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
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (t ...
diffusion and protein binding. In addition, surface deposition of a fluorescing
phospholipid bilayer (or monolayer) allows the characterization of
hydrophilic
A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press.
In contrast, hydrophobes are n ...
(or
hydrophobic
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, ...
) surfaces in terms of surface structure and free energy.
Similar, though less well known, techniques have been developed to investigate the 3-dimensional
diffusion
Diffusion is the net movement of anything (for example, atoms, ions, molecules, energy) generally from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in Gibbs free energy or chemical ...
and
binding of molecules inside the cell; they are also referred to as FRAP.
Experimental setup
The basic apparatus comprises an
optical microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of micro ...
, a
light source and some
fluorescent
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
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
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The ...
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 10
5 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
Carl Friedrich Gauss (1777–1855) is the eponym of all of the topics listed below.
There are over 100 topics all named after this German mathematician and scientist, all in the fields of mathematics, physics, and astronomy. The English eponym ...
profile for the bleaching beam, the diffusion constant ''D'' can be simply calculated from:
:
where ''w'' is the radius of the beam and ''t
D'' is the "Characteristic" diffusion time.
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.
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.
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.
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.
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.
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
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
,
mitotic spindle,
nucleus
Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to:
* Atomic nucleus, the very dense central region of an atom
*Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA
Nucl ...
, or another cellular structure. 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. 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.
This could be due to flow of the cytoplasm or nucleoplasm, or transport along filaments in the cell such as
microtubules by
molecular motors
Molecular motors are natural (biological) or artificial molecular machines that are the essential agents of movement in living organisms. In general terms, a motor is a device that consumes energy in one form and converts it into motion or mecha ...
.
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
(which involves
modified Bessel function
Bessel functions, first defined by the mathematician Daniel Bernoulli and then generalized by Friedrich Bessel, are canonical solutions of Bessel's differential equation
x^2 \frac + x \frac + \left(x^2 - \alpha^2 \right)y = 0
for an arbitrary ...
s
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