Transcript Slide 1

The method of fluorescence recovery after photobleaching (FRAP) utilizes the
phenomenon of photobleaching of fluorescent probes to measure parameters related
to molecule mobility. It was initially designed by Axelrod and coworkers to measure
two-dimensional diffusion of membrane-bound molecules (see applications of
photobleaching techniques for further application details ).
Frequently photobleaching interferes with image acquisition in fluorescence
microscopy by fading the fluorescent probes, resulting in a lower signal/noise ratio.
However in FRAP experiments, it is used to selectively photobleach a specific
area by high intensity laser pulses. Subsequently the kinetics of fluorescence
recovery are recorded by sampling images at regular time intervals with low intensity
illumination.
Closely related to FRAP is fluorescence loss in photobleaching (FLIP). In FLIP
experiments a specified region of the cell is repetitively photobleached and the
loss of fluorescence in non-bleached parts of the cell is measured.
Another variant of photobleaching techniques is iFRAP (inverse FRAP) where all the
fluorescent molecules in a cell except for a small region are bleached . The loss
of fluorescence from the unbleached region in the postbleach images is then
analyzed. With this technique qualitative information about mobility and equilibration
time can be gained. Because of the time needed to bleach large areas, this method is
especially suited to analyze the dissociation parameters of molecules which are
bound to an immobile structure for several seconds.
How FRAP works
In unbleached cells fluorescent molecules are in
equilibrium. Bleaching a fraction of the total
fluorescent molecules in a region of interest
(ROI) disturbs this equilibrium. Under optimal
conditions the recovery kinetics are dependent
only on the mobility (effective diffusion
coefficient and binding kinetics to
macromolecular structures) of the investigated
molecule.
Thus by measuring the recovery kinetics,
following properties of the molecule can be
characterized:
•the ratio between mobile and immobile
fraction
•the effective diffusion coefficient D
•the binding time (as well as
assembly/disassembly) of proteins to
macromolecular structures
•continuity of intracellular organelles (FLIP)
•formation of protein complexes (resulting in
lower D )
eff
eff
Photobleaching
Absorbtion of light by a fluorophore elevates electrons
from the ground state to an excited singlet state.
Fluorescence arises from the emission of a photon
during
relaxation
to
the
ground
state.
The exact mechanism of photobleaching is not known,
but it is assumed to be linked to a transition from the
excited singlet state to the excited triplet state. The
excited triplet state is relatively long-lived and is
chemically more reactive.
Each fluorophore has different photobleachingcharacteristics. 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.
According to the Stokes-Einstein equation
the diffusion coefficient D for a particle in a free volume depends on the Boltzmann constant (k),
the absolute temperature (T), the viscosity of the solution (h), and the hydrodynamic radius (R) of
the particle.
The mobility of a molecule in the cellular environment is affected by the following
parameters:
•The size of the molecule:an eightfold increase of the size of a soluble sperical protein decreases
D by factor 2.
•the viscosity of the cellular environment: e.g. membranes have a much higher viscosity than
cytoplasm
•protein-protein-interactions and binding to macromolecules can also slow down the
diffusion
•if flow or active transport is involved in the movement of the probed molecule, the measured
movement rate can become significantly higher than the theoretical diffusion rate
The diffusion coefficient D of the probed molecule can be measured via the halftime of the recovery
(t ). This coefficient is influenced by the above stated parameters. Therefore this value is often
termed as the effective diffusion coefficient (or apparent diffusion coefficient) Deff (in µm2 s-1)
and reflects the mean squared displacement explored by the proteins through a random walk over
time.
Changes of the effective diffusion coefficient, e.g. due to binding to a larger molecule, can be
exploited to study the function of the protein of interest. For instance the endonuclease ERCC1/XPF
is binding to DNA when damages are induced by UV light resulting in a decreased D (Houtsmuller
et al. 1999).
By performing the FRAP experiment at different temperatures (e.g. 27 and 37 °C) it is possible to
determine if energy-dependent processes are involved in the mobility of the investigated molecule.
While the difference in molecular diffusion due to a 10 K change in absolute temperature is too
small to be resolved by FRAP (D decreases only ~3%, Phair & Misteli 2000), energy-dependent
processes are more sensitive to temperature (Hoogstraten et al. 2002).
half
eff
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 t 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.
Depending on the investigated molecule the amount of
interaction with other molecules will be variable. For
example proteins which associate with relatively
immobile cellular structures such as the cytoskeleton
have a significantly reduced recovery compared to a
freely mobile molecule.
Using kinetic modeling the binding characteristics of the
examined molecule can determined by the ratio
between mobile and immobile fraction.
An idealized plot of a FRAP recovery curve.
II: initial intensity
I0: intensity at timepoint t0 (first postbleach intensity)
I1/2: half recovered intensity (I1/2 = (IE - I0) / 2)
IE: endvalue of the recovered intensity
thalf: Halftime of recovery corresponding to I1/2 (t1/2 - t0)
Mobile fraction Fm = (IE - I0) / (II - I0)
Immobile fraction Fi = 1 - Fm
The optimal fluorophore should be bright and stable under
low intensity illumination during image acquisition (preand postbleach). Under high intensity illumination it
should bleach fast and irriversibly. Another prerequisite is
that the used fluorophore must not inhibit the function of
the investigated molecule.
Fluorophores for FRAP-experiments (according to Ellenberg and
Rabut):
fluorophore
comment
fluorescein and
derivatives
probably the most used chemical fluorophore,
tends to bleach too easily and photoleaching is
partly reversible (Periasamy 1996)
fluorescent proteins
mostly irreversible photobleaching
EGFP tends to multimerize at high concentrations
Regardless of fluorophore, it is important to test it´s
photobleaching characteristics under the imaging
condition used.
The “classical approach” for FRAP measurements utilizes a widefield epifluorescence microscope to monitor the fluorescence intensity during pre- and
postbleach acquisition. For bleaching a laser beam is focused onto a small
diffraction limited spot (~1µm) in the region of interest. These systems are not
‘of the shelf’ and have to be custom-build.
In principal the standard commercially available confocal laser-scanning
microscope (CLSM) equipped with acousto-optical tuneable filter (AOTF) is
suitable for FRAP, iFRAP and FLIP experiments. The AOTF is needed to
switch the laser power rapidly between the low intensity imaging and the high
intensity bleaching modes.
The higher the diffusion constant of the investigated molecule, the more
imaging speed and laser power is required. Read more about useful confocal
equipment for FRAP experiments.
Standard features of typical CLSM necessary for FRAP experiments:
AOTF to attenuate the laser power between high intensity illumination for bleaching
and low intensity for imaging mode
Interactive definition of the bleaching area by regions of interest (ROI) or spots
including saving parameters of the ROI(s) or spot(s) for evaluation
The software should be as flexible as possible to define prebleach-, bleach- and
postbleach-settings either by special routines or by macro-programming, e.g.:
number and frequency of frames
scan speed and zoom factor
laser intensity (AOTF settings)
Depending on the required resolution and the desired depth of bleaching, different
objectives are suitable for FRAP experiments.
For homogenous bleaching in z-direction objectives with a lower magnification like
a 20x 0.7 NA lens are suitable, but result in a lower resolution.
Higher resolution with increased light intensity in the focal plane but incomplete
bleaching in z-direction can be achieved using lenses with a high numerical
aperture like a 63x 1,4NA objective. The thinner the structure subjected to
bleaching the higher the NA should be.
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
Long-term FRAP-experiments which explore molecules with very slow recovery
characteristics also requires additional properties of the imaging system:
To correct for laser-fluctuations the laser intensity can be quantified by ‘monitor
diodes’ or transmission images.
Tracking of the mobile photobleached cells using autofocus routines
There is no universal protocol for FRAP experiments
since the design of a FRAP experiment always has to
take into account the bleaching and recovery
characteristics of the molecule under investigation. The
visualized cells should not be affected by high
illumination conditions.
Therefore the optimal conditions have to be empirically
tested for each biological system.
The information in this section intends to provide general
rules and hints applicable to most photobleaching
experiments.
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.
Preconditions
Before meaningful FRAP data can be measured the following experiments should
be carried out:
First when using fluorescent protein constructs the cells should be checked for
physiological and morphological alterations compared to control cells. Only
cells which are ‘normal’ should be used for further analysis by FRAP.
Bleaching characteristics have to be checked in fixed cells to determine bleach
rates without movement of the probed molecule
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
The imaging in a FRAP experiment generally consists of 3 steps:
The sample is first imaged at low intensity illumination in a prebleach series
(usually about 10 images) to measure the fluorsecence equilibrium before
disturbance.
The second step is to bleach one or more spots or regions of interest (ROI)
with high intensity illumination to disturb the fluorescence equilibrium.
In the last imaging step a series of postbleach images is acquired to record the
fluorescence recovery kinetics.
Finally several data evaluation steps reveal mobility-related parameters of the
molecule of interest.
The prebleach series is used to:
determine the (total) fluorescence intensity at low intensity illumination prior to
bleaching to provide a reference point for fluorescence recovery; typically 310 images.
(If fluorescent proteins (FP) are imaged with more than 1 image/s a prebleach
series of 50-100 images is needed to reach a steady state of FPs in dark states
(Weber et al. 1999)).
calculate the fluorescence loss due to acquisition photobleaching
To prevent artefacts from pixel saturation, the maximum intensity (255 in 8-bit
images) should be reached only in very few pixels. The offset should be
adjusted that the background pixels show grayvalues slightly above zero
(otherwise information can be lost). To increase the dynamic range it can be
advantageous to employ the 12-bit mode.
In the pre- and postbleach-series laser intensity should be attenuated as low as
possible to get a sufficient signal - for FRAP experiments minimized acquisition
photobleaching is more important than ‘nice images’.
In the data evaluation step the fluorescence intensity during recovery will be
normalized with the prebleach values.
In the bleaching step one or more spot(s) or region(s) of
interest (ROI) are irradiated with high intensity
illumination.
Ideally the bleaching event should be instantaneous, in
practice it should not exceed a tenth of the half time of the
recovery. Therefore for analysing rapid kinetics, more
powerful lasers as well as time optimized acquisition
routines are essential.
Parameters that influence the bleaching process:
•Laser power: More laser power enables faster bleaching
but also can harm the cells.
•Zoom: Zooming in increases the effective irradiation of
the scanned area. Thus zooming in speeds up the
bleaching, but the response time for switching back to the
unzoomed imaging mode can delay the acquisition of the
postbleach series. Which is especially undesirable when
analysing rapid kinetics.
•Scan speed: The slower the scan speed the more energy
is radiated (longer pixel dwell time)
It is important to calibrate the bleached volume for each set
of parameters (laser power, objective, zoom, speed, etc.)
which is best done using fixed samples. A more precise
definition of the bleached volume along the optical axis
can be achieved using two-photon excitation.
The postbleach series monitors the dynamics and
extent of the fluorescence recovery. The following
hints help to improve the accuracy of the recovery
detection:
•The acquisition frequency should be adjusted to
resolve the dynamic range of the recovery with
good temporal resolution (rule of thumb: at least
20 data points during the time required for the half
of the recovery).
•Acquisition photobleaching should be minimized
to record the recovery dynamics as precisly as
possible.
•The ideal postbleach acquisition duration is 10 to
50 times longer than the halftime (Axelrod 1976). In
practice initial experiments should be conducted until no
noticeable further increase in fluorescence intensity is
detected.
•When using FPs the imaging frequency should not
be altered during an experiment because the
fraction of FPs driven into dark states could be
altered complicating the analysis of the data.
Depending on the experiment there are several data evaluation steps
which have to be carried out before meaningful results can be achieved:
1.Alignment of the images (only necessary if the regions of
interest moved over time).
2.Fluorescence intensity quantification (obtaining the raw data)
3.Background subtraction
4.Correct for laser fluctuations, photobleaching during acquisition
(postbleach) and total fluorescence loss caused by the bleaching
step
5.Normalization
6.Mobile/immobile fraction
7.T½ halftime of the equilibration of bleached and unbleached
molecules
8.Theoretical models to additionally determine binding
characteristics of the analysed molecule
Image Alignment
During longer FRAP experiments, e.g. analyzing very slow molecules or molecules
which bind to immobile structures, the bleached ROI can move over time. In order
to obtain the right data in these cases it is essential to correct for this
movement by alignment prior to the intensity quantification. Image Alignment
can be done with ImageJ TurboReg (freeware) or the commercial tool Autoaligner
(Bitplane AG, Switzerland).
Obtaining the ‘raw data’
To determine the “raw FRAP data” the total or average pixel values in the
bleached ROI has to be determined for each timepoint. This can be done
with the most confocal operating software (e.g. Zeiss LSM, Leica LCS) or
with other image processing software which can handle time series (e.g.
the freeware ImageJ).
Background subtraction
The image brightness not only originates from fluorescence of the fluorescently labelled
molecules of interest. For example detector readout noise, autofluorescence (medium,
glass...), and reflected light contribute to the total detected intensity. Therefore the
average background value (background measurement in an area outside the cell)
should be subtracted from the average pixel value in the bleaching region for
each timestep.
The background quantification can be carried out with the confocal operating software
(e.g. Zeiss LSM, Leica LCS) or with other image processing software which can handle
time series (e.g. the freeware ImageJ).
Further necessary corrections
Laser fluctuations, acquisition photobleaching, and fluorescence loss during
photobleaching leads to intensity changes during image acquisition. In order to
obtain data with a linear relationship between the measured fluorescence
intensity and the concentration of fluorescent molecules, the raw data has to be
corrected for these changes. One straightforward possibility to do so is to
divide the background subtracted fluorescent measurement by the total cell
intensity at each time point.
If this is not possible, e.g. when only a part of the cell can be imaged, alternative
correction methods are available:
Acquisition photobleaching can be corrected for by measurement of the
fluorescence intensity of neighboring cells, in control experiments or the
prebleach series. The fluorescence measurement at each timepoint can be
divided by a function representing the acquisition photobleaching: y(n) = exp(n/x) with n = image number can be easily determined by measuring the total
fluorescence intensity of an unbleached neighboring cell or the gradual
fluorescence decrease in the prebleach or postbleach images.
Laser intensity fluctuations can be compensated for by dividing the
fluorescence measurement at each timepoint by the corresponding value of the
laser monitor diode or the averaged intensity of the transmission channel
outside the cell (corrected for the nonzero offset of the diode or transmission
detector, respectively).
Normalization
To compare different experiments usually the fluorescence intensity of the average
prebleach intensity is normalized to one by dividing the intensity of all timepoints by
the average prebleach intensity. This can be easily done with common spreadsheet
programs.
It is also possible to normalize to numbers of fluorophores by using fluorophore
calibration standards (Ellenberg and Rabut).
Determination of the Mobile / Immobile Fraction
If the whole population of the investigated molecule is freely mobile the fluorescence
intensity (background subtracted and corrected for loss of fluorescence due to the
bleaching pulse) recovery curve should reach a plateau at 100% of the initial
fluorescence of the prebleach. Binding of a fraction of the molecules of interest to slow
or immobile structures (e.g. Nuclear envelope) reduces the recovered level of the
fluorescence, the fractions can be calculated with the following equations.
Mobile fraction Fm = (IE - I0) / (II - I0)
Immobile fraction Fi = 1 - Fm
With: IE: Endvalue of the recovered fluorescence intensity
I0: first postbleach fluorescence intensity
II: Initial (prebleach) fluorescence intensity
An additional method to measure the mobile and immobile fractions exemplified
for the nucleus is described by Houtsmuller 2001. A spot in the compartment of
interest is bleached over an extended period of time with relatively low laser intensity.
During this extended bleaching time a large percentage of mobile molecules passes
through the bleaching spot and partially will be bleached. Subsequently, the mobile
molecules are allowed to completely redistribute through the nucleus (depending on
their diffusion coefficient). The ratio of fluorescence intensity of confocal images before
and after this procedure is then plotted against distance to the laser spot. To accurately
calculate the immobile fraction from this plot one should obtain two reference curves,
representing the situations in which all molecules are immobile (fixed sample) and in
which all molecules are mobile (e.g. in an inducible system).
Determination of the halftime of the recovery (thalf)
The halftime (thalf ) of recovery is the time from the bleach to the
timepoint where the fluorescence intensity reaches the half (I1/2) of the
final recovered intensity (IE).
Fitting the recovery data to an exponential equation can be used
to determine t :
If the investigated molecule freely diffuses in the cell or
compartment a simple exponential formula should be used:
half
Where A is the endvalue of the recovered intensity (I ), t is the fitted
parameter and t is the time after the bleaching pulse.
After determination of t by fitting the above equation to the recovery curve
the corresponding halftime of the recovery can be calculated with the
following formula:
E
If the molecule binds to slow or immobile macromolecular structures or
the diffusion is partially hindered it is very likely that the recovery curve
cannot fit properly by a single exponential equation. The use of a
biexponential equation can often overcome this problem.
To compare the halftimes of a molecule under different
experimental conditions (e.g. during interphase and mitosis) it
is essential to use bleaching regions with the same size, relative
position in the cell and scanning parameters.
An idealized plot of a FRAP
recovery curve.
II: initial intensity
I0: intensity at timepoint t0 (first
postbleach intensity)
I1/2: half recovered intensity
corresponding to t1/2
(I1/2 = (IE - I0) / 2)
IE: endvalue of the recovered
intensity
thalf: Halftime of recovery (t1/2
- t0)
Mobile fraction Fm = (IE - I0) / (II
- I0)
Immobile fraction Fi = 1 - Fm
Kinetic modelling
FRAP experiments contain information about the diffusional properties of the studied
molecule, but also about it’s binding characteristics. Using kinetic modeling and
computer simulation, this information can be extracted and hypotheses can be evaluated
in comparison with experimental data (Phair 2001).
The lifetime of different molecular states (e.g. freely diffusing or bound) can be simulated
by calculating their rates of formation and decay (e.g. kon and koff). Two kinds of kinetic
models are used by cell biologists for this purpose:
Compartmental model:
Biological processes are described by a finite number of compartments that
contain a chemical species at a cellular location. Each compartment is defined as
“well mixed” (the dynamics are not diffusion limited). The crossover of molecules
between compartments represents exchange processes between different places
(e.g. cytoplasm and ER), chemical states (e.g. phosphorylation) or chemical
interactions (e.g. binding to receptormolecules).
The transfer rates can be determined by fitting to best match the experimental
data. Time is the only variable in a compartmental model.
Spatial model:
The cell is divided into a number of spatial elements. In each element the
investigated molecule can transfer between different molecular states.
Additionally these different molecular states can exchange with neighboring
elements by diffusion.
Programs for kinetic modelling
Program
Link
Comment
SAAM II
www.saam.com
Easy
compartmental
model setup
Berkeley
Madonna
www.berkeleymado compartmental
nna.com
model
Mathlab
www.mathworks.co Good graphic
m
output
possibilities
Gepasi
www.gepasi.org
Spatial model
Freeware
Virtual Cell
www.nrcam.uchc.e
du
Easy way to
write spatial
models
freeware
(academical
use)
WinSAAM
www.winsaam.com
freeware