1. FIONA: locating Single Molecules with a few nanometers accuracy
Very good accuracy: 1.5 nm, msec
Can track single fluorophore very well with FIONA.
center
width
Width is big, but can determine center. Collect ~1-10k photons.
Can see average = width/S.N.
= 250 nm/√N ~ 1.5 nm
W.E. Moerner, Crater Lake

2. 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

3. Takes 16 nm hand-over-hand steps
Kinesin
<step size> = 16.3 nm
y ~ texp(-kt)
Takes 16 nm hand-over-hand steps
16 nm
0 nm

4. finite # of photons, pixel size, background fluorescence
Effect of:
finite # of photons, pixel size, background fluorescence
Pixel size: Get small enough pixel so you have enough. At that point it’s essentially independent.