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Diffusion of CaM and CaMK-II
Andrew HarrellDr. Waxham Lab
University of Texas Medical School
Fick’s Diffusion Model
J
nVolume V
Units for D =
Fluorescence
• Excitation of a molecule to a higher energy state by photon energy.
• Subsequent lowering of energy state, accompanied by an emission of radiation.
• Ultraviolet -> Visible light.
Fluorescent Correlation Spectroscopy
• Uses multi-photon laser excitation to induce fluorescence.
• Fluorescent intensity is recorded as a function of time.
• A correlation curve is created, which relates fluorescence at a particular time to fluorescence at other times.
FCS Apparatus
• Laser light (λ = 780 nm) chosen to maximize dye activity.
Data Collection
• Measure the fluorescent intensity as a function of time.
• Computer calculates the correlation function vs. (a time delay).
Correlation Curves
• Wavelength 780 nm chosen to maximize activity of the Alexa-488 dye.
• D(CaM) = 75.00• D(CaMKII+CaM) =
15.78• ( )
10-6
10-4
10-2
100
-1
0
1
10-6
10-4
10-2
100
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
(sec)
g( )
Nac 10.0286K 2.95taud 0.00015778
epsilon 2.3011
Adjusted R2 -102.1231
Vtrue (fL) 0.057516C (M) 1.024e-007Ntrue 3.5456
D (um2/sec) 73.071
take37-take36
data
fit
Determining Diffusion Constants
• Interpolate along the curve to find G(0).– G(0) is inversely proportional to the concentration.
• Determine the x-coordinate of the point on the best-fit curve whose corresponds to half of G(0).
• The time is called .• Based on a Gaussian approximation to the
excitation volume, and the two-photon excitation method, we know that:
Procedural Concerns
• Bleaching– Possibility that molecules will be chemically
altered by the light, in a way which prevents future fluorescence.
– Two-photon excitation helps to avoid bleaching.
• Determining the “size” of the activity volume– 3-D Gaussian approximation vs. solution to
Maxwell’s equations
Related Topics
• Fluorescence Recovery After Photobleaching (FRAP) method.– “Opposite” of FCS; uses an intense pulse to
photobleach all of the molecules in a certain volume and then observes fluorescent molecules as they diffuse back into the region.
• Measuring simultaneous fluorescence of multiple molecules
Acknowledgements
• Dr. Waxham – lab director• Hugo Sanabria – supervisor• Matt Swulius – provided images• Ben Goins – thesis material
Questions???
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