This is an old revision of the document!
This page is intended to be a repository of useful information for STORM sample preparation and imaging.
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.
In order to collect good STORM imaging data sample prep is key. Below are important aspects to optimize and consider.
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. A critical exploration of how sample preparation affects image quality. Has optimized fixation protocols for STORM imaging.
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.
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.
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.