Biological example - two-photon time-domain FLIM

2 Biological example - two-photon time-domain FLIM 

Cells transfected with GFP fused to the protein of interest can he excited hy two-photon absorption of near-infrared light. As mentioned in the previous section this method of excitation reduces photo-damage and acquires inherently z-sectioned, three-dimensional information. This is achieved without the use of confocal apertures as this nonlinear absorption process is confined to a single, diffraction-limited focal plane. Photodamage is thus minimized, permitting long periods of data acquisition. 

The next example illustrates how two-photon time domain FLIM can be used for detecting the interaction of two protein kinases PDKl (3,4,5-phosphoinositide protein kinase) and PKB (protein kinase B) at the plasma membrane of NIH3T3 cells. Both PDKl and PKB associate with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 via their plecktrin homology (PH) domains. It seems that this mutual interaction with such lipids leads these enzymes to co-localize at the plasma membrane and in turn to activate PKB. However, until recently it had not been shown that these molecules actually associate at the plasma membrane. 

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