Sungchul Hohng Department of Physics & Astronomy Seoul National - - PowerPoint PPT Presentation

Sungchul Hohng

Department of Physics & Astronomy Seoul National University

1. 2. 3. 4. Fluorescence FRET Single-Molecule Localization Single-Molecule Localization Microscopy

Contents

Perrin-Jablonski diagram

Perrin-Jablonski diagram : a diagram that illustrates the electronic states of a molecule and the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity. Radiative transitions are indicated by straight arrows and nonradiative transitions by squiggly arrows. The vibrational ground states of each electronic state are indicated with thick lines, the higher vibrational states with thinner lines.

Vibrational relaxation: I nternal conversion: a non-radiative transition between two electronic states

• f the same spin multiplicity.

I ntersystem crossing: a transition to a state with a different spin multiplicity

vr

Stokes shift

Single-molecule detection!!!

P: laser power, τ: integration time, φ: quantum yield, η: detection efficiency

Diffraction Limit

“Beitrage zur Theorie des Mikroskops und der Mikroskopischen Wahrnehmung”

• Arch. Mikrosk. Anat. 9, 413 (1873).

Abbe, Ernst

~300 nm

1. Fluorescence

Contents

2. FRET 3. 4. Single-Molecule Localization Single-Molecule Localization Microscopy

FRET: Optical Method with 1-nm & 1-ms resolution

Fluorescence Resonance Energy Transfer

Some Historical Facts

1922, Cario and Frank: Observation of FRET 1927, Perrin: Resonance energy transfer, dipole-dipole interaction 1948, Förster: Derivation of FRET efficiency Active application to biological problems in ensemble level 1996, Ha: Single-molecule FRET

~

• J. D. Jackson, Classical electrodynamics 3rd ed., p.441.

n H Z E R e n ck H

D D ikR D D

ˆ ) ˆ ( 4

2

× = × =     µ π

[ ]

3 2

1 ) ˆ ( ˆ 3 4 1 1 ) ˆ ( 4 R n n E R n i H

D D D D D

µ µ πε µ π ω      − ⋅ = × =

In the radiative zone In the near zone

n R R ˆ = 

D

µ 

A

µ 

[ ]

3

1 ) ˆ )( ˆ ( 3 4 1 R n n E H

D A D A A

µ µ µ µ πε µ       ⋅ ⋅ − ⋅ = ⋅ − =

6 2 * *

1 , | | , R A D H A D kt ∝ ∝

6 0)

( 1 1 ) ( 1 1 R R k k k k k k k E

t nr r nr r t t

+ = + + = + + =

6 1 2 4 5

) 10 79 . 8 ( κ φ ⋅ ⋅ ⋅ ⋅ =

− −

J n R

D

[Å]

2 2

) cos cos 3 (cos

AR DR DA

θ θ θ κ − = :

D

φ

Donor quantum yield

1.0 0.8 0.6 0.4 0.2 0.0

Emission/Absorption

650 600 550 500 450

Wavelength (nm) Acceptor Absorption Donor Emission

Spectral Overlap

[ ] ( )

( )

∫ ∫

∞ ∞

⋅ ≡

4

) ( λ λ λ λ λ λ ε d f d f J

D D A

E = 1/ (1 + (R/R0)6) R0: 50% energy transfer distance (3 ~ 7 nm) R0 = 5.0 nm

Subnanometer Sensitivity Spectroscopic Ruler

FRET: Optical Method with 1-nm & 1-ms resolution

Intermolecular Interaction Internal Motion

TIR (Total Internal Reflection)

Donor Acceptor

단일분자 FRET의 측정장치

TIR (Objective type)

Confocal

10 20 30 40 50 60 70 80

Intensity

10 20 30 40 50 60 70 80

Intensity

10 20 30 40 50 60 70 80

Intensity Time (s)

Highly polymorphic & extremely dynamic

Sungchul Hohng et al., J. Mol. Biol. (2004).

10 20 30 40 50 200 400

Count Dwell Time (s)

10 20 30 40 50 200 400

Count Dwell Time (s)

Sungchul Hohng et al., J. Mol. Biol. (2004). Low τf = 2.4 s High τb = 3.3 s

Mg2+ stabilizes stacked conformers

10 20 30 40 50 60 70 80

Intensity Time (s)

1 10 100 0.1 1

Low High

Rate Constant (s

• 1)

[Mg

2+] (mM)

k = A exp(∆H*/RT)

3.2 3.3 3.4 3.5 3.6 0.1 1 10

1 mM 2 mM 5 mM 10 mM 20 mM 50 mM 100 mM

kf + kb (s

• 1)

1000/T (K

• 1)

1 10 100 20 40 ∆H

**

RT Ln(A) (kcal mol

• 1)

[Mg

2+] (mM)

Activation Enthalpy vs. Entropy

Arrhenius Plots Activation Energy

Correlated Motion

1 2 3 4 1 2 3 4

Cy2 Cy3 Cy5 Cy7

Lee et al. Angew. Chem. Int. Ed. (2010)

For every action there exists an equal but opposite reaction. —Sir Isaac Newton

How the trap is possible?

Paramagnetic bead

Magnetic tweezers

1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Time (ms)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

5.2 pN 2.9 pN 1.6 pN 0.9 pN

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0.5 pN

1 2 3 1 10

kb kf

Rate (s

• 1)

Force (pN)

isoI isoII tsI tsII

Lee et al. (JACS, 2013)

Magnetic tw eezers + FRET Electrom agnetic tw eezers + FRET

Uhm et al. (Bulletin of the Korean Chemical Society, 2016)

1. Fluorescence

Contents

3. Single-Molecule Localization 4. Single-Molecule Localization Microscopy 2. FRET

Although the resolution of an optical microscope is ~ 250 nm, “center of a spot, and hence the location

• f the object, can be determined to a much greater

precison.” “It’s much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide.”

Fluorescence Imaging with One Nanometer Accuracy

(1.5 nm, 1-500 msec)

Diffraction limited spot

Width of λ/2 ≈ 250 nm

40 80 120 160 200 240 280 5 10 15 20 25 5 10 15 20 25

Photons

X D a t a Y axis

center width

Enough photons (signal to noise)…Center determined to ≈ 1 nm.

• R. E. Thompson et al. Biophys. J. 82, 2775 (2002)

( )

N s N a b s N a s x

2 2 2 2 4 2 2 2

8 12 ≈ + + = ∆ π

Nanometer Localization

spot size pixel size background Photon number

By measuring head (foot)-step size using optical microscopy, we can differentiate the two models!

Hand-over-hand: Head (foot) takes 16 nm steps

16 nm Adapted from Hua, Chung, Gelles, Science, 2002

8 nm 8 nm

Inchworm: Head (foot) takes 8 nm steps

How do they walk?

Center of mass

37/2 nm

x 74 nm 37-2x 37 nm

Myosin V Labeling on Light Chain: Expected Step Sizes Expected step size

Hand-over-hand: Head = 2 x 37 nm= 74, 0, 74 nm CaM-Dye: 37-2x, 37+2x, … Inchworm: always Scm = 37 nm 0 nm 37+2x

23nm, 51 nm, 23nm, …

74nm, 0 nm, 74nm, …

) exp( 2 )) exp( ) exp( ( ) ( ) exp( ) ( : ) exp( ) ( :

2 2 1 1 2 2 1 1

kt k t k k t k k t P t k k t g A B t k k t f B A − = − + − = − = ′ → − = → ) exp( ) exp( ) ( )) ( exp( ) exp( ) ( ) ( ) ( :

2 2 2 2 1 1

kt t k du kt k t P du u t k k u k k du u t g u f t P A A

t t t

− = − = − − ⋅ − = − ⋅ = ′ →

∫ ∫ ∫

If k1= k2= k Case I Case II

Sako et al. Nat. Cell Biol. (2000)

1. Fluorescence

Contents

3. Single-Molecule Localization 4. Single-Molecule Localization Microscopy 2. FRET

Seurat, G. P .

점묘법 (Pointillism)

Sunday Afternoon on the Island of La Grande Jatte

Dark State & Activation

I m aging Procedure

Super-resolution Microscopy Good for Cell Studies, but not for Tissue Studies

Zhuang, Xiaowei (Harvard)

STORM ( STochastic Optical Reconstruction Microscopy)

Photosw itching of Organic Dyes

3 -D STORM

Huang et al. Science 2008

Multi-Color STORM

Bates et al. Science 2007 Bates et al. ChemPhysChem. 2012

STORM in Neurosciences

Neuron Contour

Lakadamyali et al. PLoS one, 20012

Actin filam ent in axon

Xu et al. Science, 2013

Chem ical Synapse

Dani et al. Neuron, 2010

The principal difficulty in this regime is how best to overcome cellular autofluorescence, i.e., emission that arises from the relatively high concentration of potentially interfering natural cellular fluorophores, such as flavins, NADH, and other molecules.” ̶ Trends in Analytical Chemistry (2003) The signal from a single FP is stronger than the autofluorescence from the thin monolayer of bacterial cells used but would be overwhelmed by thicker yeast or mammalian cells in the wide-field microscope. ̶ Annu. Rev. Biophys. (2008)

• W. E. Moerner
• X. Sunney Xie

Challenge # 1 : Huge Background

• 1. TIRF (Sako et al. Nat. Cell Biol. 2000)
• 2. HILO (Tokunaga et al. Nat. Meth. 2008)
• 3. Confocal Microscopy
• 3. SPI M (Zanacchi et al. Nat. Meth. 2011)

W ays to reduce autofluorescence

“Com m ercial scanning confocal m icroscopes suffer from low signal collection and detector efficiency.” ̶ X. Sunney Xie ( Annu Rev Biophys, 2 0 0 8 )

Com m ercial Fast Confocal Microscopes

Lee et al. Biophys. J. ( 2 0 1 2 ) , patent pending

• 30
• 25
• 20
• 15
• 10
• 5

5 10 15 20 25 30 0.0 0.2 0.4 0.6 0.8 1.0

Line scan confocal HILO Epi-fluorescence

Normailzed intensity z-axis position (µm) w / 1 0 nM free dye

HI LO m icroscopy Real-tim e confocal

Top Bottom

0.00 0.01 0.02 0.03 0.04 0.05 Diffusion coefficient (um^2/s) Bottom Top

3 μm

Z=0um Z=4um Z=7.5um Lee et al. Biophys. J. ( 2 0 1 2 ) , patent pending