1. Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School

2. Fick’s Diffusion Model J n Volume V Units for D =

4. 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.

5. 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.

6. FCS Apparatus Laser light (λ = 780 nm) chosen to maximize dye activity.

8. Data Collection Measure the fluorescent intensity as a function of time. Computer calculates the correlation function vs. (a time delay).

9. Correlation Curves Wavelength 780 nm chosen to maximize activity of the Alexa-488 dye. D(CaM) = 75.00 D(CaMKII+CaM) = 15.78 ( )

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:

11. 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

12. 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

13. Acknowledgements Dr. Waxham – lab director Hugo Sanabria – supervisor Matt Swulius – provided images Ben Goins – thesis material Questions???