FIONA: locating Single Molecules with a few nanometers accuracy Very good accuracy: 1.5 nm, 1-500 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

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

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

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.