1. Today’s Announcements
Read ECB: Will assign later today.
Homework assigned (later) today
Today’s take-home lessons (i.e. what you should be able to answer at end of lecture)
Molecular motors: What are they? (3 families, 2 which walk on microtubules; one family which walks on actin)
Super-Accuracy: FIONA (nanometer accuracy, << l/2)
Confocal microscopy (Can discriminate according to z-axis)
Super-resolution microscopy—STED, STORM, (PALM next time) microscopy (gets resolution << l/2)

2. Fluorescence Imaging with One Nanometer Accuracy
FIONA
Fluorescence Imaging with One Nanometer Accuracy
Very good accuracy:
1.5 nm, msec

3. Diffraction limited spot: Single Molecule Sensitivity
Accuracy of Center = width/ S-N
= 250 nm / √104 = 2.5 nm = ± 1.25nm
Width of l/2 ≈ 250 nm
center
width
Enough photons (signal to noise)…Center determined to ~1.3 nm
Dye lasts 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min)
Start of high-accuracy single molecule microscopy
Thompson, BJ, 2002; Yildiz, Science, 2003

4. Biomolecular Motors: Intra- & Extra-Cellular Motion
Characteristics
nm scale
Move along tracks
intracellular directional movement
cell shape changes & extracellular movement
Use ATP as energy source D
Actin, mtubules K
ATP-binding
heads
Nature Reviews
ATP  mechanical work
Cargo binding
Kinesin Myosin Dynein  Motor
Microtubule actin Microtubule  polymer

5. Motility of quantum-dot labeled Kinesin (CENP-E)+ -
Quantum Dot
Streptavidin conjugate
Streptavidin
Biotinylated
Anti-Pentahis
antibody
Six-histidine tag
Leucine zippered
CENP-E dimer
w/ six histidine-tag
Axoneme
or microtubule
8.3 nm/step from optical trap 5

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

7. Kinesin: Hand-over-hand or Inchworm?
16 nm
qs655
8.3 nm, 8.3 nm
8.3 nm
16.6 nm
16.6, 0, 16.6 nm, 0…
0 nm
8.3
[ATP] = 5 mM ; 4 msec exposure time
(Originally 0.3 mM ; 500 msec exp. time)
[ATP] (16.6x higher), 125x faster acq.
pixel size is 160nm
2 x real time
Toprak et al, PNAS, 2009

8. Takes 16 nm hand-over-hand steps (even at 5mM)
Kinesin
<step size> = 16.3 nm
y ~ texp(-kt)
Can you derive this?
Takes 16 nm hand-over-hand steps (even at 5mM)
16 nm
0 nm

9. Kinesin: H-over-H, but how does neck not twist?
Hand-over-hand: Head (foot) takes 16.6 nm steps
16 nm
Adapted from Hua, Chung, Gelles, Science, 2002
8 nm
Inchworm: Head (foot) takes 8.3 nm steps
Can you think of an experiment to figure this out?

10. Scan sample in x, y, z and reconstruct entire image
Confocal Detection
Sample is 3-D. Detectors are 2-D. How do you get z-axis sectioning with Microscopy? A pinhole allows only in-focus light through
3-D sample
Detector
(Intensity)
Light mostly
gets rejected
Focused Light creates fluorescence which
gets to detector
Smaller the pinhole, better out-of-focus discrimination but lose more signal.
Scan sample in x, y, z and reconstruct entire image

11. Confocal Microscopy Lots of different ways of arranging to get fast scanning: Moveable mirrors (only have to move sample in z-direction, Nipow disk….

12. 3-D sectioning with Confocal
Three-dimensional reconstruction of a series of 2D images of PMMA spheres

13. Super-resolution Breaking the classical diffraction limit
Can we achieve nanometer resolution?
i.e. resolve two point objects separated by d << l/2?
Idea: 1) Make Point-Spread Function smaller << l/2
2) Make one temporally or permanently disappear, find center (via FIONA) and then reconstruct image.

14. STimulated Emission Depletion (STED)
S. Hell
Sharpen the fluorescence focal spot is to selectively inhibit the fluorescence at its outer part.
200nm
Net result is a smaller Point Spread Function
Huang, Annu. Rev. Biochem, 2009

15. Biological Example of STED
The transient receptor potential channel M5
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.
Hell, PNAS, 2007

16. STochastic Optical Reconstruction Microscopy
Basics of Most Super-Resolution Microscopy Inherently a single-molecule technique
Huang, Annu. Rev. Biochem, 2009
STORM
STochastic Optical Reconstruction Microscopy
Bates, 2007 Science

17. Answer, and turn in at the end of class.
Class evaluation
1. 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.