Fluorescence You can get beautiful pictures

www.invitrogen.com

Imaging: Your eye is a Microscope!

www.olympusmicro.com

Lens Maker’s Equation: 1/f = 1/o + 1/i(Actually have two lenses: your cornea and your lens)

NoiseWhy 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/secTherefore: 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))

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

Basic Set-up of Fluorescence Microscope

Semwogerere & Weeks, Encyclopedia of Biomaterials and Biomedical Engineering, 2005

(Lasers, Arc Lamps)

Detector

Nikon, Zeiss, Olympus, Leica—Microscope ManufacturerAndor, Hamamatsu, Princeton Instruments, other…make (EM)CCDs

Why can you see so little fluorescence

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!

Basics of fluorescenceShine light in, gets absorbed, reemits at longer wavelength

Light In

Light Out

Time (nsec)

-/fY = e

Stokes Shift (10-100 nm)

ExcitationSpectra

EmissionSpectra

Photobleaching Important: Dye emits 105 107 photons, then dies!

1. Absorption [Femtosec]

4. Fluorescence& Non-radiative

[Nanosec]

3. Stays at lowest excited vibrational states for a “long” time (nsec)

What happens for non-fluorescing molecule? (in 3. nr really fast)

5. Thermal relaxation[Picosec]

2. Thermal Relaxation(heat, in I.R.)

[Picosec]

Basics of fluorescenceShine light in, gets absorbed, reemits at longer wavelength

1. Absorption [Femtosec]

4. Fluorescence (krad)& Non-radiative (knonrad)

[Nanosec]

3. Stays at lowest excited vibrational states for a “long” time (nsec)

What happens for non-fluorescing molecule?

5. Thermal relaxation[Picosec]

2. Thermal Relaxation[Picosec]

kT = knr+ krad

f = 1/ kT

What happens if krad

What happens if krnr

krnr >> krad

Quantum yield = prob fluorescing”krad /(knr+ krad)

Fluorophores & Quantum Yield

k = krad + kn.r. ; = rad + n.r.

quantum yield = krad/(krad + kn.r)

Very good dyes have q.y. approaching one (kn.r ≈ 0)

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.

“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.

(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

Green Fluorescent Protein: Genetically-encoded dye

Fluorescent protein from jelly fish

Different Fluorescent Proteins

mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry

Absorption

Fluorescence

Shaner, Tsien, Nat. Bio., 2004

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.

http://en.wikipedia.org/wiki/Diffraction-limited_system

d = /2 N.A.

How fine (small) can you see?

What determines (ultimate, i.e. best) resolution of technique… microscope, eye, etc.?

[2 parts] 1. Primarily λ (wavelength). (visible 400-700nm)

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.]

red

gre

en

blu

epurp

le

400 nm 488 514 532 633 750

Wavelength & Resolution

visible=≈ 400-700 nm

/2 N.A.: air= /2: oil= /(2)(1.4)

= 500 nm: Best resolution 200-250 nm

Modern day optical microscopes are highly optimized– perfect diffraction limited. (Electron microscopes are 1000’s of times worse.)

Short Long

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 ~ /2NA.

Biomolecular Motors: Intra- & Extra-Cellular MotionCharacteristics• nm scale • Move along tracks• intracellular directional movement• cell shape changes & extracellular

movement• Use ATP as energy source

Act

in,

tub

ule

s

ATP mechanical workNat

ure

Rev

iew

s

Microtubule actin Microtubule polymer Kinesin Myosin Dynein Motor

ATP-bindingheads

Cargo binding

K

D

Super-Accuracy: Nanometer Distances

center

width

Center can be found much more accurately than width

W.E. Moerner, Crater Lake

8 nm steps

Fluorescence Imaging with One Nanometer

Accuracy1.5 nm accuracy

1-500 msec

x = width /√N(Uncertainty in the position of the average ~ width and inversely related to √ # of photons)

≈ 250/√10k = 1.3 nm

Super-Accuracy: Nanometer Distances

Fluorescence Imaging with One Nanometer

Accuracy Center can be found much more accurately than width

W.E. Moerner, Crater Lake

8 nm steps

Step size (nm)Time (sec)

8.4 ± 0.7 nm/step

16 nm

qs655

pixel size is 160nm2 x real time

8.3 nm, 8.3 nm

8.3 nm

16.6 nm

16.6, 0, 16.6 nm, 0…0 nm

16.6 nm

8.3 8.3 nm

Kinesin: Hand-over-hand or Inchworm?

Kinesin

0 2 4 6 8 10 12 14

0

80

160

240

320

400

480

560

640

720

800

880

960

1040

1120

1200

1280

disp

lace

men

t (nm

)

time(sec)

<step size> = 16.3 nm

y ~ texp(-kt)

Takes 16 nm hand-over-hand steps

16 nm0 nm

16 nm

Can you derive this?

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.