Fluorescence loss in photobleaching

Fluorescence Loss in Photobleaching (FLIP) is a fluorescence microscopy technique used to examine movement of molecules inside cells and membranes. A cell membrane is typically labeled with a fluorescent dye to allow for observation. A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope. After each imagining scan, bleaching occurs again. This occurs several times, to ensure that all accessible fluorophores are bleached since unbleached fluorophores are exchanged for bleached fluorophores, causing movement through the cell membrane. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole.

Before photobleaching can occur, cells must be injected with a fluorescent protein, often a green fluorescent protein (GFP), which will allow the targeted proteins to fluoresce and therefore be followed throughout the process. Then, a region of interest must be defined. This initial region of interest usually contains the whole cell or several cells. In FLIP, photobleaching occurs just outside the region of interest; therefore a photobleaching region also needs to be defined. A third region, the region where measurement will take place, needs to be determined as well. A number of initial scans need to be made to determine fluorescence before photobleaching. These scans will serve as the control scans, to which the photobleached scans will be compared later on. Photobleaching can then occur. Between each bleach pulse, it is necessary to allow time for recovery of fluorescent material. It is also important to take several scans of the region of interest immediately after each bleach pulse for further study. The change in fluorescence at the region of interest can then be quantified in one of three ways. The most common is to choose the location, size and number of the regions of interest based on visual inspection of the image sets. The two other, rather new but more reliable approaches are either by detecting areas of different probe mobility on an individual image basis or by physical modeling of fluorescence loss from moving bodies.

Loss of fluorescence is defined by the mobile fraction, or the fraction of fluorophores capable of recovering into a photobleached area, of the fluorescently labeled protein. Incomplete loss of fluorescence indicates that there are fluorophores that do not move or travel to the bleached area. This allows for definition of the immobile fraction, or the fraction of fluorophores incapable of recovering into a photobleached area, of fluorescent-labeled proteins. Immobility indicates that there are proteins that may be in compartments closed off from the rest of the cell, preventing them from being affected by the repeated photobleaching.

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