Transcript Lecture 15 (3/29/10) "Fluorescence: Super Accuracy & Super

Today’s Announcements
1. Read ECB: Will assign later today.
2. Homework assigned (later) today
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, << l/2)
3. Confocal microscopy (Can discriminate according to z-axis)
4. Super-resolution microscopy—STED, STORM, (PALM next
time) microscopy (gets resolution << l/2)
Fluorescence Imaging with
One Nanometer Accuracy
Very good accuracy:
1.5 nm, 1-500 msec
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
Prism-type TIR 0.2 sec integration
center
280
240
Photons
200
160
120
width
80
40
0
5
10
Y ax
15
15
10
20
is
20
25
25
5
0
ta
X Da
Enough photons (signal to noise)…Center determined to ~1.3 nm
Z-Data from Columns 1-21
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
Actin, mtubules
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
K
Nature Reviews
ATP-binding
heads
Cargo
binding
Kinesin
Microtubule
Myosin
actin
ATP 
Dynein
 Motor
Microtubule  polymer
mechanical work
Motility of quantum-dot labeled Kinesin (CENP-E)
Streptavidin
Quantum Dot
Streptavidin conjugate
Biotinylated
Anti-Pentahis
antibody
Six-histidine tag
Axoneme
or microtubule
Leucine zippered
CENP-E dimer
w/ six histidine-tag
-
8.3 nm/step from optical trap
+
Kinesin (Center-of-Mass) Moving
Kinesin moves with 8.4 nm /ATP step size.
Kinesin: Hand-over-hand or Inchworm?
qs655
8.3 nm, 8.3 nm
8.3 nm 8.3 8.3 nm
16.6 nm
16 nm
[ATP] = 5 mM ; 4 msec exposure time
(Originally 0.3 mM ; 500 msec exp. time)
[ATP] (16.6x higher), 125x faster acq.
16.6 nm
0 nm
16.6, 0, 16.6 nm, 0…
pixel size is 160nm
2 x real time
Toprak et al, PNAS, 2009
Kinesin
100
1200
80
1120
1040
320
288
60
40
960
880
0
-32 -24 -16 -8
800
0
8
16 24 32 40 48 56 64
step size (nm)
720
<step size> = 16.3 nm
60
640
256
224
192
160
128
96
64
32
50
560
0
5.0
5.5
6.0
40
480
count
displacement (nm)
20
displacement (nm)
count
1280
400
320
240
6.5
7.0
8.0
8.5
y ~ texp(-kt)
20
0
0.0
80
0.2
0.4
0.6
0.8
Can you
derive this?
1.0
1.2
dwell time (sec)
0
0
16 nm2
164 nm
0 nm
6
9.0
30
10
160
7.5
time(sec)
8
time(sec)
10
12
14
Takes 16 nm hand-over-hand steps (even at 5mM)
Kinesin: H-over-H, but how does neck not twist?
Hand-over-hand: Head (foot) takes 16.6 nm steps
16 nm
Inchworm: Head (foot) takes 8.3 nm steps
8 nm
8 nm
Adapted from Hua, Chung, Gelles, Science, 2002
Can you think of an experiment to figure this out?
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)
Focused Light creates Light mostly
gets rejected
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
Confocal Microscopy
Lots 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
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.
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
http://www.mpibpc.gwdg.de/groups/hell/
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
Basics of Most Super-Resolution Microscopy
Inherently a single-molecule technique
Huang, Annu. Rev. Biochem, 2009
STORM
STochastic Optical
Reconstruction Microscopy
Bates, 2007 Science
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.