Fluorescence loss in photobleaching

A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope.

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.

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.

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.

The sooner a part of the cytoplasm is used in the shuttling process, the more rapidly it experiences complete loss of fluorescence.

However, if shuttling occurs slowly, the fluorescence levels will remain unaffected or decrease only slightly.

The major difference between these two microscopy techniques is that FRAP involves the study of a cell’s ability to recover after a single photobleaching event whereas FLIP involves the study of how the loss of fluorescence spreads throughout the cell after multiple photobleaching events.

Another important difference is that in FRAP, there is a single photobleaching event and a recovery period to observe how well fluorophores move back to the bleached site.

However, in FLIP, multiple photobleaching events occur to prevent the return of unbleached fluorophores to the bleaching region.

In these experiments, it is also key to have a control group in order to adjust the results and correct the recovery curve for the overall loss in fluorescence.