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???