Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling,

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Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling, in vitro, in vivo 3.Super-Accuracy (FIONA), Super-Resolution (STORM, PALM, STED) 4.Total Internal Reflection 1.HW due next Wednesday, 3/10/10; answers on Friday. 2.March 15 th, 17 th, night of 18 th : Presentations. Reports due night of 18 th. Today’s Announcements

Biophysics 101 Essentials of fluorescence Basic Fluorescence Single molecule detection methods (confocal, two-photon, TIRF) Imaging (FIONA, etc…) FRET Polarization FCS (Later) Thanks to ISS (of Champaign IL, a great place to live!) Photon Technology International (PTI)

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!

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

You can get beautiful pictures

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

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