Single-Molecule Methods I - in vitro

Bo Huang

Macromolecules

2013.03.11

F1-ATPase: a case study

ADP ATP

Membrane

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

1

2

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

Lavis & Raines, ACS Chem Biol, 2008

“Good” Organic Fluorophores

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

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bili

ty

Number of molecules0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bili

ty

Number of molecules0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bili

ty

Number of molecules

1 2 3 40.00

0.02

0.04

0.06

0.08

0.10

Navg = 1 Navg = 0.5 Navg = 0.1

The number of molecules in the detection volume follows Poisson distribution

P(k; Navg) = Navgk e-Navg / k! Ensuring single molecule detection

↓ No molecule most of the time!

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

0 500 1000 1500 2000 2500 3000 3500 4000 4500 50000

100

200

300

400

500

600

Co

un

ts

Time / us

1.5

1.4

1.3

1.2

1.1

1.0

10 -6

10 -5

10 -4

10 -3

10 -2

10 -1

10 0

Fluorescence correlation spectroscopy (FCS)

Autocorrelation

1/C

on

cen

trat

ion

Diffusion time

The simplest single molecule experiment: Fluorophore counting

Ulbrich & Isacoff, Nat Methods, 2007

Single-pair FRET

Fluorescence Resonance Energy Transfer (FRET)

Donor

Acceptor

FRET efficiency: Quantum efficiency of energy transfer

E = kFRET / (kFRET + kfl + knr)

Often approximated by “proximity ratio”

E ≈ Iacceptor / (Idonor + Iacceptor)

Fluorescence

Nonradioactive decay

kfl

knr

kFRET

Energy transfer

FRET efficiency

Donor

Acceptor

R

6

0

1

1

R

RE

0 1 2 0

0.5

1

E

R/R0

Förster Radius

R06 = 8.8 × 1023 κ2 n-4 Q0 J

Orientation factor (0 ≤ κ ≤ 4) usually 2/3 (caution!)

Refractive index

Donor quantum efficiency

Overlap integral J = ∫ fD(λ) εA(λ) λ4 dλ

The Förster radius can be calculated from measurable parameters

For common FRET pairs, R0 ≈ 4-6 nm

Donor emission Accepter absorption

Diffusion spFRET

FRET efficiency

Immobilized spFRET

What can we measure?

T. Ha Group

FRET histogram

Time

Dwell time distribution FRET transition matrix

Typical spFRET setup

Total internal reflection fluorescence microscopy

θ1 = 90°

sinθ2 ≡ sinθc = n1/n2

n1

n2

θ1

θ2

n1sinθ1 = n2sinθ2

Total internal reflection:

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: 50-100 nm.

TIRM improves S/N for surface imaging

Epifluorescence TIRF

Prism-type TIRF configuration

CCD

Quartz slide

Spacer Buffer

Coverglass

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

• High S/N

Objective

• Simple setup

• Oil immersion objective

• Ordinary coverglass

• Higher signal but even higher background

Surface Immobilization

Streptavidin Neutravidin

Streptavidin Neutravidin

Biotinylated lipid

The classic flow channel

The Full Jabłonski Diagram

hν

hν

Emission

Intersystem crossing

Triplet State

Vibration relaxation

hν

Phosphorescence Triplet

Quenching

Lifetime ≈ μs - ms

Absorption

S1

S0

T1

Non-radioactive decay

Photobleaching

hν

Absorption

Triplet State

Vibration relaxation Intersystem crossing

Most common cause: O2

S1

S0

T1

Fight against photobleaching

Donor photobleaching

The oxygen scavenging system

Glucose + O2

Glucose oxidase

Gluconic acid + H2O2

Catalase

H2O

2 ×

Triplet quencher:

β-mercaptoethanol

β-mercaptoethylamine

HO-CH2-CH2-SH

H3N-CH2-CH2-SH

Trolox

Alternated Excitation (ALEX)

Analysis

Coming up:

Single-molecule methods in cells