steven Chu’s group. [12]
  • Imaging protein expression in bacteria with a protein-binding aptamer that forms a structured RNA capable of binding a fluorescent chromophore (Spinach) on protein binding. [13]
  • A Citrine YFP variant with reduced chloride sensitivity by insertion of three glycine residues into a loop. [14]
  • An optogenetic method for inactivating proteins by localizing them away from their sites of action. [15]
  • Another publication analyzing super-resolution imaging by photon reassignment. [16]
  • A new method, Fourier ptychographic microscopy, for acquiring high resolution, wide field of view images by using a low NA objective and illuminating the sample at multiple angles. [17]
  • A light-inducible system for driving gene expression and chromatin modification by using a TALE DNA-binding domain and a light-driven protein dimerization domain. [18]
  • Systematic testing and production of FRET biosensors by screening multiple circularly permuted variants of both donor and acceptor as well as different positions within the final construct. [19]
  • A high-speed light sheet microscopy system and a sophisticated data reduction pipeline for high-speed imaging of zebrafish embryonic development. [20]


  1. D.A. Nelson, C. Manhardt, V. Kamath, Y. Sui, A. Santamaria-Pang, A. Can, M. Bello, A. Corwin, S.R. Dinn, M. Lazare, E.M. Gervais, S.J. Sequeira, S.B. Peters, F. Ginty, M.J. Gerdes, and M. Larsen, "Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation", Biology Open, vol. 2, pp. 439-447, 2013.
  2. M.J. Gerdes, C.J. Sevinsky, A. Sood, S. Adak, M.O. Bello, A. Bordwell, A. Can, A. Corwin, S. Dinn, R.J. Filkins, D. Hollman, V. Kamath, S. Kaanumalle, K. Kenny, M. Larsen, M. Lazare, Q. Li, C. Lowes, C.C. McCulloch, E. McDonough, M.C. Montalto, Z. Pang, J. Rittscher, A. Santamaria-Pang, B.D. Sarachan, M.L. Seel, A. Seppo, K. Shaikh, Y. Sui, J. Zhang, and F. Ginty, "Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue", Proceedings of the National Academy of Sciences, vol. 110, pp. 11982-11987, 2013.
  3. R. De Michele, C. Ast, D. Loqué, C. Ho, S.L. Andrade, V. Lanquar, G. Grossmann, S. Gehne, M.U. Kumke, and W.B. Frommer, "Fluorescent sensors reporting the activity of ammonium transceptors in live cells", eLife, vol. 2, 2013.
  4. S. Lee, W.A. Lim, and K.S. Thorn, "Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae", PLoS ONE, vol. 8, pp. e67902, 2013.
  5. J. Patti, and E.Y. Isacoff, "Measuring Membrane Voltage with Fluorescent Proteins", Cold Spring Harbor Protocols, vol. 2013, pp. pdb.top075804, 2013.
  6. Y. Wu, R. Christensen, D. Colón-Ramos, and H. Shroff, "Advanced optical imaging techniques for neurodevelopment", Current Opinion in Neurobiology, vol. 23, pp. 1090-1097, 2013.
  7. A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d'Este, S. Jakobs, C. Eggeling, and S.W. Hell, "Nanoscopy with more than 100,000 'doughnuts'", Nature Methods, vol. 10, pp. 737-740, 2013.
  8. P. BARBER, I. TULLIS, G. PIERCE, R. NEWMAN, J. PRENTICE, M. ROWLEY, D. MATTHEWS, S. AMEER-BEG, and B. VOJNOVIC, "The Gray Institute ‘open’ high-content, fluorescence lifetime microscopes", Journal of Microscopy, vol. 251, pp. 154-167, 2013.
  9. A. Szymborska, A. de Marco, N. Daigle, V.C. Cordes, J.A.G. Briggs, and J. Ellenberg, "Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging", Science, vol. 341, pp. 655-658, 2013.
  10. K.D. Piatkevich, F.V. Subach, and V.V. Verkhusha, "Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome", Nature Communications, vol. 4, 2013.
  11. T. Chen, T.J. Wardill, Y. Sun, S.R. Pulver, S.L. Renninger, A. Baohan, E.R. Schreiter, R.A. Kerr, M.B. Orger, V. Jayaraman, L.L. Looger, K. Svoboda, and D.S. Kim, "Ultrasensitive fluorescent proteins for imaging neuronal activity", Nature, vol. 499, pp. 295-300, 2013.
  12. A. Pertsinidis, K. Mukherjee, M. Sharma, Z.P. Pang, S.R. Park, Y. Zhang, A.T. Brunger, T.C. Sudhof, and S. Chu, "Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ", Proceedings of the National Academy of Sciences, vol. 110, pp. E2812-E2820, 2013.
  13. W. Song, R.L. Strack, and S.R. Jaffrey, "Imaging bacterial protein expression using genetically encoded RNA sensors", Nature Methods, vol. 10, pp. 873-875, 2013.
  14. J. Liang, Y. Yang, P. Yin, Y. Ding, Y. Shen, M. Qin, J. Wang, Q. Xu, Y. Cao, and W. Wang, "A Yellow Fluorescent Protein with Reduced Chloride Sensitivity Engineered by Loop-Insertion", ChemBioChem, vol. 14, pp. 1423-1426, 2013.
  15. X. Yang, A.P. Jost, O.D. Weiner, and C. Tang, "A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast", Molecular Biology of the Cell, vol. 24, pp. 2419-2430, 2013.
  16. C.J.R. Sheppard, S.B. Mehta, and R. Heintzmann, "Superresolution by image scanning microscopy using pixel reassignment", Optics Letters, vol. 38, pp. 2889, 2013.
  17. G. Zheng, R. Horstmeyer, and C. Yang, "Wide-field, high-resolution Fourier ptychographic microscopy", Nature Photonics, vol. 7, pp. 739-745, 2013.
  18. S. Konermann, M.D. Brigham, A. Trevino, P.D. Hsu, M. Heidenreich, . Le Cong, R.J. Platt, D.A. Scott, G.M. Church, and F. Zhang, "Optical control of mammalian endogenous transcription and epigenetic states", Nature, 2013.
  19. R.D. Fritz, M. Letzelter, A. Reimann, K. Martin, L. Fusco, L. Ritsma, B. Ponsioen, E. Fluri, S. Schulte-Merker, J. van Rheenen, and O. Pertz, "A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space", Science Signaling, vol. 6, pp. rs12-rs12, 2013.
  20. B. Schmid, G. Shah, N. Scherf, M. Weber, K. Thierbach, C.P. Campos, I. Roeder, P. Aanstad, and J. Huisken, "High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics", Nature Communications, vol. 4, 2013.