Formation à la microscopie photonique plein-champ et confocale 2018
Dynamic and Molecular interactions experiments:
FRAP
FCS
FRET
Formation à la microscopie photonique plein-champ et confocale 2018
Co-
localisation
Structure
and Live imaging
Morphology
Fluorescence
Microscopy
Dynamic and
3D images
Molecular
interaction
...
Formation à la microscopie photonique plein-champ et confocale 2018
Dynamic Molecular Interaction
Dynamic and
FRAP FRET
Fluorescence Recovrey After Photobleaching Molecular Förster Resonnace Energy Transfert
interaction
Principle Principle
FRAP curve (raw data) FRET by intensity
Curve analysis (fitting) FRET by acceptor
Another technique: FLIP photobleaching
Equipment FRET by FLIM
Sum up + / - Equipment
Sum up+ / -
FCS
Fluorescence Correlation Spectroscopy
Principle
System and Calibration
Example
Formation à la microscopie photonique plein-champ et confocale 2018
FRAP
Fluorescence Recovrey After Photobleaching
Pre-bleach acquisition : n images with a t interval I
t
Bleaching on the region of interest I
t
I
Post-bleach acquisition : n images with a t’ interval
t
I
t
Formation à la microscopie photonique plein-champ et confocale 2018
FRAP Background Subtraction:
Fluorescence Recovrey After Photobleaching
Ifrapbackground(t) = Ifrap(t) - Ibackground(t)
Bleaching Correction :
(∑Icellprébackground) / nimageprébleach
Ifrapcorr(t) = * Ifrapbackground(t)
Icellbackground(t)
Normalisation :
Ifrapcorr(t) - Ibleachcorr
Ifrapnorm(t) =
Ifrapprécorr - Ibleachcorr
Ifrapprécorr(t) = (∑Ifrapprébackground) / nimageprébleach
Immobile
Fraction
Mobile
Fraction
Half lifetime t1/2
Formation à la microscopie photonique plein-champ et confocale 2018
FRAP
Fluorescence Recovrey After Photobleaching
Models :
2D free diffusion
f(t) = A * (1 - e-τt )
3D free diffusion (Ellenberg model )
Chemical interaction model
I(t)=If-(If-Ii)*e-koff*t
Interaction phenomenon :
Fitting :
• transit interactions in the environment
According to the size and the form of the ROI • free diffusion then binding
• diffusion and binding
According to the diffusion model (2D, 3D, free, binding. . .)
Etc . . .
% mobile and immobile fraction
Half lifetime dissociation constant koff
Diffusion Coefficient D
Formation à la microscopie photonique plein-champ et confocale 2018
FRAP
Fluorescence Recovrey After Photobleaching
- Important bleaching during acquisition data unusable
- Moving cells loss of FRAP région
- Fluorescence recovery of one part of fluorophores after bleaching
- Choose the fit model
- Limit of time resolution depending on acquisition time
Formation à la microscopie photonique plein-champ et confocale 2018
Derivative from FRAP
Fluorescence Recovrey After Photobleaching
FLIP : Fluorescence Loss in Photobleaching
t
Formation à la microscopie photonique plein-champ et confocale 2018
FRAP
Fluorescence Recovrey After Photobleaching
Equipments :
confocal
FRAP head implemented on a spinning-disk
multiphotonique system
widefield microscope with a FRAP module
lasers or diodes
equipments for living sample
Formation à la microscopie photonique plein-champ et confocale 2018
FCS
Fluorescence Correlation Spectroscopy
Principle of FCS and dcFCCS measurements, for details see Bacia et al., Nat. Methods 2006
Analysis of fluorescence signal fluctuation in a small volume
Formation à la microscopie photonique plein-champ et confocale 2018
FCS
Fluorescence Correlation Spectroscopy
Volume excitation :
• Fluorescent beads
• Reference molecule
Pinhole alignment :
Collect a maximum of fluorescence signal
http://www.usc.es/fotofqm/en/units/single-molecule-fluorescence/fluorescence-correlation-spectroscopy-fcs
Formation à la microscopie photonique plein-champ et confocale 2018
FCS
Fluorescence Correlation Spectroscopy
Data fitting (molecule behaviour) :
Diffusion coeffcient
Formation à la microscopie photonique plein-champ et confocale 2018
FCS
Fluorescence Correlation Spectroscopy
Oligomer or monomer in presence of Ca 2+ ?
http://http://biochimej.univ-angers.fr/Page2/COURS/3CoursdeBiochSTRUCT/9TraficVesiculaire/1TraficVesiculaire.htm
Formation à la microscopie photonique plein-champ et confocale 2018
FCS
Fluorescence Correlation Spectroscopy
• Protein behaviour
• Curve fitting with the two-component
translational diffusion model
Formation à la microscopie photonique plein-champ et confocale 2018
FRET
Förster Resonnace Energy Transfert
donor
acceptor
Formation à la microscopie photonique plein-champ et confocale 2018
FRET
Förster Resonnace Energy Transfert
Conditions :
Orientation factor κ
1 fluorophore = 1 e- absorption dipole + 1 e- emission dipole
Donor emission dipole orientation / Absorption acceptor dipole
Κ²=4 or Κ²=1 Κ²=0
r distance which separates 2 fluorophores < 100 Ǻ
R0 = r for 50% of efficiency FRET (E)
R0= [2.8 * 1017 * Κ² * QD * EA * J(λ)]1/6
E= FRET efficiency
Overlapping of donor emission spectrum on acceptor excitation spectrum
Formation à la microscopie photonique plein-champ et confocale 2018
FRET by intensity / spectral detection
Förster Resonnace Energy Transfert
Controls :
negative control = donor + acceptor co-localisation but no interaction
positive controle = donor + acceptor tandem ; interaction
Spectral bleedthrough :
acceptor in donor filter Dfa
E = Ff – (Df * %Ffd) – (Af * % Ffa)
donor/acceptor in the FRET filter Ffd / Ffa
donor in acceptor filter Afd
Dd Ad Fd
Donor only
Afd Ffd
Acceptor only Da Aa Fa
Dfa Ffa
FRET Df Af Ff
Donor Filter Acceptor Filter FRET Filter
Formation à la microscopie photonique plein-champ et confocale 2018
FRET by intensity / spectral detection
Förster Resonnace Energy Transfert
Ratiometric Measurement : FRET index
« low FRET » « high FRET »
Detection of donor and acceptor
emission spectra
480 nm -> 590 nm every 10 nm 480 nm -> 590 nm every 10 nm
Donor Intensity R1 Donor Intensity
R1 = Ratio I = R2 =
Acceptor Intensity R2 Acceptor Intensity
Formation à la microscopie photonique plein-champ et confocale 2018
FRET by acceptor photobleaching
Förster Resonnace Energy Transfert
1 donor image + 1 acceptor image Acceptor photobleaching 1acceptor image + 1donor image
Excitation laser for the acceptor
100%
Region of interest
I I
λ λ
FRET index =
Formation à la microscopie photonique plein-champ et confocale 2018
FRET by FLIM
Förster Resonnace Energy Transfert
Why measure the fluorescence lifetime ?
Deexcitation kinetic constant
k1 = kr + knr + kT
τD = 1 / k1
kDr kDnr kA Fluorescence lifetime
Deexcitation kinetic constant
k2 = kr + knr
donor acceptor τD = 1 / k2
Q= FRET quantum efficiency Fluorescence lifetime
τ= Fluorescence lifetime
Formation à la microscopie photonique plein-champ et confocale 2018
FRET par FLIM
Förster Resonnace Energy Transfert
FLIM : Fluorescence Lifetime Microscopy
Intensity Domain : Donor Exc Frequential Domain : Modular frequency
laser
Pulsed Laser
Donor Em m
φ
Detection : Detection :
• measure the intensity in each time window • Analysis of the demodulation m and the phase shift φ
• count each photon in time window • and calculation
Formation à la microscopie photonique plein-champ et confocale 2018
FRET par FLIM
Förster Resonnace Energy Transfert
How measure the fluorescence lifetime ?
Intensity Domain : Frequential Domain :
I ou N logI Data in frequential domain
kDA + kD
kD = knr + kr « Phasor »
kDA = kFRET + knr + kr
Δt Δt
Fluorescence curve decline
E=1- =1-
Donor bound proportion =
Formation à la microscopie photonique plein-champ et confocale 2018
FRET
Förster Resonnace Energy Transfert
Equipments : by intensity Equipments : by FLIM
•Ratiometric measurements •Intensity measurements in time window
widefield system with donneur / acceptor / FRET pulsed laser
dichroic cube system equipped with gated camera
confocal with spectral detection module system equipped with streak camera
• Acceptor photobleaching measurements • Single photon counting measurements
system equipped with FRAP module pulsed laser
confocal system equipped with APD or GaAsP or HyD
synchronized on the laser pulse
widefield system equipped with quadrant anode
• Measurements in frequential domain
system equipped with modular frequency laser
modular detector
Formation à la microscopie photonique plein-champ et confocale 2018
FRET
Förster Resonnace Energy Transfert
FRET by ratiometric measurement FRET by FLIM
• simple equipment • expensive equipment
• easy to perform • easy to perform
• a lot of controls conditions • measurements independing on concentration
• risk of cross-talk • efficiency measurement more rigorous
• measurement depending on molecules concentration
• artefact problem
FRET by photobleaching
• simple equipment
• easy to perform
• measurements depending on molecules concentration
• artefact problem
• fixed sample
Resultats Interpretation
• False positive
•False negative
