1. Very good accuracy: 1.5 nm, 1-500 msec W.E. Moerner, Crater Lake FIONA: locating Single Molecules with a few nanometers accuracy 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 Can track single fluorophore very well with FIONA. How to get enough signal

2. 16 nm q655 8.3 nm, 8.3 nm 8.3 nm 16.6 nm 16.6, 0, 16.6 nm, 0… 0 nm 16.6 nm 8.38.3 nm Hand-over-hand or Inchworm? (kinesin)

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

4. Effect of: finite # of photons, pixel size, background fluorescence Pixel size: Get small enough pixel so you have enough.(a< 100 nm). At that point it’s essentially independent. Use modern detections where b is very small. Bottom line: photon number (s i ) is what counts!

5. What property of dyes do you need for FIONA, super- accuracy localization? Answer: photostable dyes.

6. Yes…in Drosophilia cells, individual kinesin & dynein may move cooperatively (Kural, Science, 2005) We have great x-y accuracy in vitro Can we get this accuracy in a cell? Dynein Kinesin  r = 1.5 nm  t = 1.1 msec 1. We used GFP: how to make stable enough?We used multiple GFPs—in one group. Problems?1. How to get in labels? 2. How to get out a lot of photons fast?

7. (Motor) proteinGFP Green Fluorescent Protein GFP – genetically encoded dye (fluorescent protein) Genetically encoded  perfect specificity. Came from Jelly Fish Inserted in Tobacco (plant) & in Monkeys (animals) Attach DNA for GFP onto end of DNA encoding for protein. Get DNA inside cell and DNA process takes over…perfectly Lots of FP mutants—different colors Kinesin – GFP fusion

8. eGFP: Problem: dies quickly Horse radish peroxidase-Ni 2+ -NTA immobilization < 50,000 counts before photobleaching (~20 x less) Ambient (with oxygen) oxygen free, gloxy No difference with/without oxygen

9. To be bright-enough, especially with GFPs, need many GFPs. It doesn’t so long as distribution within ball doesn’t change How to get photons out fast? If take up large size…? How does that effect localization?