Transcript Basics of Fluorescence

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.
Today’s take-home lessons
(i.e. what you should be able to answer at end of lecture)
1.
2.
3.
4.
GFP can Fluoresce.
Basics of labeling in vivo (GFP, FLASH, others…).
Super-Accuracy (FIONA)
Total Internal Reflection
Green Fluorescent Protein (Nobel, 2009)
Genetically encoded dye (fluorescent protein)
(Motor) protein
Kinesin – GFP fusion
GFP
Wong RM et al. PNAS, 2002
Genetically encoded  perfect specificity
Photo-active GFP
G. H. Patterson et al., Science 297, 1873 -1877 (2002)
Photoactivatable variant
of GFP that, after intense
irradiation with 413nanometer light,
increases
fluorescence 100 times
when excited by 488nanometer light and
remains
stable for days under
aerobic conditions
Native= filled circle
Photoactivated= Open squares
Wild-type GFP
T203H GFP: PAGFP
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 nonessential 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
Bi-Arsenic FLASH, ReASH…
Tsien, Science, 1998
Tsien, Science, 2002
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
TIR- ( > c)
Exponential
decay
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.
Experimental Set-up for TIR
(2 set-ups)
Laser
Sample
Objective
Sample
Objective
Dichroic
Laser
Filter
Lens
Filter
CCD
Detector
CCD
Detector
Lens
Wide-field, Prism-type,
TIR Microscope
Wide-field
Objective-TIR
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
Accuracy of Center = width/ S-N
= 250 nm / √104 = 2.5 nm = ± 1.25nm
Width of l/2 ≈ 250 nm
Prism-type TIR 0.2 sec integration
center
280
240
Photons
200
160
120
width
80
40
0
5
10
15
Y ax
is
15
10
20
20
25
25
5
0
ta
X Da
Enough photons (signal to noise)…Center determined to ~1.3 nm
Z-Data from Columns 1-21
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
How well can you localize?
Depend on 3 things
1. # of Photons Detected (N)
Prism-type TIR 0.2 sec integration
center
280
2. Pixel size of Detector (a)
240
Photons
200
3. Noise (Background) of Detector (b)
160
120
width
(includes background fluorescence and detector noise)
80
40
0
5
10
Y ax
15
is
15
20
20
25
25
10
ta
X Da
5
0

=
i
 s i2 a 2 12 8ps i4 b 2
 
 2 2
N
N
a N





Z-Data from Columns 1-21
derived by Thompson et al. (Biophys. J.).
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.