Today: FIONA: localizing single dyes to a few nanometers

If a dye is attached to something, and that something moves over time, one can track it

very well with FIONA.

Fluorescence Imaging with One Nanometer Accuracy

Very good accuracy: 1.5 nm, 1-500 msec

W.E. Moerner, Crater Lake

Diffraction limited spot: Single Molecule Sensitivity

center

widthCollect from ~ 1-10k photons

How accurately can you see the center?

The width of the distribution (the standard deviation) is ≈ 250 nm (/2).[Doesn’t matter how many photons you have: never less than this…it does depend on ]

10,000 photons.Uncertainty = √Accuracynm/ sem= 250 nm/ 100 = 2.5 nm

But this doesn’t tell you about how well you can tell about the center.For this, you want the standard error of the mean sem = sd/√N

Width= /2

center

So, with ~10k photons, can tell where center is to 1.25 nm accuracy.

How accurately can you localize a fluorophore?Depend on 3 things

1. # of Photons Detected (N)

3. Noise (Background) of Detector (b)(includes background fluorescence and detector noise)

2. Pixel size of Detector (a)If “a” is too big, can’t localize it better than “a” nm.Make sure that Gaussian has like 10 x 10 pixels.

i =

derived by Thompson et al. (Biophys. J., 2002)Corrected by (Mortensen et al, Nat. Methods, 2010)

center

width

Biomolecular Motors: Intra- & Extra-Cellular MotionCharacteristics• nm scale • Move along tracks• intracellular directional movement• cell shape changes & extracellular

movement• Use ATP as energy source

Act

in,

tub

ule

s

ATP mechanical workNat

ure

Rev

iew

s

Microtubule actin Microtubule polymer Kinesin Myosin Dynein Motor

ATP-bindingheads

Cargo binding

K

D

+-

Quantum DotStreptavidin conjugate

Streptavidin

BiotinylatedAnti-Pentahisantibody

Six-histidine tag

Leucine zipperedCENP-E dimerw/ six histidine-tagAxoneme

or microtubule

Motility of quantum-dot labeled Kinesin (CENP-E)

Super-Accuracy: Nanometer Distances

Kinesin (Center-of-Mass) MovingKinesin moves with 8.4 nm /ATP step size.

Super-Accuracy: Nanometer Distances

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

8.3 8.3 nm

Hand-over-hand or Inchworm? (kinesin)

Kinesin

0 2 4 6 8 10 12 14

0

80

160

240

320

400

480

560

640

720

800

880

960

1040

1120

1200

1280

disp

lace

men

t (nm

)

time(sec)

<step size> = 16.3 nm

y ~ texp(-kt)

Takes 16 nm hand-over-hand steps

16 nm0 nm

16 nm

Can you derive this?

Kinesin (HHMI/Harvard)

The End