Laser Light Scattering

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# Laser Light Scattering - PowerPoint PPT Presentation

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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Presentation Transcript
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

Laser at fo

Narrow line incident laser

scattered light

Df

f

fo

0 is way off scale

Df ~ 1 part in 1010 - 1015

Light Scattering Experiment

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

t

For small t

t

For larger t

g(t)

t

tc

Intensity autocorrelation
• g(t) = <I(t) I(t + t)>t
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)
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)
Why Laser Light Scattering?
• Probes all motion
• Non-perturbing
• Fast
• Study complex systems
• Little sample needed

Problems: Dust and

best with monodisperse samples

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

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

Change pH

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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

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

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?

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 \$\$