(In random order), students giving mid-term talks:Thuy Ngo

Wylie Ahmed Charles Wilson

Mohamed Ghoneim Xin Tang

Claire MathisPengfei 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.

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

(Motor) protein GFP

Green Fluorescent Protein (Nobel, 2009)Genetically encoded dye (fluorescent protein)

Kinesin – GFP fusion Wong RM et al. PNAS, 2002

Genetically encoded perfect specificity

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, increasesfluorescence 100 times when excited by 488-nanometer light and remainsstable for days under aerobic conditions

Native= filled circle

Photoactivated= Open squares

GFP: How protein makes color

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.

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) , NH2CH2COOH, is the smallest of the 20 amino acids.

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, 2002Tsien, Science, 1998

Bi-Arsenic FLASH, ReASH…

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 = dp = 58 nm

dp=(/4)[n12sin2i) - n2

2]-1/2

With dp = 58 nm , can excite sample and not much background.

TIR- (> c) Exponential decay

Experimental Set-up for TIR(2 set-ups)

Wide-fieldObjective-TIR

Laser

Objective

Filter

Dichroic

Sample

CCDDetector

Lens

Wide-field, Prism-type, TIR Microscope

Sample

Laser

ObjectiveFilter

CCDDetector

Lens

Objective TIR: better S/N

www.olympusmicro.com

Fluorescence Imaging with One Nanometer Accuracy

Very good accuracy: 1.5 nm, 1-500 msec

Diffraction limited spot: Single Molecule Sensitivity

center

width

Enough photons (signal to noise)…Center determined to ~1.3 nmDye lasts 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min)

Accuracy of Center = width/ S-N = 250 nm / √104 = 2.5 nm = ± 1.25nm

Thompson, BJ, 2002; Yildiz, Science, 2003Start of high-accuracy single molecule microscopy

Width of /2 ≈ 250 nm

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)

i

22

2422 812

Na

bs

N

a

N

s ii =

derived by Thompson et al. (Biophys. J.).

center

width

Class evaluation1. 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.