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. Fluorescence Imaging with One Nanometer Accuracy Very good accuracy: 1.5 nm, 1-500 msec W.E. Moerner, Crater Lake Diffraction limited spot: Single Molecule Sensitivity 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 ( /2). [Doesn’t matter how many photons you have: never less than this…it does depend on  ] 10,000 photons. Uncertainty = √  Accuracy  nm/ sem = 250 nm/ 100 = 2.5 nm (actually 1.3 nm) 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 = /2 cente r So, with ~10k photons, can tell where center is to 1.3 nm accuracy.

4. How accurately can you localize a fluorophore? Depend on 3 things 1. # of Photons Detected (N) 3. Noise (Background) of Detector (b) (includes background fluorescence and detector noise) 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. = derived by Thompson et al. (Biophys. J., 2002) Corrected by (Mortensen et al, Nat. Methods, 2010) cente r width

5. Imaging (Single Molecules) with very good S/N (at the cost of seeing only a thin section very near the surface) Total Internal Reflection (TIR) Fluorescence Microscopy For glass (n=1.5), water (n=1.33): TIR angle = >57° Penetration depth = d p = 58 nm d p =( /4  )[n 1 2 sin 2  i ) - n 2 2 ] -1/2 With d p = 58 nm, can excite sample and not much background. TIR- (  >  c ) Exponential decay You (or Marco!) must align microscope in TIR before you can take FIONA data To get such super-wide angle = high numerical aperture, need oil objective NA > 1.34. Therefore need 1.4 NA

6. 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 Actin,  tubules ATP  mechanical work Nature Reviews Microtubule actin Microtubule  polymer Kinesin Myosin Dynein  Motor ATP-binding heads Cargo binding K D

7. Motility of quantum-dot labeled Kinesin (CENP-E) Super-Accuracy: Nanometer Distances

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