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storm [2012/10/10 14:50]
kthorn
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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 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 =====
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 A nice review of photoactivatible fluorescent proteins for localization microscopy as of 2009. 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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 ==== Multicolor imaging ==== ==== 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.}} {{:​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.}}
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 {{:​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.}} {{:​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. 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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 {{:​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.}} {{:​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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