Fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) is a

method for determining the kinetics of diffusion through
tissue or cells. It is capable of quantifying the two
dimensional lateral diffusion of a molecularly thin film
containing fluorescently labeled probes, or to examine single
cells. This technique is very useful in biological studies of
cell membrane diffusion and protein binding. In addition,
surface deposition of a fluorescing phospholipid bilayer (or
monolayer) allows the characterization of hydrophilic (or
hydrophobic) surfaces in terms of surface structure and free
energy.

Similar, though less well known, techniques have been

developed to investigate the 3-dimensional diffusion and
binding of molecules inside the cell; they are also referred to
as FRAP.

Contents
Experimental Setup
Applications
Supported Lipid Bilayers
Protein Binding
Applications Outside the Membrane
Diffusion-limited fluorescence recovery
Principle of FRAP A) The bilayer is uniformly labeled
Reaction-limited recovery
with a fluorescent tag B) This label is selectively
Diffusion and reaction photobleached by a small (~30 micrometre) fast light
See also pulse C) The intensity within this bleached area is
References monitored as the bleached dye diffuses out and new
dye diffuses in D) Eventually uniform intensity is
restored

Experimental Setup
The basic apparatus comprises an optical microscope, a light source and some fluorescent probe. Fluorescent emission is
contingent upon absorption of a specific optical wavelength or color which restricts the choice of lamps. Most commonly, a broad
spectrum mercury or xenon source is used in conjunction with a color filter. The technique begins by saving a background image
of the sample before photobleaching. Next, the light source is focused onto a small patch of the viewable area either by switching
to a higher magnification microscope objective or with laser light of the appropriate wavelength. The fluorophores in this region
receive high intensity illumination which causes their fluorescence lifetime to quickly elapse (limited to roughly 105 photons
before extinction). Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot. As
Brownian motion proceeds, the still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes
in the bleached region. This diffusion proceeds in an ordered fashion, analytically determinable from the diffusion equation.
Assuming a Gaussian profile for the bleaching beam, the diffusion constant D can be simply calculated from:
where w is the radius of the beam and tD is the "Characteristic" diffusion time.[1][2]

Applications

Supported Lipid Bilayers

Originally, the FRAP technique was intended for use as a means to characterize the mobility of individual lipid molecules within
a cell membrane.[1] While providing great utility in this role, current research leans more toward investigation of artificial lipid
membranes. Supported by hydrophilic or hydrophobic substrates (to produce lipid bilayers or monolayers respectively) and
incorporating membrane proteins, these biomimetic structures are potentially useful as analytical devices for determining the
identity of unknown substances, understanding cellular transduction, and identifying ligand binding sites.

Protein Binding
This technique is commonly used in conjunction with green fluorescent protein (GFP) fusion proteins, where the studied protein
is fused to a GFP. When excited by a specific wavelength of light, the protein will fluoresce.[3] When the protein that is being
studied is produced with the GFP, then the fluorescence can be tracked. Photodestroying the GFP, and then watching the
repopulation into the bleached area can reveal information about protein interaction partners, organelle continuity and protein
trafficking.[4]

If after some time the fluorescence doesn't reach the initial level anymore, then some part of the fluorescence is caused by an
immobile fraction (that cannot be replenished by diffusion). Similarly, if the fluorescent proteins bind to static cell receptors, the
rate of recovery will be retarded by a factor related to the association and disassociation coefficients of binding. This observation
has most recently been exploited to investigate protein binding.[3][5][6] Similarly, if the GFP labeled protein is constitutively
incorporated into a larger complex, the dynamics of fluorescence recovery will be characterized by the diffusion of the larger
complex.[7]

Applications Outside the Membrane

FRAP can also be used to monitor proteins outside the membrane. After the protein of interest is made fluorescent, generally by
expression as a GFP fusion protein, a confocal microscope is used to photobleach and monitor a region of the cytoplasm,[3]
mitotic spindle, nucleus, or another cellular structure.[8] The mean fluorescence in the region can then be plotted versus time
since the photobleaching, and the resulting curve can yield kinetic coefficients, such as those for the protein's binding reactions
and/or the protein's diffusion coefficient in the medium where it is being monitored.[9] Often the only dynamics considered are
diffusion and binding/unbinding interactions, however, in principle proteins can also move via flow, i.e., undergo directed motion,
and this was recognized very early by Axelrod et al.[1] This could be due to flow of the cytoplasm or nucleoplasm, or transport
along filaments in the cell such as microtubules by molecular motors.

The analysis is most simple when the fluorescence recovery is limited by either the rate of diffusion into the bleached area or by
rate at which bleached proteins unbind from their binding sites within the bleached area, and are replaced by fluorescent protein.
Let us look at these two limits, for the common case of bleaching a GFP fusion protein in a living cell.

Diffusion-limited fluorescence recovery

For a circular bleach spot of radius and diffusion-dominated recovery, the fluorescence is described by an equation derived by
Soumpasis[10] (which involves modified Bessel functions and )

with the characteristic timescale for diffusion, and is the time. is the normalized fluorescence (goes to 1 as goes to
infinity). The diffusion timescale for a bleached spot of radius is , with D the diffusion coefficient.

Note that this is for an instantaneous bleach with a step function profile, i.e., the fraction of protein assumed to be bleached
instantaneously at time is , and , for is the distance from the centre of the
bleached area. It is also assumed that the recovery can be modelled by diffusion in two dimensions, that is also both uniform and
isotropic. In other words, that diffusion is occurring in a uniform medium so the effective diffusion constant D is the same
everywhere, and that the diffusion is isotropic, i.e., occurs at the same rate along all axes in the plane.

In practice, in a cell none of these assumptions will be strictly true.

1. Bleaching will not be instantaneous. Particularly if strong bleaching of a large area is required, bleaching may
take a significant fraction of the diffusion timescale . Then a significant fraction of the bleached protein will
diffuse out of the bleached region actually during bleaching. Failing to take account of this will introduce a
significant error into D.[11][12][13]
2. The bleached profile will not be a radial step function. If the bleached spot is effectively a single pixel then the
bleaching as a function of position will typically be diffraction limited and determined by the optics of the confocal
laser scanning microscope used. This is not a radial step function and also varies along the axis perpendicular to
the plane.
3. Cells are of course three-dimensional not two-dimensional, as is the bleached volume. Neglecting diffusion out of
the plane (we take this to be the xy plane) will be a reasonable approximation only if the fluorescence recovers
predominantly via diffusion in this plane. This will be true, for example, if a cylindrical volume is bleached with the
axis of the cylinder along the z axis and with this cylindrical volume going through the entire height of the cell.
Then diffusion along the z axis does not cause fluorescence recovery as all protein is bleached uniformly along
the z axis, and so neglecting it, as Soumpasis' equation does, is harmless. However, if diffusion along the z axis
does contribute to fluorescence recovery then it must be accounted for.
4. There is no reason to expect the cell cytoplasm or nucleoplasm to be completely spatially uniform or isotropic.
Thus, the equation of Soumpasis is just a useful approximation, that can be used when the assumptions listed above are good
approximations to the true situation, and when the recovery of fluorescence is indeed limited by the timescale of diffusion .
Note that just because the Soumpasis can be fitted adequately to data does not necessarily imply that the assumptions are true and
that diffusion dominates recovery.

Reaction-limited recovery
The equation describing the fluorescence as a function of time is particularly simple in another limit. If a large number of proteins
bind to sites in a small volume such that there the fluorescence signal is dominated by the signal from bound proteins, and if this
binding is all in a single state with an off rate koff, then the fluorescence as a function of time is given by[14]

Note that the recovery depends on the rate constant for unbinding, koff, only. It does not depend on the on rate for binding.
Although it does depend on a number of assumptions[14]

1. The on rate must be sufficiently large in order for the local concentration of bound protein to greatly exceed the
local concentration of free protein, and so allow us to neglect the contribution to f of the free protein.
2. The reaction is a simple bimolecular reaction, where the protein binds to localised sites that do not move
significantly during recovery
3. Exchange is much slower than diffusion (or whatever transport mechanism is responsible for mobility), as only
then does the diffusing fraction recovery rapidly and then acts as the source of fluorescent protein that binds and
 replaces the bound bleached protein and so increases the fluorescence. With r the radius of the bleached spot,
this means that the equation is only valid if the bound lifetime .

If all these assumptions are satisfied, then fitting an exponential to the recovery curve will give the off rate constant, koff.
However, other dynamics can give recovery curves similar to exponentials, so fitting an exponential does not necessarily imply
that recovery is dominated by a simple bimolecular reaction. One way to distinguish between recovery with a rate determined by
unbinding and recovery that is limited by diffusion, is to note that the recovery rate for unbinding-limited recovery is independent
of the size of the bleached area r, while it scales as , for diffusion-limited recovery. Thus if a small and a large area are
bleached, if recovery is limited by unbinding then the recovery rates will be the same for the two sizes of bleached area, whereas
if recovery is limited by diffusion then it will be much slower for the larger bleached area.

Diffusion and reaction

In general, the recovery of fluorescence will not be dominated by either simple isotropic diffusion, or by a single simple
unbinding rate. There will be both diffusion and binding, and indeed the diffusion constant may not be uniform in space, and
there may be more than one type of binding sites, and these sites may also have a non-uniform distribution in space. Flow
processes may also be important. This more complex behavior implies that a model with several parameters is required to
describe the data; models with only either a single diffusion constant D or a single off rate constant, koff, are inadequate.

There are models with both diffusion and reaction.[2] Unfortunately, a single FRAP curve may provide insufficient evidence to
reliably and uniquely fit (possibly noisy) experimental data. Sadegh Zadeh et al. [15] have shown that FRAP curves can be fitted
by different pairs of values of the diffusion constant and the on-rate constant, or, in other words, that fits to the FRAP are not
unique. This is in three-parameter (on-rate constant, off-rate constant and diffusion constant) fits. Fits that are not unique, are not
generally useful.

Thus for models with a number of parameters, a single FRAP experiment may be insufficient to estimate all the model
parameters. Then more data is required, e.g., by bleaching areas of different sizes,[13] determining some model parameters
independently, etc.

See also
Fluorescence microscope
Photobleaching
Fluorescence loss in photobleaching (FLIP)

References
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