Transcript Laser Light Scattering

Laser Light Scattering
- Basic ideas – what is it?
- The experiment – how do you do it?
- Some examples systems – why do it?
Double Slit Experiment
Coherent beam
Extra path length
screen
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=
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Light Scattering Experiment
Scatterers in solution (Brownian motion)
Scattered light
Laser at fo
Narrow line incident laser
Doppler broadened
scattered light
Df
0 is way off scale
fo
Df ~ 1 part in
f
1010
-
1015
More Detailed Picture
detector
q
Inter-particle interference
Detected
intensity
Iaverage
time
How can we analyze the fluctuations in intensity?
Data = g(t) = <I(t) I(t + t)>t = intensity autocorrelation function
Intensity autocorrelation
• g(t) = <I(t) I(t + t)>t
t
For small t
For larger t
t
g(t)
tc
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
k BT
D
6h R
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?
1.
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Probes all motion
Non-perturbing
Fast
Study complex systems
Little sample needed
Problems: Dust and
best with monodisperse samples
Some Examples
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
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
Antibody molecules
• Technique to make 2-dimensional crystals
of proteins on an EM grid (with E. Uzgiris
at GE R&D)
Change pH
60o
120o
Conformational change with pH results in a 5% change in
D – seen by LLS and modeled as a swinging hinge
Aggregating/Gelling Systems
Studied 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
– BSA (bovine serum
 b amyloid
- insulin
– Chaperones
what factors cause or promote
albumin) aggregation?
what is the structure of the
aggregates?
how can proteins be protected
from aggregating?
Focus on the onset of gelation –
• Polysaccharides:
– Agarose
– Carageenan
what are the mechanisms causing gelation?
how can we control them?
what leads to the irreversibility of gelation?
Collaborators and $$
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Nate Poulin ’14 & Christine Wong ‘13
Michael Varughese ’11 (med school)
Anna Gaudette ‘09
Bilal Mahmood ’08 & Shivani Pathak ’10 (both in med school)
Amy Serfis ‘06 & Emily Ulanski ’06 (UNC, Rutgers )
Shaun Kennedy (U Michigan, Ann Arbor in biophysics)
Bryan Lincoln (PhD from U Texas Austin, post-doc in Dublin)
Jeremy Goverman (medical school)
Shirlie Dowd (opthamology school)
Ryo Fujimori (U Washington grad school)
Tomas Simovic (Prague)
Ken Schick, Union College
J. Estes, L. Selden, Albany Med
Gigi San Biagio, Donatella Bulone, Italy
Thanks to NSF, Union College for $$