FIONA II & AFM II

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

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

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 106 photons/sec. Noise = 103 photons/sec Therefore: if star puts out 103 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. 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…

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

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 TIR- (q > qc) Exponential decay dp=(l/4p)[n12sin2i) - n22]-1/2 For glass (n=1.5), water (n=1.33): TIR angle = >57° Penetration depth = dp = 58 nm With dp = 58 nm , can excite sample and not much background. To get such super-wide angle = high numerical aperture, need oil objective NA > 1.34. Therefore need 1.4 NA You (or Marco!) must align microscope in TIR before you can take FIONA data

7. How long can you look for? Determined by photobleaching (time). Good organic dyes (Cy3-DNA) If you hit it with a lot of laser light, emits a lot of light, doesn’t last as long. Hit it such that it emits 5,000 photons per time interval, has 200 frames. If it’s 1 second/frame = 20 sec If it’s 0.1 sec/frame = 20 sec Photostability = 1.3M (!) 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

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 msec/pt : 400nm  5 mM) Toprak, PNAS, 2009

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

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

12. eGFP oxygen free, gloxy Ambient (with oxygen) < 50,000 counts before photobleaching (~20 x less) No difference with/without oxygen Horse radish peroxidase-Ni2+-NTA immobilization

13. mEGFP stability vs. illumination intensity * Power measured at objective (Illumination area ~ 6.71e-5 m2) 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