Paper Roundup – June 2014

  • A method for recovering GFP fluorescence after resin embedding by treament with alkaline buffers [1]
  • A 3D-STORM analysis tool using compressed sensing [2]
  • A method for imaging single-copy splice variants of RNAs using plasmonic coupling of gold nanoparticles [3]
  • Improving the performance of multifocal multiphoton microscopy by reassignment of scattered photons [4]
  • A review of photoswitchable fluorescent proteins for super-resolution imaging [5]
  • 6nm resolution imaging of DNA origami using DNA-PAINT [6]
  • A temporal focusing, two-photon line microscope for rapid volumetric imaging, used to imaging neuronal activity in 3D neuronal cultures [7]
  • A review of plasmonic alternatives to FRET for determining the distance between particles at the 10 – 100 nm distance scale. [8]
  • Improved deconvolution performance using a spatially varying regularization term [9]
  • Organelle-specific calcium indicators in a variety of different colors [10]
  • A multiplexing scheme for improving the speed of Fourier Ptychographic miroscopy [11]
  • Lasing from the fluorescent protein Venus, suspended in droplets or in bacteria [12]
  • An optimized protocol for CLARITY clearing of tissues, including details of a CLARITY-optimized light sheet microscope [13]
  • A review of the uses of flatbed scanners in biomedical imaging [14]
  • Ribozymes that catalyze their own labeling with fluorescein iodoacetamide [15]
  • A comparison of probes for tracking acidic organelles [16]
  • A review of fluorescence cryo-microscopy [17]
  • The current issue of Nature Chemical Biology focuses on light, with reviews of fluorescent labeling strategies [18], photoswitchable fluorescent proteins [19], and single molecule tracking [20]
  • Measurements of Dendra2 photoconversion, blinking, and bleaching rates in cells [21]
  • A paper describing construction of a whole slide imaging microscope [22]
  • Two new fluorescent voltage sensor proteins [23], [24]
  • A light-field microscope for high speed neuronal imaging of C. elegans and zebrafish [25]
  • Correlated cryogenic PALM and cryo-electron microscope [26]


  1. H. Xiong, Z. Zhou, M. Zhu, X. Lv, A. Li, S. Li, L. Li, T. Yang, S. Wang, Z. Yang, T. Xu, Q. Luo, H. Gong, and S. Zeng, "Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging", Nature Communications, vol. 5, 2014.
  2. L. Gu, Y. Sheng, Y. Chen, H. Chang, Y. Zhang, P. Lv, W. Ji, and T. Xu, "High-Density 3D Single Molecular Analysis Based on Compressed Sensing", Biophysical Journal, vol. 106, pp. 2443-2449, 2014.
  3. K. Lee, Y. Cui, L.P. Lee, and J. Irudayaraj, "Quantitative imaging of single mRNA splice variants in living cells", Nature Nanotechnology, vol. 9, pp. 474-480, 2014.
  4. J.W. Cha, V.R. Singh, K.H. Kim, J. Subramanian, Q. Peng, H. Yu, E. Nedivi, and P.T.C. So, "Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy", Scientific Reports, vol. 4, 2014.
  5. D.M. Shcherbakova, P. Sengupta, J. Lippincott-Schwartz, and V.V. Verkhusha, "Photocontrollable Fluorescent Proteins for Superresolution Imaging", Annual Review of Biophysics, vol. 43, pp. 303-329, 2014.
  6. M. Raab, J.J. Schmied, I. Jusuk, C. Forthmann, and P. Tinnefeld, "Fluorescence Microscopy with 6 nm Resolution on DNA Origami", ChemPhysChem, vol. 15, pp. 2431-2435, 2014.
  7. H. Dana, A. Marom, S. Paluch, R. Dvorkin, I. Brosh, and S. Shoham, "Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks", Nature Communications, vol. 5, 2014.
  8. P.C. Ray, Z. Fan, R.A. Crouch, S.S. Sinha, and A. Pramanik, "Nanoscopic optical rulers beyond the FRET distance limit: fundamentals and applications", Chemical Society Reviews, vol. 43, pp. 6370, 2014.
  9. J. SEO, S. HWANG, J. LEE, and H. PARK, "Spatially varying regularization of deconvolution in 3D microscopy", Journal of Microscopy, pp. n/a-n/a, 2014.
  10. J. Suzuki, K. Kanemaru, K. Ishii, M. Ohkura, Y. Okubo, and M. Iino, "Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA", Nature Communications, vol. 5, 2014.
  11. L. Tian, X. Li, K. Ramchandran, and L. Waller, "Multiplexed coded illumination for Fourier Ptychography with an LED array microscope", Biomedical Optics Express, vol. 5, pp. 2376, 2014.
  12. A. Jonáš, M. Aas, Y. Karadag, S. Manioğlu, S. Anand, D. McGloin, H. Bayraktar, and A. Kiraz, "In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities", Lab on a Chip, vol. 14, pp. 3093, 2014.
  13. R. Tomer, L. Ye, B. Hsueh, and K. Deisseroth, "Advanced CLARITY for rapid and high-resolution imaging of intact tissues", Nature Protocols, vol. 9, pp. 1682-1697, 2014.
  14. Z. Göröcs, and A. Ozcan, "Biomedical imaging and sensing using flatbed scanners", Lab on a Chip, vol. 14, pp. 3248, 2014.
  15. A.K. Sharma, J.J. Plant, A.E. Rangel, K.N. Meek, A.J. Anamisis, J. Hollien, and J.M. Heemstra, "Fluorescent RNA Labeling Using Self-Alkylating Ribozymes", ACS Chemical Biology, vol. 9, pp. 1680-1684, 2014.
  16. A. Pierzyńska-Mach, P.A. Janowski, and J.W. Dobrucki, "Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles", Cytometry Part A, vol. 85, pp. 729-737, 2014.
  17. R. Kaufmann, C. Hagen, and K. Grünewald, "Fluorescence cryo-microscopy: current challenges and prospects", Current Opinion in Chemical Biology, vol. 20, pp. 86-91, 2014.
  18. K.M. Dean, and A.E. Palmer, "Advances in fluorescence labeling strategies for dynamic cellular imaging", Nature Chemical Biology, vol. 10, pp. 512-523, 2014.
  19. V. Adam, R. Berardozzi, M. Byrdin, and D. Bourgeois, "Phototransformable fluorescent proteins: Future challenges", Current Opinion in Chemical Biology, vol. 20, pp. 92-102, 2014.
  20. A. Kusumi, T.A. Tsunoyama, K.M. Hirosawa, R.S. Kasai, and T.K. Fujiwara, "Tracking single molecules at work in living cells", Nature Chemical Biology, vol. 10, pp. 524-532, 2014.
  21. S. Avilov, R. Berardozzi, M.S. Gunewardene, V. Adam, S.T. Hess, and D. Bourgeois, "In cellulo Evaluation of Phototransformation Quantum Yields in Fluorescent Proteins Used As Markers for Single-Molecule Localization Microscopy", PLoS ONE, vol. 9, pp. e98362, 2014.
  22. G. Bueno, O. Déniz, M.D.M. Fernández-Carrobles, N. Vállez, and J. Salido, "An automated system for whole microscopic image acquisition and analysis", Microscopy Research and Technique, vol. 77, pp. 697-713, 2014.
  23. Y. Gong, M.J. Wagner, J. Zhong Li, and M.J. Schnitzer, "Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors", Nature Communications, vol. 5, 2014.
  24. F. St-Pierre, J.D. Marshall, Y. Yang, Y. Gong, M.J. Schnitzer, and M.Z. Lin, "High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor", Nature Neuroscience, vol. 17, pp. 884-889, 2014.
  25. R. Prevedel, Y. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E.S. Boyden, and A. Vaziri, "Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy", Nature Methods, vol. 11, pp. 727-730, 2014.
  26. Y. Chang, S. Chen, E.I. Tocheva, A. Treuner-Lange, S. Löbach, L. Søgaard-Andersen, and G.J. Jensen, "Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography", Nature Methods, vol. 11, pp. 737-739, 2014.

How to make easily portable videos

As long as I’ve been doing microscopy, it’s been tricky to make videos that are easily playable cross-platform. A lot of microscopy software packages support only a few video output formats, possibly with codecs that either aren’t available on the machine you want to play videos from or that produce bad compression artifacts.

To avoid these problems I currently use Handbrake to transcode videos to H264 video in an mp4 container, which appears to be playable on just about any machine. It’s how I’ve produced all the videos on this blog. It also produces pretty good compression and nice looking movies. In general, I first use the microscopy software to produce an uncompressed AVI file. This is huge, but avoids introducing compression artifacts. I then open this in Handbrake and transcode it to H264 video. The default settings produce good results and converting a 10 second video only takes a few seconds. You can see some of the results in these posts.

The Optical Invariant, or: Why LEDs make bad light sources for light sheet microscopy

I want to build a light sheet microscope, so recently I’ve been thinking about optical designs for light sheets. This paper [1] describes about the simplest light sheet you can make: collimate a laser beam and use a cylindrical lens to focus it to a sheet in your sample. If you want higher resolution or magnification, you can focus the sheet on the back focal plane of an objective, but if you only need a few micron resolution, the cylindrical lens is alone is enough to achieve that. Since the application I’m interested is the same as that of the paper (imaging whole cleared mouse brains and other large samples), a few micron resolution is enough for many applications, and I’m planning on building a very similar system (partly because we have an unused AZ100 to work with).

Done this way, you only really need two lenses: the cylindrical lens and a second lens to collimate the output of an optical fiber from your laser. With such a simple optical system, the laser is by far the largest cost, and so someone I was talking to recently suggested using an LED instead. I thought this seemed like a good idea – even though the LED doesn’t produce a nice collimated beam, you don’t need a ton of power for a light sheet system – the paper above uses 1.5 mW per mm of beam width, or 15 mW for a 1 cm wide beam, which is probably as big as we’d ever need. It’s no problem to get a 1W (or even multiwatt) LED, so we should be able to throw away a bunch of light to collimate it and still have enough light to illuminate our specimen, right? Continue reading


  1. 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.

Lots of new fluorescent proteins at Addgene

Michael Davidson, at Florida State University, is contributing his entire collection of over 3000 fluorescent protein plasmids to Addgene. This is going to be a great resource for fluorescent imaging; currently there are 136 fluorescent protein vectors with no tags, and 139 fusions of different proteins to mEmerald alone.  Many more will be posted as they are curated.

Mike is one of the unsung heroes of fluorescent microscopy: in addition to maintaining the Molecular Expressions, MicroscopyU, Zeiss Campus, and a number of other websites, you will also see that he is an author on many fluorescent protein publications. He’s made a career out of testing the performance of fluorescent proteins by fusing them to as many as 40 different mammalian proteins and testing their localization and behavior. It’s the plasmids from these tests (among others) that are being deposited at Addgene.

Take a look at the plasmids at Addgene, and if they’re useful to you, thank Mike for all the hard work his group has put in to bring us better fluorescent proteins.

Paper roundup – May 2014

  • A new SIM reconstruction algorithm that minimizes negative pixel values [1]
  • A microfluidic chemostat that traps cells in the fluid phase without contact with the device walls [2]
  • The forthcoming issue of Current Opinion in Chemical Biology is focused on imaging, including reviews on high speed biological imaging [3], quantitative super-resolution microscopy [4], and live cell reporters [5], as well as many others.
  • A description of a software system for automated mosaicing and and segmentation of large (250 GB) data sets, using the Farsight Toolkit [6]
  • Neuron labeling with fluorescent nanodiamonds [7]
  • A protocol for generating DNA origami structures for super-resolution calibration [8]
  • Building a simple light sheet microscope for imaging C. elegans [9]
  • A review of live imaging in Drosophila [10]
  • An improved infrared fluorescent protein, iFP2.0, and coexpression of heme oxygenase, give improved near-infrared imaging in neurons and in animals [11]
  • A simulator for partially coherent imaging [12]
  • Simultaneous 3D imaging of neuronal activity using light-field microscopy [13]
  • An exhortation for biologists doing fluorescence imaging to quantify the number of molecules they observe [14]
  • A review of methods for optical control of protein function [15]
  • A smartphone-based microscope using lensless imaging, with multiple illumination angles acquired by tilting the device by hand [16]
  • A new peptide-lipid conjugate, mCLING, that can be used for labeling cell membranes. It is fixable, and can be functionalized with many different dyes. [17]
  • A protein-complementation assay using an infrared fluorescent protein [18]
  • A simple flat-fielding approach for well by well correction in high throughput imaging [19]
  • Reflective confocal imaging of myelinated axons [20]
  • New photoactivatible proteins for super-resolution imaging with minimal aggregation [21]


  1. C.H. Righolt, S. Mai, L.J. van Vliet, and S. Stallinga, "Three-dimensional structured illumination microscopy using Lukosz bound apodization reduces pixel negativity at no resolution cost", Optics Express, vol. 22, pp. 11215, 2014.
  2. E.M. Johnson-Chavarria, U. Agrawal, M. Tanyeri, T.E. Kuhlman, and C.M. Schroeder, "Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution", Lab on a Chip, vol. 14, pp. 2688, 2014.
  3. P.W. Winter, and H. Shroff, "Faster fluorescence microscopy: advances in high speed biological imaging", Current Opinion in Chemical Biology, vol. 20, pp. 46-53, 2014.
  4. N. Durisic, L.L. Cuervo, and M. Lakadamyali, "Quantitative super-resolution microscopy: pitfalls and strategies for image analysis", Current Opinion in Chemical Biology, vol. 20, pp. 22-28, 2014.
  5. I.R. Corrêa, "Live-cell reporters for fluorescence imaging", Current Opinion in Chemical Biology, vol. 20, pp. 36-45, 2014.
  6. N. Rey-Villamizar, V. Somasundar, M. Megjhani, Y. Xu, Y. Lu, R. Padmanabhan, K. Trett, W. Shain, and B. Roysam, "Large-scale automated image analysis for computational profiling of brain tissue surrounding implanted neuroprosthetic devices using Python", Frontiers in Neuroinformatics, vol. 8, 2014.
  7. T. Hsu, K. Liu, H. Chang, E. Hwang, and J. Chao, "Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds", Scientific Reports, vol. 4, 2014.
  8. J.J. Schmied, M. Raab, C. Forthmann, E. Pibiri, B. Wünsch, T. Dammeyer, and P. Tinnefeld, "DNA origami–based standards for quantitative fluorescence microscopy", Nature Protocols, vol. 9, pp. 1367-1391, 2014.
  9. C. Chardès, P. Mélénec, V. Bertrand, and P. Lenne, "Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development", Journal of Visualized Experiments, 2014.
  10. E. Rebollo, K. Karkali, F. Mangione, and E. Martín-Blanco, "Live imaging in Drosophila: The optical and genetic toolkits", Methods, vol. 68, pp. 48-59, 2014.
  11. D. Yu, W.C. Gustafson, C. Han, C. Lafaye, M. Noirclerc-Savoye, W. Ge, D.A. Thayer, H. Huang, T.B. Kornberg, A. Royant, L.Y. Jan, Y.N. Jan, W.A. Weiss, and X. Shu, "An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging", Nature Communications, vol. 5, 2014.
  12. S.B. Mehta, and R. Oldenbourg, "Image simulation for biological microscopy: microlith", Biomedical Optics Express, vol. 5, pp. 1822, 2014.
  13. R. Prevedel, Y. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E.S. Boyden, and A. Vaziri, "Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy", Nature Methods, vol. 11, pp. 727-730, 2014.
  14. V.C. Coffman, and J. Wu, "Every laboratory with a fluorescence microscope should consider counting molecules", Molecular Biology of the Cell, vol. 25, pp. 1545-1548, 2014.
  15. A.S. Baker, and A. Deiters, "Optical Control of Protein Function through Unnatural Amino Acid Mutagenesis and Other Optogenetic Approaches", ACS Chemical Biology, vol. 9, pp. 1398-1407, 2014.
  16. S.A. Lee, and C. Yang, "A smartphone-based chip-scale microscope using ambient illumination", Lab on a Chip, vol. 14, pp. 3056, 2014.
  17. N.H. Revelo, D. Kamin, S. Truckenbrodt, A.B. Wong, K. Reuter-Jessen, E. Reisinger, T. Moser, and S.O. Rizzoli, "A new probe for super-resolution imaging of membranes elucidates trafficking pathways", The Journal of Cell Biology, vol. 205, pp. 591-606, 2014.
  18. E. Tchekanda, D. Sivanesan, and S.W. Michnick, "An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions", Nature Methods, vol. 11, pp. 641-644, 2014.
  19. A.D. Coster, C. Wichaidit, S. Rajaram, S.J. Altschuler, and L.F. Wu, "A simple image correction method for high-throughput microscopy", Nature Methods, vol. 11, pp. 602-602, 2014.
  20. A.J. Schain, R.A. Hill, and J. Grutzendler, "Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy", Nature Medicine, vol. 20, pp. 443-449, 2014.
  21. S. Wang, J.R. Moffitt, G.T. Dempsey, X.S. Xie, and X. Zhuang, "Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging", Proceedings of the National Academy of Sciences, vol. 111, pp. 8452-8457, 2014.