1. Today: FIONA: localizing single dyes to a few nanometers
If a dye is attached to something, and that something moves over time, one can track it very well with FIONA.

2. Diffraction limited spot: Single Molecule Sensitivity
W.E. Moerner, Crater Lake
FIONA
Fluorescence Imaging with One Nanometer Accuracy
Very good accuracy:
1.5 nm, msec
center
width
Collect from ~ 1-10k photons

3. How accurately can you see the center?
The width of the distribution
(the standard deviation)
is ≈ 250 nm (l/2).
[Doesn’t matter how many photons you have: never less than this…it does depend on l.]
Width
= l/2
center
But this doesn’t tell you
about how well you can tell about the center.
For this, you want the
standard error of the mean
sem = sd/√N
10,000 photons.
Uncertainty = √10,000
Accuracy = 250 nm/ sem
= 250 nm/ 100 = 2.5 nm
So, with ~10k photons, can tell where center is to 1.25 nm accuracy.

4. How accurately can you localize a fluorophore? Depend on 3 things
center
width
1. # of Photons Detected (N)
2. Pixel size of Detector (a)
If “a” is too big, can’t localize it better than “a” nm.
Make sure that Gaussian has like 10 x 10 pixels.
3. Noise (Background) of Detector (b)
(includes background fluorescence and detector noise) =
derived by Thompson et al. (Biophys. J., 2002)
Corrected by (Mortensen et al, Nat. Methods, 2010)

5. Biomolecular Motors: Intra- & Extra-Cellular Motion
Characteristics
nm scale
Move along tracks
intracellular directional movement
cell shape changes & extracellular movement
Use ATP as energy source D
Actin, mtubules K
ATP-binding
heads
Nature Reviews
ATP  mechanical work
Cargo binding
Kinesin Myosin Dynein  Motor
Microtubule actin Microtubule  polymer

6. Super-Accuracy: Nanometer Distances+ -
Quantum Dot
Streptavidin conjugate
Streptavidin
Biotinylated
Anti-Pentahis
antibody
Six-histidine tag
Leucine zippered
CENP-E dimer
w/ six histidine-tag
Axoneme
or microtubule
Motility of quantum-dot labeled Kinesin (CENP-E) 6

7. Kinesin (Center-of-Mass) Moving
Super-Accuracy: Nanometer Distances
Kinesin (Center-of-Mass) Moving
Kinesin moves with 8.4 nm /ATP step size.

8. Hand-over-hand or Inchworm? (kinesin)
16 nm
q655
8.3 nm, 8.3 nm
8.3 nm
16.6 nm
16.6, 0, 16.6 nm, 0…
0 nm
8.3

9. Takes 16 nm hand-over-hand steps
Kinesin
<step size> = 16.3 nm
y ~ texp(-kt)
Can you derive this?
Takes 16 nm hand-over-hand steps
16 nm
0 nm

10. Kinesin
(HHMI/Harvard)

11. The End