Upgrading to five-color imaging

We’ve recently been working on upgrading one of our microscopes to do five-color imaging, using a DAPI / FITC / Cy3 / Cy5 / Cy7 filter set. To do so, we replaced our Sutter Lambda XL with a Sutter Lambda LS xenon arc lamp, containing a special 1100 nm cold mirror (thanks Sutter!) so that more of the infrared emission makes it to the output. The spectra for both lamps, measured at the liquid light guide output, are shown below.new LS

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Filter and Filter Wheel Miscellany

We’ve been working on setting up a microscope for five color imaging recently, and as a result I’ve learned a few tips about filters and filter wheels I wanted to pass on.

First, we’re using the Sutter-threaded Semrock filters. These are standard Semrock filters, threaded to mount directly in a Sutter filter wheel (without the filter cups).  They reduce the mass of the filter wheel and allow you to operate it faster. However, the size of the filters and the installation notches (for the spanner wrench) is slightly different than the Sutter cups, meaning that they can’t be installed with the standard Sutter wrenches. Fortunately Sutter makes a special tool for installing the filters; it’s part number X100555, and if you’re going to use these filters, you should definitely get one from Sutter.

Second, I’ve learned that Finger Lakes Imaging makes high speed filter wheels. They have a six position wheel with 23 ms switching times between adjacent positions (this compares to 40 ms for the standard Sutter filter wheel). More interestingly, apparently there is a version of this six position wheel that can be driven by digital inputs, allowing hardware-triggered acquisition. I haven’t seen this wheel yet, but it seems intriguing.

Theatrical gels as color filters

A recent chat with a colleague about cheap sources for optical filters and a similar conversation on facebook reminded me of an old trick for getting cheap fluorescence filters: theatrical gels. These are the filters used to give theatrical lights their color and they are sheets of dyed plastic. While they don’t have the performance of dichroic filters (or even absorptive glass filters), they are much cheaper – a 2 foot square piece costs less than $10. Conveniently, Rosco, one of the major manufacturers of gels, provides absorption spectra for all of them. They also have an easy-to-use web tool for browsing through different color filters.

For example, here’s the spectrum of Roscolux #15, which makes a not bad long pass filter with a cut-on wavelength of about 550nm:


Transmission spectrum of Roscolux #15.

Their low cost makes them useful for situations where you need a large area filter and don’t need the performance of a dichroic filter. For instance, I originally discovered them for screening GFP libraries on a 22 cm plate – we plated out thousands of colonies, illuminated them with a filtered arc lamp, and then covered a pair of goggles with gel filters to see the fluorescence of each colony.  They’ve also been used for building cheap blue transilluminators for visualizing DNA gels.

These gels aren’t going to replace dichroic filters, but have their place. If you have a clever use for them, post a comment.

Fluorescent Protein Photobleaching and Light Source

In the recent paper on the new Zoanthus derived fluorescent protein, mPapaya1 [1], I saw a figure in the supplementary material that gives a good illustration of the challenges comparing photobleaching rates between proteins  The figure is reproduced here:


Photobleaching data on four different fluorescent proteins. A&B: excitation with a mercury arc lamp; 494/41 nm filter for mPapayas; 500/24 nm filter for mCitrine and mVenus. C&D: LED excitation; 525 nm LED with 494/41 nm filter for mPapayas; 460 nm LED for mCitrine and mVenus. E&F: Confocal laser scanning with a 515 nm laser. From [1].

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  1. H. Hoi, E. Howe, Y. Ding, W. Zhang, M. Baird, B. Sell, J. Allen, M. Davidson, and R. Campbell, "An Engineered Monomeric Zoanthus sp. Yellow Fluorescent Protein", Chemistry & Biology, vol. 20, pp. 1296-1304, 2013. http://dx.doi.org/10.1016/j.chembiol.2013.08.008

Online Microscopy Course

The iBiology Microscopy Course is now live!  This is an online course consisting of about 60 lectures from experts in microscopy. It covers all aspects of microscopy and should be an excellent way to learn more about microscopy, whether you are a beginner in microscopy or want to learn more about cutting-edge super-resolution techniques. The course was organized by Ron Vale, with help from myself and Nico Stuurman and the lectures are by and large given by experts in each field (in many cases the inventor of a technique is the lecturer).

I’m proud to have contributed in a small way to making this course a reality and I hope it is as useful to others as I imagine it will be.

Paper Roundup: September 2013

  • A short review of tissue clearing methods. [1]
  • Optimizing FRET probes by driving interaction of the donor and acceptor in a favorable FRET geometry, either by electrostatic interaction or by using weak heterodimerization domains. [2]
  • A review on alternating laser excitation (ALEX) for FRET microscopy. [3]
  • Silicon nanoparticles: a novel bright, nonblinking probe for single molecule studies (essentially a non-blinking Qdot replacement). [4]
  • Fluorescent nanodiamonds have been used for long-term tracking of lung stem cells in mice. [5]
  • A new red-shifted channelrhodopsin. [6]
  • Journal of Optics has a special issue on high-resolution (super-resolution) optical imaging.
  • A comparison of single particle tracking and temporal image correlation spectroscopy for monitoring diffusion and transport. [7]
  • A comprehensive review of fluorescent proteins for super-resolution imaging. [8]
  • SuperNova: a monomeric version of KillerRed for chromophore-assisted laser inactivation. [9]
  • A frequency domain approach for very high speed imaging, where different pixels are excited at different frequencies. The fluorescence emission is recorded on a PMT and signals at different frequencies are extracted to recover the image. It runs at over 4000 fps for 200 x 92 pixels. [10]
  • A wide-field two-photon approach for neuronal imaging in C. elegans. [11]


  1. D.A. Yushchenko, and C. Schultz, "Tissue Clearing for Optical Anatomy", Angewandte Chemie International Edition, vol. 52, pp. 10949-10951, 2013. http://dx.doi.org/10.1002/anie.201306039
  2. R. Grünberg, J.V. Burnier, T. Ferrar, V. Beltran-Sastre, F. Stricher, A.M. van der Sloot, R. Garcia-Olivas, A. Mallabiabarrena, X. Sanjuan, T. Zimmermann, and L. Serrano, "Engineering of weak helper interactions for high-efficiency FRET probes", Nature Methods, vol. 10, pp. 1021-1027, 2013. http://dx.doi.org/10.1038/nmeth.2625
  3. J. Hohlbein, T.D. Craggs, and T. Cordes, "Alternating-laser excitation: single-molecule FRET and beyond", Chem. Soc. Rev., vol. 43, pp. 1156-1171, 2014. http://dx.doi.org/10.1039/C3CS60233H
  4. H. Nishimura, K. Ritchie, R.S. Kasai, M. Goto, N. Morone, H. Sugimura, K. Tanaka, I. Sase, A. Yoshimura, Y. Nakano, T.K. Fujiwara, and A. Kusumi, "Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging", The Journal of Cell Biology, vol. 202, pp. 967-983, 2013. http://dx.doi.org/10.1083/jcb.201301053
  5. T. Wu, Y. Tzeng, W. Chang, C. Cheng, Y. Kuo, C. Chien, H. Chang, and J. Yu, "Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds", Nature Nanotechnology, vol. 8, pp. 682-689, 2013. http://dx.doi.org/10.1038/nnano.2013.147
  6. J.Y. Lin, P.M. Knutsen, A. Muller, D. Kleinfeld, and R.Y. Tsien, "ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation", Nature Neuroscience, vol. 16, pp. 1499-1508, 2013. http://dx.doi.org/10.1038/nn.3502
  7. F. LUND, and D. WÜSTNER, "A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility", Journal of Microscopy, vol. 252, pp. 169-188, 2013. http://dx.doi.org/10.1111/jmi.12080
  8. K. Nienhaus, and G. Ulrich Nienhaus, "Fluorescent proteins for live-cell imaging with super-resolution", Chem. Soc. Rev., vol. 43, pp. 1088-1106, 2014. http://dx.doi.org/10.1039/c3cs60171d
  9. K. Takemoto, T. Matsuda, N. Sakai, D. Fu, M. Noda, S. Uchiyama, I. Kotera, Y. Arai, M. Horiuchi, K. Fukui, T. Ayabe, F. Inagaki, H. Suzuki, and T. Nagai, "SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation", Scientific Reports, vol. 3, 2013. http://dx.doi.org/10.1038/srep02629
  10. E.D. Diebold, B.W. Buckley, D.R. Gossett, and B. Jalali, "Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy", Nature Photonics, vol. 7, pp. 806-810, 2013. http://dx.doi.org/10.1038/nphoton.2013.245
  11. T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, "Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light", Nature Methods, vol. 10, pp. 1013-1020, 2013. http://dx.doi.org/10.1038/nmeth.2637