Living up to Life
Introduction to FCS and FRAP
University of Edinburgh, November 2013
Paul McCormick
Leica Microsystems
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FRAP
Fluorescence Recovery After
Photobleaching
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Application
To measure molecular diffusion and active
processes in time.
Can be Fast or Slow processes – measured
in XY:
Movement and localization of
macromolecules in living cell (RNA and
protein dynamics in the nucleus, mobility of
macromolecular drugs).
Molecule trafficking in ER and Golgi
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FRAP Principle
Excitation
e.g 488 nm
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Bleached Area
beam
fluorescent
molecules
Movement of fluorescent
molecules into the
bleached
area (recovery)
Complete Recovery
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FRAP: Mode of operation
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1.
Determination of pre-bleach levels.
2.
Photobleaching (short excitation pulse) of selected cells / areas.
3.
Recovery: diffusion of unbleached molecules into the bleached area and increase
of fluorescence intensity.
Record the time course of fluorescence recovery at various time intervals, using a
light level sufficiently low to prevent further bleaching.
4.
Quantification: graph shows the time course of fluorescence recovery (calculated
as average percentage recovery of initial fluorescence).
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Experimental Set-up
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Each fluorophore has different photobleaching characteristics. For
FRAP experiments it is important to choose a dye which bleaches
minimally at low illumination power (to prevent photobleaching during
image acquisition) but bleaches fast and irreversibly at high
illumination power.
If molecules with rapid kinetics are investigated, advanced features
can be necessary for FRAP experiments :
•
higher laser power to bleach faster (to minimize diffusion during
bleaching)
•
time optimized FRAP modules (switching delays between bleach
and postbleach image aquisition should be minimized)
•
small formats and fast acquisition speed
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Experimental Set-up
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One general consideration in FRAP experiments is to minimize the bleaching during
acquisition instead of acquiring “nice” images. The data has to be averaged over the
selected area anyway to diminish statistical distributed noise.
To minimize photobleaching during acquisition these parameters should be
adjusted:
–
decreasing the pixel resolution by zooming out or by lowering the pixel
number (e.g. 128x128 instead of 512x512)
–
decreasing the pixel dwell time using a faster scan speed (this is also
preferable to monitor rapid recovery kinetics)
–
decreasing the laser power during image acquisition to a minimum
–
using fluorophores which are less susceptible to photobleaching at low
laser intensities
–
frame or line averaging should be avoided to reduce undesired
photobleaching in the imaging mode
–
opening the pinhole leads to a brighter signal with less laser power
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FRAP wizard
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Bleach tools of Leica for FRAP:
• ROI-Scan
• Fly Mode
• Zoom In ROI
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FRAP with LAS AF: Guided Steps
of Work
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FRAP with LAS AF: Guided Steps
of Work
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FRAP with LAS AF: Guided Steps
of Work
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FRAP with LAS AF: Guided Steps
of Work
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FRAP-wizard - Analysis of data
ROI based
ROI 4
ROI
3
ROI
1
ROI
2
FLIP slow
FLIP fast
FRAP
Reference
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FRAP: mobile fraction vs. immobile
fraction in ER
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Fluorescence recovery after photobleaching
A) Plot of fluorescence intensity in a region of interest versus time after photobleaching a fluorescent protein. The
prebleach (F i ) is compared with the recovery (F ∞) to calculate the mobile and immobile fractions. Information
from the recovery curve (from F o to F ∞) can be used to determine the diffusion constant of the fluorescent
protein.
B) Cells expressing VSVG–GFP were incubated at 40 °C to retain VSVG–GFP in the endoplasmic reticulum (ER)
under control conditions (top panel) or in the presence of tunicamycin (bottom panel).
Fluorescence recovery after photobleaching (FRAP) revealed that VSVG–GFP was highly mobile in ER
membranes at 40 °C but was immobilized in the presence of tunicamycin.
Lippincott-Schwartz, et al. - JUNE 2001 VOLUME 2 www.nature.com/reviews/molcellbio
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Data Analysis…
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For qualitative determination of the recovery dynamics, e.g. to compare
differences of one molecule at different conditions, a simple exponential equation
can be used as a first approximation:
• After determination of τ by fitting the above equation to the recovery curve the
corresponding halftime of the recovery can be calculated with the following formula:
• If the molecule binds to a slow or immobile macromolecular structure it is very
likely that the recovery curve does not fit a single exponential equation. To
overcome this problem, a biexponential equation can be used.
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FRAP wizards – how to go
faster….
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Possibilities to minimize delay of time between bleaching
and recovery:
• Reduce scan format in y 512 ≥ … ≥ 32 : flexible y formats
•
Use 1400Hz scan speed
•
Use bidirectional scan
•
Wizard minimizes automatically in time, additionally different time scales can
added for multistep kinetics (postbleach 2&3).
•
So e.g.1400Hz bidirectional scan with 256 square format results in 118
msec/frame.
•
Use FlyMode
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FLIP
Fluorescence Loss In
Photobleaching
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FLIP
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• In this photobleaching technique, loss of fluorescence rather
than fluorescence recovery is monitored.
• Fluorescence in one area of the cell is repeatedly bleached with
high laser power while images of the entire cell are collected
with low laser power.
• Using FLIP you can measure the dynamics of 2D or 3D
molecular mobility.
– e.g diffusion, transport or any other kind of movement of
fluorescently labeled molecules in living cells. The time course of
fluorescence loss is monitored here.
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FLIP: What’s around an ROI
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FLIP: Quantify Kinetics within
the ER
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Photoactivation – use the
FRAP wizard
Photoactivation is a photo-induced alteration of the
excitation or emission spectrum of a fluorophore (e.g.
fluorescent proteins).
PA-GFP:
Irradiation at ~400nm results in a 100x increase in
fluorescence when excited at 488nm (Patterson et al.,
2002, Science, 297:1873-77)
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Photoactivation – Principle of
PA-GFP
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FCS
Fluorescence Correlation Spectroscopy
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What is FCS?
Fluorescence Correlation Spectroscopy - FCS
-
fluorescence based measurement method
analyses the movement of single molecules into and out of a small illuminated
observation volume (focus of confocal SP5 – about 0.15-0.2 fl).
The movement of the molecules leads to fluctuations of fluorescence intensity that are
analyzed by statistical methods.
FCS read out parameter
• Mean Number of Molecules
• Diffusion times
• Fraction of components
•
Triplet and other dark states
=> Concentration
=> Molecule size, Viscosity
=> Bound/free ratio
=> Kinetic parameters of or chemical reactions
=> Equilibrium parameters
=> Inherent properties of molecules
=> Environmental parameters (pH, …)
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FCS data acquisition and analysis
1.
Beam park at position of
interest
=> Particles moving in and
out of confocal volume
2.
Registration of intensity
fluctuations
3.
Calculation of correlation
function
4.
Fit of corresponding
biophysical model to
correlation function
=> Get parameters
I(t)
<I>
t
G()
log 
29
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Kuschel
Calculation of autocorrelation
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Photons over time (photon mode data = time between photons)
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
Number of photons in time bin (time mode data)
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 1 1 4 4 4 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
 (0) = 20
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 1 2 4 4 2 1 0 0 0 0 0 0 1 1 1 0 0 0 0 0
 (1) = 17
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 2 2 4 2 2 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0
 (2) = 14
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 2 2 2 2 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
 (3) = 9
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 2 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 (4) = 5
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 (5) = 2
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 (6) = 1
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0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 (7) = 0
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Calculation of autocorrelation

correlation function
25
20
15
10
5
0
0
2
4
6
8
tau
G ( ) 
F (t )  F (t   )
F (t )
2
 Normalization of correlation function
 Logarithmic scale
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Results from FCS experiments
G()
1/N  1/c
corr  1/D
• Amplitude of fluctuations  concentration
• Curve shape  diffusion model
• Time of half maximal amplitude  Length of fluctuations
 diffusion coefficient of fluorescently labeled molecules
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log 
Theoretical approach
Properties of
the optical
system

I (r) = ...
Analytical
autocorrelation
function 
concentration,
brightness, diffusion
properties of up to 3
species
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Properties of
the diffusion
process
=
c (r,t) = ...
G()
log 
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Theoretical approach
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Properties of the
optical system
I (r) = ...
assuming that the product of the illumination PSF
and the detection PSF can be approximated as a 3D
Gaussian
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Theoretical approach
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Properties of the
diffusion process
c (r,t) = ...
solving the diffusion equation for different cases:
1D, 2D, 3D diffusion; anomalous/obstructed diffusion;
directed motion; confined diffusion; diffusion and binding;
intramolecular fluctuations
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Model application: Difference in diffusion
small molecules generate
short fluctuations...
I(t)
larger complexes generate
longer fluctuations...
I(t)
t
t
G()
... and rapidly
decaying
correlation
functions
log 
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... and slowly
decaying
correlation
functions
FCCS: Fluorescence cross correlation
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Extended concept:
• labeling of potential binding partners with spectrally different fluorophores
• register intensity fluctuations with two spectrally separated channels
• looking for correlations (similarities) between the corresponding signals
I(t)
No correlation
t
I(t)
Good correlation!
t
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Calculation of crosscorrelation
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
2 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 2 2 2 1 1
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
x
0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0
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Distinguish bound from unbound state


kas
+
kdis
G()
The higher the cross
correlation amplitude
in relation to the
autocorrelation
amplitudes, the higher
the degree of binding.
log 
47
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Kuschel
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Example: In vitro biochemistry
•
•
•
Reactants: Atto590-Biotin, Atto488anti-Biotin-IgG
Goals: Estimate bound fraction and Kd
from cross-correlation amplitude
Conditions:
–Ex: 488 nm, 594 nm
–Em1: 500-550 nm
–Em2: 607-683 nm
–sampling rate: 1 MHz
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IgG structure by Gareth White
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In vitro biochemistry
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Prepare experiment: Choice of dyes
for covalent labeling
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•
Criteria for suitable dyes:
– High photostability.
– Low triplet transition rate.
– Amino- and/or thiol-reactive derivatives should be available.
– Fluorescence lifetime within the lower ns-range (small against
diffusion time)
•
Excitation wavelength criterion: availability of laser line.
•
Emission wavelength criterion: avoid range of autofluorescence.
•
Avoid non-specific binding of the dye to buffer components (as BSA, detergents, ...), and the
interaction partners, especially the unlabelled partner. Hydrophobic dyes (as rhodamine) tend to
bind to chamber surfaces, membranes and proteins.
•
Regard dependence of photochemical properties of some dyes on measurement conditions as
pH, light intensity, …(like GFP depends on pH and light intensity).
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Prepare experiment: Choice of
dyes
List of FCS suitable dyes:
Alexa dyes
Cy dyes
Rhodamin Green, 6G, B, Lissamin
EvoBlue
DY dyes
TAMRA
ROX
TMR
Texas Red
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Molecular Probes
Amersham Pharmazia
Sigma, …
Evotec
Dyomics
- GFP
- YFP
- RFP (from Roger Tsien)
- Cross correlation pair: GFP-RFP
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Leica FCS: the optics
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FCS adaption for X1 port
avalanche photodiodes for
photon counting
TCS SP5 AOBS scanner
with X1 extension port
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Leica FCS Setup
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FCS control unit
filter block
FCS adaption
for X1 port
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Coverslip Correction
Not corrected
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Corrected
HCX PL APO 63x/1.2W Corr CS water immersion
lens with correction collar
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Leica FCS – FCS Wizard Overview
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Beam Park Calibration
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Measurement in solution –
Determine differences in diffusion time
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FAM-Labeled DNA,
27 bp,
D =153µs
Free dye:
Alexa488,
D = 33µs
Increase in mass => increase in diffusion time => right shift of the curve
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Measurement of EYFP in living
cells
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HeLa cells expressing pure EYFP
which is expected to be freely mobile
(Cells courtesy T. A. Knoch,
K. Rippe, German
Cancer Research Center and KIP,
University of Heidelberg)
FCS measurement spot
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Measurement of EYFP in living
cells
0.025
fit
0.02
0.015
0.01
0.005
1.00E-06
1.00E-05 1.00E-04
1.00E-03 1.00E-02
0
1.00E-01 1.00E+00
lag time [sec]
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autocorrelation function
meas.
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Results:
Autocorrelation function of
free EYFP in HeLa cell
nucleus:
• ~45 molecules in the focus,
concentration of ~60 nM for
focal size of 0.15 fl
• Diffusion correlation time
~500 sec, i.e., diffusion
coefficient of ~30 m2/sec,
i.e., viscosity ~3fold higher
than in water
Example: Measurement of EYFP in
living cells
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Diffusion in the nucleus
Molecules start at red spot:
Covered area after x seconds
0.0 sec
0.1 sec
0.2 sec
0.3 sec
0.4 sec
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Comparison between confocal imaging
and FCS
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CLSM
FCS
FCCS
PCH
Object of
investigation
Data acquisition
Moving or fixed
fluorescent molecules
x/y scanning laser beam
Moving fluorescent molecules
Data type
Image (grey values)
Light source
Detector
Laser
PMT, (APD)
Measurement at point of interest
(beampark)
Raw data: Counts over time;
Processed data: Correlation
function, histogram
Laser
APD
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Summary: the method
Observation of mobile, fluorescent particles
• Concentration range: >100 pM, <1 μM
• Dynamic range: >1 μsec, <1 sec
• Spatial resolution like the confocal microscope
• Sensitivity down to single molecule level
• Specificity
• Not imaging or scanning method
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Summary: applications
•
•
•
•
•
•
•
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Measurement of absolute concentrations at well-defined positions
(e.g. location and rate of expression, ...)
Binding studies:
reaction kinetics, equilibrium constants
Transport/diffusion
(active/passive, restricted/free, directed/non-directed)
Aggregation
Conformational changes, environmental sensor (e.g. pH)
Biophysical parameters
(e.g. membrane phases, viscosity, ... )
Mechanics/dynamics of cellular structures
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Thank you…..
Any questions?
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