Slide 1

1.HW on rough-draft of your 5 minute oral presentation due today at 5pm. 2.Another HW (regular, written assignment due Wednesday in class). The students simply have to practice a lot and make things very clear and concise. Todays Announcements Slide 2 Individual Projects 5 minute lecture! April 17 th and 19 th If you canNOT make it on April 19 th send me an email with subject title: I must have talk on April 17 th. 1. What is title of paper and talk. 2. Introduction (What is main general subject.) [How things move inside of a cell.] 3. Detail subject : [Molecular motors, in particular,how myosin V moves; Walks or inchworm.] 4. What is major question/problem. [Measure steps that are below the diffraction limit.] 5. What did they do to solve the problemnew techniques? [FIONA] 6. Solution [MyoV walks, hand-over-hand!] 7. One sentence summary of what the audience should take home/ remember. [Technique made that is very general, FIONA, for measuring step mechanism; showed that MyoV walks.] Todays Announcements Slide 3 Grading 1.Did you follow outline? 2.Were you clear? a. Not too complex, but not too simple. b. How was your eye contact? c. Was your speaking clear? 3. Did audience get a one sentence, main message? Slide 4 For visible microscopy, Resolution is limited to ~250 nm Ernst Abbe & Lord Rayleigh Recent microscopy: 1-100 nm, Ernst Abbe How fine can you see? The Limits of Microscopy Here we present techniques which are able to get super-accuracy (1.5 nm) and/or super-resolution ( Imaging (Single Molecules) with very good S/N (at the cost of seeing only a thin section very near the surface) Total Internal Reflection (TIR) Microscopy For glass (n=1.5), water (n=1.33): TIR angle = >57 Penetration depth = d p = 58 nm d p =( /4 )[n 1 2 sin 2 i ) - n 2 2 ] -1/2 With d p = 58 nm, can excite sample and not much background. TIR- ( > c ) Exponential decay Slide 15 SHRImP Super High Resolution IMaging with Photobleaching In vitro Super-Resolution: Nanometer Distances between two (or more) dyes 132.9 0.93 nm 72.1 3.5 nm 8.7 1.4 nm Distance can be found much more accurately than width (250 nm) Resolution now: Between 2-5 molecules:5-40 molecules ~ 20-100 nm Via 2-photon: ~ 35 nm (next time) Slide 16 Most Super-Resolution Microscopy Inherently a single-molecule technique Huang, Annu. Rev. Biochem, 2009 Bates, 2007 Science STORM STochastic Optical Reconstruction Microscopy PALM PhotoActivation Localization Microscopy (Photoactivatable GFP) Slide 17 PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP) 1 m PALMTEM Mitochondrial targeting sequence tagged with mEOS TIRF Patterson et al., Science 2002 Slide 18 3-D (z) resolution Slide 19 Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to todays subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.