Encyclopedia of Experiments: Biological Techniques
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Transcript
To study specific protein-protein interactions in live bacteria using fluorescence lifetime imaging microscopy, FLIM, combined with Förster resonance energy transfer, FRET, begin with a recombinant bacterial suspension. The bacteria express specific cytoplasmic proteins involved in metabolic pathways, labeled with donor and acceptor fluorescent proteins suitable for FRET.
Spot the bacterial suspension on an agarose pad, which immobilizes the bacteria on a microscope slide. Place a coverslip on the slide and seal.
Position the slide on the stage of a two-photon excitation inverted scanning microscope for FLIM-FRET microscopy. Use a fluorescence lamp to select and scan a region of interest on the bacterial monolayer.
The interaction between the expressed cytoplasmic proteins brings the donor and acceptor fluorescent proteins closer in appropriate orientation. Upon laser illumination with a suitable wavelength, the donor fluorescent protein gets excited.
The close proximity of fluorescent proteins triggers FRET, leading to non-radiative energy transfer from donor to acceptor fluorescent proteins, which gets excited and eventually returns to the ground state, emitting fluorescence.
The non-radiative energy transfer reduces the time taken for the excited donor fluorescent protein to return to its ground state, the donor fluorescence lifetime — measured by FLIM. The decrease in the donor fluorescent protein's mean fluorescence lifetime owing to FRET confirms the protein-protein interaction in the bacteria.
