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Introduction to Fluorescence Recoveryafter Photobleaching (FRAP)
Timo Zimmermann, Advanced Light Microscopy FacilityEuropean Molecular Biology Laboratory, Heidelberg
http://www.embl-heidelberg.de/almf/
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Overview
1) Introduction
2) FRAP principles
3) FRAP data analysis
4) Related techniques (FLIP, FLAP, Photoactivation, -conversion
5) Possible limitations
6) New technology developments
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λ2n (sin θ)Resolution limit R =
1 nm10-9m
1 µm10-6m
1 mm10-3m
1 cm10-2m
1 m1 Å10-10m
Cells Worm HouseflyOrganelles
LMlimit
Human
FRAPFCS
FRET
Molecular dynamics,molecular interactions
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Fluorescence Recovery after Photobleaching (FRAP)
Bastiaens and Pepperkok (2000), TIBS 25/12
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Timeline
1973: 1st application of the FRAP method (Poo and Cone)
1976: Mathematics for quantitative FRAP of focusedlaser spots in two dimensions (Axelrod et al.)
1996: Resurrection of FRAP using GFP and confocalmicroscopes (Cole et al., Lippincott-Schwartz..)
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Overview
1) Introduction
2) FRAP principles
3) FRAP data analysis
4) Related techniques (FLIP, FLAP, Photoactivation, -conversion
5) Possible limitations
6) New technology developments
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Schematic of a FRAP experimentI: Pre-bleach II: Bleach III: Post-bleach
Curve:K.Miura, Heidelberg
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Execution of a FRAP experiment
1) Take a series of images before bleach (samesettings as after the bleach)
2) Apply short local bleach
3) Take images after bleach until the recovery in thebleached area reaches a plateau
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Intensity of bleaching light
AOTF upregulation (0-100%):
Linear
Zoom In:
Exponential2zoomfactor
Speed limitation due to switching of the scanfield
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FRAP experimental data
Kappel and Eils, Leica App.Letter 2004
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Overview
1) Introduction
2) FRAP principles
3) FRAP data analysis
4) Related techniques (FLIP, FLAP, Photoactivation, -conversion
5) Possible limitations
6) New technology developments
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Correction of the experimental data
1) Background subtraction
2) Correction for photobleaching during themeasurement (whole cell or neighboring cell as reference)
3) Data normalization (alternative methods)
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Kappel and Eils, Leica App.Letter 2004
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The time constant and mobile / immobile fractions
Mobile Fraction
Immobile Fraction
Half Life (τ1/2)
In the FRAP curve, the immobile & mobile fraction can be measured by determining the plateau level.
Slide:K.Miura, Heidelberg
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Curve Fitting ( ) ( )teAtf τ−−= 1
A
1-A
Half Life (τ1/2)
½Aτ
τ−
=5.0ln
2/1
Half Life is the time when the recovery is the half of A, by definition.
Slide:K.Miura, Heidelberg
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Estimated parameters by exponential fit:
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
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Free diffusion vs. binding
Phair and Mistelli, Nature Reviews MolCellBio, 2001 Lippincott-Schwartz et al. Nature CellBio Supp. 2003
Multiple populations with differing diffusion rates => multi-component equations
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Possible FRAP artifacts
Lippincott-Schwartz et al. Nature CellBio Supp. 2003
Photo-induced immobile fraction
Problem: Potential explanation
Partial recovery: e.g. immobile fraction, physical separation
Reversible photobleaching: fixed samples, varition of the bleach spot size
Non-diffusive behaviour: binding, active transport => modelling
Different values in consecutivemeasurements: photodamage
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Overview
1) Introduction
2) FRAP principles
3) FRAP data analysis
4) Related techniques (FLIP, FLAP, Photoactivation, -conversion
5) Possible limitations
6) New technology developments
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Fluorescence Loss in Photobleaching (FLIP)
Phair and Mistelli, Nature Reviews MolCellBio, 2001
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Fluorescence Loacalisation After Photobleaching (FLAP)
based on Phair and Mistelli, Nature Reviews MolCellBio, 2001
CFPYFP
FLAP
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Photoactivatable GFP
Photoisomerization
Wavelength (nm)
GFP ExcitationGFP Emission
Lippincott-Schwartz et al. Nature CellBio Supp. 2003
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Photoactivatable GFP (PA-GFP)Excitation at 488 nm
Irradiation at405 nm
Patterson and Lippincott-Schwartz (2002), Science 297:1873-1877
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Kindling (KFP)
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488 nm
O2
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488 nm
O2
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543 nm
O2
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Spectral change after photoconversion
400 600500 700
0.5
1
Wavelength (nm)
Rel
ativ
e In
tens
ity
Ex Em
Ex
Em
after Elowitz et al. (1997), Curr. Biol. 7:809-812
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PhotoconvertedGFP
Cy5-labelledmicrotubules
Microtubule binding proteins (TPX2)
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Kaede
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Advantages of photoactivation
⇒Direct measurement of the Off-RateNegligible background
Off + OnBackground of unbound molecules
Off
FRAP
iFRAP, Photoactivation, Photoconversion
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Advantages of photoconversion
GFP channel
Photoconverted GFP channel
Binding measurements
[C]
⇒No background correctionHigh signal to background
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Overview
1) Introduction
2) FRAP principles
3) FRAP data analysis
4) Related techniques (FLIP, FLAP, Photoactivation, -conversion
5) Possible limitations
6) New technology developments
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Present limitations of quantitative FRAP analysis
- The experimental system does not correspond to a 2D diffusion model => 3D FRAP models have beendeveloped
- Diffusion during the bleach period is neglected, leading to underestimation of diffusion coefficients=> calculation models, technical solutions
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Intensity of bleaching light
AOTF upregulation (0-100%):
Linear
Zoom In:
Exponential2zoomfactor
Speed limited, does not workwith ‘Fly’-Mode
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Leica AOBS SP2
Available laser lines
Argon Laser
458 488 514 543 633405 476 496
Argon laser
100 mW => 500 mw
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Braga et al. Mol Biol Cell, 2004
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Olympus FluoView 1000
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Leica AOBS SP2‘Fly-back’ FRAP detection
=> readout within milliseconds of bleaching
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Renaissance of widefield microscopes with sensitive CCD cameras and laser bleaching modules(Deltavision RT Quantifiable Laser module)