Superresolution Imaging with Standard Fluorescent Probes

Bryan A. Millis1, Dylan T. Burnette1, Jennifer Lippincott‐Schwartz1, Bechara Kachar1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.8
DOI:  10.1002/0471143030.cb2108s60
Online Posting Date:  September, 2013
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For more than 100 years, the ultimate resolution of a light microscope (∼200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today's light microscopes, it is the combination of high‐end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access information at an order of magnitude greater resolution than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but, as with many cutting‐edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking‐assisted localization microscopy (BaLM) makes use of the inherent blinking and bleaching properties of almost all fluorophores as a means to generate superresolution images. Curr. Protoc. Cell Biol. 60:21.8.1‐21.8.17. © 2013 by John Wiley & Sons, Inc.

Keywords: superresolution; BaLM; microscopy; bleaching; blinking

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Setup of Microscopy Hardware for BaLM Acquisition
  • Basic Protocol 2: Sample Preparation for BaLM: Immunofluorescence Labeling of α‐Tubulin in Cos‐7 Cells
  • Basic Protocol 3: Optimization of BaLM Dataset Acquisition
  • Basic Protocol 4: Image Arithmetic and Localization Analysis for BaLM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Setup of Microscopy Hardware for BaLM Acquisition

  MaterialsGeneral microscope hardware
  • Vibration isolation table (e.g., Technical Manufacturing Corporation, Kinetic Systems Inc., Newport Corporation)
  • Motorized inverted fluorescence microscope (e.g., Nikon Ti‐E, Olympus IX‐81/83, Zeiss Axio Observer, Leica DMI6000) with motorized and encoded Z‐axis motor and switchable tube lens element. For reference, the authors use a Nikon Ti‐E.
  • Motorized and encoded specimen stage with appropriate insert(s) for desired imaging chamber(s). As an alternative, a basic non‐moving stage bolted to the stand offers stability of the sample without the cost of a motorized stage, but is not recommended due to the difficulties in navigating the sample.
  • Epi‐fluorescence illuminator
  • Filter cube turret
  • Appropriate wide‐field epi‐fluorescence filter cubes for visual examination of sample
  • Wide‐field excitation source (e.g., mercury arc lamp, EXFO Excite 120)
  • Automated focus assist (e.g., Nikon PFS, Olympus ZDC, Zeiss Definite Focus, Leica Adaptive Focus Control)
  • Appropriate laser lines and launch for desired probes (e.g., 405 nm, 440 nm, 488 nm, 561 nm, 640 nm, 647 nm). For stability and lifetime, the authors recommend diode lasers capable of producing at least 25 mW (out of the fiber), which is suitable for initiation of most bleach/blink events in a reasonable time frame. As a reference, the authors use 405 nm, 488 nm, 561 nm, and 640 nm diode lasers with respective powers out of the fiber of 19.1 mW, 38.9 mW, 44.7 mW, and 21.9 mW.
  • Appropriate laser safety mechanisms (interlocks to prevent laser light directed to eyepieces, scope‐mounted laser safety modules to prevent stray laser light emission, appropriate personal protective equipment, etc.)
  • High‐performance workstation (≥2.0 GHz processor, ≥8 gigabytes of RAM) and monitor
TIRF‐specific microscope hardware
  • High‐numerical‐aperture objective lens (NA ≥1.45)
  • Motorized TIRF illuminator for variable angle illumination (can also incorporate an epi‐fluorescence illuminator)
  • EMCCD (electron multiplying charge coupled device) camera
  • An Andor DU‐897 as well as a liquid‐cooled Photometrics Evolve have both been used successfully by the authors for BaLM imaging
  • 4× or 2.5× magnification optics (see protocol below for details)
  • Emission filter wheel (e.g., Sutter Instrument, Prior Scientific, Ludl Electronic Products)
  • Appropriate laser clean up filter and dichroic mirror for desired laser lines (mounted in fluorescence cube turret). For reference, the authors used a quad TIRF cube purchased through Nikon, which housed Chroma filters and dichroic mirror corresponding to: Zet405/488/561/635× (quad laser clean up filter), and Zt405/488/561/640rpc polychroic mirror.
  • High‐quality emission filters (mounted in the emission filter wheel) appropriate for the desired imaging probes. For reference, the authors used Chroma ET emission filters which passed 447 nm (60 nm band‐pass), 525 nm (50 nm band‐pass), 600 nm (50 nm band‐pass), and 700 nm (75 nm band‐pass). When these emission filters were paired with the above mentioned dichroic mirror, clean‐up filter, and lasers, it enabled proper excitation and emission of Alexa Fluor 405, 488, 568, and 640.
  • Optional: Additional laser blocking notch filters may be added to the emission light path (mounted with the emission filter) to ensure no bleed‐through of laser emission

Basic Protocol 2: Sample Preparation for BaLM: Immunofluorescence Labeling of α‐Tubulin in Cos‐7 Cells

  • Fibronectin from human plasma (Sigma)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐015)
  • COS‐7 (African Green monkey kidney) cells (ATCC)
  • Cytoskeletal stabilization buffer (see recipe)
  • Wash buffer (see recipe)
  • 4% (w/v) paraformaldehyde/0.2% (v/v) glutaraldehyde prepared by diluting stock solutions of 16% paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710) and 25% glutaraldehyde (Electron Microscopy Sciences, cat. no. 16220), respectively
  • Blocking buffer: 5% bovine serum albumin (fraction V) powder in PBS
  • Primary antibody: anti‐α‐tubulin antibody (Sigma)
  • Anti–mouse IgG conjugated to Alexa Fluor 488 (Molecular Probes)
  • Four‐well chamber slides (Lab‐Tek)

Basic Protocol 3: Optimization of BaLM Dataset Acquisition

  • Sample
  • 100‐nm TetraSpeck microspheres (Molecular Probes)
  • Multidimensional imaging software: imaging software capable of experiment management of X/Y location (for automated stages), Z‐axis position, wavelength of excitation (laser), emission filter position, laser power, TIRF angle, camera settings, and timing of frame acquisition; this software is sold by a variety of microscope vendors and third‐party companies, as well as in freeware format (while each have their benefits and limitations, the researcher is encouraged to ensure compatibility with their system, and a good understanding of this software is required before acquiring datasets for BaLM)

Basic Protocol 4: Image Arithmetic and Localization Analysis for BaLM

  • ImageJ software (
  • Stacks T‐functions ImageJ plugin (
  • TIFF stack of bleaching/blinking data
  • Stacks Building ImageJ plugin (
  • StackReg ImageJ plugin (
  • QuickPALM ImageJ plugin (
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Literature Cited

Literature Cited
  Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott‐Schwartz, J., and Hess, H.F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642‐1645.
  Burnette, D.T., Manley, S., Sengupta, P., Sougrat, R., Davidson, M.W., Kachar, B., and Lippincott‐Schwartz, J. 2011a. A role for actin arcs in the leading‐edge advance of migrating cells. Nat. Cell Biol. 13:371‐382.
  Burnette, D.T., Sengupta, P., Dai, Y., Lippincott‐Schwartz, J., and Kachar, B. 2011b. Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules. Proc. Natl. Acad. Sci. U.S.A. 108:21081‐21086.
  Gordon, M.P., Ha, T., and Selvin, P.R. 2004. Single‐molecule high‐resolution imaging with photobleaching. Proc. Natl. Acad. Sci. U.S.A. 101:6462‐6465.
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  Henriques, R., Lelek, M., Fornasiero, E.F., Valtorta, F., Zimmer, C., and Mhlanga, M.M. 2010. QuickPALM: 3D real‐time photoactivation nanoscopy image processing in ImageJ. Nat. Methods 7:339‐340.
  Hess, S.T., Girirajan, T.P.K., and Mason, M.D. 2006. Ultra‐high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91:4258.
  Klar, T.A., Jakobs, S., Dyba, M., Egner, A., and Hell, S.W. 2000. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. U.S.A. 97:8206‐8210.
  Murphy, D.B. and Davidson, M.W. 2012. Fundamentals of light microscopy and electronic imaging. In Fundamentals of Light Microscopy and Electronic Imaging, 2nd ed. (D.B. Murphy and M.W. Davidson) pp. 105‐109. Wiley‐Blackwell, Hoboken, N. J.
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  Simonson, P.D., Rothenberg, E., and Selvin, P.R. 2011. Single‐molecule‐based super‐resolution images in the presence of multiple fluorophores. Nano Lett. 11:5090‐5096.
  Thompson, R.E., Larson, D.R., and Webb, W.W. 2002. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82:2775‐2783.
  Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E., and Selvin, P.R. 2003. Myosin V walks hand‐over‐hand: single fluorophore imaging with 1.5‐nm localization. Science 300:2061‐2065.
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