1. FIONA II & AFM II TIRF Choice of labels-organic Fluorophores Fluorescent Proteins Quantum Dots In vivo FIONA and GFPs Imaging Mode: don’t be at zero frequency (because of Noise) Force Mode: Worm-Like Chain (WLC): very good for proteins and DNA. FIONA AFM

2. Very good accuracy: 1.5 nm, 1-500 msec W.E. Moerner, Crater Lake FIONA: locating Single Molecules to a few nanometers accuracy center width Collect from ~ 1-10k photons. Can see average = w/S.N. = 250 nm/√N ~ 1.5 nm If a dye is attached to something, and that something moves over time, one can track it very well with FIONA.

3. Noise Why can’t you see starlight in the day? (The stars are just as bright during the day as at night.) You have a “bright” background (sun)... which has a lot of noise. If you have N photons, then you have √N noise. (This is important to remember!) Example: Sun puts out a 10 6 photons/sec. Noise = 10 3 photons/sec Therefore: if star puts out 10 3 photons/sec, can just barely “see it” with Signal/Noise =1 (Really want to “see it” with S/N of at least a few >2-5)) With fluorescence, background is often practically zero, so can see down to a single molecule!

4. 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)

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

6. 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

7. How long can you look for? Determined by photobleaching (time). Good organic dyes (Cy3-DNA) Photostability = 1.3M (!) If you hit it with a lot of laser light, emits a lot of light, doesn’t last as long. If it’s 1 second/frame = 20 sec If it’s 0.1 sec/frame = 20 sec Depending on the [ATP] you may or may-not be able to see multiple steps. Organic dyes fine for in vitro, not usually good for in vivo Hit it such that it emits 5,000 photons per time interval, has 200 frames.

8. How long can you look for? Quantum Dots (inorganic binary mixtures): Infinite photostability Extremely bright (~10-100x as bright as organic fluorophores) Extremely photo-resistant ( ∞ photostable?) But…they tend to be large (15-35 nm) Recently made with <7 nm (still large) And difficult to label in vivo.

9. Can go to higher [ATP] with QDs (2  sec/pt : 400nm  5  M) Toprak, PNAS, 2009

10. Dynein Kinesin Yes…in Drosophilia cells, individual kinesin & dynein moving cooperatively (Kural, Science, 2005) We have great x-y accuracy in vitro with fluorescent dyes and quantum dots… Can we get this accuracy in vivo?  r = 1.5 nm  t = 1.1 msec

11. (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

12. eGFP 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

13. power (mW)*t 1/2 (total Photons)Avg flux (per s)t 1/2 (s) 1.24954061846.2 0.55306348787.1 0.34967345229.4 0.1346137159419.8 0.0121987437869.5 * Power measured at objective (Illumination area ~ 6.71e -5 m 2 ) mEGFP stability vs. illumination intensity No dependence on Intensity

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

15. The End