Diffraction limited spot: Single Molecule Sensitivity FIONA Fluorescence Imaging with One Nanometer Accuracy Very good accuracy: 1.5 nm, 1-500 msec Diffraction limited spot: Single Molecule Sensitivity If a dye is attached to something, and that something moves over time, one can track it very well with FIONA. W.E. Moerner, Crater Lake Collect ~ 10k photons Dx = width/√N = 250/100 nm ~ 2.5 nm (actually 1.3 nm) 8.3 nm 16.6 nm 16.6, 0, 16.6 nm, 0… 0 nm 8.3 center width

Super-Accuracy: Nanometer Distances + - Quantum Dot Streptavidin conjugate Streptavidin Biotinylated Anti-Pentahis antibody Six-histidine tag Leucine zippered CENP-E dimer w/ six histidine-tag Axoneme or microtubule (movie) Motility of quantum-dot labeled Kinesin (CENP-E) 2

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

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

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

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

How long can you look for? Determined by photobleaching (time). Photostability = 1.3M (!) 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 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

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.

Can go to higher [ATP] with QDs (2 msec/pt : 400nm  5 mM) Toprak, PNAS, 2009

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

Can you look at bunch of dyes? (A single dye might photobleach really quickly). (Why might this be case?) < diffraction limit; Size = ?? > diffraction limit; Size = ??

To be bright-enough, especially with GFPs, need many GFPs 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

Aggregated Melanosomes Dispersed Melanosomes High Resolution Organelle Tracking in Non-fluorescent Pigment Cells Possible? Example of sunscreen and animals fighting ● Xenopus Melanophores with Melanin Filled Dark Melanosomes. Aggregated Melanosomes (+ Caffeine) Dispersed Melanosomes (+ Adrenalin) ~20 min. (movie) (movie) Borisy et al.,Curr. Biol., 1998

Bright Field Imaging with One Nanometer Accuracy (bFIONA) Kural, PNAS, 2007

Microtubule-Based Transport Actin-Based Transport bFIONA: Molecular Motors in Action Microtubule-Based Transport Kinesin 2 and Dynein Actin-Based Transport Myosin V fast slow Heterotrimeric kinesin: 8 nm steps; Myosin V: 36 nm steps in vivo.

Actin Dependent Movement Melanosomes switch between myosin (actin-based) & kinesin/dynein (microtubule-based) motors. Microtubule Dependent Movement Actin Dependent Movement 36nm 50 nm X vs Y x vs time x position Diffusion time not significant (Kural et al, PNAS, 2007)

Genetically encoded  perfect specificity. Labeling in vivo: Green Fluorescent Protein GFP – genetically encoded dye (fluorescent protein) (Motor) protein GFP 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 Kinesin – GFP fusion Inserted in Tobacco (plant) & in Monkeys (animals) Lots of FP mutants—different colors Genetically encoded  perfect specificity.

http://science. wonderhowto http://science.wonderhowto.com/inspiration/fluorescent-puppies-you-can-turn-and-off-0125885/

The End