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HW due today. Another HW assigned today. Due next Wednesday. Mid-term Exam.

Today ’ s Announcements. HW due today. Another HW assigned today. Due next Wednesday. Mid-term Exam. Mid-term Test. Average = 77%. Can be reviewed at TA office hrs. Today ’ s Announcements. Individual Projects– Change in agenda

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HW due today. Another HW assigned today. Due next Wednesday. Mid-term Exam.

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

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

  3. Today’s Announcements • Individual Projects– Change in agenda • 5 minute lecture! April 17th and 19th (# 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. By Monday, 5pm, turn in (electronically) a rough draft of 5 minute lecture. Either a Word document laid out as above, or a Powerpoint.

  4. You can get beautiful pictures www.invitrogen.com

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

  6. Time (nsec) Basics of fluorescence Shine light in, gets absorbed, reemits at longer wavelength Thermal relaxation Stokes Shift (10-100 nm) Light In Excitation Spectra Emission Spectra [Picosec] Light Out Fluorescence & Non-radiative Absorption [Nanosec] [Femtosec] Thermal relaxation Fluorescence [Picosec] -t/tf Y = e Photobleaching Important: Dye emits 105 107 photons, then dies!

  7. 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. Thermal relaxation Very good dyes have q.y. approaching one (kn.r ≈ 0) Fluorescence & Non-radiative Absorption Triplet State (?) (Long lifetime: ) [Picosec: 10-12] [O2] reaction Thermal relaxation Non-Fluorescent state > 1ms krad kn.r. trad tn.r. Thermal relaxation [Nanosec: 10-9] [Femtosec] 10-15 [Picosec] [Picosec] “Good dyes: QY ≈ 1]; Absorption ≈ 100,000 cm-1M-1: A = ebc

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

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

  10. Basics of Labeling: In vitro Bind to: free cysteines (R-SH) (often only one or a few in proteins) : free lysines (R-NH2) (many per protein) Amine Reactive Cysteine Reactive Succinymidal Ester Iodoacetamide Maleimide Isothiocyanate (ITC)

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

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

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

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

  15. 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.

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