Methods Implemented in iSMS


  • Single-molecule FRET (smFRET) TIRF-microscopy
  • Quantitative smFRET
    • Calculate correction factors
    • Molecule-to-molecule correction factors (or globals)
    • Correction factor vs. FRET 2D plots
  • Single- or two-color excitation (ALEX)
    • FRET-histogram analysis
  • FRET dynamics:
    • Trace dynamics analysed automatically by Hidden Markov Modeling (the algorithm of vbFRET)
    • Transition density plots
    • Dwell-time histograms
    • Mean time plots
  • Tethered Fluorophore Motion (TFM)

Excitation schemes:

  • Single-color excitation
  • Two-color alternating laser excitation (ALEX)

Peak finder:

  • Local maximum method (fast) with background subtraction and intensity based thresholds

Photon counting methods:

  • Intensity masks (aperture photometry)
  • Gaussian point spread functions (PSFs) fitted using least-squares (LSQ) and constant weights
  • Gaussian PSFs fitted using maximum likelihood estimation (MLE)

Background subtraction:

  • Molecules:
    • Background ring (mask)
    • Intensity after bleaching or during blinking
    • Specify background pixels manually
  • Camera background:
    • Constant offset
    • Stored image

Automated algorithms:

  • Automated alignment of channels
  • Drift compensation (post acquisition, horizontal)
  • FRET-pair localization
  • Automated bleach detection
  • Molecule filters
  • Molecule grouping


  • Loads >120 different raw image file formats
  • Save/open/merge and share sessions of data with peers and collaborators
  • Customize default settings
  • Quick export of figures and data
  • Interactively explore raw data
  • Sort molecules according to properties
  • Group molecules into subpopulations
  • Fully interactive - no programming skills needed