Deconvolution, Visualization and

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• Deconvolution, Visualization and
• Analysis Software for
• Widefield and Confocal Microscopy

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• Media Cybernetics is committed to delivering the
  most advanced algorithms available on the market
  today by the cutting edge research to develop the
  most advanced algorithms for tomorrow.

Media Cybernetics provides uncompromised results
with faster performance than ever before.
The best just keeps getting better and faster
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FRET
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FRET Definition

• Fluorescence resonance energy transfer (FRET) is
  the radioationless energy transfer from the
  excited fluorescent donor to the acceptor when
  the absorption spectra of the acceptor overlaps
  with the emission spectra of the donor, and the
  two molecules are within the proximity of 10 to
  100Å with each other and have an approximately
  parallel transition dipole orientation.

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FRET Applications

• In biological applications, FRET has become
  popular to qualitatively map protein-protein
  interactions, especially after series of
  fluorescence proteins, such as YFP, GFP, etc are
  discovered in recent yeas, because researchers
  can use these fluorescence proteins to study
  living specimens without disrupting biological
  functions as those traditional dyes did.

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FRET Conditions

• Donor and Acceptor must be close (10100 Å
  apart),
• The Donor and Acceptor transition dipole
  orientations must be approximately parallel
• The fluorescence emission spectrum of Donor must
  overlap the absorption spectrum of Acceptor .
• The Donor probe must have a high quantum yield.

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FRET Measurement Artifacts

• The bleed-through, i.e. when a donor is excited
  by the acceptors excitation wavelength or vice
  versa
• the cross talk, i.e. when the emission of the
  donor is measured in a detector that is set up
  for the observation of acceptor signals, and vice
  versa.

Figure 2. The uncorrected FRET signal actually
includes Donor Cross-talk(DBST), Acceptor
bleed-through (ABST) and the real FRET signal.
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FRET Measurement Artifacts
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FRET Algorithms

• Precision FRET (PFRET) data analysis from
  Periasamys Lab
• Gordons FRET data analysis from Hermans Lab
  --- MetaMorph
• Media Cybernetics FRET

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Media Cybernetics FRET Model
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Media Cybernetics FRET Equations
µlm(x) fluorescence seen from donor
fluorophore fluorescence seen from acceptor
fluorophore concentration of donor
that is excited and fluorescing C emission filter
sensitivity to the donor concentration
of acceptor that is excited and fluorescing C
emission filter sensitivity to the
acceptor
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AQI MLE Method
Image Intensity Joint Probability The
set of parameters to be estimated includes C1(x),
C2(x), F(x) and three intermediate system
coefficients. Steepest Ascent Gradient method is
used by taking the partial derivative of the
log-likelihood expression with respect to the
relevant parameters
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AQI FRET Module

• Three Algorithms
• Option of Input of Calibration Images or
  Coefficients
• Background Subtraction
• Use ROIs on FRET and Calibration Images to
  Correct Crosstalk

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AQI FRET Module

• Automatic Loading of corrected FRET images from
  last screen
• FRET Statistics, Efficiency, Foster Distance on
  ROIs
• Thresholding function

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FRET Results
After any of the 3 algorithms are run and 3
images are generated, we use the Calculate FRET
Efficiency tab to generate statistics the ROI
statistics are shown on the grid in the GUI.
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FRET Algorithms Comparison
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Precision (3 different concentrations, 4 or 5
tests for each concentration) AQI algorithm
average CV 0.155 Gordon's algorithm average CV
0.150 Peri's algorithm average CV 0.464
FRET Algorithms Comparison
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Advantages of AQI FRET Module

• Robust application includes choice of 3
  algorithms
• Includes proprietary AQI MLE algorithm
• Automated alignment feature
• Handles 2D, 3D and 2D time-series data
• Interactive ROIs and thresholding
• Easy to use - intuitive interface
• Patent Pending

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Why Media Cybernetics

• Established technological focus in deconvolution
  over 17 years of dedicated research
• Breadth of algorithms that are well known and
  accepted world-wide optimized for maximum
  robustness
• Media Cybernetics stands behind all of its
  products and provides world class education and
  support.
• Committed focus to deconvolution, visualization
  and analysis RD ensures customer access to the
  latest technologies for today and tomorrow.

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Media CyberneticsA complete post-processing
solution

• Image improvement, restoration, visualization and
  analysis that facilitates more accurate and
  compelling results.
• Media Cybernetics creates the tools to help you
  do the science.
• AQI Product Line
• AutoDeblur (2D, Silver, Gold)
• AutoVisualize (with improved 3D-Viewer)
• AutoAnalyze (FRET, Ratio, Object
  Counting/Tracking, Colocalization)
• Tools (automatic, manual and channel-channel
  alignment)
• Image-Pro Product Line
• Image-Pro Plus and Image-Pro Plug-In module
  bundles
• Express
• Analyzer