EPFL–SV–PTBIOP

BIOP COURSE 2015

FROM LOCALIZATION

TO INTERACTION

EPFL–SV–PTBIOP

COLOCALIZATIONTYPICAL EXAMPLE

Colocalization: The presence of two or more fluorophores on

the same physical structure (in a cell).

http://www.olympusconfocal.com/applications/colocalization.html

Actin

Alexa488Vinculin

Alexa568

MICROSCOPYLOW PASS FILTERING

Object

Imag

e

EPFL–SV–PTBIOP

COLOCALIZATION

• Two different molecules can never be at the same physical spot at the same time.

• Colocalization seen in images is coming from the low-pass filtering of the image formation in light microscopy.

• Colocalization is an artificial phenomenon and therefore always relative.

EPFL–SV–PTBIOP

FÖRSTER RESONANCE

ENERGY TRANSFER

S0

S1

Donor

S0

S1

Acceptor

distance

EPFL–SV–PTBIOP

FÖRSTER RESONANCE

ENERGY TRANSFER

S0

S1

Donor

S0

S1

Acceptor

distance

FRET is the non-radiative transfer of excitation energy from a donor

fluorophore to a nearby acceptor.

(Förster, Ann. Phys. 1948 : 55-75)

EPFL–SV–PTBIOP

ENERGY TRANSFER

EFFICIENCY

0

0.2

0.4

0.6

0.8

1

0 5 10 15

distance r / nm

En

erg

ytr

an

sfe

re

ffic

ien

cy R0<R0<R0

𝐸 =1

1 +𝑟𝑅0

6

ENERGY TRANSFER

EFFICIENCY

𝑹𝟎 = 𝟎. 𝟐𝟏𝟏 ∙ 𝜿𝟐 ∙ 𝒏−𝟒 ∙ 𝑸𝒀𝑫 ∙ 𝜺𝑨 ∙ 𝟎

∞𝒇𝑫 𝝀 𝒇𝑨 𝝀 𝝀𝟒𝒅𝝀

𝟎

∞𝒇𝑫 𝝀 𝒅𝝀

𝟏𝟔

Förster distance (𝑅0) is dependant on a number of factors,

including:

• the fluorescence quantum yield of the donor in the absence

of acceptor (𝑄𝑌𝐷)

• the extinction coefficient of the acceptor (𝜀𝐴)• the refractive index of the solution (𝑛)

• the dipole angular orientation of each molecule (κ2)

• the spectral overlap integral of the donor and acceptor

EPFL–SV–PTBIOP

ENERGY TRANSFER

EFFICIENCY

Lam et al. Nature Meth 2012 p1005

See also: Sun at al. Cytometry PartA 2013 p780

FRET APPLICATIONS IN BIOLOGYPROTEIN-PROTEIN INTERACTION

3 nm

FRET Low/No

FRETNo

FRET

FRET APPLICATIONS IN BIOLOGYINTRAMOLECULAR SENSOR

3 nm

FRETNo

FRET

Ligand, small molecule, analyte

Miyawaki A, et al. Nature. 1997 Aug 28;388(6645):882-7.

Fluorescent indicators for Ca2+ based on green fluorescent proteins and

calmodulin

FRET APPLICATIONS IN BIOLOGYINTRAMOLECULAR SENSOR

Masharina A, et al. J Am Chem Soc. 2012 Nov 21;134(46):19026-34.

A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands.

FRET EFFICIENCYSUMMARY

FRET efficiency is only apparent… but can be very precise as

well…The measured FRET efficiency (E) depends on a number of factors:

Ro

the affinity of the interaction between donor and acceptor

the stoichiometry of donor and acceptor-labeled proteins

the presence of unlabeled molecules

the ratio of the number of labels per protein

the saturation of the donor

The calculated FRET efficiency is an apparent FRET efficiency

EPFL–SV–PTBIOP

FRET DETECTION

S0

S1

Donor

S0

S1

Acceptor

τDA

S0

S1

S0

S1

τD

𝐸 = 1 −𝜏𝐷𝐴𝜏𝐷

FLIM MEASUREMENTTCSPC

Time-correlated single-photon counting (TCSPC)

Records times at which individual photons are detected by photo-multiplier tube (PMT) or an

avalanche photo diode (APD) after a single pulse. The recordings are repeated for additional

pulses

Histogram of the number of events across all of these recorded time points.

From TCSPC Technical note, PicoQuant

FLIM MEASUREMENTPHASE MODULATION

Phase modulation of excitation light source

(typically LED, laser AOTF)

𝜏𝜑= 𝜔−1. 𝑡𝑎𝑛Δ𝜑, 𝜏𝑚= 𝜔−1. 𝑀−2 − 1, where 𝜔 is the frequence

Advantage: camera-based detection fast lifetime image acquisition

possible

In acceptor

photobleaching, the

acceptor molecule of

the FRET pair is

bleached, resulting in

a brightening

(unquenching) of the

donor fluorescence.

Prebleach

Image

B

l

e

a

c

h

i

n

g

Postbleach

Image

Cy3

GF

PACCEPTOR PHOTOBLEACHING

Median

Filtering

Subtraction:

Postbleach – PrebleachDivision:

Subtraction/ Postbleach

An apparent FRET efficiency (product of the efficiency of the FRET

pair and the amount of interacting donor) can be calculated

ACCEPTOR PHOTOBLEACHING

𝐸𝐴 𝑖 = 1 −𝐹𝐷 𝑖

𝐹𝑏𝑙𝑒𝑎𝑐ℎ 𝑖𝐷 = 𝐸 ∙ 𝛼𝐷(𝑖)

CFP

only

YFP

only

CFP+

YFP

Channel 1:

460-500 nm

Channel 2:

520-570 nm

RGB

Overlay

CROSS-TALK AND CROSS-

EXCITATION

D

A

D

A

Ratiometric imaging can

only be done in samples

with a fixed stochiometry

of donor and acceptor

(e.g. Cameleons)

D

D

D

D

D

A

A

A

A

A

A

A

A

A

In samples with variable

stochiometries, the detected

acceptor fluorescence has to

be corrected for emission

cross-talk and for cross-excitation

SENSITIZED EMISSION

DETECTION

Donor channel

Donor excitation

FD

Acceptor channel

Donor excitation

FDA

Acceptor channel

Acceptor excitation

FA

Required images:

=>

FDA corr/FA

Predetermined factors with pure samples of donor and acceptor:

Donor cross-talk : RD

Acceptor cross-excitation : RE

SENSITIZED EMISSION DETECTION

Donor

cross-talk

correction

Acceptor

cross-excitation

correction

FDA corr/FA

SENSITIZED EMISSION DETECTION

Predetermined factors with pure samples of donor and acceptor:

Donor cross-talk : RD

Acceptor cross-excitation : RE

𝐸𝑎 =𝐹𝑐𝑜𝑟𝑟𝐷𝐴

𝐹𝐴

𝐹𝑐𝑜𝑟𝑟𝐷 = 𝐹𝐷𝐴 − 𝐹𝐷 ∙ 𝑅𝐷 − 𝐹𝐴 ∙ 𝑅𝐸

𝐸𝑎 = 𝐶 ∙ 𝐸 ∙ 𝛼𝐴

EPFL–SV–PTBIOP

FRET DETECTION

SUMMARY

• Fluorescence Lifetime Measurement

• Absolute Energy Transfer Efficiency• Dedicated Hardware needed• Complex Data Analysis

• Intensity based Measurements

• Sensitized Emission• Can be realized on wide-field and confocal systems.• Fast and suitable for live-cell imaging• Complex data analysis• Apparent energy transfer efficiency

• Acceptor Photobleaching• Can be realized on any confocal system• Easy data analysis• Potential Artefacts with fluorescent proteins

DYNAMIC CELLULAR PROCESSES

MOVEMENT OF PARTICLES

t=0 t=Dt

N=12 N=12

Transport/movement of particles from left to right and right to left

(steady-state)

N=12 N=12

No transport/movement of particles

MOVEMENT OF PARTICLES

t=0

N=12N=12

V=0 N=12N=12

V=0

No transport/movement of particles

t=Dt

MOVEMENT OF PARTICLES

t=0

N=12

V=12

N=12

V=0

N=12

V=6

N=12

V=6

Transport/movement of particles from left to right and right to left

t=Dt

Bastiaens and Pepperkok (2000), TIBS 25/12

FLUORESCENCE RECOVERY

AFTER PHOTOBLEACHING (FRAP)

1) Introduction

2) FRAP principles

3) FRAP data analysis

4) Related techniques (FLIP, FLAP,

Photoactivation, -conversion

5) Possible limitations

6) New technology developments

OVERVIEW

I: Pre-bleach

II: Bleach

III: Post-bleach

SCHEMATIC OF A FRAP

EXPERIMENT

1) Take a series of images before bleach (same

settings as after the bleach)

2) Apply short local bleach

3) Take images after bleach until the recovery in

the bleached area reaches a plateau

FRAP EXPERIMENT IN

PRACTICE

AOTF upregulation (0-100%):

Linear

Zoom In:

Exponential

2zoomfactor

Speed limitation due to

switching of the scanfield

INTENSITY OF BLEACHING LIGHT

Kappel and Eils, Leica App.Letter 2004

FRAP EXPERIMENTAL DATA

1) Background subtraction

2) Correction for photobleaching during the measurement (whole cell

or neighboring cell as reference)

3) Data normalization (alternative methods)

DATA PROCESSING

TYPICAL FRAP EXPERIMENT

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

-30 -10 10 30 50 70 90 110

time / s

no

rma

lize

din

ten

sity

TYPICAL FRAP EXPERIMENT

0

0.2

0.4

0.6

0.8

1

1.2

-10 10 30 50

time / s

no

rma

lize

din

ten

sity

time / s

no

rma

lize

din

ten

sity

Mobile

Fraction

Immobile

Fraction

Half Life

(t1/2)

FRAP PARAMETERS

( ) ( )teAtf t 1

A

1-A

Half Life

(t1/2)

½At

t

5.0ln

2/1

CURVE FITTING

1) Mobile and immobile fraction

2) Recovery half-time

Estimation of diffusion coefficient (Axelrod et al.)

w: bleach radius

Assumptions:

- bleached area is disk shaped

- diffusion occurs only in 2D

PARAMETERS OF EXPONENTIAL FIT

𝐷 = 0.88 ∙𝑤2

4 ∙ 𝑡1/2

BINDING REACTIONS

Binding

Site(s)

Diffusion

Binding

Un-

binding

Diffusion: Df

Binding: kon

Unbinding:koff

PURE DIFFUSION

0.00 0.25 0.50 0.75 1.00

0.0

0.2

0.4

0.6

0.8

1.0

no

rma

lize

d i

nte

ns

ity

time / s

w=0.5 m

w=1.0 m

w=2.0 m

w=4.0 m

𝑓 𝑥 = exp −𝜏𝐷2 ∙ 𝑡

∙ 𝐼0𝜏𝐷2 ∙ 𝑡

+ 𝐼1𝜏𝐷2 ∙ 𝑡

𝜏𝐷 =𝑤2

𝐷𝑓

• Circular bleach area

• Analytical Solution (Soumpasis)

• Recovery depends on bleach area

REACTION DOMINATED

0.00 0.25 0.50 0.75 1.00

0.0

0.2

0.4

0.6

0.8

1.0

no

rma

lize

d i

nte

ns

ity

time / s

koff

=16 s-1

koff

=16 s-1

koff

=16 s-1

koff

=16 s-1

𝑓 𝑥 = 𝐶𝑒𝑞𝑢 1 − exp −𝑘𝑜𝑓𝑓 ∙ 𝑡 𝐶𝑒𝑞𝑢 =𝑘𝑜𝑛

𝑘𝑜𝑛 + 𝑘𝑜𝑓𝑓

Lippincott-Schwartz et al. Nature CellBio Supp. 2003Phair and Mistelli, Nature Reviews MolCellBio, 2001

Multiple populations with differing diffusion rates => multi-component

equations

DIFFUSION VS. BINDING

Lippincott-Schwartz et al. Nature CellBio Supp. 2003

Potential explanation

e.g. immobile fraction, physical

separation

fixed samples, varition of the bleach

spot size

binding, active transport => modelling

photodamage

Problem:

Partial recovery:

Reversible photobleaching:

Non-diffusive behaviour:

Different values in consecutive

measurements:

POTENTIAL FRAP ARTEFACTS

Phair and Mistelli, Nature Reviews MolCellBio, 2001

FLUORESCENCE LOSS IN

PHOTOBLEACHING

Patterson and Lippincott-Schwartz (2002), Science 297:1873-1877

Irradiation at

405 nm

Excitation at 488 nm

PHOTOACTIVATION

PHOTOCONVERSION

Other popular

photomanipulatable Proteins:

Kaede

mEOS

DRONPA

But oligomerization especially

when expressed in cells is

often an issue.

PHOTOACTIVATIONPA-GFP

Patterson and Lippincott-Schwartz (2002), Science 297:1873-1877

FRAP OUTLOOK

Mueller F. et al. 2009

Curr. Opin. Cell Biology

Diffusion

Enzymatic Activity

Phase Fluctuations

Conformational Dynamics

Rotational Motion

Protein Folding

Example of processes that could generate fluctuations

FCS

FLUCTUATIONS ARE THE SIGNAL

EPFL–SV–PTBIOP

FLUORESCENCE CORRELATION

SPECTROSCOPY (FCS)

Schwille P. and Haustein E. 2007 Annu. Rev. Biophy. Biomol Struct.

http://www.cellmigration.org/resource/imaging/imaging_

approaches_correlation_microscopy.shtml

Sample Space

Observation

Volume

1. The Rate of Motion

2. The Concentration

of Particles

3. Changes in the Particle

Fluorescence while under

Observation, for example

conformational transitions

What is Observed?

GENERATING FLUCTUATIONS

BY MOTION

t1

t2

t3

t4

t5

0 5 10 15 20 25 30 3518.8

19.0

19.2

19.4

19.6

19.8

Time (s)

De

tect

ed

Inte

nsi

ty (

kcp

s)

10-9

10-7

10-5

10-3

10-1

0.0

0.1

0.2

0.3

0.4

Time(s)

G(t

)

G(t) F(t)F(t t)

F(t)2

G(0) 1/NAs time (tau) approaches 0

Diffusion

THE AUTOCORRELATION

FUNCTION

EPFL–SV–PTBIOP

FRAP & FCS SUMMARY

•FRAP and FCS can be used to investigate the movement (e.g. Diffusion) of cellular components.

•FRAP can be realized on every commercial microscope.

•FRAP requires overexpression of the component to investigate.

•Photobleaching is toxic!Better: Usage of photo-convertebale variants.

•FCS requires a dedicated microscope setup.

•FCS works with (very) low concentrations.

•Data analyis is complex for FRAP & FCS.