Fluorescence You can get beautiful pictures www.invitrogen.com

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) www.olympusmicro.com

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))

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 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

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 fluorescence Shine light in, gets absorbed, reemits at longer wavelength Stokes Shift (10-100 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!

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

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.

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

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

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

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

What determines (ultimate, i. e 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.]

Wavelength & Resolution visible=≈ 400-700 nm /2 N.A.: air= l/2: oil= l/(2)(1.4) = 500 nm: Best resolution 200-250 nm red green blue purple 400 nm 488 514 532 633 750 Short l Long l Modern day optical microscopes are highly optimized– perfect diffraction limited. (Electron microscopes are 1000’s of times worse.)

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.

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

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

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

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

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

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.