Single-Molecule Methods I - in vitro Bo Huang Macromolecules 2014.03.10
F 1 -ATPase: a case study Membrane ADP ATP
Rotation of the axle when hydrolyzing ATP Kinosita group, 1997-2005
Single Molecule Methods Noji group, 2005
Why look at molecules, one at a time?
An ensemble picture
Single Object Measurement: Resolving Heterogeneities Most people are eating... But some are cooking
Single Object Measurement: Resolving Heterogeneities Conformation states of protein folding Laurance, Kong, Jager and Weiss, PNAS, 2005 (102), p 17348
Single Object Measurement: Avoiding Synchronization Single object movies Myosin walk on actin filament F 1 ATPase rotation
Single Object Measurement: Avoiding Synchronization 2 1 3
Single Object Measurement: Detecting Rare Species
Single Object Measurement: Detecting Rare Species RNA polymerase at work Backtracking (0.1% probability) to correct for base pair mismatch Abbondanzieri, Greenleaf, Shaevitz, Landick and Block., Nature 2005 (438), p 460
Brief history of single molecule methods 1989 - W.E. Moerner - Absorption 1990 - M. Oritt - Fluorescence 1993 - Room temperature (NSOM) 1994 - Confocal 1995 - TIRF 1998 - Optical trap 1996-2001 Single pair FRET 2003 - Single molecule localization 2000-2006 - Super-resolution microscopy
Single-Molecule Experiments Fluorescence Force
Pulling a Molecule Atomic force microscope Optical trap Magnetic tweezers Buffer flow
Protein Unfolding by AFM
Optical trap
Magnetic Tweezers
Flow-stretching Labeled DNA (free) Labeled DNA (flow) DNA+nucleosome (flow)
How to detect single molecule fluorescence? Great fluorophores Low background Sensitive detection Single molecule concentration
Good Organic Fluorophores Lavis & Raines, ACS Chem Biol, 2008
Reducing background: confocal Plan Apo 100x Oil NA 1.4 APD or PMT
Reducing background: TIRF CCD
Sensitive detection High NA Objective $5,000 ~ $15,000 EMCCD $20,000 ~ $40,000 APD $3,000 ~ $10,000
Single molecule concentration The number of molecules in the detection volume follows Poisson distribution P(k; N avg ) = N avgk e -N avg / k! Ensuring single molecule detection No molecule most of the time! N avg = 1 N avg = 0.5 N avg = 0.1 Probability 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 Number of molecules Probability 1.0 0.8 0.6 0.4 0.2 0.0 Probability 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 Number of molecules 0.10 0.08 0.06 0.04 0.02 0.00 1 2 3 4 0 1 2 3 4 Number of molecules
Single molecule concentration Confocal detection volume 1 µm 3 = 1 fl N avg = 1 1/(6 10 23 ) mol / 1 10-15 L 2 nm N avg = 0.1 200 pm Single molecule concentration 100 pm TIRF detection volume 0.1 fl N avg = 1 20 nm
Few molecule spectroscopy Counts 1/Concentration Fluorescence correlation spectroscopy (FCS) 1.5 1.4 1.3 1.2 Diffusion time 1.1 1.0 10-6 10-5 10-4 10-3 10-2 10-1 10 0 600 500 400 300 200 100 Autocorrelation 0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Time / us
The simplest single molecule experiment: Fluorophore counting Ulbrich & Isacoff, Nat Methods, 2007
Single-pair FRET
Fluorescence Resonance Energy Transfer (FRET) FRET efficiency: Quantum efficiency of energy transfer Donor k fl k nr Fluorescence Nonradioactive decay k FRET Energy transfer E = k FRET / (k FRET + k fl + k nr ) Acceptor Often approximated by proximity ratio E I acceptor / (I donor + I acceptor )
FRET efficiency Donor E 1 E 1 1 R R 0 6 R 0.5 Acceptor 0 0 1 2 R/R 0
Förster Radius The Förster radius can be calculated from measurable parameters Orientation factor (0 κ 4) usually 2/3 (caution!) Refractive index R 06 = 8.8 10 23 κ 2 n -4 Q 0 J Donor quantum efficiency Overlap integral J = f D (λ) ε A (λ) λ 4 dλ Donor emission Accepter absorption For common FRET pairs, R 0 4-6 nm
Diffusion spfret FRET efficiency
Immobilized spfret
What can we measure? FRET histogram Time Dwell time distribution FRET transition matrix T. Ha Group
Typical spfret setup
Total internal reflection fluorescence microscopy n 1 n 1 sinθ 1 = n 2 sinθ 2 θ 1 Total internal reflection: n 2 θ 2 θ 1 = 90 sinθ 2 sinθ c = n 1 /n 2 n 1 = 1.33 (water), n 2 = 1.52 (glass), θ c = 61
The evanescent wave in TIR The energy of the evanescent wave is localized near the interface. The strength of the evanescent wave field decreases exponentially. The penetration depth is a function of the wavelength and the incident angle. Typical penetration depth: 50-100 nm.
TIRM improves S/N for surface imaging Epifluorescence TIRF
Prism-type TIRF configuration Quartz slide Spacer Buffer Coverglass CCD
Through-the-objective TIRF
Requirement for TIRF objective NA = n glass sinθ max n water = n glass sinθ c θ max θ c NA n water 1.33
1.40 NA TIRF compatible objectives Barely enough for laser TIRF 1.45 / 1.49 NA TIRF objective More homogenous illumination field Higher efficiency for lamp TIRF Compromised image quality 1.65 NA Sapphire coverglass Toxic oil
Prism vs. Objective type TIRF Prism More complicated illumination Water immersion objective Quartz slides Objective Simple setup Oil immersion objective Ordinary coverglass High S/N Higher signal but even higher background
Surface Immobilization Streptavidin Neutravidin Streptavidin Neutravidin Biotinylated lipid
The classic flow channel
The Full Jabłonski Diagram Vibration relaxation Intersystem crossing Lifetime μs - ms S 1 hν Triplet State hν Absorption Emission Non-radioactive decay T 1 Triplet Quenching hν Phosphorescence S 0
Photobleaching Vibration relaxation Intersystem crossing S 1 Triplet State hν T 1 Absorption Most common cause: O 2 S 0
Fight against photobleaching Donor photobleaching
The oxygen scavenging system Glucose + O 2 H 2 O Glucose oxidase Catalase Gluconic acid + H 2 O 2 2 Triplet quencher: β-mercaptoethanol β-mercaptoethylamine HO-CH 2 -CH 2 -SH H 3 N-CH 2 -CH 2 -SH Trolox
Alternated Excitation (ALEX)
Coming up: Single-molecule methods in cells
Analysis