Transcript Diffusion of CaM and CaMK-II

Diffusion of CaM and CaMK-II
Andrew Harrell
Dr. Waxham Lab
University of Texas Medical School
Fick’s Diffusion Model
J
Units for D =
Volume
V
n
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
take37-take36
1.11
data
fit
1.1
1.09
epsilon
1.08
2.3011
Adjusted R2
-102.1231
1.07
g()
• Wavelength 780
nm chosen to
maximize activity
of the Alexa-488
dye.
• D(CaM) = 75.00
• D(CaMKII+CaM) =
15.78
• (
)
Nac 10.0286
K 2.95
taud 0.00015778
Vtrue (fL) 0.057516
C (M) 1.024e-007
Ntrue 3.5456
1.06
1.05
D (um2/sec) 73.071
1.04
1.03
1.02
1.01
-6
10
-4
-2
10
10
0
10
 (sec)
1
0
-1
-6
10
-4
10
-2
10
0
10
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???