Download Single-Molecule Methods

Single-Molecule Methods
I - in vitro
Bo Huang
Macromolecules
2013.03.11
F1-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
F1 – 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
EnsuringP(k;
single
molecule
Navg) =
Navgk e-N /detection
k!
↓
No molecule most of the time!
avg
Navg = 0.5
Navg = 0.1
1.0
1.0
0.8
0.8
0.8
0.6
0.4
0.2
Probability
1.0
Probability
Probability
Navg = 1
0.6
0.4
0.2
0
1
2
3
4
Number of molecules
0.6
0.4
0.2
1
2
3
4
0.0
0.0
0.0
0.10
0.08
0.06
0.04
0.02
0.00
0
1
2
3
4
Number of molecules
0
1
2
3
4
Number of molecules
Single molecule concentration
Confocal detection volume ≈ 1 µm3 = 1 fL
Navg = 1  1/(6 × 1023) mol / 1 × 10-15L ≈ 2 nM
Navg = 0.1  200 pM
Single molecule concentration ≤ 100 pM
TIRF detection volume ≈ 0.1 fL
Navg = 1  20 nM
“Few molecule” spectroscopy
1/Concentration
Fluorescence correlation spectroscopy (FCS)
1.5
1.4
1.3
Diffusion time
1.2
1.1
1.0
10
-6
10
-5
10
-4
10
-3
10
-2
10
-1
10
0
Autocorrelation
600
Counts
500
400
300
200
100
0
0
500
1000
1500
2000
2500
Time / us
3000
3500
4000
4500
5000
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
kfl
knr
kFRET
Fluorescence
Nonradioactive decay
Energy transfer
E = kFRET / (kFRET + kfl + knr)
Acceptor
Often approximated by “proximity ratio”
E ≈ Iacceptor / (Idonor + Iacceptor)
FRET efficiency
E
Donor
1
R
0.5
Acceptor
0
E
0
1
1
 R
1   
 R0 
2
6
R/R0
Förster Radius
The Förster radius can be calculated from measurable parameters
Orientation factor (0 ≤ κ ≤ 4)
usually 2/3 (caution!)
Refractive index
R06 = 8.8 × 1023 κ2 n-4 Q0 J
Donor quantum
efficiency
Overlap integral
J = ∫ fD(λ) εA(λ) λ4 dλ
Donor emission Accepter absorption
For common FRET pairs, R0 ≈ 4-6 nm
Diffusion spFRET
FRET efficiency
Immobilized spFRET
What can we measure?
Time
FRET histogram
Dwell time distribution
FRET transition matrix
T. Ha Group
Typical spFRET setup
Total internal reflection fluorescence microscopy
n1
n1sinθ1 = n2sinθ2
θ1
Total internal reflection:
θ1 = 90° 
n2
θ2
sinθ2 ≡ sinθc = n1/n2
n1 = 1.33 (water), n2 = 1.52 (glass), θc = 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: 50100 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 = nglass sinθmax
nwater = nglass sinθc
θmax ≥ θc
NA ≥ nwater ≈ 1.33
TIRF compatible objectives
• 1.40 NA
– “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
• High S/N
• Higher signal but even
higher background
• Oil immersion objective
• Ordinary coverglass
Surface Immobilization
Streptavidin
Neutravidin
Streptavidin
Neutravidin
Biotinylated lipid
The classic flow channel
The Full Jabłonski Diagram
Vibration relaxation
S1
hν
Absorption
S0
Intersystem
crossing
Triplet State
hν
Emission
Non-radioactive
decay
Lifetime ≈ μs - ms
T1
hν
Triplet
Quenching
Phosphorescence
Photobleaching
Vibration relaxation
Intersystem
crossing
S1
hν
Triplet State
T1
Absorption
Most common cause: O2
S0
Fight against photobleaching
Donor photobleaching
The oxygen scavenging system
Glucose + O2
H2O
Glucose oxidase
Gluconic acid + H2O2
Catalase
2×
Triplet quencher:
β-mercaptoethanol
HO-CH2-CH2-SH
β-mercaptoethylamine
H3N-CH2-CH2-SH
Trolox
Alternated Excitation (ALEX)
Analysis
Coming up:
Single-molecule methods in cells