Techniques:

• 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

Practical:

• 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