1. (In random order), students giving mid-term talks: Thuy Ngo Wylie Ahmed Charles Wilson Mohamed Ghoneim Xin Tang Claire Mathis Pengfei Yu Matthias Smith Anthony Hung Yu Ho Vishal Soni Joshua Glaser Eric Johnson Kiran Girdhar Today’s Announcements Email me your Powerpoint presentation to me at least 1 hr before class ! (Put your name as filename.) You may use your own computers but it may be harder.

2. Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.GFP can Fluoresce. 2.Basics of labeling in vivo (GFP, FLASH, others…). 3.Super-Accuracy (FIONA) 4.Total Internal Reflection

3. (Motor) proteinGFP Green Fluorescent Protein (Nobel, 2009) Genetically encoded dye (fluorescent protein) Kinesin – GFP fusion Wong RM et al. PNAS, 2002 Genetically encoded  perfect specificity

4. G. H. Patterson et al., Science 297, 1873 -1877 (2002) Photo-active GFP Wild-type GFP T203H GFP: PA- GFP Photoactivatable variant of GFP that, after intense irradiation with 413- nanometer light, increases fluorescence 100 times when excited by 488- nanometer light and remains stable for days under aerobic conditions Native= filled circle Photoactivated= Open squares

5. GFP: How protein makes color Threonine (Thr or T)is an α-amino acid, HO 2 CCH(NH 2 )CH(OH)CH 3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar. Tyrosine (abbreviated as Tyr or Y) is a non- essential amino acid with a polar side group. The word "tyrosine" is from the Greek tyros, meaning cheese, as it was first discovered in 1846 by German chemist Justus von Liebig in the protein casein from cheese. Glycine (Gly or G), NH 2 CH 2 COOH, is the smallest of the 20 amino acids.

6. Basics of Labeling In vivo (inside cell) Cell has a membrane, which is, in general, impermeant to dyes! Bi-Arsenic FLASH, Fluorescent Proteins, SNAP-tag, Halo-tag Tsien, Science, 2002 Tsien, Science, 1998 Bi-Arsenic FLASH, ReASH…

7. 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

8. Experimental Set-up for TIR (2 set-ups) Wide-field Objective-TIR Laser Objective Filter Dichroic Sample CCD Detector Lens Wide-field, Prism-type, TIR Microscope Sample Laser Objective Filter CCD Detector Lens Objective TIR: better S/N www.olympusmicro.com

9. Fluorescence Imaging with One Nanometer Accuracy Very good accuracy: 1.5 nm, 1-500 msec

10. Diffraction limited spot: Single Molecule Sensitivity 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) Accuracy of Center = width/ S-N = 250 nm / √10 4 = 2.5 nm = ± 1.25nm Thompson, BJ, 2002; Yildiz, Science, 2003 Start of high-accuracy single molecule microscopy Width of /2 ≈ 250 nm

11. How well can you localize? Depend on 3 things 1. # of Photons Detected (N) 3. Noise (Background) of Detector (b) (includes background fluorescence and detector noise) 2. Pixel size of Detector (a) = derived by Thompson et al. (Biophys. J.). center width

12. 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.