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storm [2014/05/05 11:27]
kthorn [Protocols]
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.
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- 
  
 ===== 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.]]   * [[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.]]
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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 ====
/var/www/html/dokuwiki/data/attic/storm.1399314455.txt.gz · Last modified: 2014/05/05 11:27 by kthorn