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.

Diffraction limited spot: Single Molecule Sensitivity W.E. Moerner, Crater Lake FIONA Fluorescence Imaging with One Nanometer Accuracy Very good accuracy: 1.5 nm, 1-500 msec center width Collect from ~ 1-10k photons

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.

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)

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

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

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

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) Can you derive this? Takes 16 nm hand-over-hand steps 16 nm 0 nm

Kinesin (HHMI/Harvard)

The End