This page is intended to be a repository of useful information for STORM sample preparation and imaging.

• Fluorescent proteins, SNAP-tags, other genetically encodable molecules.
• Indirect immunofluorescence: The added bulk of two antibodies attached to your protein of interest has been shown to degrade the effective resolution.
• Direct immunofluorescence: Direct immunofluorescence, ideally with Fab fragments or nanobodies, results in your dyes being much closer to your protein of interest and thereby giving higher resolution images.
• Vital dyes: Mitotracker, ER-tracker, and DiI, among others, have recently been shown to photoswitch. See Shim et. al. 2012.

Any dye which can be switched from an off state to an on state can be used for super-resolution imaging by localization microscopy. This has led to a large number of different experimental designs in the literature. Here we highlight a few commonly used approaches.

• Photoactivatible fluorescent proteins: This is the method that was originally described as PALM and FPALM. A fluorescent protein that can be switched from off to on or from green to red is used. One of the most commonly used proteins is mEos or tdEos, but a large number of photoswitchable and photoconvertible proteins can be used. We have put together a table of some of the more commonly used fluorescent proteins for superresolution.
• Stochastic switching of small molecule dyes: often called direct STORM (dSTORM) or GSDIM (ground-state depletion with individual molecule return). High power excitation of a number of small molecule dyes in a buffer containing an oxygen scavenging system and a thiol results in there reversible conversion to a dark state. This then stochatically return to the emitting state and are localized. Typically they cycle between the on and off states many times before photobleaching. Alexa647 or Cy5 have the best performance in this imaging modality, but Atto488 and Cy3B can be used for multicolor imaging. The paper by Dempsey et al. extensively characterizes 26 dyes in this imaging modality.
• Combined reporter/activator dyes: classic STORM imaging. This uses the Alexa647 reporter above, but instead of waiting for spontaneous return from the dark state, it is paired with an activator dye (typically Alexa405, Alexa488, or Alexa568). Excitation of the activator dye triggers the return of nearby Alexa647 molecules to the on state. Using this method requires labeling your own antibodies with the activator/reporter combination.
• Caged / Photoswitchable small molecule dyes: Stefan Hell's group has developed sets of caged dyes and photoswitchable dyes that start in a non-fluorescent state but that can be converted to a fluorescent state (see papers below). These are commercially available from Abberior.

In order to collect good STORM imaging data sample prep is key. Below are important aspects to optimize and consider.

• Fixation protocol. Proper fixation to preserve sample ultrastructure is critical for good super-resolution imaging. A recent paper characterized the effect of different fixation protocols on STORM image quality and has optimized protocols for the best image quality. See Whelan et al. 2015.

• Signal-to-Noise Ratio: High background and/or weak signal makes it significantly harder to obtain molecule localizations.
  • Optimize your fixation and staining to reduce background and increase signal. The use of techniques such as reduction with Sodium Borohydride can greatly reduce some of the autofluorescence associated with fixation
  • Pick the best dye/protein to do the job. The paper by Dempsey et al. (see link below) characterizes 26 dyes, make sure the dye you have picked will work well for your application.

• Dishes vs. Slides: If at all possible we recommend that you prepare your samples for imaging in glass-bottomed petri dishes.
  • Allows for easy exchange of imaging buffer. In order to use stochastic switching of small molecule dyes for STORM imaging a special imaging buffer is required (see protocols below) that needs to be added fresh right before imaging.
  • Allows for the use Perfect Focus to help prevent z-drift during imaging.

• STORM protocols from the Huang lab
• A protocol from the Cell Imaging Center at U. Alberta
• Nikon N-STORM sample preparation manual
• These protocols for GSDIM sample prep from Leica may also be useful.

Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl. 2008;47(33):6172-6. The paper describing dSTORM.

Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods. 2008 Nov;5(11):943-5. The paper describing GSDIM.

Lana Lau, Yin Loon Lee, Steffen J. Sahl, Tim Stearns, and W. E. Moerner. STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein. Biophysical Journal. 2012 June 102:2926–2935 This paper has a nice test of the effect of antibody labeling density on image resolution using STED microscopy. While they study the effect of labeling density on STED imaging, I suspect much of what they find is relevant to STORM and SIM imaging as well.

Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods. 2012 Apr 29 This paper demonstrates the use of a single chain antibody (nanobody) against GFP for STORM imaging of GFP tagged proteins, by binding an Alexa 647 labeled nanobody to GFP-tagged proteins. The nanobody is commercially available from here: http://www.chromotek.com/home/

Lew MD, Lee SF, Ptacin JL, Lee MK, Twieg RJ, Shapiro L, Moerner WE. Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):E1102-10. A localization microscopy experiment combining single molecule blinking of eYFP and localization of Nile Red by random insertion of the dye into the membrane at very low concentration.

Jones SA, Shim SH, He J, Zhuang X. Fast, three-dimensional super-resolution imaging of live cells. Nat Methods. 2011 Jun;8(6):499-508. This paper demonstrates fast STORM imaging of live cells; it also compares the performance of a number of photoswitchable dyes and mEos2 and tdEos.

Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, Olenych S, Davidson MW, Betzig E. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20308-13. This paper describes a number of methods for two-color PALM imaging using genetically encoded fluorescent proteins.

Lippincott-Schwartz J, Patterson GH. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 2009 Nov;19(11):555-65. A nice review of photoactivatible fluorescent proteins for localization microscopy as of 2009.

Whelan DR, Bell TD. Image artifacts in Single Molecule Localization Microscopy: why optimization of sample preparation protocols matters. Scientific Reports. 2015 . 10.1038/srep07924

Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods. 2011 Nov 6;8(12):1027-36. This paper directly compares the performance of 26 different dyes for single dye localization microscopy (dSTORM). An excellent resource for choosing dyes for multicolor imaging.

Belov VN, Wurm CA, Boyarskiy VP, Jakobs S, Hell SW. Rhodamines NN: a novel class of caged fluorescent dyes. Angew Chem Int Ed Engl. 2010 May 3;49(20):3520-3.

Belov VN, Bossi ML, Fölling J, Boyarskiy VP, Hell SW. Rhodamine spiroamides for multicolor single-molecule switching fluorescent nanoscopy. Chemistry. 2009 Oct 19;15(41):10762-76.

Shim SH, Xia C, Zhong G, Babcock HP, Vaughan JC, Huang B, Wang X, Xu C, Bi GQ, Zhuang X. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci U S A. 2012 Aug 28;109(35):13978-83. This paper demonstrates that a number of commonly used vital dyes, including DiI and dyes from the Mito-tracker, ER-tracker, and Lyso-tracker families, photoswitch and can be used for localization microscopy.

Bates M, Huang B, Dempsey GT, Zhuang X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science. 2007 Sep 21;317(5845):1749-53. Multi-color STORM imaging using dye-pair labeled antibodies.

Dani A, Huang B, Bergan J, Dulac C, Zhuang X. Superresolution imaging of chemical synapses in the brain. Neuron. 2010 Dec 9;68(5):843-56. Describes using three color STORM imaging to localize proteins within synapses in 10 um brain sections.

Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Fölling J, Jakobs S, Schönle A, Hell SW, Eggeling C. Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J. 2010 Oct 20;99(8):2686-94. Three color imaging of Alexa 488, Alexa 514, Atto 532, and Cy3 in PVA-embedded samples at the relatively high laser power of 10 kW/cm2.

Vaughan JC, Jia S, Zhuang X. Ultrabright photoactivatable fluorophores created by reductive caging. Nat Methods. 2012 Oct 28. doi: 10.1038/nmeth.2214. Conversion of Atto488, Cy3, Cy3B, Alexa647, and Cy5.5 to a dark stage that can be photoactivated by UV illumination.

Fernández-Suárez M, Ting AY. Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43.

Huang B, Babcock H, Zhuang X. Breaking the diffraction barrier: super-resolution imaging of cells. Cell. 2010 Dec 23;143(7):1047-58.

Moerner WE. Microscopy beyond the diffraction limit using actively controlled single molecules. J Microsc. 2012 Jun;246(3):213-20.

Manley S, Gunzenhäuser J, Olivier N. A starter kit for point-localization super-resolution imaging. Curr Opin Chem Biol. 2011 Dec;15(6):813-21.

van de Linde S, Sauer M. How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem Soc Rev. 2013 Aug 13.

Dempsey, Wang, and Zhuang. Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy. In Handbook of Single-Molecule Biophysics, Hinterdorfer and Van Oijen, eds.