Transcript Nano-130709-Ross

Seeing at the Nanoscale
New Microscopies for the Life Sciences
Copyright Jim Gipe/Pivot Media
Dr. Jennifer Ross, Department of Physics
University of Massachusetts Amherst
July 9, 20131
Biology: Bridging Meters to Nanometers
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Brain
Worse than the internet
Very complicated
100 Billion cells
10 Trillion+ interconnections
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Nerve Cells
Cell Body
Dendrites
Axon
Axon of nerve cell, Baas Lab, Drexel
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Structure and
Function
Growth Cone
Axon
Cell Body
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Bones
of the Cell
Microtubules
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Girders
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The nerve of you,
Body Worlds
3 feet = 1 meter
Long Distance
Support
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Long Distance
Support
Axon
Cell
Body
Growth Cone
Communications
Hub
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Long Distance
Communications
Axon
Growth Cone
Communications
Hub
Cell Body
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Long Distance
Maintenance
Growth Cone
Communications
Hub
Axon
Cell Body
Factory
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Long Distance
Maintenance
Growth Cone
Communications
Hub
Final Destination
Axon
Throughway
Cell Body
Factory
How can we get from Point A to Point B?
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Diffusion
3 feet = 1 meter
Robert Brown
Brownian Motion,
1827
Albert Einstein
Diffusion Equation,
1905
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3000 Years Later...
3 feet = 1 meter
Some neuronal cells can be up to 1 m long to connect your toes to your
spinal cord
If particles diffuse with a diffusion coefficient of:
How long will it take a 4 nm protein to diffuse down the 1-meter axon
filled with cytoplasm that is 5x more viscous than water?
Hint: What are the units of the diffusion coefficient?
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Highways
Axon
Throughway
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Big Rigs
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Big Rigs
Some neuronal cells can be up to 1 m long to connect your toes to your
spinal cord
Motor proteins transport goods up and down the axon taking 8 nm
steps.
How many steps do they need to take to go from the cell body (soma) to
the axon terminals, 1 m away?
If they travel at a velocity of 1  m/s, how long will the trip take?
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Nano Motors
Kinesin = Motion protein
NO: Walking Backward
NO: Turning Around
Feet are stuck to the highway in a specific direction.
Uses ATP as an energy source (1 ATP per step)
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Big Rig Cargo
Motors walk cargo:
Organelles (mitochondria)
Vesicles carrying signaling molecules to talk to the
other cells.
Protein structures (other microtubules, RNA)
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Big Rig Cargo
Multiple Motors bind to cargo
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Big Rig Cargo
Dynein = force protein
Multiple Motors bind to cargo
Can have different types of motors that walk in
different directions = TUG OF WAR
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Motor Experiments
Filament Gliding
QuickTime™ and a
GI F decompressor
are needed to see this picture.
Motor Experiments
Single Molecule
Single Molecule
Imaging
Qui ck Ti me™and a
decompre ss or
are needed to s ee th i s pi c tu re.
Micro-parasol
Black Particles:
melanosomes
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
Dark pigment-filled
vesicles (10-100 nm).
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Micro-parasol
Micro-parasol
Black Particles:
melanosomes
Dark pigment-filled vesicles
(10-100 nm).
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
Protect your cells’ nuclei
from harmful UV rays
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Micro-parasol
Micro-parasol
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
Powered by motor proteins that walk on microtubule and
actin filaments
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How do we Visualize Single Molecules at Nanoscale?
When you illuminate a sample in epi-fluorescence, a rather large volume
is illuminated
Causes background fluorescence
Many molecules in the field
Slide
How can we see single molecules?
Cover glass
Inverted objective
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Visualizing Single Molecules
1) Dilute the sample
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Visualizing Single Molecules
1) Dilute the sample
•
We could use a confocal spot with apertures to block out-of-plane
fluorescence
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Visualizing Single Molecules
1) Dilute the sample
•
We could use a confocal spot with apertures to block out-of-plane
fluorescence
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Total Internal Reflection Fluorescence
My method of choice
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Total Internal Reflection Fluorescence Microscopy
Focus laser on back-focal plane of objective
It comes out collimated
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Total Internal Reflection Fluorescence Microscopy
Why does the light bend?
Move focused spot to edge of back focal plane
Collimated beam tilts
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Total Internal Reflection Fluorescence Microscopy
Snell’s Law says: n1 sin ( 1) = n2 sin ( 2)
Calculate:
If nglass = 1.51 and nwater = 1.38, what is the “critical angle” for total
internal reflection? (When does  2 = 90°?)
Move to far edge so that angle > critical angle for
total internal reflection
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Total Internal Reflection Fluorescence Microscopy
Zoom in on Evanescent Wave
Decays exponentially in z
Only about 100 nm into sample
Brighter is closer to cover glass
Only molecules within 100 nm are visible
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Total Internal Reflection Fluorescence Microscopy
Zoom in on Evanescent Wave
Decays exponentially in z
Only about 100 nm into sample
What does an exponential decay look like?
Plot it.
If it dies off after 100 nm, what does that
tell you about the decay constant??
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Resolution Limits to Imaging Single Molecules
A motor protein takes an 8 nm step, can we measure that in our single
molecule assay?
Please vote by holding up the number of fingers you want to vote
for: (1) Yes. (2) No.
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Resolution Limits to Imaging Single Molecules
A motor protein takes an 8 nm step, can we measure that in
our single molecule assay?
Ideally, the motor is only about 4 nm, so an
8 nm step should be visible
But it’s not resolvable…
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Resolution Limits to Imaging Single Molecules
Why?? Why can’t we see the step?
What is resolution??
What is the resolution of our microscope?
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Resolution Limits to Imaging Single Molecules
Why?? Why can’t we see the step?
What is resolution??
The quantifiable ability to resolve or distinguish between two items. In
this case, the items are the images of the same molecule at two
positions.
Why does it happen??
Because light is a wave, we cannot focus it infinitely well. This is a
fundamental physical limit of all imaging systems.
What is the resolution of our microscope?
Distance resolvable:
Microscope resolution:
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Resolution Limits to Imaging Single Molecules
Water waves diffracting
through a hole in barrier
Why?? Why can’t we see the step?
The objective diffracts the light, because it is a
wave. Water waves diffract, too:

This is the diffraction limit
NA = n*sin max
n = index of refraction
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Resolution Limits to Imaging Single Molecules
Why?? Why can’t we see the step?
Calculate:
What is  max if the NA of
my objective is 1.49 and
the index of refraction, n,
is 1.51?

NA = n*sin max
n = index of refraction
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Resolution Limits to Imaging Single Molecules
Diffraction limited spot for a high-NA objective
Calculate:
What is d if the NA
of my objective is
1.49 and the
wavelength of light
is 508 nm?
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Resolution Limits to Imaging Single Molecules
Diffraction limited spot for a high-NA objective
How can we improve
our resolution?
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Super-resolution
Intensity
Use math tricks!
Intensity of diffraction-limited spot
highest at center
Actually a Bessel function, but is
well-fit by a 2-D Gaussian
Fit the shape of the intensity to find
the center with high accuracy
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FIONA: Fluorescence Imaging with One Nanometer Accuracy
Replace the fuzzy spot with a 2-20 nm dot
More photons (brighter spot) leads to better “resolution” and a
smaller dot.
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Effect of pixel size on resolution
Here, resolution is limited by pixel size - not fundamental
properties of light
Not resolvable
Large enough to
be resolved
Gaussian fitting gives better than 1 pixel
accuracy
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FIONA: Fluorescence Imaging with One Nanometer Accuracy
Follow the motor for multiple frames
More photons, better fitting
The diffraction limit is broken!
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Summary
Molecular biology exists, organizes, and engineers on the
nano-scale
Amazingly, these organizational principles are at the core
of all animals from bacteria and yeast to people and
elephants!
New optical techniques requiring math tricks are required
to see these nano-processes
Thank you for your attention!
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