1. Fluorescence You can get beautiful pictures

2. Imaging: Your eye is a Microscope!
Lens Maker’s Equation: 1/f = 1/o + 1/i
(Actually have two lenses: your cornea and your lens)

3. Noise Why can’t you see starlight in the day?
(The stars are just as bright during the day as at night.)
You have a “bright” background (sun)...
which has a lot of noise.
If you have N photons, then you have √N noise.
(This is important to remember!)
Example: Sun puts out a 106 photons/sec. Noise = 103 photons/sec
Therefore: if star puts out 103 photons/sec,
Can just barely “see it” with Signal/Noise =1
(Really want to “see it” with S/N of at least a few >2-5))

4. Example of Noise con’t Let’s say star puts out 100 photon/sec.
(It turns out you (your eye) can see about 1 photon!!)
S/N day?
At night?
Fluorescence vs standard light Microscopy

5. Basic Set-up of Fluorescence Microscope
Detector
Why can you see so little fluorescence
(Lasers, Arc Lamps)
Nikon, Zeiss, Olympus, Leica—Microscope Manufacturer
Andor, Hamamatsu, Princeton Instruments, other…make (EM)CCDs
Semwogerere & Weeks, Encyclopedia of Biomaterials and Biomedical Engineering, 2005

6. Why can you see so little?
Fluorescence: Background is zero!
Notice that in fluorescence, you are looking at reflection of signal. Therefore if no absorption (the first step in fluorescence), you get no fluorescence.
Hence background is zero!

7. Basics of fluorescence
Shine light in, gets absorbed, reemits at longer wavelength
Stokes Shift ( nm)
Excitation
Spectra
Emission
Light In
Light Out
2. Thermal Relaxation
(heat, in I.R.)
[Picosec]
1. Absorption
[Femtosec]
3. Stays at lowest excited vibrational states for a “long” time (nsec)
4. Fluorescence
& Non-radiative
[Nanosec]
Time (nsec)
Fluorescence
-t/tf
Y = e
5. Thermal relaxation
[Picosec]
What happens for non-fluorescing molecule? (in 3. nr really fast)
Photobleaching Important: Dye emits 105 107 photons, then dies!

8. Basics of fluorescence
Shine light in, gets absorbed, reemits at longer wavelength
kT = knr+ krad
2. Thermal Relaxation
[Picosec]
tf = 1/ kT
1. Absorption
[Femtosec]
Quantum yield = prob fluorescing”
krad /(knr+ krad)
3. Stays at lowest excited vibrational states for a “long” time (nsec)
4. Fluorescence (krad)
& Non-radiative (knonrad)
[Nanosec]
What happens if krad 
What happens if krnr 
5. Thermal relaxation
[Picosec]
What happens for non-fluorescing molecule?
krnr >> krad

9. Fluorophores & Quantum Yield
k = krad + kn.r. ; t = trad + tn.r.
quantum yield = krad/(krad + kn.r)
Have ≥ 1 electron that is free to move. Excitation light moves e’s around, i.e. a dipole, and it can re-radiate, often with polarization.
Very good dyes have q.y.
approaching one (kn.r ≈ 0)
“Good dyes: QY ≈ 1]; Absorption ≈ 100,000 cm-1M-1: A = ebc
In general, to make something fluorescent, it must either naturally be fluorescent, or you have to make it fluorescent. Can do latter by adding a fluorescent dye (see above), or you can do molecular biology to make something fluorescent by adding a fluorescent protein.

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

11. Green Fluorescent Protein: Genetically-encoded dye
Fluorescent protein from jelly fish

12. Different Fluorescent Proteins
Absorption
Fluorescence
mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry
Shaner, Tsien, Nat. Bio., 2004

13. How fine (small) can you see?
d = l/2 N.A.
Memorial to Ernst Karl Abbe, who approximated the diffraction limit of a microscope as , where d is the resolvable feature size, λ is the wavelength of light, n is the index of refraction of the medium being imaged in, and θ (depicted as α in the inscription) is the half-angle subtended by the optical objective lens.

14. What determines (ultimate, i. e
What determines (ultimate, i.e. best) resolution of technique… microscope, eye, etc.?
[2 parts]
1. Primarily λ (wavelength). (visible nm)
Why? Uncertainty principle (Homework).
2. Collection Angle/focal length/ Numerical Aperture
Resolution ≈ # λ/N.A.
# = a factor = ½ (details not important)
Resolution ≈ λ/[2N.A.]

15. Wavelength & Resolution
visible=≈ nm
/2 N.A.: air= l/2: oil= l/(2)(1.4)
= 500 nm: Best resolution nm
red
green
blue
purple
400 nm
Short l
Long l
Modern day optical microscopes are highly optimized– perfect diffraction limited. (Electron microscopes are 1000’s of times worse.)

16. Accuracy & Resolution:
How accurately can you tell where one object is:
How well can you resolve two nearby (point) objects?
Point-Spread Function (PSF)
A single light-emitting spot will be smeared out, no matter how small the spot is, because of the wavelength of light to ~ l/2NA.

17. Biomolecular Motors: Intra- & Extra-Cellular Motion
Characteristics
nm scale
Move along tracks
intracellular directional movement
cell shape changes & extracellular movement
Use ATP as energy source D
Actin, mtubules K
ATP-binding
heads
Nature Reviews
ATP  mechanical work
Cargo binding
Kinesin Myosin Dynein  Motor
Microtubule actin Microtubule  polymer

18. Super-Accuracy: Nanometer Distances
FIONA
Fluorescence Imaging with One Nanometer Accuracy
1.5 nm accuracy
1-500 msec
Center can be found much more accurately than width
W.E. Moerner, Crater Lake
center
width
Quantum
Dot
Kinesin
(CENP-E)
Axoneme
or microtubule
8 nm steps
Dx = width /√N
(Uncertainty in the position of the average ~ width and inversely related to √ # of photons)
≈ 250/√10k = 1.3 nm

19. Super-Accuracy: Nanometer Distances
FIONA
Fluorescence Imaging with One Nanometer Accuracy
W.E. Moerner, Crater Lake
Center can be found much more accurately than width
Quantum
Dot
Kinesin
(CENP-E)
Axoneme
or microtubule
8 nm steps
Step size (nm)
Count
Time (sec)
Position (nm)
8.4 ± 0.7 nm/step

20. Kinesin: Hand-over-hand or Inchworm?
16 nm
qs655
8.3 nm, 8.3 nm
8.3 nm
16.6 nm
16.6, 0, 16.6 nm, 0…
0 nm
8.3
pixel size is 160nm
2 x real time

21. Takes 16 nm hand-over-hand steps
Kinesin
<step size> = 16.3 nm
y ~ texp(-kt)
Can you derive this?
Takes 16 nm hand-over-hand steps
16 nm
0 nm

22. Answer, and turn in at the end of class.
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.