Laser Light Scattering

1
Laser Light Scattering

• - Basic ideas what is it?
• - The experiment how do you do it?
• - Some examples systems why do it?

2
Double Slit Experiment
3
Light Scattering Experiment
4
More Detailed Picture
detector
q
Inter-particle interference
How can we analyze the fluctuations in
intensity? Data g(t) ltI(t) I(t t)gtt
intensity autocorrelation function
5
Intensity autocorrelation

• g(t) ltI(t) I(t t)gtt

6
What determines correlation time?

• Scatterers are diffusing undergoing Brownian
  motion with a mean square displacement given by
  ltr2gt 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
of D 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
Antibody molecules

• Technique to make 2-dimensional crystals of
  proteins on an EM grid (with E. Uzgiris at GE RD)

Y
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, cont
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