1. Fluorescence You can get beautiful pictures

2. X-ray Diffraction Quiz
Quiz next Tuesday covering fluorescence lectures
(I will send out a list of questions via .)

3. (Accuracy << 250 nm: 1.5 nm, 1-500 ms)
Super-Accuracy
(Accuracy << 250 nm: 1.5 nm, ms)
FIONA
Fluorescence Imaging with One Nanometer Accuracy
center
width
Center can be found much more accurately than width
S/N ≈ width /√N
≈ 250/√104 ≈ 1.3 nm
When light gets dim,
what happens to your ability to find the center?

4. We have great x-y accuracy in vitro
with fluorescent dyes and quantum dots…
Can we get this accuracy in vivo?
Yes…in Drosophilia cells, individual kinesin & dynein moving cooperatively (Kural, Science, 2005)
Dynein
Kinesin
dr = 1.5 nm
dt = 1.1 msec

5. Accuracy & Resolution:
How accurately can you tell where one object is:
How well can you resolve two nearby (point) objects?
Point-Spread Function (PSF)
A single light-emitting spot will be smeared out, no matter how small the spot is, because of the wavelength of light to ~ l/2NA.

6. Nanometer Distances between two (or more) dyes
Super-Resolution:
Nanometer Distances between two (or more) dyes
SHRImP
Super High Resolution IMaging with Photobleaching
132.9 ± 0.93 nm
8.7 ± 1.4 nm
72.1 ± 3.5 nm

7. Extending SHRImP to more than 2 dyes.
Can use technique with 3, 4, 5…up to about 10 dyes.
e.g. with 3 dyes, take image of 3 dyes; one photobleaches and have 2 remaining dyes; take image, then one of two remaining dye photobleaches. Then you have 1 dye left, take image and finally that too dies.
Then analyze data starting “backwards”, i.e. with the one dye that photobleached last. Call this Image 1. You do FIONA on it and get the position with nanometer accuracy. Then you go to the image of 2 dyes,(call it Image 2), and subtract off the image of Image 1. From (Image 2 – Image 1) you can get the position of dye 2. Then you go to Image 3 and subtract off Image 2 and determine via FIONA the position of dye 3.
The same technique with 4 or more dyes.
Problem:It becomes more difficult as the # of dyes increases. At some point will be very difficult to see one dye photobleaching. Why?

8. Question: The Limit of SHRImP
At most you can activate dyes such that there is one active dye per diffraction-limited spot.
Why?
Want each point-spread-function of dye to be clearly resolved. If more than one per diffraction-limited spot, then it is difficult to locate with nanometer resolution using FIONA.

9. Make photobleaching temporary or activate a non-fluorescent dye
Let dye blink. Or even better, have dye be non-fluorescent, then activate it with a different wavelength of dyes, then shine the activate dye with another wavelength which makes it fluoresce.

10. On class of Super-Resolution Microscopy Inherently a single-molecule technique that relies on blinking or on/off behavior
Huang, Annu. Rev. Biochem, 2009
STORM
STochastic Optical Reconstruction Microscopy
PALM
PhotoActivation Localization Microscopy (Photoactivatable GFP)
Bates, 2007 Science

11. PALM - Photo-activated localization super-resolution microscopy
Huang et al. Science 2008
3D super-resolution
Weak near-UV light
Read out with visible light
After many cycles
EOS-FP>1000 photons
The PALM cycle
Betzig et al. Science 2006
PALM imaging with 10 ~ 20 nm resolution (localization precision).

12. PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP)
1 mm
TIRF
PALM
TEM
Mitochondrial targeting
sequence tagged with mEOS
Patterson et al., Science 2002

13. Nerve Receptors & Synapses How you remember, learn; effect of stroke
Your brain: 100 billion neurons, 100 trillion synapses
Pre-synaptic Bouton
Post-synaptic Spine
Valtschanoff & Weinberg, 2003
Pre-synapse
Post-synapse
Nerve 1
Nerve 2
Information flow
PSD95
Homer1
Synapse
(30 nm) nm
Idea is to measure the dynamics (via FIONA) and structure (via PALM/STORM) as a function of mutation (associated with diseases such as schizophrenia and autism) of the neuronal synapases, with nanometric spatial resolution and about msec resolution temporal resolution for a long time (about an hour or more).
Need 3-D super-accuracy & resolution fluorescence,

14. More STORM Images
Dani, Zhuang, Neuron, 2010

15. More STORM Images
Dani, Zhuang, Neuron, 2010

16. 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.