1.HW due today. 2.Another HW assigned today. Due next Wednesday. 3.Mid-term Exam. Today’s Announcements

Mid-term Test Average = 77% Can be reviewed at TA office hrs.

3.Individual Projects– Change in agenda 5 minute lecture! April 17 th and 19 th (# that can make it?) Intro: what is main subject. (How things are moved about a cell.) Detail subject: Molecular motors. Question; How myosin v moves: does it walk hand-over- hand or inchworm. Problem: Steps are tiny so needed new technique. Solution: Invented FIONA: capable of seeing 1 nm steps. Overall Solution: Moves by walking, i.e. “hand-over-hand. Today’s Announcements By Monday, 5pm, turn in (electronically) a rough draft of 5 minute lecture. Either a Word document laid out as above, or a Powerpoint.

You can get beautiful pictures

1.How to label things so they fluoresce—ext. labels, GFP. 2.Total Internal Reflection Fluorescence (TIRF)- near surface. 3.Accuracy and Resolution—how fine can you see. 4.Fluorescence. What is it (amplitude, time-scale)? Today’s take-home lessons (i.e. what you should be able to answer at end of lecture)

Basics of fluorescence Shine light in, gets absorbed, reemits at longer wavelength Light In Light Out Thermal relaxation Fluorescence & Non-radiative Absorption Thermal relaxation [Femtosec] [Picosec] [Nanosec] [Picosec] Time (nsec) Fluorescence -/f-/f Y = e Stokes Shift ( nm) Excitation Spectra Emission Spectra Photobleaching Important: Dye emits 10 5  10 7 photons, then dies!

Fluorophores & Quantum Yield Thermal relaxation Fluorescence & Non-radiative Absorption Thermal relaxati on [Femtosec] [Picosec: ] [Nanosec: ] [Picosec] k n.r. k rad  n.r.  rad k = k rad + k n.r. ;  =  rad +  n.r. quantum yield = k rad /(k rad + k n.r ) Very good dyes have q.y. approaching one (k n.r ≈ 0) Triplet State (?) (Long lifetime: ) Thermal relaxati on [O 2 ] reaction Non-Fluorescent state 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. > 1  s [Picosec] “Good dyes: QY ≈ 1]; Absorption ≈ 100,000 cm -1 M -1 : A = ebc

Basic Set-up of Fluorescence Microscope Semwogerere & Weeks, Encyclopedia of Biomaterials and Biomedical Engineering, 2005 (Lasers, Arc Lamps) (Electronic Detectors: CCD, EMCCDs, PMTs, APDs) Nikon, Zeiss, Olympus, Leica—Microscope Manufacturer Andor, Hamamatsu, Princeton Instruments, other…make EMCCDs

Fluorescence vs. standard light Microscopy Which is more sensitive? Answer: Fluorescence…no background!

Basics of Labeling: In vitro Bind to: free cysteines (R-SH) (often only one or a few in proteins) : free lysines (R-NH 2 ) (many per protein) Cysteine Reactive Amine Reactive Maleimide Isothiocyanate (ITC) Succinymidal Ester Iodoacetamide

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, 2002 Tsien, Science, 1998 Bi-Arsenic FLASH, ReASH…

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

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

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

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.