Skip to main content

STORM - Stochastic Optical Reconstruction Microscopy

Learn how to build a super-resolution STORM (Stochastic Optical Reconstruction Microscopy) system using UC2 components. STORM enables imaging below the diffraction limit by sequentially activating individual fluorescent molecules and precisely localizing them.

What is STORM?​

STORM is a super-resolution microscopy technique that achieves nanometer-scale resolution (typically 20-50nm) by:

  1. Imaging individual fluorescent molecules one at a time
  2. Precisely determining their positions (typically <20nm accuracy)
  3. Reconstructing a high-resolution image from thousands of localizations

This bypasses the classical diffraction limit (~200nm) of conventional optical microscopy.

System Components​

Illumination​

High-intensity laser illumination is critical for STORM. The system uses:

  • 638nm Red Laser Module (500mW) for photoactivation and imaging
  • Optional beam magnifier with rotating diffuser for homogeneous illumination
  • Telescopic lens arrangement to shape the laser profile

Stability​

Nanometer-scale imaging requires exceptional mechanical stability:

  • Vibration isolation systems
  • Temperature-controlled environment
  • Drift correction mechanisms
  • Stable sample mounting

Software​

Specialized software for:

  • Real-time molecule localization
  • Image reconstruction algorithms
  • Drift correction
  • 3D STORM data processing

Electronics​

  • Camera triggering and synchronization
  • Laser intensity control via TTL modulation
  • Motorized focus control
  • Automated acquisition sequences

What You'll Learn​

  • Super-resolution microscopy principles
  • Single-molecule localization techniques
  • High-intensity laser illumination systems
  • Beam shaping and homogenization
  • Mechanical stability requirements
  • STORM image reconstruction
  • Drift correction methods
  • Sample preparation for STORM

Tutorials in this Section​

  • Main Introduction - Overview of STORM system
  • Illumination Setup - Laser configuration and beam shaping
  • Stability Considerations - Achieving nanometer-scale stability
  • Software Configuration - Acquisition and reconstruction software
  • Electronics Integration - Synchronization and control
  • Results and Examples - STORM imaging examples and analysis

Key Techniques​

Laser Beam Shaping​

A telescopic lens arrangement with a rotating diffuser creates homogeneous illumination:

  • Removes hot spots in laser profile
  • Ensures even excitation across field of view
  • Critical for quantitative single-molecule localization

Single-Molecule Localization​

Individual fluorophores are:

  1. Stochastically activated
  2. Imaged until photobleached
  3. Localized with sub-pixel precision (<20nm)
  4. Compiled into super-resolution image

Drift Correction​

Long acquisition times (minutes to hours) require:

  • Fiducial marker tracking
  • Cross-correlation algorithms
  • Real-time drift compensation

Applications​

  • Cell Biology: Visualizing cellular nanostructures (cytoskeleton, organelles)
  • Neuroscience: Mapping synaptic proteins at nanoscale
  • Membrane Biology: Studying protein clustering and organization
  • DNA Imaging: Visualizing chromatin structure
  • Materials Science: Characterizing nanostructured materials

Technical Specifications​

  • Resolution: 20-50nm lateral, 50-100nm axial (3D)
  • Laser Intensity: High power density required (1-10 kW/cm²)
  • Acquisition Time: Minutes to hours depending on density
  • Localization Precision: <20nm per molecule
  • Frame Rate: 20-100 Hz typical
  • Field of View: Typically 50-100 μm

Required Components​

Optical​

  • 638nm high-power laser (500mW)
  • Telescopic lens arrangement
  • Rotating diffuser (modified fan + cling film)
  • High-NA objective (>1.4 NA recommended)
  • Emission filters matched to fluorophore

Mechanical​

  • Vibration isolation table or platform
  • Stable sample mounting stage
  • Optional motorized Z-stage for 3D imaging
  • UC2 cubes and mounting components

Electronic​

  • High-sensitivity EMCCD or sCMOS camera
  • TTL control for laser modulation
  • Synchronization electronics
  • Computer for acquisition and processing

Software​

  • Molecule localization algorithms
  • Image reconstruction software
  • Drift correction tools

Challenges and Solutions​

High Laser Intensity: Use beam expander and diffuser for homogeneous illumination Stability: Implement vibration isolation and drift correction Long Acquisition: Optimize buffer conditions and fluorophore selection Data Processing: Use GPU-accelerated reconstruction algorithms

Perfect for researchers interested in pushing the boundaries of optical microscopy to achieve nanometer-scale resolution!