Stochastic Optical Reconstruction Microscopy (STORM)

Jianquan Xu1, Hongqiang Ma1, Yang Liu2

1 Biomedical and Optical Imaging Laboratory, Departments of Medicine and Bioengineering, University of Pittsburgh, Pittsburgh, 2 Biomedical and Optical Imaging Laboratory, Departments of Medicine and Bioengineering, University of Pittsburgh, University of Pittsburgh Cancer Institute, Pittsburgh
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.46
DOI:  10.1002/cpcy.23
Online Posting Date:  July, 2017
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Super‐resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten‐fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects—sample preparation, image acquisition, and image reconstruction—to ensure a high‐quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc.

Keywords: single molecule localization microscopy (SMLM); stochastic optical reconstruction microscopy (STORM); super‐resolution fluorescence microscopy

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Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling of Photo‐Switchable Fluorophores
  • Basic Protocol 2: Image Acquisition
  • Basic Protocol 3: Image Reconstruction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Labeling of Photo‐Switchable Fluorophores

  • Poly‐D‐lysine (PDL)
  • Gold nanoparticle suspension (100 nm particle size; BBI Solutions, cat. no. EM.GC100)
  • Alexa Fluor 405 carboxylic acid succinimidyl ester (ThermoFisher)
  • Alexa Fluor 647 carboxylic acid succinimidyl ester (ThermoFisher)
  • Cy2 and Cy3B reactive dye (GE Healthcare)
  • Dimethylsulfoxide (DMSO; anhydrous)
  • Sodium bicarbonate (NaHCO 3)
  • Secondary antibodies:
    • Donkey anti‐rabbit antibody (Jackson ImmunoResearch)
    • Donkey anti‐mouse antibody (Jackson ImmunoResearch)
  • Phosphate‐buffered saline (PBS; Sigma‐Aldrich)
  • Cytoskeleton buffer (see recipe)
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • 0.1% (v/v) Triton X‐100 in PBS (permeabilization buffer)
  • Blocking buffer: 3% (w/v) BSA plus 0.1% (v/v) Triton X‐100 in PBS
  • Primary antibody:
    • Rabbit anti‐histone H2B antibody (Abcam, cat. no. ab1790)
    • Rabbit anti‐alpha tubulin antibody (Abcam, cat. no. ab18251)
    • Rabbit anti‐H3K4me3 antibody (EMD Millipore, cat. no. 07‐473)
    • Mouse anti‐H3K9ac antibody (Abcam, cat. no. ab12179)
  • Washing buffer (see recipe)
  • Glass bottom cell culture dishes (World Precision Instruments, cat. no. FD3510) or #1.5 coverslips
  • Ultrasound bath sonicator
  • Shaking platform
  • NAP‐5 size‐exclusion columns
  • Nanodrop 2000 microspectrophotometer

Basic Protocol 2: Image Acquisition

  • Glucose oxidase from Aspergillus niger, type VII, lyophilized powder, ≥ 100,000 U/g solid (Sigma‐Aldrich)
  • 17 mg/ml catalase from bovine liver, lyophilized powder, ≥ 10,000 units/mg protein (Sigma‐Aldrich) in buffer A
  • Buffer A (see recipe)
  • Cysteamine (MEA; Sigma‐Aldrich)
  • 0.25 N HCl
  • Buffer B (see recipe)
  • Dish with cells from protocol 1, step 22
  • Immersion oil for microscopy
  • Cyclooctatetraene (COT; Sigma‐Aldrich; optional)
  • Fluorescence microscope

Basic Protocol 3: Image Reconstruction

  • Raw images from protocol 2
  • ThunderSTORM software (
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Literature Cited

Literature Cited
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