Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/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  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.