Today’s take-home lessons(i.e. what you should be able to answer at end of lecture)

1. Molecular motors: What are they? (3 families, 2 which walk on microtubules; one family which walks on actin)

2. Super-Accuracy: FIONA (nanometer accuracy, << /2)3. Confocal microscopy (Can discriminate according to z-axis)4. Super-resolution microscopy—STED, STORM, (PALM next

time) microscopy (gets resolution << /2)

1. Read ECB: Will assign later today.2. Homework assigned (later) today

Today’s Announcements

Fluorescence Imaging with One Nanometer Accuracy

Very good accuracy: 1.5 nm, 1-500 msec

Diffraction limited spot: Single Molecule Sensitivity

center

width

Enough photons (signal to noise)…Center determined to ~1.3 nmDye lasts 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min)

Accuracy of Center = width/ S-N = 250 nm / √104 = 2.5 nm = ± 1.25nm

Thompson, BJ, 2002; Yildiz, Science, 2003Start of high-accuracy single molecule microscopy

Width of /2 ≈ 250 nm

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)

8.3 nm/step from optical trap

Kinesin (Center-of-Mass) Moving

Kinesin moves with 8.4 nm /ATP step size.

16 nm

qs655

pixel size is 160nm2 x real time

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

Kinesin: Hand-over-hand or Inchworm?

[ATP] = 5 M ; 4 msec exposure time(Originally 0.3 M ; 500 msec exp. time)

[ATP] (16.6x higher), 125x faster acq.

Toprak et al, PNAS, 2009

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 (even at 5M)

16 nm0 nm

16 nm

Can you derive this?

Kinesin: H-over-H, but how does neck not twist?

Can you think of an experiment to figure this out?

Hand-over-hand: Head (foot) takes 16.6 nm steps

16 nm

Adapted from Hua, Chung, Gelles, Science, 20028 nm 8 nm

Inchworm: Head (foot) takes 8.3 nm steps

Sample is 3-D. Detectors are 2-D.How do you get z-axis sectioning with Microscopy?

A pinhole allows only in-focus light through3-D sample

Detector(Intensity)

Confocal Detection

Focused Light creates fluorescence which gets to detector

Light mostly gets rejected

Smaller the pinhole, better out-of-focus discrimination but lose more signal.

Scan sample in x, y, z and reconstruct entire image

Confocal MicroscopyLots of different ways of arranging to get fast scanning:

Moveable mirrors (only have to move sample in z-direction, Nipow disk….

3-D sectioning with Confocal

Three-dimensional reconstruction of a series of 2D images of PMMA spheres

Can we achieve nanometer resolution?i.e. resolve two point objects separated by d <</2?

Idea: 1) Make Point-Spread Function smaller << /2 2) Make one temporally or permanently disappear, find

center (via FIONA) and then reconstruct image.

Super-resolutionBreaking the classical diffraction limit

STimulated Emission Depletion (STED)

http://www.mpibpc.gwdg.de/groups/hell/Net result is a smaller Point Spread Function

Sharpen the fluorescence focal spot is to selectively inhibit the fluorescence at its outer part.

Huang, Annu. Rev. Biochem, 2009

S. Hell

200nm

Biological Example of STED

Hell, PNAS, 2007

Analysis of spot size for Confocal (A) and STED (B) images of TRPM5 immunofluorescence layer of the olfactory epithelium. (A, C Inset) Confocal image at a lower (higher; box) magnification taken with a confocal microscope. (B) STED image. Effective point-spread function in the confocal (189 nm) and STED (35 nm) imaging modes.

The transient receptor potential channel M5

Basics of Most Super-Resolution MicroscopyInherently a single-molecule technique

Huang, Annu. Rev. Biochem, 2009

Bates, 2007 Science

STORM STochastic Optical

Reconstruction Microscopy

Class evaluation1. What was the most interesting thing you learned in class today?

2. What are you confused about?

3. Related to today’s subject, what would you like to know more about?

4. Any helpful comments.

Answer, and turn in at the end of class.