Today: FIONA: localizing single dyes to a few nanometers

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. FIONA Fluorescence Imaging with One Nanometer Accuracy center width W.E. Moerner, Crater Lake Diffraction limited spot: Single Molecule Sensitivity Very good accuracy: 1.5 nm, 1-500 msec 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.] 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 Width = l/2 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. center width = derived by Thompson et al. (Biophys. J., 2002) Corrected by (Mortensen et al, Nat. Methods, 2010) How accurately can you localize a fluorophore?Depend on 3 things 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)

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 K Actin, mtubules ATP-binding heads Nature Reviews ATP  mechanical work Cargo binding Kinesin Myosin Dynein  Motor Microtubule actinMicrotubule polymer

6. Super-Accuracy: Nanometer Distances Streptavidin Quantum Dot Streptavidin conjugate 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)

7. 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) 8.3 nm 8.3 8.3 nm q655 16.6 nm 16.6 nm 8.3 nm, 8.3 nm 16 nm 0 nm 16.6, 0, 16.6 nm, 0…

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

10. Kinesin (HHMI/Harvard)

11. The End