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Laser Light Scattering

Laser Light Scattering. - Basic ideas – what is it? - The experiment – how do you do it? - Some examples systems – why do it?. Coherent beam. Extra path length. screen. +. +. =. =. Double Slit Experiment. Scatterers in solution (Brownian motion). Scattered light.

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Laser Light Scattering

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  1. Laser Light Scattering - Basic ideas – what is it? - The experiment – how do you do it? - Some examples systems – why do it?

  2. Coherent beam Extra path length screen + + = = Double Slit Experiment

  3. Scatterers in solution (Brownian motion) Scattered light Laser at fo Narrow line incident laser Doppler broadened scattered light Df f fo 0 is way off scale Df ~ 1 part in 1010 - 1015 Light Scattering Experiment

  4. Detected intensity Iaverage time More Detailed Picture detector q Inter-particle interference How can we analyze the fluctuations in intensity? Data = g(t) = <I(t) I(t + t)>t = intensity autocorrelation function

  5. t For small t t For larger t g(t) t tc Intensity autocorrelation • g(t) = <I(t) I(t + t)>t

  6. What determines correlation time? • Scatterers are diffusing – undergoing Brownian motion – with a mean square displacement given by <r2> = 6Dtc (Einstein) • The correlation time tc is a measure of the time needed to diffuse a characteristic distance in solution – this distance is defined by the wavelength of light, the scattering angle and the optical properties of the solvent – ranges from 40 to 400 nm in typical systems • Values of tc can range from 0.1 ms (small proteins) to days (glasses, gels)

  7. Diffusion • What can we learn from the correlation time? • Knowing the characteristic distance and correlation time, we can find the diffusion coefficient D • According to the Stokes-Einstein equation where R is the radius of the equivalent sphere and h is the viscosity of the solvent • So, if h is known we can find R(or if R is known we can find h)

  8. Why Laser Light Scattering? • Probes all motion • Non-perturbing • Fast • Study complex systems • Little sample needed Problems: Dust and best with monodisperse samples

  9. Some Examples

  10. Superhelical DNA where = Watson-Crick-Franklin double stranded DNA pBR322 = small (3 million molecular weight) plasmid DNA Laser light scattering measurements ofD vs q give a length L = 440 nm and a diameter d = 10 nm DNA-drug interactions: intercalating agent PtTS produces a 26o unwinding of DNA/molecule of drug bound Since D ~ 1/size, as more PtTS is added and DNA is “relaxed,” we expect a minimum in D

  11. As drug is added DNA first unwinds to open circle and then overwinds with opposite handedness. At minimum in D the DNA is unwound. This told us that there are 34 superhelical turns in native pBR pBR is a major player in cloning – very important to characterize well

  12. Change pH Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 60o 120o Antibody molecules Y • Technique to make 2-dimensional crystals of proteins on an EM grid (with E. Uzgiris at GE R&D) Conformational change with pH results in a 5% change in D – seen by LLS and modeled as a swinging hinge

  13. Aggregating/Gelling SystemsStudied at Union College • Proteins: • Actin – monomers to polymers and networks Study monomer size/shape, polymerization kinetics, gel/network structures formed, interactions with other actin-binding proteins Why? Epithelial cell under fluorescent microscope Actin = red, microtubules = green, nucleus = blue

  14. Aggregating systems, con’t what factors cause or promote aggregation? what is the structure of the aggregates? how can proteins be protected from aggregating? • BSA (bovine serum albumin) • b amyloid • insulin • Chaperones • Polysaccharides: • Agarose • Carageenan Focus on the onset of gelation – what are the mechanisms causing gelation?how can we control them?what leads to the irreversibility of gelation?

  15. Collaborators and $$ • Nate Poulin ’14 & Christine Wong ‘13 • Michael Varughese ’11 (med school) • Anna Gaudette ‘09 • Bilal Mahmood ’08 & Shivani Pathak ’10 (both in med school) • Amy Serfis ‘06 & Emily Ulanski ’06 (UNC, Rutgers ) • Shaun Kennedy (U Michigan, Ann Arbor in biophysics) • Bryan Lincoln (PhD from U Texas Austin, post-doc in Dublin) • Jeremy Goverman (medical school) • Shirlie Dowd (opthamology school) • Ryo Fujimori (U Washington grad school) • Tomas Simovic (Prague) • Ken Schick, Union College • J. Estes, L. Selden, Albany Med • Gigi San Biagio, Donatella Bulone, Italy Thanks to NSF, Union College for $$

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