a single region of the cell is bleached continuously to assess the direction of intracellular trafficking
The technique requires the preparation of cells labeled with fluorophores, usually GFP and related proteins, specific to the targets of interest. Continuous bleaching of a defined area of the cell requires precise targeting of laser illumination sufficient to inactivate all fluorophores entering the area. Time-lapse imaging may be used to capture the loss of fluorescence in the direction of transport. The terms FLIP and FLOP may be used also to describe the movement into (FLIP) and out (FLOP) of a region.
Similar to FRAP, FLIP is useful tool in understanding protein dynamics in living cells. It can be used to assess receptor motility, study signalling events and determine the continuity of membranes such as the endoplasmic reticulum.
FLIP can be carried out on any of Nikon's research-level inverted widefield epi-fluorescence microscopes used for live cell imaging or the FN1 electrophysiology microscope system. It is often carried out in association with FRAP and TIRF imaging. FLIP is also carried out on confocal microscope systems (A1R+, C2+, C2 si+) where optical sectioning allows fluorophore dynamics to be monitored in three dimensions and without the interference of out of focus light above and below the imaging plane. AOM and AOTF technology (available with A1R+, C2+, C2 si+ confocal systems) allows precise control of laser illumination.
The Ti inverted microscope is ideal for any live cell study. A modular design allows configuration with confocal (A1R+, C2+, C2 si+) systems allowing users to switch between widefield and confocal imaging modes. Time-lapse parameters can be defined using NIS-Elements, which also enables 3-D rendering, if required. Image resolution is enhanced through the use of specialist Plan Fluor, Super Fluor and Plan Apochromat VC objectives. Images may also be sharpened using deconvolution software.