New Sutter LED combiner

Sutter recently announced a clever new LED combiner that allows you to combine multiple LED light sources into a single output beam. It relies on the fact that interference filters are very good reflectors of the wavelengths they don’t transmit, so you can use a filter to simultaneously pass the output of one LED while reflecting wavelengths from other LEDs. The concept is shown in the diagram below:

The Sutter 421 LED combiner. The number at each position indicates how many reflections that LED undergoes before reaching the output. Image courtesy of Sutter.

One of the many nice things about this concept is that changing LED wavelengths is really easy: you just replace the LED and the filter in front of it. You can also mount a second pentagon on the first to combine up to seven wavelengths (in principle you could even cascade a third pentagon to get 10 wavelengths, but at some point the filter designs get pretty tricky and the losses add up). You can also combine light sources other than LEDs, provided you can find appropriate interference filters.

We demoed a six color version of this a few weeks ago, using two pentagons and LEDs for DAPI, FITC, Cy3, Cy5, CFP, and YFP. We tested it with a Semrock Sedat Quad filter set and Chroma GFP/RFP and CFP/YFP filter sets. At all wavelengths tested it was as bright or brighter (in some cases as much as 10-fold brighter) than the Lambda XL we were using as a reference.  We’re now working with Sutter to get a seven color version of this (including 340 nm excitation for Fura-2) to install on our microscope. This will allow us to synchronize the LEDs to the camera, so that the LEDs are only on when the camera is exposing, minimizing photobleaching and phototoxicity. This should be a very nice LED illumination option for microscopy, particularly for users who want a modular system that’s easy to modify as needed.

Preprint: Review of Genetically Encoded Fluorescent Tags

I was recently asked to write a brief Technical Perspective on fluorescent tags for Molecular Biology of the Cell. These are meant to be introductions to a topic for novices in the field; I previously wrote one on light microscopy.

I’ve posted a preprint of the fluorescent tag review here; please send me any comments and I will incorporate them into the final version. I would have posted the preprint on BioRxiv, but it seems that they don’t host reviews.

Paper Roundup – November 2016

  • A detailed investigation of ER structure by multiple super-resolution methods [1]
  • Using deep convolutional neural networks to segment cells automatically with high accuracy [2]
  • A light sheet microscope that automatically adjusts the illumination plane to correct for sample-induced distortion [3]
  • Tools for scanning angle interference microscopy (SAIM) acquisition and analysis [4]
  • Super-resolution mapping of fluorophore orientation [5]
  • Isotropic point spread functions for fast cellular resolution 2-photon imaging [6]
  • CyRFP1, a long Stokes shift fluorescent protein co-excited with GFP but with separable emission [7]
  • mMaroon1, a new far-red fluorescent protein, and a four-color Fucci cell cycle sensor [8]
  • Multi-color electron microscopy [9]
  • A detailed review of fluorescent proteins [10]
  • A nice discussion of challenges in live cell time lapse imaging [11]
  • Ni2+ as a triplet state quencher for improved light output from Cy3 and Cy5 [12]
  • A python tool for image analysis [13]
  • Optimal reconstruction of 2D-SIM data [14]
  • A new bright monomeric red fluorescent protein, mScarlet [15]
  • A review of fluorescent tagging methods [16]
  • Adaptive SIM microscopy to reduce bleaching [17]
  • Tools for cluster analysis of single molecule localization microscopy methods [18]
  • A super-resolution microscope based on incoherent holography [19]


  1. J. Nixon-Abell, C.J. Obara, A.V. Weigel, D. Li, W.R. Legant, C.S. Xu, H.A. Pasolli, K. Harvey, H.F. Hess, E. Betzig, C. Blackstone, and J. Lippincott-Schwartz, "Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER", Science, vol. 354, pp. aaf3928-aaf3928, 2016.
  2. D.A. Van Valen, T. Kudo, K.M. Lane, D.N. Macklin, N.T. Quach, M.M. DeFelice, I. Maayan, Y. Tanouchi, E.A. Ashley, and M.W. Covert, "Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments", PLOS Computational Biology, vol. 12, pp. e1005177, 2016.
  3. L.A. Royer, W.C. Lemon, R.K. Chhetri, Y. Wan, M. Coleman, E.W. Myers, and P.J. Keller, "Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms", Nature Biotechnology, 2016.
  4. C.B. Carbone, R.D. Vale, and N. Stuurman, "An acquisition and analysis pipeline for scanning angle interference microscopy", Nature Methods, vol. 13, pp. 897-898, 2016.
  5. K. Zhanghao, L. Chen, X. Yang, M. Wang, Z. Jing, H. Han, M.Q. Zhang, D. Jin, J. Gao, and P. Xi, "Super-resolution dipole orientation mapping via polarization demodulation", Light: Science & Applications, vol. 5, pp. e16166, 2016.
  6. R. Prevedel, A.J. Verhoef, A.J. Pernía-Andrade, S. Weisenburger, B.S. Huang, T. Nöbauer, A. Fernández, J.E. Delcour, P. Golshani, A. Baltuska, and A. Vaziri, "Fast volumetric calcium imaging across multiple cortical layers using sculpted light", Nature Methods, vol. 13, pp. 1021-1028, 2016.
  7. T. Laviv, B.B. Kim, J. Chu, A.J. Lam, M.Z. Lin, and R. Yasuda, "Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins", Nature Methods, vol. 13, pp. 989-992, 2016.
  8. B.T. Bajar, A.J. Lam, R.K. Badiee, Y. Oh, J. Chu, X.X. Zhou, N. Kim, B.B. Kim, M. Chung, A.L. Yablonovitch, B.F. Cruz, K. Kulalert, J.J. Tao, T. Meyer, X. Su, and M.Z. Lin, "Fluorescent indicators for simultaneous reporting of all four cell cycle phases", Nature Methods, vol. 13, pp. 993-996, 2016.
  9. S. Adams, M. Mackey, R. Ramachandra, S. Palida Lemieux, P. Steinbach, E. Bushong, M. Butko, B. Giepmans, M. Ellisman, and R. Tsien, "Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species", Cell Chemical Biology, vol. 23, pp. 1417-1427, 2016.
  10. E.A. Rodriguez, R.E. Campbell, J.Y. Lin, M.Z. Lin, A. Miyawaki, A.E. Palmer, X. Shu, J. Zhang, and R.Y. Tsien, "The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins", Trends in Biochemical Sciences, 2016.
  11. S. Skylaki, O. Hilsenbeck, and T. Schroeder, "Challenges in long-term imaging and quantification of single-cell dynamics", Nature Biotechnology, vol. 34, pp. 1137-1144, 2016.
  12. V. Glembockyte, J. Lin, and G. Cosa, "Improving the Photostability of Red- and Green-Emissive Single-Molecule Fluorophores via Ni2+ Mediated Excited Triplet-State Quenching", The Journal of Physical Chemistry B, vol. 120, pp. 11923-11929, 2016.
  13. T.S.G. Olsson, and M. Hartley, "jicbioimage: a tool for automated and reproducible bioimage analysis", PeerJ, vol. 4, pp. e2674, 2016.
  14. V. Perez, B. Chang, and E.H.K. Stelzer, "Optimal 2D-SIM reconstruction by two filtering steps with Richardson-Lucy deconvolution", Scientific Reports, vol. 6, pp. 37149, 2016.
  15. D.S. Bindels, L. Haarbosch, L. van Weeren, M. Postma, K.E. Wiese, M. Mastop, S. Aumonier, G. Gotthard, A. Royant, M.A. Hink, and T.W.J. Gadella, "mScarlet: a bright monomeric red fluorescent protein for cellular imaging", Nature Methods, 2016.
  16. E.A. Specht, E. Braselmann, and A.E. Palmer, "A Critical and Comparative Review of Fluorescent Tools for Live Cell Imaging", Annual Review of Physiology, vol. 79, 2016.
  17. N. Chakrova, A.S. Canton, C. Danelon, S. Stallinga, and B. Rieger, "Adaptive illumination reduces photobleaching in structured illumination microscopy", Biomedical Optics Express, vol. 7, pp. 4263, 2016.
  18. J. Griffié, M. Shannon, C.L. Bromley, L. Boelen, G.L. Burn, D.J. Williamson, N.A. Heard, A.P. Cope, D.M. Owen, and P. Rubin-Delanchy, "A Bayesian cluster analysis method for single-molecule localization microscopy data", Nature Protocols, vol. 11, pp. 2499-2514, 2016.
  19. N. Siegel, V. Lupashin, B. Storrie, and G. Brooker, "High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers", Nature Photonics, vol. 10, pp. 802-808, 2016.

Access problems with https://

I’ve had a couple of reports of people unable read the posts by clicking on the front page links. At least some of this is due to the blog only being set up to work with HTTP.  If  you try and access pages through HTTPS, you’ll run into problems. The way to fix this seems to be to switch all links over to HTTPS.  I’ll do that in the next few weeks (unfortunately, it’s not totally straightforward), but in the meantime, if you can’t access a page, try accessing it via http:// instead of https://.

Testing a Point Grey Camera for Fluorescence Microscopy

About two years ago, I mentioned Point Grey cameras. These are cameras sold to the machine vision and industrial inspection market, and are much cheaper than typical microscopy cameras – most are <$1000. Point Grey puts out very nice spec sheets listing all of their cameras, and the specifications for some are pretty impressive – cameras with < 3e- read noise for ~$500. Nico Stuurman has recently written a Micro-manager driver for these cameras, and was kind enough to let me test one of these cameras. We mounted it opposite a Hamamatsu Flash4.0 (an older Flash4.0, with ~72% QE), and did a qualitative comparison by taking sequential images of the same test slide on both cameras.

The Point Grey camera we tested was a Chameleon3 CM3-U3-31S4M. This uses a Sony IMX265 sensor, which has 2048 x 1536 3.45 μm pixels, with 71% QE, <3e- read noise, and sells for ~$500. It can run at up to 55 fps. On paper, this camera should perform almost as well as the Flash 4.0. The images below are of a Texas red-phalloidin stained cell, captured with a 20x / 0.75 NA objective and a 10 ms exposure on both cameras. Click on the images to see the full size image.


The Flash 4.0 camera, 10 ms exposure. Click for full size.


The Point Grey camera, 10 ms exposure. Click for full size.

Continue reading

New Nikon Stand

Nikon has just announced a new stand, the Ti2.  Some noteworthy features, including a 25mm camera port with an F-mount (with a new tube lens and larger filter cubes; it looks like the Plan Apo λ objectives are flat across this field), an LED brightfield illuminator with a fly-eye lens for uniform illumination, a motorized correction collar, an internal camera for back focal plane imaging, and encoding of all microscope components.

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, pp. 12471, 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, pp. 34486, 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, 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. I’ll probably continue to keep the NIC page the authoritative one, but updating on Github is so easy that it’s tempting to move it there.

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.