Seizure Detection by FRAP

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Seizure Detection by FRAP

• Bio-analytics
• Prof Dr. Klemer
• Presenter by
• Mohammad Habibi

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Outlines

• FRAP
• ECS
• Seizure Detection by FRAP
• Technical Tips
• Results and conclusion

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What is FRAP?

• FRAP (Fluorescence Recovery After Photobleaching)
• Measuring the fluorescence in a defined region
  of a sample after a bleaching process

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How does it work?

• Tag molecules with fluorescein material
• Illuminate the region by very low intensity light
  to obtain emitted light
• Illuminate the region with very high intensity
  short pulse laser (photo bleaching)
• (basically the signal will be cut off by HI
  laser)
• Recording the progress of fluorescence recovery
  in the bleached area with high temporal
  resolution

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FRAP Process
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FRAP Process
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Diffusion Coefficient Calculation

• Curve fitting

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What kind of information does it provide?

• Changes in intensity in the bleached region
  represent the sum of all movements of the
  fluorescent molecules, whether passive (e.g.,
  diffusion) or active (e.g., transport).
• The regeneration time (half-recovery period) is a
  measure of the speed of fluorescent molecules
  movement

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Schematic
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FRAP Equipment
CCD camera
Argon laser
Laser optics
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Why do we need to know about diffusion?

• Many types of diseases can be diagnosed by the
  diffusion coefficient information
• Such as
• Brain edema
• Alzheimer's disease
• Epilepsy (Seizure)

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Is the FRAP only way to measure diffusion?

• NO,
• Diffusion can be measured by
• Radiotracer
• Diffusion-weighted MRI
• TMA (tetramethylammonium)

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FRAP Advantages

• Minimally invasive (avoid damage related to
  micropipette)
• Can be applied to study of many types of
  macromolecules such as DNA, Protein,
  carbohydrates
• Excellent temporal and spatial resolution
• well suited for measurement in vivo

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Key Elements in FRAP

• Laser intensity before bleaching (detecting
  signal with acceptable SNR)
• Laser intensity during bleaching (4000 fold)
• Laser pulse time bleaching (1-5 msec)
• Sampling 500 msec after bleaching

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Extracellular Space (ECS)

• 20 of the brain volume
• Jelly-like matrix ( ions, neurotransmitter,
  metabolites, peptides, ECS molecules)
• Forming the microenvironment for all cells,
  mediating glia-neuron communication via
  diffusible metabolites, ions
• Non-synaptic communication or volume
  transmission
• ECS plays important role in pathological
  condition like brain edema or seizure activity

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Detecting seizure by FRAP

• The experiment has been done on a mouse
• 1) exposing the intact dura
• 2) loading fluorescein-dextrans
• (dissolve with aCSF)
• 3) loading time from 1hr to 2 hrs

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Loading of ECS by Fluorescein
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Detecting (Cont)

• 4) Recording the signal at detector before and
  after bleaching
• 5) injection of PTZ to model generalized seizure
• 6) Recording the signal before bleaching
• 7)Photobleaching and recording signal
• (before seizure , during seizure and after
  seizure)
• Results
• Seizure caused changes in diffusion
  coefficient before and after seizure activity

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Detecting Seizure Result
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Schematic
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Instruments characters

• Laser first order beam, 2 Watt, 488 nm, argon
  ion
• Acousto-optic modulator
• Dichronic mirror , 510 nm
• Objective lens 50,numerical aperture 0.55,
  working distance 8 mm, Nikon air
• Photomultiplier, amplifier, A/D(14 bits)

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Experiment process

• Sampling fluorescence continuously for 200 msec
  before bleaching
• Sampling at 1 Mhz after bleaching and over 500
  msec after bleaching
• Repeating process for 10 to 60 sec
• Curve fitting - using non-linear least squares to
  find half-time recovery t 1/2
• F(t) is fluorescent recovery function

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Key Points

• Type of fluorescein material (4,70,500 kDa)
• Loading time
• Timing issue
• Laser power
• Calibration is required in this method
• Excellent Temporal resolution (less than 1 min)
• Simple signal processing

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Conclusion

• FRAP can be applied to detect and predict
  seizures
• However its an invasive technique

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• Questions?