Fluorescence loss in photobleaching
Encyclopedia
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 cell
s or membrane
s. Typically a cell membrane is labelled with a fluorescent dye
, and a specific area of the labeled membrane is bleached
using the beam from a confocal laser scanning microscope. The fluorescence intensity from that region of the membrane is measured over time. Motion of fluorescent molecules into and along the membrane slowly restores the fluorescence in the bleached region, while depleting the fluorescence in other regions (by exchange of bleached for unbleached fluorophores).
Measurement of the rate of this recovery provides an estimate of the lateral membrane fluidity. Changes in the size and shape of the bleached region can also indicate directional flow along the cell membrane.
FLIP is also useful in verifying the continuity of membranous organelles (e.g., the Golgi apparatus
). A small circumscribed region of the organelle is continuously bleached. As fluorophores diffuse along the membrane into the illuminated spot, they are bleached; eventually, the fluorescence of the entire organelle is depleted.
It is closely related to another technique, Fluorescence recovery after photobleaching
(FRAP). The difference between FLIP and FRAP is that FLIP follows the path of the bleached fluorophores, while FRAP follows the recovery of the bleached region.
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s or membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
s. Typically a cell membrane is labelled with a fluorescent dye
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
, and a specific area of the labeled membrane is bleached
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...
using the beam from a confocal laser scanning microscope. The fluorescence intensity from that region of the membrane is measured over time. Motion of fluorescent molecules into and along the membrane slowly restores the fluorescence in the bleached region, while depleting the fluorescence in other regions (by exchange of bleached for unbleached fluorophores).
Measurement of the rate of this recovery provides an estimate of the lateral membrane fluidity. Changes in the size and shape of the bleached region can also indicate directional flow along the cell membrane.
FLIP is also useful in verifying the continuity of membranous organelles (e.g., the Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
). A small circumscribed region of the organelle is continuously bleached. As fluorophores diffuse along the membrane into the illuminated spot, they are bleached; eventually, the fluorescence of the entire organelle is depleted.
It is closely related to another technique, Fluorescence recovery after photobleaching
Fluorescence recovery after photobleaching
Fluorescence recovery after photobleaching 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 membrane...
(FRAP). The difference between FLIP and FRAP is that FLIP follows the path of the bleached fluorophores, while FRAP follows the recovery of the bleached region.