(In random order), students giving mid-term talks: Thuy Ngo Wylie Ahmed Charles Wilson

1. Today’s Announcements (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 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) • GFP can Fluoresce. • Basics of labeling in vivo (GFP, FLASH, others…). • Super-Accuracy (FIONA) • Total Internal Reflection

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

4. Photo-active GFP G. H. Patterson et al., Science 297, 1873 -1877 (2002) 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 T203H GFP: PA-GFP Wild-type GFP

5. GFP: How protein makes color 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. Threonine (Thr or T)is an α-amino acid, HO2CCH(NH2)CH(OH)CH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar. Glycine (Gly or G), NH2CH2COOH, 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 Bi-Arsenic FLASH, ReASH… Tsien, Science, 1998 Tsien, Science, 2002

7. Imaging (Single Molecules) with very good S/N (at the cost of seeing only a thin section very near the surface) TIR- (q > qc) Exponential decay Total Internal Reflection (TIR) Microscopy dp=(l/4p)[n12sin2i) - n22]-1/2 For glass (n=1.5), water (n=1.33): TIR angle = >57° Penetration depth = dp = 58 nm With dp = 58 nm , can excite sample and not much background.

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

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

10. center width 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 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) Start of high-accuracy single molecule microscopy Thompson, BJ, 2002; Yildiz, Science, 2003

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

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.