1.hw due today. 2.another hw assigned today. due next wednesday. 3.mid-term exam. today’s...
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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.TRANSCRIPT
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1. HW due today.2. Another HW assigned today. Due next Wednesday.3. Mid-term Exam.
Today’s Announcements
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Mid-term TestAverage = 77%
Can be reviewed at TA office hrs.
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3. Individual Projects– Change in agenda5 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.
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.
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You can get beautiful pictures
www.invitrogen.com
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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)
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Basics of fluorescenceShine 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)
-/fY = e
Stokes Shift (10-100 nm)
ExcitationSpectra
EmissionSpectra
Photobleaching Important: Dye emits 105 107 photons, then dies!
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Fluorophores & Quantum Yield
Thermal relaxation
Fluorescence& Non-radiative
Absorption
Thermal relaxation[Femtosec]
10-15
[Picosec: 10-12]
[Nanosec: 10-9]
[Picosec]
kn.r.krad
n.r.rad
k = krad + kn.r. ; = rad + n.r.
quantum yield = krad/(krad + kn.r)
Very good dyes have q.y. approaching one (kn.r ≈ 0)
Triplet State (?)(Long lifetime: )
Thermal relaxation
[O2] 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.
> 1s
[Picosec]
“Good dyes: QY ≈ 1]; Absorption ≈ 100,000 cm-1M-1: A = ebc
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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 ManufacturerAndor, Hamamatsu, Princeton Instruments, other…make EMCCDs
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Fluorescence vs. standard light Microscopy
Which is more sensitive?
Answer: Fluorescence…no background!
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Basics of Labeling: In vitroBind to: free cysteines (R-SH) (often only one or a few in proteins)
: free lysines (R-NH2) (many per protein)
Cysteine Reactive Amine Reactive
MaleimideIsothiocyanate (ITC)
Succinymidal Ester
Iodoacetamide
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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, 2002Tsien, Science, 1998
Bi-Arsenic FLASH, ReASH…
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(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
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Different Fluorescent Proteins
mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry
Absorption
Fluorescence
Shaner, Tsien, Nat. Bio., 2004
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Green Fluorescent Protein: Genetically-encoded dye
Fluorescent protein from jelly fish
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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.