Fluorescence recovery after photobleachingPhotobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
(FRAP) denotes an optical technique 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 membraneThe cell membrane is the biological membrane separating the interior of a cell from the outside environment....
diffusion and protein binding. In addition, surface deposition of a fluorescing
phospholipidPhospholipids are a class of lipids and are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...
bilayer (or monolayer) allows the characterization of
hydrophilicHydrophile, from the Greek "water" and φιλια "friendship," refers to a physical property of a molecule that can transiently bond with water through hydrogen bonding. This is thermodynamically favorable, and makes these molecules soluble not only in water, but also in other polar solvents...
(or
hydrophobicIn chemistry, hydrophobicity is the physical property of a molecule that is repelled from a mass of water....
) surfaces in terms of surface structure and free energy.
Similar, though less well known, techniques have been developed to investigate the 3-dimensional
diffusionMolecular diffusion, often called simply diffusion, is a net transport of molecules from a region of higher concentration to one of lower concentration by random molecular motion. The result of diffusion is a gradual mixing of material...
and
bindingBinding may refer to:* Binding , a property relating to anaphors and c-command* Legally binding, in lawJoining physical objects together:* Foot binding* Ski binding, a device for connecting a foot to a ski...
of molecules inside the cell; they are also referred to as FRAP.
Experimental Setup
The basic apparatus comprises an
optical microscopeThe optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes...
, a
light source and some fluorescent probe.
Fluorescent emissionFluorescence is the emission of visible light by a substance that has absorbed light of a differing, usually invisible, wavelength. Absorption of a photon triggers the emission of a photon with a longer wavelength. A shorter wavelength emission is sometimes observed from multiple photon absorption...
is contingent upon absorption of a specific optical wavelength or color which restricts the choice of lamps. Most commonly, a broad
spectrumA spectrum is a condition that is not limited to a specific set of values but can vary infinitely within a continuum. The word saw its first scientific use within the field of optics to describe the rainbow of colors in visible light when separated using a prism; it has since been applied by...
mercuryMercury , also called quicksilver or hydrargyrum , is a chemical element with the symbol Hg and atomic number 80...
or
xenonXenon is a chemical element represented by the symbol Xe. Its atomic number is 54. A colorless, heavy, odorless noble gas, xenon occurs in the Earth's atmosphere in trace amounts...
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
laserA laser is a device that emits light through a process called stimulated emission. Laser light is usually spatially coherent, which means that the light either is emitted in a narrow, low-divergence beam, or can be converted into one with the help of optical components such as lenses...
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 equationThe diffusion equation is a partial differential equation which describes density fluctuations in a material undergoing diffusion. It is also used to describe processes exhibiting diffusive-like behaviour, for instance the 'diffusion' of alleles in a population in population genetics.The equation...
. Assuming a
GaussianIn optics, a Gaussian beam is a beam of electromagnetic radiation whose transverse electric field and intensity distributions are described by Gaussian functions. Many lasers emit beams with a Gaussian profile, in which case the laser is said to be operating on the fundamental transverse mode, or...
profile for the bleaching beam, the diffusion constant
D can be simply calculated from:
where
w is the width of the beam and
t1/2 is the time required for the bleach spot to recover half of its initial intensity.
Supported Lipid Bilayers
Originally, the FRAP technique was intended for use as a mean 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 proteinThe green fluorescent protein is protein composed of 238 amino acids , which exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish...
(GFP)
fusion proteinFusion proteins, AKA chimeric proteins, are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
s, 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.
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
cytoplasmThe cytoplasm is the part of a cell that is enclosed within the cell membrane. In eukaryotic cells, the cytoplasm contains organelles, such as mitochondria, which are filled with liquid that is kept separate from the rest of the cytoplasm by biological membranes. The contents of the cell nucleus...
,
mitotic spindleIn cell biology, the spindle apparatus is the structure that separates the chromosomes into the daughter cells during cell division. It is part of the cytoskeleton in eukaryotic cells...
,
nucleusIn cell biology, the nucleus , also sometimes referred to as the "control center", is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as...
, 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 for the protein's binding reactions and/or the protein's diffusion coefficient in the medium where it is being monitored. The analysis is most simple when the curve is dominated by only the diffusional or only the binding components. For a circular bleach spot and diffusion-dominated recovery, the fluorescence is described by the Soumpasis equation and involves modified Bessel functions:
where
h=r
2/(2*D
f*t);
r=radius of bleach spot;
t=time;
Df=diffusion coefficient;
f(t) is the normalized fluorescence (goes to 1 as t goes to infinity).
For a binding-dominated reaction, in which the diffusion is much faster than the unbinding of the bleached protein and subsequent binding of unbleached protein, it is given by
where
Beq is the fraction of the protein that is bound to other structures in the photobleached region at equilibrium, and
koff is the dissociation constant for the binding. Sometimes there are multiple binding states in which case there are just more exponential terms of the same form. Many FRAP recoveries are not dominated overwhelmingly by just diffusion or just binding, so their curves are more complex; FRAP recoveries are analyzed in much more detail in Sprague and Pego et al. (see References below).
See also
- Fluorescence microscope
A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.- Technique :In most cases, a component of interest in the specimen is...
- Photobleaching
Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
- Fluorescence loss in photobleaching
Fluorescence Loss in Photobleaching, or FLIP, is a technique in fluorescence microscopy which can be used to examine the movement or diffusion of molecules inside cells or membranes. Typically a cell membrane is labelled with a fluorescent dye, and a specific area of the labeled membrane is ...
(FLIP)