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

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.

NoiseWhy 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/secTherefore: 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!

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

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880

960

1040

1120

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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

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 = dp = 58 nm

dp=(/4)[n12sin2i) - n2

2]-1/2

With dp = 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

How long can you look for?Determined by photobleaching (time).

Good organic dyes (Cy3-DNA)

0 500000 1000000 1500000 2000000 2500000 30000000

10

20

30

40

50

60

Data: Count3_CountModel: ExpDec1Equation: y = A1*exp(-x/t1) + y0Weighting: y No weighting Chi^2/DoF = 0.99756R^2 = 0.99857 y0 -6.42969 ±2.36237A1 76.53159 ±1.87272t1 1322629.15612±114117.66856

Y A

xis

Titl

e

X Axis Title

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 ormay-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.

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 sec/pt : 400nm 5 M)

Toprak, PNAS, 2009

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 nmt = 1.1 msec

(Motor) protein GFP

Green Fluorescent ProteinGFP – 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 colorsKinesin – GFP fusion

eGFP

Horse radish peroxidase-Ni2+-NTA immobilization

< 50,000 counts before photobleaching (~20 x less)

Ambient (with oxygen) oxygen free, gloxy

No difference with/without oxygen

power (mW)* t1/2 (total Photons) Avg flux (per s) t 1/2 (s)

1.2 49540 6184 6.2

0.5 53063 4878 7.1

0.3 49673 4522 9.4

0.13 46137 1594 19.8

0.012 19874 378 69.5

* Power measured at objective (Illumination area ~ 6.71e-5 m2)

mEGFP stability vs. illumination intensity

No dependence on Intensity

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

The End