(in random order), students giving mid-term talks: thuy ngo wylie ahmed charles wilson mohamed...
TRANSCRIPT
(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
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