Fluorescence You can get beautiful pictures www.invitrogen.com

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

(Accuracy << 250 nm: 1.5 nm, 1-500 ms) Super-Accuracy (Accuracy << 250 nm: 1.5 nm, 1-500 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?

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

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.

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

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?

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.

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.

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

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).

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

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) 250-500 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 10-100 msec resolution temporal resolution for a long time (about an hour or more). Need 3-D super-accuracy & resolution fluorescence,

More STORM Images Dani, Zhuang, Neuron, 2010

More STORM Images Dani, Zhuang, Neuron, 2010

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.