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storm [2012/09/14 15:15]
kthorn [Labeling Method]
storm [2016/06/23 12:24] (current)
kthorn
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   * Fluorescent proteins, SNAP-tags, other genetically encodable molecules.   * 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 significantly ​degrade the effective resolution.+  * 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.   * 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.   * Vital dyes: Mitotracker,​ ER-tracker, and DiI, among others, have recently been shown to photoswitch. See Shim et. al. 2012.
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 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. 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. +  * 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 [[storm:​FPs|a table of some of the more commonly used fluorescent proteins for superresolution]]
-  * Stochastic switching of small molecule dyes: often called direct STORM (dSTORM). 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.+  * 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.   * 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 [[http://​www.abberior.com/​products/​productlist/​cat/​labels-by-function/​|Abberior]].   * 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 [[http://​www.abberior.com/​products/​productlist/​cat/​labels-by-function/​|Abberior]].
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 In order to collect good STORM imaging data sample prep is key. Below are important aspects to optimize and consider. 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 [[http://​www.nature.com/​articles/​srep07924|Whelan et al. 2015]].
 +
   * Signal-to-Noise Ratio: High background and/or weak signal makes it significantly harder to obtain molecule localizations.   * 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         * 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
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         * 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 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.         * Allows for the use Perfect Focus to help prevent z-drift during imaging.
- 
- 
  
 ===== Protocols ===== ===== Protocols =====
-  * [[http://​craterlake.ucsf.edu:​18080/​cm/​wiki/?​id=359|STORM protocols from the Huang lab]] 
-  * [[http://​www.microscopy.med.ualberta.ca/​techniques/​storm-immunofluorescence-protocol/​|A protocol from the Cell Imaging Center at U. Alberta]] 
   * {{:​nikon_storm_sample_preparation.pdf|Nikon N-STORM sample preparation manual}}   * {{:​nikon_storm_sample_preparation.pdf|Nikon N-STORM sample preparation manual}}
 +  * [[http://​www.leica-microsystems.com/​science-lab/​sample-preparation-for-gsdim-localization-microscopy-protocols-and-tips/​|These protocols for GSDIM sample prep from Leica may also be useful.]]
  
 ===== References ===== ===== References =====
  
-{{:dani_huang_synapse_storm.pdf|Dani AHuang B, Bergan JDulac CZhuang XSuperresolution ​imaging ​of chemical synapses in the brainNeuron2010 Dec 9;68(5):843-56.}} +{{:dstorm.pdf|Heilemann M, van de Linde S, Schüttpelz M, Kasper RSeefeldt ​B, Mukherjee ATinnefeld PSauer MSubdiffraction-resolution fluorescence ​imaging ​with conventional fluorescent probesAngew Chem Int Ed Engl2008;47(33):​6172-6.}} The paper describing dSTORM. 
-Describes using three color STORM imaging to localize proteins within synapses in 10 um brain sections.+ 
 +{{:​gsdim.pdf|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.
  
 {{:​moerner_sted_labeling.pdf|Lana Lau, Yin Loon Lee, Steffen J. Sahl, Tim Stearns, and W. E. Moerner. STED Microscopy with Optimized Labeling Density Reveals 9-Fold {{:​moerner_sted_labeling.pdf|Lana Lau, Yin Loon Lee, Steffen J. Sahl, Tim Stearns, and W. E. Moerner. STED Microscopy with Optimized Labeling Density Reveals 9-Fold
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 {{:​jones2011faststorm.pdf|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.}} {{:​jones2011faststorm.pdf|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. This paper demonstrates fast STORM imaging of live cells; it also compares the performance of a number of photoswitchable dyes and mEos2 and tdEos.
 +==== Fluorescent Proteins ====
  
 {{:​shroff2007.pdf|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.}} {{:​shroff2007.pdf|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. This paper describes a number of methods for two-color PALM imaging using genetically encoded fluorescent proteins.
 +
 +{{:​jls_ticb.pdf|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.
 +
 +{{::​sci_rep_2014_whelan_dr.pdf|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.
 +
 ==== Single dye imaging ==== ==== Single dye imaging ====
  
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 {{:​papers:​belov2009.pdf|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.}} {{:​papers:​belov2009.pdf|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.}}
  
 +==== Other dyes ====
 +
 +{{:​shim_et_al_2012.pdf|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.
 +
 +==== Multicolor imaging ====
 +
 +{{:​bates_et_al_2007.pdf|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_huang_synapse_storm.pdf|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_et_al_2010.pdf|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.
 +
 +{{:​reductivecaging.pdf| 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.
  
 ==== Reviews ==== ==== Reviews ====
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 {{:​moerner.pdf|Moerner WE. Microscopy beyond the diffraction limit using actively controlled single molecules. J Microsc. 2012 Jun;​246(3):​213-20.}} ​ {{:​moerner.pdf|Moerner WE. Microscopy beyond the diffraction limit using actively controlled single molecules. J Microsc. 2012 Jun;​246(3):​213-20.}} ​
 +
 +{{:​manley_review.pdf|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.}}
 +
 +{{:​lindesauer2013.pdf|van de Linde S, Sauer M. How to switch a fluorophore:​ from undesired blinking to controlled photoswitching. Chem Soc Rev. 2013 Aug 13.}}
 +
  
 [[http://​books.google.com/​books?​id=pT3WaiL5YNkC&​pg=PA96&​dq=storm+microscopy&​hl=en&​sa=X&​ei=pLbfT4nuJoq42wWr_tCyCg&​ved=0CEcQ6AEwAg#​v=onepage&​q&​f=false|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.]] [[http://​books.google.com/​books?​id=pT3WaiL5YNkC&​pg=PA96&​dq=storm+microscopy&​hl=en&​sa=X&​ei=pLbfT4nuJoq42wWr_tCyCg&​ved=0CEcQ6AEwAg#​v=onepage&​q&​f=false|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.]]
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