1. Diffraction limited spot: Single Molecule Sensitivity
FIONA
Fluorescence Imaging with One Nanometer Accuracy
Very good accuracy:
1.5 nm, 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

2. 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

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

4. 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)

5. 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

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).
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

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. 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

11. 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 = ??

12. 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

13. 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

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

15. 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.

16. 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)

17. 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.

18. http://science. wonderhowto

19. The End