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

Today’s Announcements

Mid-term TestAverage = 77%

Can be reviewed at TA office hrs.

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.

You can get beautiful pictures

www.invitrogen.com

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 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!

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

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

Fluorescence vs. standard light Microscopy

Which is more sensitive?

Answer: Fluorescence…no background!

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

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…

(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

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 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.